[From the U.S. Government Printing Office, www.gpo.gov]



               The 1990 State/Federal
     Natural Resource Damage Assessment
                and Restoration Plan
           for the Exxon Valdez Oil Spill
            Volume 1: Assessment and Restoration Plan
                      Appendices A, B, C

















                                                              August 1990





          Dear Reviewer:

          This document describes the second year of studies being undertaken
          to determine the injury to natural resources resulting from the
          Exxon Valdez oil spill. These studies are being conducted by the
          State of Alaska and the United States to assess related damages and
          develop restoration plans.

          The 1990 plan has benefitted greatly from the many thoughtful
          public comments on the "State/Federal Natural Resources Damage
          Assessment Plan for the Exxon Valdez Oil Spill, August 1989. 11 The
          current plan was assembled through the-cooperative efforts of the
          State of Alaska acting through the Department of Fish and Game and
          the United States acting through the Federal Departments of
          Agriculture and the Interior, the National Oceanic and Atmospheric
          Administration, and the U.S. Environmental Protection Agency.

          Public comment on this document will assist the Trustee Council in
          developing future injury assessment and restoration efforts.

          Questions concerning the plan and its distribution should be
          directed to U.S. Department of Agriculture, Forest Service Public
          Affairs Office (907) 586-8806.

          Comments should be received by October 15, 1990, at the following
          address:

                               Trustee Council
                               P. 0. Box 20792
                               Juneau, AK 99802










          We appreciate your interest and look forward to your participation
          in this important process.

          Sincerely,




                                                    Walter Stieglitz
          Commissioner                              Director
          Alaska Department of Fish and Game        Alaska Region
                                                    Fish and Wildlife Service
                                                    Department of the Interior


                                                         n P'
                                                     teve JSoyer
          Regional Forester                         Director
          Alaska Region                             Alaska Region
          Forest Service                            National Marine Fisheries
          Department of Agriculture                 Service
                        r
                                                     teve


































                       THE 1990 STATE/FEDERAL NATURAL RESOURCE
                       DAMAGE ASSESSMENT AND RESTORATION PLAN
                            FOR THE EXXON VALDEZ OIL SPILL












                                     TABLE-OF CONTENTS






           INTRODUCTION  ..................................................      1


           PART I

           Injury Determination/Quantification,

                  Coastal Habitat Injury Assessment  .........................  10
                  Air/Water Injury Assessment  ...............................  21
                  Fish/Shellfish Injury Assessment  ..........................  52
                  Marine Mammal Assessment  .................................. 199
                  Terrestrial Mammal Injury Assessment  ..................... 245
                  Bird Injury Assessment  ................................... 272
                  Historic Properties and Archaeological Resources  .......... 308
                  Technical services  ....................................... 311


           PART II


           Economics  ......................................................  318


           PART III

           Restoration Planning    .........................................  333

           PART IV


           Budget  ........................................................   354

           APPENDICES

           A.     Quality Assurance/Quality Control
           B.     Histopathology Procedures
           C.     Glossary of Terms and Acronyms
           D.     Response to Comments on 1989 Plan
                  (Bound Separately)











                                   INTRODUCTION



           The March 24, 1989, grounding of the tanker Exxon Valdez in
           Alaska's Prince William Sound caused the largest oil spill in U.S.
           history. Approximately 11 million gallons of North Slope crude oil
           moved through the southwestern portion of the Sound and along the
           coast of the western Gulf of Alaska (see map, Fig. 1). The spill
           resulted in injury to fish, birds and mammals and a variety of
           other forms of marine life and habitats.

           This plan describes the second year of the process by which damages
           will be assessed so that funds to restore impacted resources or the
           services the resources provided, can be sought from those
           responsible for the Exxon Valdez oil spill (EVOS). The State of
           Alaska acting through the Alaska Department of Fish and Game
           (ADF&G) and the United States acting through the federal
           Departments of Agriculture (DOA), Commerce (DOC), through the
           National oceanic and Atmospheric Administration (NOAA), and
           Interior (DOI) are acting together as Natural Resource Trustees as
           provided by the Comprehensive Environmental Response, Compensation,
           and Liability Act (CERCLA), and the Clean Water Act (CWA), and
           other state and federal authorities. The Environmental Protection
           Agency (EPA) is assisting in damage assessment and is coordinating
           the federal restoration efforts with those of the State of Alaska.

           The 1990 damage assessment studies plan builds on the 1989 damage
           assessment studies.   These studies-are designed to determine the
           nature and extent of the injuries, loss or destruction to resources
           and will lead to a determination of damages.     The assessment of
           damages for injury to natural resources requires consideration of
           (1) the nature of the resources at risk, (2) the nature of the oil
           in the aquatic environment, (3) the exposure of the resources to
           the oil, and (4) oil-related damages to important resources. The
           data provides a base for developing a restoration plan.

           The purpose of determining damages--the estimated monetary value of
           the injured resources and the cost to restore those resources and
           the services they provided--is to pursue a claim against parties
           responsible for the spill.    Funds received as the result of the
           claim will be used to restore, replace or acquire the equivalent of
           the injured natural resources and services and to reimburse
           agencies for relevant costs incurred.      The U.S. Department of
           Justice and Alaska Department of Law represent the federal and
           state governments, respectively, in pursuits of claims.

           In 1989 the Trustees developed a damage assessment plan
           incorporating 63 studies in ten categories. The Trustee Council
           monitored the assessment process to ensure that study objectives
           were met.

           In order to identify studies that should be continued, terminated








          or new studies that should be initiated, the Trustee Council
          considered the extensive public comments on the initial plan, and
          consulted damage assessment investigators, other agency scientific
          staff, legal counsel, , and independent outside expert reviewers.
          The studies were evaluated from five perspectives: (1) immediate
          injury, (2) long-term alteration of populations, (3) sublethal
          effects, (4) ecosystem-wide effects and (5) habitat degradation.
          As a result of the review, 47 of the studies were continued, 26 of
          the studies were discontinued or merged into other studies, and 4
          new studies were initiated (Table 1).      Many of the continuing
          studies were modified.

          The studies described in this plan fall into ten categories: (1)
          Coastal Habitat, (2) Air/Water, (3) Fish/Shellfish, (4) Marine
          Mammals, (5) Terrestrial Mammals, (6) Birds, (7) Technical Services
          (including chemistry, histopathology, and an integrated geographic
          information system, complete with mapping) to support the resource
          studies, (8) Restoration, (9) Historic Properties and Archeological
          Resources, and (10) Economic Studies. The cost f or the studies f or
          the 1990 oil spill year (March 1, 1990       February 28, 1991) is
          approximately $37 million.

          The Coastal Habitat study measures spill-related changes in the
          supratidal, intertidal, and. shallow subtidal zones.          it is
          designed to document injury to resources that rely on these
          habitats, and to assess damages for the loss of services provided
          by these habitats.

          The Air/Water studies determine the distribution and composition of
          petroleum hydrocarbons or their environmental conversion products
          in water,, sediments, and living resources. Information gathered on
          the distribution and nature of the hydrocarbons and their
          conversion products provides a basis for documenting exposure and
          for determining injury to resources. The combined results of the
          Coastal Habitat and Air/Water studies also form a basis for
          estimating rates of recovery of natural resources and the potential
          for accelerating recovery.

          The Fish/Shellf ish studies focus on identifying potential injury to
          their various life stages in areas affected by the oil spill.
          Species were selected for study based on their respective niche or
          overall importance within the ecosystem, ability to be sampled, and
          the existence of an historic data base.

          Marine mammal studies include direct observations of injury (e.g.,
          through carcass counts) as well as estimates of population effects
          based on pathologic and toxicologic indicators (as is being
          undertaken with otters and seals). In addition, the direct
          observational data allows for inferences to be made about injuries
          to populations.

          Terrestrial mammals near the coast may have been exposed to

                                           2








          hydrocarbons by breathing fumes and eating oiled carcasses or
          vegetation. The studies will determine the presence of hydrocarbons
          in tissues of dead animals, and the ef f ects, if any, of oil
          exposure on local populations of brown bears, Sitka black-tailed
          deer and river otters. Studies of reproduction in laboratory mink
          are also being conducted to serve as a model for assessing injury
          to other potentially affected species.

          The plan for determining injury to birds is organized into four
          units: (1) surveys and censuses, (2) raptors, (3) sea birds, and
          (4) waterfowl, shorebirds, and passerines.        The information
          obtained will contribute to an understanding of mortality,
          population changes, and other factors essential for the damage
          assessment process. Studies proposed for birds focus on improving
          the accuracy of mortality estimates and collecting data on survival
          and reproductive success in relation to exposure to hydrocarbons
          and conversion products. These and other data will be gathered on
          birds potentially most affected by the spill or best serving as
          indicators for impacts on other important ecological components.

          The technical services category includes activities which provide
          process support or information services to all studies in the areas
          of analytical chemistry, quality assurance/quality control for the
          damage assessment process, histopathology, and an integrated
          geographic information system, complete with mapping.

          The restoration plan describes the strategy and scope of the
          restoration process and feasibility studies planned for the second
          oil spill year. Restoration measures will be implemented as soon
          as it becomes ecologically feasible, appropriate methods are
          identified, and funds are available.

          Studies on historic properties and archaeological resources will
          proceed in    two steps:       (1)  inventory, description, and
          classification; and (2) qualitative and quantitative descriptions
          and measurements of changes detrimental to the archeological
          resources related to the spill.

          The value of lost or injured natural resources, and the goods and
          services they provide humans, are based on results from economic
          studies. In this regard, damages forming the basis of the
          Trustees' claim against the potentially responsible parties are
          calculated by considering (1) the reduction of these goods and
          services, including intrinsic values, resulting from the spill, and
          (2) the cost of restoring these goods and services to their
          pre-spill level, replacing them or acquiring their equivalent.

          The Trustees emphasized in a March 1990 letter to the Exxon
          Corporation and Exxon Shipping, Inc. their desire to place all
          state and federal injury assessment data in a public repository,
          providing that the two corporations do likewise. In a letter dated
          June 6, 1990 to the Trustees, Exxon proposed a more limited data

                                           3









            sharing arrangement.   On July 19, 1990, the Trustees and Exxon
            representatives discussed the creation of a public data repository.
            The parties agreed to establish a technical committee to seek
            agreement on an exchange of detailed study plans and to develop a
            plan based on an initial set of damage assessment studies that
            could be used as an example of how data would be placed in the
            repository.












































                                             4













                   TABLE ONE: STUDIES AUTHORIZED IN 1989 AND 1990




          Study
          Category       Number          Title               1989      1990


          Coastal          CH1      comprehensive              X         X
          Habitat                     Assessment

          Air/Water        AW1      Geographical Extent        X
                                       in Water

                           AW2      Injury to Subtidal         X         X
                                    Sediments

                           AW3      Hydrocarbons in            X         X
                                       Water

                           AW4      Injury to Deep             X        1/
                                       Water

                           AW5      Injury to Air              X

                           AW6      Oil Toxicity                         X

          Fish/Shellfish   FS1      Salmon Spawning            X         X
                                       Area Injury

                           FS2      Egg and Preemergent        X         X
                                       Fry Sampling

                           FS3      Coded-Wire Tagging         X         X

                           FS4      Early Marine Salmon        X         X
                                        Injury

                           FS5      Dolly Varden Injury        X         X

                           FS6      Sport Fishery              X
                                      Harvest & Effort

                           FS7      Salmon Spawning Area       X         X
                                      Injury, outside PWS

                           FS8      Egg & Preemergent Fry      X         X
                                      Sampling, Outside PWS

                           FS9      Early Marine Salmon        X
                                      Injury, Outside PWS

                                           5















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                                                                                WOUF
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                                                                                AEFUCE:


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                                                                                                                                                   Refuges, and Critical 'Habitats
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           Study
           Category        Number          Title                 1989        1990


                             FS10     Dolly Varden & Sockeye       x
                                      Injury, Lower Cook Inlet

                             FS11     Herring Injury               x          x

                             FS12     Herring Injury,              x
                                        Outside PWS

                             FS13     Clam Injury                  x          x

                             FS14     Crab Injury                  x

                             FS15     Spot Shrimp  Injury          x          x

                             FS16     Injury to oysters            x

                             FS17     Rockfish Injury              x          x

                             FS18     Trawl Assessment             x          x

                             FS19     Larvae Fish Injury           x

                             FS20     Underwater Observations      x

                             FS21     Clam Injury, Outside PWS     x          2/

                             FS22     Crab Injury, outside PWS     x          x

                             FS23     Rockfish Injury,             x          3/
                                        Outside PWS


                             FS24     Trawl Assessment,            x          x
                                        Outside PWS

                             FS25     Scallop Mariculture          x
                                        Injury

                             FS26     Sea Urchin Injury            x

                             FS27     Sockeye Over-Escapement                 x

                             FS28     Run Reconstruction                      x

                             FS29     Life History Modeling                   4/

                             FS30     Salmon Database Mgmt                    x

                                              6














         Study
         Category       Number          Title               1989      1990


         Marine          MM1       Humpback Whale             x         x
         Mammals
                         MM2       Killer Whale               x         x


                         MM3       Cetacean Necropsy          x

                         MM4       Sea Lion                   x         x


                         MM5       Harbor Seal                x         x


                         MM6       Sea Otter Impact           x         x

                         MM7       Sea Otter Rehabilitation   x         x

         Terrestrial     TM1       Injury to Sitka Black-     x         x
         Mammals                     Tail Deer

                         TM2       Injury to Black Bear       x         x

                         TM3       Injury to River Otter      x         x
                                     and Mink

                         TM4       Injury to Brown Bear       x         x

                         TM5       Injury to Small Mammals    x

                         TM6       Reproduction of Mink       x         x

         Birds           B1        Beached Bird Survey        x         x

                         B2        Censuses & Seasonal        x         x
                                    Distribution

                         B3        Seabird Colony Surveys     x         x

                         B4        Bald Eagles                x         x

                         B5        Peale's Peregrine Falcons  X         x

                         B6        Marbled Murrelets          x


                         B7        Storm Petrels              x

                         B8        Black-legged Kittiwakes    x


                                          7













         Study
         Category         Number        Title                 1989      1990


                          B9        Pigeon Guillemots           X

                          B10       Glaucous-winged Gulls       X

                          B11       Sea Ducks                   X          X


                          B12       Shorebirds                  X

                          B13       Passerines                  X          X

                          B14       Exposure to North           X
                                     Slope Oil

         Technical        TS1       Hydrocarbon Analysis        X          X
         services
                          TS2       Histopathology              X          X

                          TS3       Mapping                     X          X

         Archeology       ARCH1     Archeological Resources     5/         X


         Restoration      RP1       Restoration Planning        X          X

         Economics        ECON1     commercial Fisheries        X          X
                                    Losses

                          ECON2     Fishing Industry Costs      X          6/

                          ECON3     Bioeconomic Models          X          6/

                          ECON4     Public Land Effects         X          X

                          ECON5     Recreation Damages          X          X

                          ECON6     Subsistence Losses          X          X

                          ECON7     Intrinsic Values            X          X

                          ECON8     Research Program Effects    X          X

                          ECON9     Archeological Damage        X          X
                                    Quantification




                                           8









            1/  AW4 Combined with AW2
            2/  FS21 Combined with FS 13
            3/  FS23 Combined with FS17
            4/  FS29 Combined with FS28
            5/  Part of Econ. 9
            6/  Combined with Econ. I












































                                           9












































                                       PART I


                         INJURY DETERMINATION/QUANTIFICATION












           COASTAL HABITAT STUDY NUMBER I

           Study Title: Comprehensive Assessment of Injury to Coastal Habitats

           Lead Agency:   USFS

           Cooperating Agencies: NOAA, DEC, NPS, FWS, ADF&G, DNR


                                       INTRODUCTION

           The purpose of the Coastal Habitat Injury Assessment is to document
           and quantify injuries to biological resources found in the shallow
           subtidal, intertidal, and supratidal zones throughout the shoreline
           areas affected by EVOS.

           Study sites are selected and ground-truthed during Phase I. Phase
           II is an intensive evaluation of the study sites to determine the
           extent of injury to natural resources. The objective of this study
           is to estimate the effects of various degrees of oiling on the
           quantity (abundance and biomass), quality (reproductive condition
           and growth rate), and composition (diversity and proportion of
           population) of key species in the critical trophic levels of
           coastal communities. These data are expected to provide evidence
           of injury to the overall health and productivity of these critical
           coastal habitats, and provide information necessary to the more
           species-specific studies on the effects of the oil spill on
           affected mammals, birds and fish that use these habitats.

                                         PHASE I

           This study uses a stratified random sample design to select basic
           experimental units called study sites.      Oiled study sites were
           selected from shorelines which were affected by the oil spill and
           control study sites were selected from shorelines which were not
           oiled. The shoreline was subdivided into ten strata; five habitats
           multiplied by two oiling types.     The study sites are grouped by
           strata within three geographic regions: Prince William Sound (PWS) ,
           Cook Inlet/Kenai Peninsula (CIK) , and Kodiak Archipelago/Alaska
           Peninsula (KAP). Consolidating a wide range of habitat and oiling
           characteristics into ten strata, coupled with the relatively coarse
           resolution of the available habitat and oiling data base, resulted
           in variances between ground-truthed classifications and mapped
           classifications. These variances were addressed by confining the
           additional, inductively selected sites in 1990 from the top 40
           randomly ranked sites in PWS and the top 50 ranked sites in CIK and
           KAP, respectively.    These sites now represent a simple random
           sample of the shoreline and will continue to be used to make
           inductive inferences to the universe of all possible sites. There
           are a total of 102 sites to be studied in 1990.

           Sites in the very lightly and lightly oiled strata are not included

                                            10









         for additional site selection or comprehensive sampling in 1990.
         This will enable the assessment,to focus on moderately and heavily
         oiled sites and their respective controls.

         Approximately 40 additional study sites will be deductively (non-
         randomly) selected in 1990 to provide additional spatial and
         habitat coverage in strata where a full array of inductive study
         sites could not be obtained in 1989.      Approximately 18 of the
         additional sites will be new control sites selected to match the
         physical and biological characteristics of existing inductively
         selected oiled sites. The site selection process for control sites
         in 1990 will be confined to those shorelines which match, as
         closely as possible, the biological and physical attributes of
         their respective oiled sites.

         In the CIK and KAP regions, additional sites (approximately 8) will
         be selected, where appropriate, from sites that were occupied by
         the National Park Service in 1989.    The remaining sites will be
         selected using the original data base, as supplemented with
         September oiling classifications, aerial photos, and additional
         ground-truthing.

                                     OBJECTIVES

         1.   To maintain a statistically valid study site selection
              strategy and identify additional study sites using existing
              map-based coastal habitat and oil impact classification
              schemes.

         .2.  To ground-truth potential study sites to evaluate map-based
              habitat and oil impact classifications.

         3.   To describe and mark approximately 45 study sites in addition
              to the 57 sites that have been identified for comprehensive
              sampling in 1990.



                                       METHODS

         Four sites, representative of habitat types appropriate to each
         region in the original stratified random sample for the 1989 data
         base and ranked moderately and heavily oiled, are selected for
         study in 1990.    Each site will be matched with a control site
         deductively chosen to approximate closely the physical and
         biological attributes of the oiled site. The selection of control
         sites will begin with the existing pool of randomly selected non-
         oiled sites that were identified and surveyed in 1989.
         Photographs, maps, and geomorphology and biological descriptions of
         the sites will be used as the bases for selection.

         if a suitable control cannot be found from the existing pool of
         1989 sites, candidate control sites will be identified by









         consulting the original data base and oiling classifications and by
         locating equivalent habitat types in closest proximity to the oiled
         sites. Approximately 5 candidate control sites will be identified
         for each oiled site.      Sites will be visited in order of their
         distance from the oiled site until a suitable match is found.
         Sites will also be examined for any physical characteristics unique
         to the oiled site that have not been accounted for in the five
         habitat types. In PWS, control sites must be located on islands to
         match oiled sites that are located on islands.

         Supplemental oiled sites and matched controls will be sought and
         added if there are fewer than three replicates of a particular
         habitat type in the existing sample within each region (excluding
         f ine-textured habitats in PWS) .        Supplemental sites will be
         deductively selected to include intertidal study sites occupied by
         the NPS in 1989, "set-aside" (untreated) sites in PWS, and areas
         which provide additional spatial distribution of a particular
         stratum in a region. Data from supplemental sites will not be used
         to expand inductively estimates of injuries to the universe of
         potential sites. Rather, it will be used to estimate the effects
         of oiling and treatment on a given habitat type.

         The methods for locating and marking additional study sites will
         follow the methods used in the 1989 reconnaissance survey.


                                    . BIBLIOGRAPHY

         Alaska Department of Environmental Conservation. 1989. Oil spill
               impact maps.    Unpublished preliminary data being developed
               under Air/Water Study Number 1.

         Cochran, William G. 1977. Sampling techniques. Third Edition.
               John Wiley and Sons. New York. Chapter 11, pp. 292-324.

         Environmental Systems Research Institute            (ESRI).       ARCINFO
               geographic systems software.          Version 5.0.         Redland,
               California.

         Gundlach, E.R. and M.O. Hayes.       1989.   Vulnerability of coastal
               environments to oil pollution. 12 Marine Technology Society
               Journal pp. 18-27.

         Hayes, M.O. and C.H. Ruby. 1979. Oil spill vulnerability index
               maps, Kodiak Archipelago. Unpublished maps. 47 leaves.

         Hayes, M.O., E.R. Gundlach, and C.D. Getter. 1980. Sensitivity
               ranking of energy port shorelines. Proceedings of a specialty
               conference on ports.     American Society of Civil Engineers.
               New York. Pp. 697-709.



                                            12










            Research Planning Institute (RPI), Inc. 1983a. Sensitivity of
                 coastal environments and wildlife to spilled oil, Prince
                 William Sound, Alaska, and atlas of coastal resources.
                 Prepared f or National Oceanic and Atmospheric Administration.
                 Office of Oceanography and Marine Services, Seattle,
                 Washington. 48 leaves.

            Research Planning Institute (RPI), Inc. 1983b. Sensitivity of
                 coastal environments and wildlife to spilled oil, Shelikof
                 Strait Region, Alaska, and atlas of coastal resources.
                 Prepared for National Oceanic and Atmospheric Administration,
                 Office of Oceanography and Marine Services, Seattle,
                 Washington. 431eaves.

            Research Planning Institute (RPI), Inc. 1985. Sensitivity of
                 coastal environments and wildlife to spilled oil, Cook
                 Inlet/Kenai Peninsula, Alaska, and atlas of coastal resources.
                 Prepared for National Oceanic and Atmospheric Administration,
                 Office of Oceanography and Marine Assessment, Seattle,
                 Washington. 64 leaves.

            Research Planning Institute (RPI), Inc. 1986. Sensitivity of
                 coastal environments and wildlife to spilled oil, Southern
                 Alaska Peninsula, an atlas of coastal resources. Prepared for
                 National Oceanic and Atmospheric Administration, National
                 Ocean Service, Alaska Office and U.S. Department of the
                 Interior, Minerals Management Service, Alaska OCS Region. 69
                 leaves.


            BUDGET: ADF&G


            salaries                        $     14.7
            Travel                                 3.0
            Contracts                            131.5
            Equipment & supplies                   7.5

            Total                           $ 156.7
















                                              13












                                        PHASE II

                             PART A: Injury Determination

           Coastal habitats are unique areas of high productivity supporting
           a diverse array of organisms, including many commercially and
           ecologically important species.   These habitats are particularly
           vulnerable to oil spill impacts because of the grounding of oil in
           the intertidal zone, the persistence of oil in intertidal and
           subtidal sediments, and the effects of associated clean-up
           activities.

           Oil may affect coastal organisms directly by coating or ingestion,
           with toxic effects leading to death or reproductive failure.
           Indirectly, oiling may cause decreased productivity, accumulation
           of toxic effects through the food chain, and loss of microhabitat
           such as algae beds.    Assessment of injuries to coastal habitat
           resources and determination of rates of recovery require
           consideration of the various coastal geomorphologic types, the
           degree of oiling, the affected habitat, and their trophic
           interactions. coastal habitats consist of three interactive zones
           (supra-, inter-, and subtidal) . Animals may use multiple zones,
           necessitating a coordinated study of the effects of oiling over the
           entire habitat. The complexity of this system requires expertise
           in many disciplines. Therefore, an interdisciplinary team with the
           appropriate expertise, including plant and systems ecology, marine
           biology, and statistical analysis, has been established.

           Initial field studies were completed by November 1, 1989.
           Processing of samples and data analysis is being conducted to
           determine the variance and magnitude of changes between non-oiled
           and moderately and heavily oiled sites.

                                       OBJECTIVES

           A.   Estimate the quantity (abundance and dry weight biomass),
                quality (reproductive condition and growth rate), and
                composition (diversity and proportion of standing crop) of
                critical trophic levels (and subsequent impact on trophic
                interactions) in moderately and heavily oiled sites relative
                to non-oiled sites.

           B.   Estimate hydrocarbon concentrations in sediments and soils.

           C.   Establish the response of these parameters to varying degrees
                of oiling and subsequent clean-up procedures.

           D.   Extrapolate impact results to the entire spill-affected area.

           E.   Estimate the rate of recovery of the habitats studied and
                their potential for restoration.


                                           14









         F.   Provide linkages to other studies by demonstrating the
              relationships between oil, trophic level impacts, and higher
              organisms.

                                      METHODS

         Vertical transects will be established at each of the study sites
         selected in Phase I. Work will be conducted along these transects
         in the supratidal and intertidal zones.       For this study, the
         intertidal extends from the 11011 tide mark to Mean High High Water
         (MHHW), and the supratidal is from MHHW or where terrestrial
         vegetation begins (if below MHHW) to the highest extent of possible
         oil occurrence. The intertidal transects will be extended into the
         supratidal zone at locations in the KAP where coastal plant
         communities occur.    Primarily, this will be in fine-textured,
         coarse-textured, and sheltered estuarine habitats. Beach sediment
         texture will be determined as part of Phase I.             Community
         composition, cover, and standing crop by trophic level will be
         estimated. Key species (dominant producers and food sources) will
         be determined and studied according to the methods listed below, to
         estimate the quantity, quality, and composition at each trophic
         level, and to collect samples for determination of hydrocarbon
         contamination.   Sediment samples will be collected by DEC for
         analyses of hydrocarbon composition and changes in concentration
         over time. Using a geographic information approach, the impact (by
         habitat type and degree of oiling) over the entire area affected by
         the oil spill will be integrated and field-verified.

         In 1990, sampling in the supratidal zone will occur in the KAP
         where there is extensive vegetation and sufficient wave exposure to
         move oil above the tide line.

         Subtidal sites will be selected independent of the       supra- and
         intertidal sites.    Subtidal sites will include three physio-
         geographic types:   bays, points and runs (straight lines).      The
         physio-geographic areas will be further divided into three habitat
         types: Nereocystis beds, Zostera beds, and Laminaria beds. These
         will be further divided into strata selected according to
         ecological importance, potential impact, and extent of habitat
         within the oiled region.

         Specific methods for each component of the study were developed as
         follows:


              Coastal
                   1.   Initial Site Survey
                   2.   Locating Transects
                   3.   Sample Identification and Chain of Custody

              Supratidal
                   1.   Quadrant Location
                   2.   Determination of Plant Productivity

                                          15









                              a.   Analysis of Vegetation Nutrient
                                   Content
                              b.   Analysis of In Vitro Digestibility
                   3.   Analysis of Soil/Sediment Microbial Activity
                   4.   Sampling of Soils and Sediments for
                        Hydrocarbon Concentration

               Intertidal


                   Invertebrates
                   1.   Locating 1 Quadrants
                   2.   Swath Surveys
                   3.   Reproductive Condition
                   4.   Growth and Survivorship
                   5.   Hydrocarbon Sampling Procedures
                   6.   Experimental Work
                   7.   General Laboratory Sorting Procedures
                   8.   Subsampling of Intertidal Samples
                   9.   Processing of Histological Samples

                   Fish
                   1.   Locating Transects
                   2.   Locating Quadrants
                   3.   Sampling Quadrants
                   4.   Minnow Trap Sampling
                   5.   Sample Storage and Identification
                   6.   Fish for Hydrocarbon Analysis

                   Plants
                   1.   Introduction
                   2.   Study Plan
                        a.    Stratified Sampling
                        b.    Site Experiments at Selected
                              Habitats
                        C.    Field Experiments

              Subtidal
                   1.   Sampling
                   2.   Field Schedule
                   3.   Laboratory Procedure for Benthic
                        Invertebrates
                   4.   Hydrocarbon Sampling Procedures
                   5.   Data Analysis

         Analysis of samples obtained in 1989 is still underway and will
         continue as additional samples are collected. Samples from 1990
         will be processed as rapidly as possible after they are returned
         from the field.   The data from all of the component studies are
         being entered into the INGRES database management system.       This
         system is widely used, and has good data security features. Use of
         this data base system will therefore maximize both internal
         integration and availability of the data to related damage

                                          16









            assessment projects.

                                         BIBLIOGRAPHY

            AOAC. 1980. official Methods of Analysis of the A.O.A.C., 13th ed.
                  Chipperfield, P.N.J. 1953. Observations on the breeding and
                  settlement of Mytilus edulis (L.) in British waters. J. Mar.
                  Biol. Ass. U.K. 32:449-476.

            Johnson, R.D. and H.L. Bergman.      1984. Use of histopathology in
                  aquatic-toxicology: A Critique. Pp. 19-36. In Containment
                  Effects on Fisheries, V.W. Cairns, P.V. Hodson and J.0.
                  Nriagu, eds. John Wiley and Sons.

            A.L. Page, R. H. Miller, and D.R. Keeney (eds.). 1982. Methods of
                  Soil Analysis, Part 2.          Chemical and Microbiological
                  Properties. Second Edition. Am. Soc. Agron.

            Ropes, J.W. 1968. Reproductive cycle of the surf clam, Spisula
                  solidissima, in offshore New Jersey. Biol. Bull. 135:349-365.

            Seed, R. 1969. The ecology of Mytilus edulis L.
                  (Lame 11 ibranch iata) . I. Breeding and Settlement. Oecologia.
                  3:277-350.

            Sheehan, D.C. and B.B. Hrapchak. 1980. Theory and Practice of
                  Histotechnology. 2nd Ed. C.V. Mosby Co.

            Tietge, J.E., R.D. Johnson and H.L. Bergman. 1988. Morphometric
                  changes in gill secondary lamellae of brook trout (Salvelinus
                  fontainalis) after long-term exposure to acid and aluminum.
                  Can. J. Fish Aquat. Sci. 45: 1643-1648.

            Tranter, D.J. 1958. Reproduction in Australian pearl oysters. II.
                  Pinctada albina (Lamarck):      gametogenesis.     Aust. J. Mar.
                  Freshwtr. Res. 9: 144-158.

            United States Department of Agriculture Research Service.           1970.
                  Forage Fiber Analysis. Agric. Handbook No. 379.

            Wilson, B.R. and E.P. Hodgkin. 1967. A comparative account of the
                  reproductive cycle of 5 species of marine mussels (Bivalvia:
                  Mytilidae) in the vicinity of Freemantle, W. Australia. Aust.
                  J. Mar. Freshwtr. Res. 18: 175-203.












                                               17












                 PART B: PRE-SPILL AND POST-SPILL CONCENTRATIONS OF
          HYDROCARBONS IN SEDIMENTS AND MUSSELS AT INTERTIDAL SITES WITHIN
                    PRINCE WILLIAM SOUND AND THE GULF OF ALASKA.

          Damage assessment of the oil spill in PWS and the GOA requires
          information on hydrocarbon contamination levels in water, sediment
          and biota prior to the spill (baseline), and at various times after
          the spill in order to determine the potential impact and duration
          of impact.    Hydrocarbon baseline information is available for
          several sites in PWS prior to oil transport and for the first 4
          years of oil shipment.    The intertidal baseline for hydrocarbon
          levels in mussels, sediment, water, and fish had been established
          at 10 sites from 1977 to 1981.       All sites are located on low
          energy, low gradient beaches, often at the head of embayments, and
          most sediments transects are associated with eel grass. All sites
          have adjacent bands of mussels (Mytilus trossulus).

          Because of the potential persistence of hydrocarbons in sediments
          in temperate and subarctic intertidal and subtidal environments,
          sampling may be continued to document depuration and recovery.
          Concentrations of the full range of individual aliphatic and
          aromatic hydrocarbons in sediments and mussels from intertidal
          sites will be reported. Abundance of mussels and other epifauna
          along sediment and mussel transects will be photographically
          recorded during each sampling period. These data will provide a
          basis for estimating temporal and spatial impact to other biota of
          the nearshore environment.


                                       OBJECTIVES

          A.   Sample and estimate hydrocarbon concentrations in mussels and
               sediment from 20 sites within 10% of the actual concentration
               95% of the time, when total aromatic concentrations are
               greater than 200 ng/g dry wt.

          B.   Test the null hypothesis that hydrocarbon contamination of
               sediments and mussels is the same for the pre-spill and post-
               spill period.

          C.   Document changes in abundance and distribution of intertidal
               epifauna and test the null hypothesis that no differences
               occur at oiled and non-oiled sites.


                                        METHODS

          Ten intertidal sites in PWS and Port Valdez were sampled for
          sediments, mussels, water, and fish annually from 1977 to 1981 to
          establish a baseline against which future changes in hydrocarbon
          concentrations can be compared.    Sites were initially sampled in
          spring, summer and fall to determine if short-term changes occurred
          during the warm season.    These sites were resampled in March of
          1989, immediately before several of them were impacted by the EVOS.

                                           18










         Ten additional sites were established to cover areas in the
         trajectory of the oil path. Four of these sites were on the Kenai
         Peninsula (KP) and the remaining six were in PWS.       Sediment and
         mussel samples were taken. Photo documentation was initiated along
         mussel and sediment transects at each site. These sites were re-
         sampled several times during the summer of 1989 to document the
         appearance of and changes in hydrocarbon contamination from the
         EVOS.

         Sediments: Transect lines thirty meters (m) in length are located
         parallel to the water line at -0.75 m to +0.75 m (depending on
         specific site). Sediment samples will be collected in triplicate
         at each site by compositing 10 cores (dia 3.2 cm x depth 1.25 cm)
         taken at random along a 30-meter transect for each sample.
         Composite sediments will be placed in chemically clean 4-oz. jars,
         *placed in an ice chest with artificial ice and transported. These
         will be frozen within 2-3 hours of collection. one blank sample
         will be taken at each site.

         Mussels: These transects are located in mussel bands, parallel to
         the water line, usually just above (+1 m tide level) the sediment
         transects. Triplicate mussel samples will be collected by taking
         approximately 30 2-5 cm. mussels (enough to produce >10 gms tissue)
         at random along the 30-meter transect. Samples in 16 oz. jars will
         be cooled, transported and frozen in the same manner as the
         sediment samples.

         Photo Documentation: Close-range views will be photographed of the
         strata, macroflora and epifauna. Photos will be taken every 4 or
         8 m along the sediment transect and every 2 or 4 m along the mussel
         transect line beginning at 1 meter. Macrophyte cover as well as
         epifaunal occurrence and density will be recorded from photographs
         taken of 625 cm2 quadrants placed along the sediment and mussels
         transect lines. A grid of 100 random dots projected on each slide
         will be used to estimate the occurrence and percentage of surface
         area covered by macrophytes and epifauna. Macrophytes and epifauna
         will be identified to species where possible.

         Data Analysis:

         Random sample and subsample collection will ensure that
         hydrocarbons present in the sample represent the average
         concentration at each site.           "Hot spots" of hydrocarbon
         concentration over the 30 meter transects should be canceled out by
         this procedure. Selected triplicate samples will be analyzed, the
         mean concentrations and deviations from these means determined, and
         appropriate statistical tests applied - either ANOVA or paired
         comparisons (Tukey or Shef f e I tests). Digital tables of individual
         hydrocarbons will be reported.

         Macrophyte and epifauna occurrence and cover will be analyzed using
         one-way ANOVA or paired comparisons (oiled versus non-oiled where

                                           19









         strata are similar).      They will be tested at the .05 level of
         significance.

                                      BIBLIOGRAPHY

         Connell, Joseph H. 1970.     A predator-prey system in the marine
               intertidal region. 1. Balanus qladula and several predatory
               species of Thais. Ecol. Monog. 40:49-78.

         Gundlach, Erich R., Paul D. Boehm   I Michel Marchand, Ronald M.
               Atlas, David M. Ward, and Douglas Wolfe. 1983. The fate of
               Amoco Cadiz oil. Science 221:122-129.

         Karinen, John F., L. Scott Ramos, Patty G. Prohaska, and William D.
               MacLeod, Jr. In Preparation. Hydrocarbon distribution in the
               marine environment of Port Valdez and Prince William Sound,
               Alaska.

         Warner, J.S. 1976. Determination of aliphatic and aromatic
               hydrocarbons in marine organisms. Anal. Chem. 48:578-583.



         BUDGET: USFS


         PART A:


         Salaries                    $    56.0
         Travel                           14.0
         Contracts                     8,818.0
         Equipment                        65.0

         Total                       $ 8,953.0



         PART B:


         Salaries                    $    34.0
         Travel                           36.0
         Contracts                        82.0
         Equipment  & Supplies              8.0

         Total                       $    160.0


         BUDGET SUMMARY


         ADF&G                       $    156.7
         USFS                          9,113.0

         Total                       $ 9,269.7




                                            20










                          AIR/WATER RESOURCES INJURY ASSESSMENT


           The evaluation of injury to air, water, and sediment resources is
           a critical component in assessing the overall damage to natural
           resources caused by the EVOS. Three studies addressing impacts on
           water column and bottom sediments will continue in the second year
           of the damage assessment effort. Air resources were studied in the
           first year immediately after the oil spill, but will not be
           continued because of the short term impact to the air resource.


           Water and Sediment Resources

           Assessment of the concentrations,of petroleum hydrocarbons in the
           water column of PWS and the Kenai Fjords region began almost
           immediately after the EVOS.     oil affected pelagic and nearshore
           waters, benthic sediments, intertidal habitats and adjoining
           habitats above high tide. Quantifying hydrocarbon levels in the
           water column was most critical during the first few weeks following
           the EVOS, when dissolution of soluble components was most rapid and
           the likelihood of toxic exposure was highest. As wind and current
           spread the oil and carried it farther away from the spill site,
           concern shifted from immediate impacts in the water column to
           longer-term effects from shoreline oiling on nearshore and subtidal
           sediments, and to chronic low-level hydrocarbon contamination of
           the water column.

           Marine water quality is protected under state and federal water
           quality standards which include classifications for such uses as
           growth and propagation of fish and wildlife, aqua'culture, and human
           uses such as recreation. Moreover, State of Alaska water quality
           standards for petroleum hydrocarbons establish criteria for water
          .habitats.                                 I

           The three water and sediment studies funded for this year are
           designed   to reveal the continuing extent of hydrocarbon
           contamination remaining after the spill.         Chronic, low-level
           contamination of the water column is expected to continue through
           the bleeding-off of oil from impacted shorelines. Nearshore and
           offshore sediments continue to be exposed to further contamination
           from the suspension and sinking of oily beach materials.
           Documenting the extent of continuing contamination will assist in
           demonstrating injury to the water resource along with chemical
           exposure of marine mammals, birds, intertidal and shallow subtidal
           communities, fisheries, and terrestrial mammals dependent on beach
           habitats.

           The Air/Water (A/W) studies are integrated with the Coastal Habitat
           studies to provide data on injury to habitats for other studies
           that address injury to biological resources. A/W studies will also
           help establish the basis for restoration.

                                            21









           The three continuing water quality studies focus on:

           1.   Petroleum hydrocarbon-induced injury to subtidal marine
                sediment resources and injury to benthic infauna.

           2.   Geographic and temporal distribution of dissolved and
                particulate petroleum hydrocarbons in the water column.

           3.   The toxicity of weathered oil and the fate and effects of oil
                transformation compounds within the marine environment.

           A/W Study 1, which is not continued in 1990, documented the extent
           of the surface oiling, and mapped the results for use by other
           studies.    Further oil fingerprinting will be conducted under
           response activities, or by Technical Services Study Number 1. A
           final report mapping the distribution of surface slicks in the
           first year of the spill will be produced this year.

           A/W Study 2 is now combined with elements of A/W Study 4 into one
           integrated sediment contamination study. This study will continue
           to document the presence, persistence, and chemical composition of
           petroleum hydrocarbons in subtidal marine sediments. These data
           will assist in quantifying injury to the sediment and will provide
           the chemical linkage needed to assess biological injury. Shallow
           subtidal   oil   concentrations    will   be   compared   with    oil
           concentrations in adjacent intertidal areas to better understand
           the fate of oil. This study will continue to determine the degree
           of injury to the benthic infaunal resource and the duration of any
           documented injury. Additionally, microbial screening techniques of
           subtidal sediments will be employed to determine the presence,
           toxicity, and degradation rates of oil. Sediment sampling stations
           extend outside PWS to include the Kenai Fjords, Katmai, Cook Inlet,
           Kodiak, and the Aleutian chain.

           A/W Study 3 will continue to document hydrocarbon concentrations in
           the water column at a range of depths and locations.       Trends in
           ambient water quality will be determined using the blue mussel as
           a biological indicator of low-level, chronic water quality
           contamination to supplement chemical measurements. Sediment traps
           will be deployed to measure sedimentation and associated
           hydrocarbon inputs to subtidal sediments.

           A/W Study 5 was completed.

           A/W Study 6 is a new study designed to address the concerns f or
           long-term contamination and toxicity of weathered oil and its
           degradation products to selected test organisms, and to integrate
           the results of several projects into a mass-balance budget for the
           distribution, transport, transformation, and persistence of spilled
           oil in Alaska coastal environments.




                                            22










          AIR/WATER STUDY NUMBER 2

          Study Title: Petroleum Hydrocarbon-Induced Injury to Subtidal
                        Marine Sediment Resources

          Lead Agency: NOAA, State of Alaska



                                      INTRODUCTION

          A proportion of -the oil that entered the water (either the original
          crude oil derived from the spill, oil leaching from contaminated
          shorelines, and/or oil dispersed into receiving waters via
          shoreline remediation procedures) probably has reached, or will
          reach, the bottom as a result of physical (Boehm et al. 1987) and
          biological processes.

          Benthic data collected in polluted waters elsewhere suggest that
          changes in number and diversity of species, as well as abundance
          and biomass of species, can be expected if sizable amounts of oil
          settle to the bottom.     These changes can have serious trophic
          implications since many subtidal benthic invertebrates are
          important food resources for bottom-feeding species such as
          pandalid shrimps, crabs, bottomfishes and sea otters. Further, the
          larvae of most benthic organisms in PWS move into the water column
          (in March through June) and are utilized as food by large
          zooplankters and larval and juvenile stages of pelagic f ishes,
          small salmon f ry, and herring. Thus, damage to the benthic system
          by hydrocarbon contamination can af f ect f eeding interactions of
          important species both on the bottom and in the water column.

          Continuation of this study will evaluate the extent of subtidal
          hydrocarbon contamination in PWS, along the LKP, and near Kodiak
          Island.  The purpose of this study will be to determine to what
          depth petroleum hydrocarbons have been transported over the winter
          months of 1989/90, to continue the time-course of data acquisition
          necessary to answer the question of persistence of petroleum
          hydrocarbons in subtidal sediments, and to determine the impact of
          oiling upon subtidal resources. Fewer sites will be studied this
          year. However, intensity of sampling will be increased.

          Three projects formerly funded under A/W Study 4 will be included
          in this study. The first enumerates hydrocarbon oxidizing bacteria
          and assesses the maximum potential for 'in situ' biodegradation of
          selected hydrocarbons at various sites within and outside of PWS.
          Coupled with data on ambient hydrocarbon concentrations, the
          microbial data will allow a gross estimate of the maximum possible
          rate of bacterial hydrocarbon oxidation 'in situ' to be made. The
          second project will screen sediments for petroleum hydrocarbons
          using ultra-violet fluorescence spectrophotometry and will assess
          the toxicity of marine sediments using the luminescent marine
          bacterium Photobacterium phosphoreum to test for aqueous toxicants

                                           23









        (Schiewe et al. 1985). The third project titled "Injury to Deep
        Benthos" will examine the injury, if any, to infaunal communities
        below a depth of 20m in bays adjacent to eel grass beds.           The
        sampling for all projects included in A/W Study #2 will be
        conducted from the same vessel and time (June, July). The present
        study will also coordinate closely with the subtidal project of the
        Coastal Habitat Study. Sediment and microbiological samples will
        be collected at the identical eelgrass sites where the Coastal
        Habitat study will sample shallow subtidal benthos.

                                      OBJECTIVES

        A.    Determine occurrence, persistence, and chemical composition of
              petroleum hydrocarbons in subtidal marine sediments.

        B.    Provide marine sediment data to assist agencies in mass
              balance calculations on the fate of oil in the marine
              environment.

        C.    Relate subtidal oil concentrations to adjacent intertidal
              concentrations.

        D.    Screen sediments for oil contamination and estimate the toxic
              ef f ects of petroleum hydrocarbons using bacterial bioassays of
              sediment samples collected from oiled and nonoiled habitats.

        E.    Enumerate hydrocarbon oxidizing bacteria and assess the
              maximum potential for 'in situ' biooxidation of selected
              hydrocarbon substrates in subtidal marine sediments at oiled
              and nonoiled sites within and outside of PWS.

        F.    Determine if changes occurred in the macro-benthos by
              comparing species richness, species diversity, general
              abundance and biomass, and trophic composition of the benthic
              biota living on similar substrata at approximately 40, 100,
              and >100m below sea grass beds between oiled and unoiled bays.

        G.    Determine if temporal changes will occur in the macro-benthos
              between oiled and unoiled bays by comparing species richness,
              species diversity, general abundance and biomass, and trophic
              composition of the benthic biota at specific stations.

        H.    If changes are detected in the infauna, examine the
              relationship between the accumulation and retention of
              hydrocarbons in sediments and the effect on the benthic biota.





                                       METHODS

        The methods employed by the three agencies cooperating in this

                                          24









           study are described separately below.

           National Marine Fisheries Service:

           Auke Bay Laboratory

           Sediments will be sampled at 16 sites in PWS (four reference sites
           and 12 contaminated sites).     Sampling will be conducted during
           three periods (May, June/July and September) . Six sites will be the
           same as those   to be sampled by the subtidal project of Coastal
           Habitat Study.  Outside PWS eight sites will be sampled. Six sites
           will be on the  Kenai Peninsula and two sites will be near Kodiak
           Island. These   sites will be sampled in July.

           Three samples, each a composite of eight subsa*mples collected
           randomly along  a 30 m transect laid parallel to the shoreline will
           be taken at each intertidal site. These samples will be collected
           at low tide or by divers. Intertidal collections will be made at
           a single tidal height in the range of +1 to -1 m relative to mean
           lower low water (MLLW) depending on the distribution of fine
           sediments.

           Subtidal sediment collections will be made at depths of 3, 6 and 20
           m below MLLW in May and September and at 3, 6, 20, 40 and 100 m in
           June/July. Collections at 3, 6 and 20 m will be made by divers on
           transects laid along the appropriate isobath and sampled in the
           same way as described above for the intertidal transects.         The
           subtidal project of Coastal Habitat Study Number I will sample
           sediments, infauna and epifauna in the same depth range at six of
           the PWS sites.     Samples taken at depths below 20 m will be
           collected with a Haps corer. A Smith-McIntyre grab will be used to
           sample those sediments which cannot be effectively sampled with the
           Haps corer.    Three cores will be taken at each depth.          Four
           subsamples will be removed at randomly selected points within each
           core. The subsamples will be combined to form one sample per core.
           The samples will be taken at the same sites as the benthos (see
           deep benthos sampling methods below) , however sediments will not be
           taken from the same core/grab as the benthos samples because the
           volume removed for sediment hydrocarbon analysis will jeopardize
           the quality of the benthos samples.

           Northwest Fisheries Center

           Surface sediment samples for establishing levels of petroleum
           hydrocarbon residues and sediment-associated toxicity will be
           collected June through July, 1990 (Table 1) . Sites will be located
           in potentially oil-impacted areas and also in unimpacted areas in
           PWS and LCI.

           Selected sediment samples will be analyzed for petroleum
           hydrocarbons and other organic contaminants.           After rapid
           extraction of sediments, relative aromatic hydrocarbon levels in

                                            25









          sediment extracts will be measured using liquid chromatography
          coupled to a fluorescence detector.     Sediment toxicity will be
          estimated using the Microtox bioassay that utilizes the luminescent
          marine bacterium Photobacterium Rhosphoreum (Schiewe et al. 1985).
          The test involves exposing suspensions of the bacterium to saline
          solutions of organic solvent extracts of sediment samples and
          measuring the effect of exposure on the amount of light emitted
          f rom the bacteria. The results of the test can be used to rank the
          relative toxicity of sediment samples. The relationship between
          Microtox results and contaminant levels in sediments will be used
          to provide support f or the rankings of sediment toxicity by the
          Microtox bioassay.

          Sampling activities will be conducted at 27 sites in PWS and LCI,
          including oiled and nonoiled sites (Table 1) .     Samples will be
          collected at water depths of 0 (intertidal), 3, 6, 20, 40, and 100
          meters. At each site, sediment samples will be collected with a
          box corer, Van Veen or Smith-McIntyre grab.         Each of three
          replicate sediment subsamples for each depth will be placed in two
          20 ml scintillation vials and stored at - 200 C. The coordinates
          and depths of each station will be recorded.

          Three sediment replicates are composited, the excess water is
          decanted and the sediment is stirred to homogenize and placed into
          a tared 100-ml centrifuge tube.         Sodium sulfate, methylene
          chloride, and activated copper are added.     Clumps are broken up
          with a tef lon stirring rod, if necessary.     Each tube is capped
          tight enough to prevent leakage.    Each mixture is placed in a
          sonic bath or sonicated with a sonic probe. The sonicated samples
          are centrifuged for 5 minutes at 1,500 rpms. Each extract is then
          decanted into 50-ml labeled concentrator tubes. To the sediment
          remaining in the centrifuge tubes, another 10 ml of methylene
          chloride is added.    The mixture is stirred with a teflon rod,
          capped, and sonicated in bath or with probe and centrifuged for 5
          minutes. The solution is decanted into the original concentrator
          tube, another 10 ml methylene chloride is added, stirred, and
          sonicated and centrifuged again as described previously. The third
          extract is added to the first two, a boiling chip is added and the
          solution concentrated to exactly 10 ml. The sample is divided into
          two 5.0-ml portions, one for HPLC screening and one for Microtox
          analysis.

          To the sediment portion for high pressure liquid chromatography
          (HPLC), polystyrene internal standard is added and about I ml of
          the mixture is transferred to a vial for the autosampler (the
          remainder is stored in another vial in the freezer).             The
          analytical procedure for the detection of aromatic hydrocarbons
          (AH) in the sediment is similar to that of Krahn et al. (1988a,b),
          but analytical columns, are used instead of preparatory columns.
          The sediment extract (150 ul) is injected onto the HPLC columns and
          isocratically eluted with methylene chloride.         The internal
          standard is detected with a UV detector and the aromatic compounds

                                           26









         with a fluorescence detector (phenanthrene wavelengths--260/380 nm
         and an additional wavelength pair to be selected). To quantitate
         the total Ahs, the total f luores6ence area is integrated during the
         time when the fraction would be collected for the prep cleanup
         (Krahn et al. 1988b) and converted to phenanthrene equivalents (the
         concentration of phenanthrene that would result in an equivalent
         integrated area) or to other AH equivalents, respectively (as
         determined from above).

         Initially, dose-response studies using oil and o i 1 -contaminated
         sediments will be evaluated by the Microtox bioassay in conjunction
         with the ultraviolet fluorescence (UVF) screening methods.
         Subsequently, five ml of each sediment extract in methylene
         chloride will be obtained as described. Samples (volumes will be
         determined in the dose-response studies) will be exchanged into 1
         ml of ethanol by solvent evaporation under constant heat. Microtox
         assays of the organic extracts will then be conducted as described
         in the Puget Sound Estuary Program protocols for sediment bioassays
         (EPA 1988).

         Alaska Department of Environmental Conservation:

         Microbiology sediment samples from the intertidal and shallow
         subtidal areas will be obtained from the shore parties and divers
         collecting    samples    for   hydrocarbon    chemistry     analysis.
         Microbiology samples from deeper subtidal areas will be obtained
         f rom the core or grab sampler at the same stations and times as
         those collected for chemistry analysis.

         Sediment samples for microbiological analysis will be collected in
         sterile Whirlpak bags as composites in triplicate along the same
         horizontal transects from which the chemistry samples are obtained.
         Care will be taken to avoid contamination of samples by the
         sampling personnel and cross-contamination between different
         sediment samples. Sampling apparatus should be thoroughly rinsed
         with water between samples and, where possible, disinfected with
         alcohol or other disinfectant. Samples obtained from the deeper
         water grabs will be collected from the center of the core to avoid
         surface contamination incidental to sample handling.

         Hydrocarbon biodegradation potential associated with sediment
         microbes will be assayed by adding radiolabelled aliphatic and
         aromatic substrates to sediment samples.     (14C) -hexadecane, (14C) _
         phenanthrene and  (14C) -benzo(a]pyrene will be the three hydrocarbons
         substrates used.         Each substrate will be monitored for
         biodegradation by the evolution of radio-C02 from the samples after
         two incubation periods.     The incubation periods will be chosen
         appropriately to show biodegradation activity for the given
         substrate (e.g., the benzopyrene incubations will be longer than
         for hexadecane).

         A total of 20 grams of sediment from each sample will be needed for

                                          27









         this assay.       Each sediment sample assayed for hydrocarbon
         degradation will first be mixed 1:10 with sterile seawater
         augmented with mineral nutrients. Ten ml aliquots of the resulting
         slurry will then be placed in sterile 40-ml incubation vials fitted
         with silicone septa.      For each substrate, on selected sediment
         samples, two concentrations will be used to investigate the effect
         of hydrocarbon concentration on biooxidation rates. The substrate
         of interest will be added at either 1 or 10 ppm (ug/ml slurry)
         concentrations by injection via syringe through the septa.           The
         substrates will be added in an acetone carrier (Bauer and Capone,
         1988).      Two replicate vials       for   each    substrate/sediment
         sample/incubation time combination will be prepared with a "time
         zero" killed control also prepared for each substrate and
         triplicate set. All vials will be placed on a rotatory shaker for
         24 hours and then incubated at ambient temperatures for the
         duration of the incubation period.

         Following incubation of the sample for the appropriate period (or
         initially in the case of the controls) , substrate biodegradation in
         the sample vials will be halted by the addition of 1 ml 1ON NaOH
         through the septum.     This will result in a Ph greater than 13,
         killing the culture of degraders and sequestering any evolved C02
         in the form of carbonates in solution. The extent of hydrocarbon
         degradation will be monitored by measuring the radio-CO evolved
         from each vial (Foght et al., 1989).         After transport to the
         analytical facility at the University of Alaska, the sample vial
         contents will be acidified by addition of concentrated HC1 via
         syringe through the septum. The headspace will be purged of radio-
         C02 and the effluent gas will be passed first through an organic
         vapor trap and then through phenethylamine scintillation cocktail
         to trap the evolved C02 (Fedorak et al., 1982). The mean of each
         set of biodegradation samples for each substrate, concentration and
         incubation period will be compared with the "time zero" controls to
         assess for losses due to volatilization in transit or any possible
         abiotic C02 evaluation.      The extent of biodegradation will be
         expressed as a percentage of the total radiocarbon added to the
         sample after correction for abiotic losses and ambient hydrocarbon
         concentrations.

         In addition to the biooxidation potential assay, populations of
         hydrocarbon oxidizing bacteria will be enumerated using a "dilution
         to extinction" technique.         The Most Probable Number (MPN)
         statistical enumeration technique, as modified for oil degrading
         bacteria and shipboard space constraints (e.g., using sterile, 24-
         well tissue culture plates), will be used.

         Aliquots of slurry taken from the dilution bottles generated for
         the biooxidation potential study will be serially diluted ten-fold
         several times, giving a range of dilutions from 1:10 to 1:109.
         Five replicate 100-ul aliquots of these dilutions will be placed
         into the microliter plates' wells filled with sterile, carbon-free
         marine broth, producing five identical inoculations of each

                                            28









           dilution. Then a "drop" of sterile Prudhoe Bay crude oil will be
           added to each inoculation well. The crude oil serves as the sole
           carbon source for any bacteria in the inoculum, selecting only
           those able to grow on crude oil. A positive indication of growth
           will be emulsification of the slick formed on addition of the oil
           to the wells. The most probable number of hydrocarbon degraders
           will then be calculated using a standard MPN table.

           University of Alaska:

           Five replicate samples will be taken at each of three stations
           within six bays identified as oil-exposed sites and at three
           stations within six bays determined to have been      uncontaminated
           (control) sites.     All stations sampled will be     at approximate
           depths of 40, 100, and >100m on a transect extending below seagrass
           (Zostera) beds within each of the identified bays. The intertidal
           and shallow subtidal stations on the transect will be sampled for
           biota.    A total of 36 deep stations x 5 replicates will be
           collected on a single cruise in early July in conjunction with
           microbiological and hydrocarbon sampling projects that will be
           underway from the same ship platform at the same or at
           approximately the same time. Benthic samples at oil-exposed and
           unexposed sites will be collected on bottoms that are as physically
           similar as possible, based on chart and fathometer data and
           preliminary grab samples to be made before actual sampling occurs.
           Considerable amounts of ship time might occasionally be required
           at some sites to ensure that similar bottom types are compared
           between oiled and control sites.

           The six oil-exposed sites that will be sampled for deep benthos are
           Northwest Bay, Disk Island, Herring Bay, Bay of Isles, Snug Harbor,
           and Sleepy Bay. The six unexposed (control) sites to be sampled
           are West Bay, Rocky Bay, Zaikof Bay, MacLeod Harbor, and two sites
           to be selected prior to the July cruise.

           The benthic biological samples from approximately 40, 100, and
           >100m will be collected with a O.lm2 van Veen grab weighted with
           31.7 kg of lead to facilitate penetration. Five replicate samples
           will be taken at all stations.     Material from each grab will be
           washed on nested 1.0 mm steel screens and preserved in 10%
           formalin-seawater solution buffered with hexamine.



                                       DATA ANALYSIS

           The null hypotheses to be tested will depend on which of the
           objectives listed above is under consideration.      In general, for
           sediment analyses the null hypothesis will state that the
           concentration of petroleum hydrocarbons at particular depths or the
           distribution of petroleum hydrocarbons with depth at oiled sites
           does not differ from that at reference sites.       All data will be
           tested for heteroscedasticity with Bartlett's test or equivalent.

                                             29









       Data will be reported as means and 95% confidence intervals
       calculated according to a standard formula (Sokal and Rohlf 1981).
       Parametric statistics (Model I analysis.of variance with site and
       depth as fixed factors and Scheffe's a posteriori test) will be
       used to test for differences in hydrocarbon concentrations between
       sites and depths if underlying assumptions of the parametric
       procedures are met (with data transformation if required),
       otherwise nonparametric tests (eg. the Kruskal-Wallis test) will be
       employed.   Key petroleum weathering and source ratios will be
       calculated (Boehm et al. 1987).

       The relationship of sediment toxicity to luminescent bacteria at
       the study sites and hydrocarbon concentrations determined in
       sediment will be compared statistically using appropriate tests
       (Sokal and Rohlf 1981). Where significant differences are found,
       the a value will be understood to be < 0.05.

       Analysis of the data on the deep benthos will be completed using
       previously written programs at the University of Alaska for
       comparison of species (taxa), rank abundance and rank biomass of
       species (taxon) . A diversity program will also be used to examine
       differences and similarities between stations. Station groups and
       species (taxon) assemblages for each year and for the combined data
       collected on cruises in future years will be identified using the
       technique of hierarchical cluster analysis. Principal coordinate
       analysis will be used as an aid in interpretating of the cluster
       analysis of the data and in identifying misclassifications of
       stations by cluster analysis. Use of'both of these multivariate
       techniques makes it possible to examine similarities (or
       dissimilarities) between groups of stations, and will be useful
       when comparing oiled vs unoiled bays.

       A Kruskal-Wallis and a multiple comparison test for significance
       will be used to test for differences in the total abundance and
       biomass between the stations sampled in each year and in the multi-
       year data sets. These same tests will be made on the abundance and
       biomass of selected, dominant taxa at stations between years. The
       taxa will be chosen from the rank abundance and biomass printouts
       for each station, and taxa selected will generally be those
       commonly present within bays being compared. However, taxa that are
       common at stations within unoiled bays, but rare or missing at
       stations within oiled bays, will also be tested. other statistical
       tests, such as the two-tailed Wilcoxon signed ranks test for
       pairwise observations, will be used to test differences between
       stations at similar depths and bottom type within unoiled and oiled
       bays.

       Various measures of diversity will be calculated, and compared
       qualitatively between stations at similar depths within unoiled and
       oiled bays.    The indices to be calculated and presented are:
       Shannon Diversity (measures total diversity), Simpson Dominance
       (useful f or identifying dominance by one or a f ew taxa at a

                                        30









             station), Evenness, and Species Richness.

             The calculation of K-dominance curves f or the abundance and biomass
             data will be used in an attempt to assess the effect of
             hydrocarbons on benthic organisms in oiled bays.          This is a
             technique designed to detect pollution-induced disturbance on
             marine benthic communities. Distributions of geometric classes of
             abundance of species will also be calculated. Assessment of the
             distribution of taxa in these abundance classes is often useful to
             identify indicator species within a disturbed area.

             The goal of the data analysis in this study is to determine the
             effects of short and long-term accumulations of petroleum
             hydrocarbons on benthic species composition, species diversity,
             abundance, biomass, and trophic composition. The critical aspect
             of the study is whether concentrations of petroleum contaminants
             from the EVOS are present at concentrations which cause deleterious
             effects on benthic organisms.     Because the "deleterious effects"
             criteria are complex and often require subjective interpretation,
             a detailed comparison is ultimately required of the hydrocarbon
             concentrations    at   which   various    biochemical,     behavioral,
             physiological, organismal, population, and ecological effects
             occur.   This only addresses certain aspects of the organismal,
             population, and ecological effects on the benthic infauna.


                                         BIBLIOGRAPHY

             Abelson, P. H. 1989. Oil spills. Science 244:629.

             Bauer, J.E. and D.G. Capone. 1988. Effects of co-occurring
                  aromatic hydrocarbons on degradation of polycyclic aromatic
                  hydrocarbons in marine sediment slurries.            Appl. Env.
                  Micrbiol. 54:1644-1655.

             Boehm, P. D., M. S. Steinhauer, D. R. Green, B.           Fowler, B.
                  Humphrey, D. L. Fiest, and W. J. Cretney. 1987.       Comparative
                  fate of chemically dispersed and beached crude oil   in subtidal
                  sediments of the arctic nearshore. Arctic 40, supp. 1: 133-
                  148.

             Environmental Protection Agency. 1987. Recommended protocols for
                  sampling and analyzing subtidal benthic macroinvertebrate
                  assemblages in Puget Sound. Environmental Protection Agency,
                  Final Report TC-3991-04.

             Fedorak, P.M., J.M. Foght, and D.W.S. Westlake. 1982. A method for
                  monitoring mineralization of 14C-labeled compounds in aqueous
                  samples. Water Res. 16:1285-1290.

             Foght, J.M., D.L. Gutnick, and D.W.S. Westlake. 1989. Effect of
                  Emulsan on biodegradation of crude oil by pure and mixed

                                               31









             bacterial cultures. Appl. Env. Microbiol. 55:36-42.

        Hoberg, M. K. 1986. A numerical analysis of the benthic infauna
             of three bays in Prince William sound, Alaska. M.A. Thesis,
             Humboldt State University, Arcata, CA 153 pp.

        Krahn, M.M., L.K. Moore,    R.G. Bogar, C.A. Wigren, S-L, Chan, and
             D.W. Brown. 1988a. A rapid high-pressure liquid chromato-
             graphic method f or    isolating contaminants from tissue and
             sediment extracts.    J. Chromatography. 437:161-175.

        Krahn, M.M., C.A. Wigren, R.W. Pearce, L.K. Moore, R.G. Bogar,
             W.D. MacLeod, Jr., S-L, Chan, and D.W. Brown.                1988b.
             Standard analytical procedures of the NOAA National Analytical
             Facility. New HPLC cleanup and revised extraction procedures
             for organic contaminants. U.S. Dep. Commer., NOAA Tech. Memo
             NMFS F/NWC-153, 52 p.

        Schiewe, M.H., E.G. Hawke, D.J. Actor, and M.M. Krahn. 1985. Use
             of a bacterial bioluminescence assay to assess toxicity of
             contaminated marine sediments.       Can. J. Fish. Aquat. Sci.
             42:1244-1248.

        Science Applications International Corporation. 1989. Screening
             analysis f or petroleum hydrocarbons in sediments and sediment
             pore    waters    by    use    of   ultra-violet      fluorescence
             spectrophotometry for Exxon Valdez damage assessment. Draft
             Final Report No. SAIC-89/7570&230* to the National Oceanic and
             Atmospheric Administration. 36p.

        Sokal, R. R. and F. J. Rohlf 1981. Biometry. W. H. Freeman and
             company, San Francisco. 859pp.




















                                          32














             Table 1.   Location of sites in PWS and the GOA where intertidal
             and subtidal sediment and biological samples will be collected in
             1990. Samples of hydrocarbon-degrading bacteria will be collected
             at all sites in each sampling period.         Samples for microtox
             bioassay will be collected at all sites in June/July only. Deep
             benthos (D) will be collected at selected sites in June/July.
             Depths to be sampled are: A   intertidal (1)   3, 6 and 20 m; C    I,
             3, 6, 20, 40 and 100 m.

             Location               May     June/July      Sept


             Prince William Sound'
             Bay of Isles            A         C,D           A
             Block Island            A          C            A
             Chenega Island          A          C            A
             Disk Island             A         C,D           A
             Fox Farm                A          C            A
             Green Island            A          C            A
             Herring Bay             A         C,D           A
             Macleod Harbor          A         C,D           A
             NE Knight Island        A          C            A
             NE Port Fidalgo         A          C            A
             Northwest Bay           A         C,D           A
             Olsen Bay               A          C            A
             Rocky Bay               A         C,D           A
             Sleepy Bay              A         C,D           A
             Smith Island            A          C            A
             Snug Harbor             A         C,D           A
             West Bay                A         C,D           A
             Zaikof Bay                        C,D           A

             Gulf of Alaska
             Agnes Cove                          C
             Black Bay                           C
             Chugach Bay                         C
             Hallo Bay                           C
             Katmai Bay                          C
             Sunny Cove                          C
             Tonsina Bay                         C
             Windy Bay                           C

             1. Two additional sites will be selected before June 1990 to
             provide additional control sites for the deep benthos project.






                                               33












                                         BUDGET


         NOAA

         Salaries                            $159.8
         Travel                                20.9
         Contracts                             53.0
         Supplies                              50.5
         Equipment                             32.6
         Vessel                                150.0

         Total                               $466.8

         ADF&G

         Salaries                            $174.7
         Travel                                  5.7
         Contracts                             124.9
         Supplies                              23.1
         Equipment                               5.1

         Total                               $333.5





























                                          34











           AIR/WATER STUDY NUMBER 3

           Study Title: Geographic and Temporal Distribution of Dissolved
                         and Particulate Petroleum Hydrocarbons in the
                         Water Column

           Lead Agencies: NOAA, DEC

                                      INTRODUCTION



           This study will continue to assess the geographic and temporal
           distribution of dissolved and particulate hydrocarbons in the water
           column and deposited in sediments resulting from the EVOS.
           Knowledge of these concentrations will determine whether violations
           of State of Alaska Water Quality Criteria have occurred, and will
           allow estimation of the exposure risk of subsurface marine biota to
           petroleum hydrocarbons. This study extends work begun within one
           week of the grounding of the Exxon Valdez and continued to date.

           A. DEC

           During the autumn of 1989 (November/December), DEC collected
           interstitial water samples at target sampling sites in PWS and
           deployed sediment traps at selected sites. It was determined that
           no further interstitial water sampling would be conducted in 1990.
           Studies related to hydrocarbonoclastic bacteria will be continued
           by AW 2. An increased number and distribution of sediment traps is
           planned.

           B. NOAA

           Trends in hydrocarbon concentration in the water column will be
           studied by analyzing the hydrocarbon body burden of transplanted
           bay mussels Mytilus trossulus.      The use of a bioaccumulator
           provides a time integrated indication of hydrocarbons available in
           the water column.    No further direct sampling of the nearshore
           water column will be done because hydrocarbon concentrations in the
           water column will likely be below detection levels in field samples
           that are practical to analyze.

           The products of this study will consist of estimates of aliphatic
           and aromatic hydrocarbons in the matrices examined. These data will
           be used to determine biological resource exposure to petroleum
           hydrocarbons.

           This study is coordinated with the other A/W studies and with the
           CH 1 to provide information on petroleum hydrocarbon distribution
           and movement in the nearshore water column to researchers assessing
           biological and economic damage.    In the 1990 f ield season this

                                           35









         study will share research platforms with AW 2 and FS 24. Several
         sites of this study will coincide with sites from these two studies
         and with at least one CH 1 subtidal control site. Data gathered at
         these joint sites will provide a comprehensive picture of damage
         and will be especially valuable to studies assessing biotic and
         economic damage. Selection of NOAA AW 3 study sites was aided by
         information produced by AW 1 and AW 4 (now part of AW 2).
         Information on beach cleanup at study sites will be obtained from
         the DEC Spill Response Office and NOAA HAZMAT.


                                      OBJECTIVES

         A.    To determine if sediments settling out of the water column in
               nearshore subtidal environments contain absorbed hydrocarbons
               (DEC).

         B.    Determine hydrocarbon inputs in nearshore environments and
               evaluate trends in ambient water quality using mussels
               (Mytilus trossulus) as bioaccumulators (NOAA/NMFS).



                                        METHODS


         A.    DEC

         Subtidal particulate samples will be collected with sediment traps
         for hydrocarbon analysis.       sampling arrays containing three
         sediment traps each will be placed in the subtidal zone adjacent to
         target shorelines at no more than 20 meters below mean lower low
         water.   Results will generate information on sedimentation and
         associated hydrocarbon inputs to subtidal sediments.

         Currently, five sediment trap arrays are in place in four locations
         in PWS: Sleepy Bay, Snug Harbor, Northwest Bay (2), and Northeast
         Port Fidalgo. Each platform consists of three removable long-term
         sediment traps. These traps will be picked up, and the number and
         distribution of the traps in PWS will be increased. Sediment traps
         will be placed in as close proximity as possible to the mussel
         cages being deployed in the NMFS segment of this study.
         Approximately fifteen additional emplacements (3 traps per site)
         are proposed.    Sediment trap arrays will be deployed in relation
         to shoreline habitat types, according to the Environmental
         Sensitivity Index (Gundlack and Hays 1982), in conjunction with
         bioaccumulation where possible, and in relation to shoreline
         treatment methods as deemed feasible.

         Particulate samples from sediment traps will be screened for
         hydrocarbon content by ultraviolet fluorescence spectrophotometry
         after methylene chloride extraction of the samples in the field.
         UVF is a semiquantitative method of analysis for hydrocarbons
         (ASTM, 1982). Samples showing significant quantities of petroleum

                                          36









           hydrocarbons will be further analyzed for polynuclear aromatic
           hydrocarbons (PAH) and total petroleum hydrocarbons (PHC) according
           to procedures established by TS 1.

           B. NOAA/NMFS

           Mussels will be placed at all 1989 sites within PWS (Figure 1)
           except Squire Island and The Needle.        Except for Olsen Bay
           (control), all sites were in the spill trajectory and subject to
           varying degrees of oiling. Redeployment this year will indicate
           changes in water column hydrocarbon concentrations at these sites
           since deployed mussels were last collected in September 1989.
           Seven additional sites are proposed: four at sites of maximum
           original oiling as indicated by preliminary analysis of water
           column samples (AW 3) and sediment pore water samples (AW 4), and
           at Disk Island and Black Island where extensive cleanup activity is
           anticipated.    Mussels will also be deployed at a second control
           site, McCleod Bay.

           Outside PWS, redeployment is proposed at Sunny Cove (Resurrection
           Bay), Black Bay, Tonsina Bay, Blue Fox Bay (Af ognak Island), Hallo
           Bay and Kukak Bay (control) (Figure 2). The two new sites, Agnes
           Cove and Windy Bay, are coincident with AW 2 sites.

           Local siting will ensure a site depth of 34 m (to accommodate the
           deepest mussel cage) and the best available protection from
           prevailing weather and currents. Site substrates will be cobbled
           to finer sediments to ensure that mooring anchors are set securely.
           If treatment activity occurs, siting may be adjusted or additional
           sites may be added so that beaches adjacent to sites represent both
           treated and untreated beaches.

           Physical data on location (geographic coordinates) , site depth,
           sampling time, tidal stage, and temperature and salinity at
           deployment depths will be recorded at each site.

           Bay mussels will be collected from a hydrocarbon free site,
           Admiralty Island in southeast Alaska, a few days before each new
           deployment cruise. Mussels will be held in living stream tanks,
           that have been rinsed with dichloromethane and flushed with ambient
           unfiltered seawater at the rate of 2 liters/minute        at least
           overnight. Since mussel size influences hydrocarbon uptake (Bayne
           et al., 1981), only mussels with shell length of 45-50 mm. will be
           selected for deployment. A sample from each collection of at least
           30 individuals will be measured for shell length, width, and height
           and whole wet versus dry weight.    Another 40-50 animals will be
           taken immediately prior to shipment of mussels to a deployment
           vessel as a reference sample of the population's base hydrocarbon
           level and condition.

           Mussels will be shipped to the field in layers of healthy Fucus sp.
           seaweed in insulated coolers whose lids have been drilled with air


                                            37










        holes.   Mussels will be kept aboard the deployment/ collection
        vessel in coolers and the blue ice changed daily for up to 6 days.
        on longer cruises, mussels will be resupplied by air or possibly
        irrigated with the seawater. Samples of irrigation water will be
        taken daily, extracted with dichloromethane,and frozen. A mussel
        baseline sample will be taken before deployment of irrigated
        mussels at each new site.

        A deployment "cage" is a nylon mesh diver   collecting bag held open
        by a perforated polypropylene sheet that has been rinsed with
        dichloromethane and fitted into the bag bottom.       Twenty mussels
        will be placed in each bag separately (i.e. byssal connections to
        other mussels will be separated so that byssal development observed
        when mussels are collected will have occurred during field
        exposure.)   Assuming some mortality during exposure, this number
        was chosen to provide at least triplicate samples of 3 ï¿½ .5 g of
        issue for hydrocarbon analysis (Krahn et al 1988).     At 4 sites in
        PWS an extra cage containing 40 mussels will be deployed at 1 m to
        be exposed for 8 to 10 weeks so that hydrocarbon uptake over the
        longer period may be compared with uptake over the 2 shorter
        periods at the same site. Filled bags will be closed and attached
        to the mooring line with a halibut snap. At each site, bags will
        be attached at the I m, 5 m, and 25 m depths. The 2 shallower cage
        depths were chosen to correspond to water column depths sampled by
        this study in the f irst 6 weeks after the spill; mussels at the
        third depth will be exposed to the water column about 10 m above
        the bottom at low tide.      Reference samples of mussels will be
        taken just after the final deployment on a cruise to determine any
        hydrocarbon uptake or deterioration of general condition during
        holding of mussels on the vessel. Exposure times will be 4 to 5
        weeks, and 8 to 10 weeks at the four selected sites. There will be
        three periods of exposure at PWS sites and one period at Kenai,
        Afognak and Alaska Peninsula sites.

        After exposed mussels are retrieved, the number of clumps of
        mussels, the number of individuals per clump, comments regarding
        the strength and elasticity of byssal threads, and the number of
        alive, dead, or gaping animals will be recorded. Dead or gaping
        animals will discarded. At least 1 hydrocarbon free 16 oz jar with
        a Teflon lid will be filled with live animals from each bag, kept
        in a cooler, and frozen at -18 OC as soon as possible. A field air
        blank will be taken at the site and on the vessel, if sample jars
        are filled aboard the vessel. Mussel cages will then be refilled
        and redeployed.

        Naturally occurring adult mussels will be collected in intertidal
        areas adjacent to some deployment sites. These will be packed in
        clean 16 oz jars and handled similarly to caged mussel samples.

        Sample estimates are: 236 caged exposed mussel samples, 59 air
        blanks, 20 native mussel samples, and 15 reference samples.


                                          38














                                      DATA ANALYSIS


            A. DEC

            Hydrocarbon    concentration    data    will    be    tested     for
            heteroscedasticity (Bartlett's test) and reported as means and 95%
            confidence intervals calculated according to a standard formula
            (Sokal and Rohlf, 1981).    Parametric statistics will be used to
            test for differences between hydrocarbon concentrations between
            sites, if the assumptions of 'parametric procedures are met.
            Otherwise, nonparametric tests (e.g., the Kruskal-Wallis test) will
            be employed.

            B. NOAA/NMFS

            ANOVA will be used to determine the significance of differences of
            any hydrocarbons found in the collected samples.

            Products of this study will consist of tables containing lists of
            hydrocarbons found in the samples collected.



                                       BIBLIOGRAPHY

            ASTM D-3650-78. Standard Test Method for Comparison of Waterborne
                 Petroleum Oils by Fluoresence Analysis.

            Bayne, B.L., K.R. Clarke and M.N. Moore. 1981. Some practical
                 considerations in the measurement of pollution effects on
                 bivalve molluscs and some possible ecological consequences.
                 Aquatic Toxicology 1:159-174.

            Gundlach, E.R. and M.O. Hayes. 1982. The oil spill environmental
                 sensitivity index applied to the Alaskan coast:        in 1ï¿½82
                 Arctic Marine Oil Pollution (AMOP) Seminar.           EcTi-onton,
                 Alberta, Canada. pp.311-323

            Krahn, M.M., C.A. Wigren, R.W. Pearce, L.K. Moore, R.G. Bogar, W.D.
                 MacLeod,Jr., S. Chan, and D.W. Brown. 1988. Standard
                 analytical procedures of the NOAA National Analytical
                 Facility, 1988, New HPLC cleanup and revised extraction
                 procedures for organic contaminants.          NOAA Technical
                 Memorandum NMFS F/NWC-153. 52 pp.

            Sokal, R. R. and R. J. Rohlf. 1981. Biometry. Freeman, San
                 Francisco.








                                            .39













        BUDGET: DEC

        Salaries                          $ 19.6
        Travel                                1.4
        Contracts                           17.5
        Supplies & Equipment                  9.0


        TOTAL                             $ 47.5



        BUDGET: NOAA


        Salaries                          $161.5
        Travel                              15.3
        Contracts                           12.3
        Supplies                            40.2
        Equipment                           43.2
        Ship Costs:                         200.0

        TOTAL                             @-472.5

        TOTAL both Projects               $520.0




























                                              40










            AIR/WATER STUDY NUMBER 6

            Study Title: Fate and Toxicity of Spilled Oil from the EVOS

            Lead Agency: NOAA

                                        INTRODUCTION


            Overview and Relation to other Studies

            This study is designed to:     a) assess the toxicity of weathered
            EXXON VALDEZ oil and its degradation products to selected test
            organisms; and     b) integrate the results from selected other
            projects into an overall budget for the distribution, transport,
            transformation, and persistence of spilled oil in Alaskan coastal
            environments.     The study is very closely coordinated with A/W
            Study 2 for its field work and toxicity studies, and will require
            close interaction with all of the present and past A/W studies, the
            Coastal Habitat study, and with related spill response studies for
            completion of the spilled oil budget.


            Toxicity of Crude Oil in Relation to the Weathering Process

            Currently, limited information is available on the significance of
            either the polar constituents of crude oil or the intermediate
            oxidation products of petroleum hydrocarbons (whether from
            photooxidation or biodegradation) in terms of their potential for
            bioaccumulation and toxicity to resource organisms in the marine
            environment.    Since these compounds have undergone preliminary
            oxidation and (sometimes) conjugation, they are more polar than
            their parent hydrocarbons, and will as a result generally be more
            subject   to    excretion   or    depuration,   less    subject    to
            bioaccumulation, more susceptible to further oxidation (or
            biodegradation if accumulated), and more susceptible to dilution
            and dispersion in the water column.      Studies proposed here are
            designed to help determine whether such polar constituents pose a
            significant risk of toxicity or mutagenicity to Alaskan marine
            organisms as a result of the EVOS.


            Acute and Sublethal Toxicity of oil to Marine Organisms

            A very considerable body of literature exists on the toxicity of
            Alaskan crude oil to Arctic and subarctic marine organisms. The
            data base is probably adequate for assessing the relative
            sensitivities of different marine species to exposure and for
            estimating the range of potential responses (at the organismic
            level) that may result from a particular level of exposure in the
            environment. However, very little of this prior toxicity research
            has been directed specifically at the contribution of either
            hydrocarbon metabolites or other oxidation products of oil that may

                                             41









         be produced by the processes.of biological or chemical weathering
         in the environment.



         Sources of Toxicity in Crude Oils

         By the mid-1970's, it had been concluded that much of the acute
         toxicity of oil was accountable directly to the content of soluble
         aromatic compounds (Moore and Dwyer 1975; Neff et al. 1976), and
         attention was being directed towards determining which fractions of
         petroleum were most responsible for the toxicity observed in
         laboratory and f ield exposures to oil.       Based on the relative
         concentrations of the low-molecular weight constituents in crude
         oill, it has become generally accepted that most of the acute
         toxicity effected by oil in the environment is derived from the
         mono- and di-nuclear aromatics.      When a water soluble fraction
         (WSF) was simulated by mixing the 10 predominant aromatic
         hydrocarbons at the same concentrations and proportions found a
         true WSF of crude oil, however, the toxicity of the resulting
         mixture was only 20-30% of the true WSF, suggesting that either
         minor aromatic constituents, or components other than aromatic
         hydrocarbons, also contribute significantly to the observed
         toxicity (Rice et al 1984).


         Polar Constituents and Oxidation Products of Oil

         Petroleum in the marine environment is decomposed primarily through
         the processes of microbial biodegradation and photooxidation or
         autooxidation     These processes are effective for oil in surface
         slicks, in the water.column, in sediments, and in the atmosphere
         (photooxidation of evaporated compounds) .       In addition, parent
         petroleum compounds are bioaccumulated, and metabolized by
         macro-organisms.     While the. eventual major products of these
         oxidative reactions are carbon dioxide and water, some of the
         oxygenated intermediates produced along the way may be more toxic
         than their precursors.

         oxidation products of photooxidation include fatty acids, alkylated
         naphthols, and substituted 1- to 3-ring aromatic and heteroaromatic
         acids, as well as alkylated benzothiophene sulfoxides (Overton et
         al.   19790,  1980).      Microbial    biodegradation    of    alkanes,
         cycloalkanes, and monoaromatics leads to the production of
         alcohols, aldehydes, and carboxylic acids that are generally of
         little concern from a toxicity standpoint. Condensed polyaromatic
         hydrocarbons, however, may be transformed by microbial metabolism
         to potential carcinogens or mutagens.             Materials such as
         benzo (a) pyrene and benzo (a) -anthracene, f or example, are oxidized
         by eucaryotic organisms (macroorganisms, yeasts and molds) to
         trans-dihydrodiols which are subsequently activated into oxides
         that bind to DNA and are powerful mutagens.


                                           42









           Some metabolic products of high-molecular weight aromatics are
           demonstrated mutagens or carcinogens and have been shown to bind to
           DNA (Ahokas et al. 1979; Lech And Bend 1980; Varanasi et al. 1981).
           These same materials are also associated with the prevalence of
           liver lesions, including neoplasms (Varanasi and Stein 1990).

           Asphaltenes and resins are two heterogeneous and poorly
           characterized assortments of (non-hydrocarbon) compounds that
           comprise only about 2% and 6%, respectively, of Prudhoe Bay crude
           oil (Clark and Brown 1977). Asphaltenes are constituents of tar
           that are highly resistent to biodegradation, and are not generally
           considered to pose a risk of toxicity to marine organisms. Resins
           include the polar and heterocyclic nitrogen sulfur oxygen (NSO)
           compounds, such as phenols, cresols, thiophenes, dibenzothiophenes,
           pyridines, and pyrroles.     Some of them are likely to undergo
           biodegradation, and very broad suites of NSO compounds have been
           Adentified in contaminated marine environments (Krone et al. 1986,
           Wolfe et al. 1981). Like hydrocarbon metabolites, many of these
           compounds are moderately water-soluble and therefore subject to
           dispersion in the water column.       While some of these polar
           compounds could be toxic at high concentrations, no studies have
           been made of the levels of toxicity exerted by these materials
           under oil spill conditions in the marine environment.


           Fate of Spilled Oil: Budgets and "Mass Balance"

           An accurate and complete mass balance has yet to be prepared for
           any major oil spill in a marine environment.        The quality of
           estimates of the quantities and locations of oil affected by
           different processes of transport or transformation have varied from
           spill to spill, depending on the local circumstances of the spill
           and the effort devoted to any particular process.           Selected
           observations at past spills have been summarized by Mackay (1981),
           Gundlach et al. (1983), Jordan and Payne (1980), National Academy
           of Sciences (1985), and Wolfe (1985, 1987). Information especially
           pertinent for summarizing the fate of oil from the EXXON VALDEZ
           spill has been and is being gathered by the Interagency Response
           Team and the ADEC, and by certain projects under the NRDA.


                                       OBJECTIVES

           A.     Document the toxicity of contaminated sediments and related.
                  environmental samples to selected marine biota

           B.     At selected sites, document and quantify the occurrence of
                  oxidized derivatives of EXXON VALDEZ oil

           C.     Determine the extent to which the observed toxicity of
                  oil-contaminated environmental samples may be attributable
                  to oxidation products of petroleum.

                                            43









        D.     Construct a summary budget or "mass balance" summarizing the
               fate of the spilled oil.


                                      METHODS


        A.     Toxicity   of   oil-Contaminated    Sediments    and    other
        .Environmental Samples

        Toxicity tests will be performed on sediment samples taken at
        selected sites sampled by A/W Study 2.    Two specific tests, both
        following well-established protocols, are proposed:      a sediment
        elutriate test using larval mussels Mytilus edulis, and a whole
        sediment test using Ampelisca abdita (or other suitable Alaskan
        species of Ampelisca).     Mytilus is an intertidal species whose
        larval recruitment is vulnerable to interruption by toxic oil
        residues remaining in intertidal sediments.     Ampelisca inhabits
        soft nearshore sediments that are possible sinks for petroleum.
        Subtidal ampeliscid amphipods exhibited considerable sensitivity to
        oil in the aftermath of the AMOCO CADIZ spill (Cabioch et al.
        1982). Use of these two species should provide a direct measure of
        the toxicity of the residual oil to actual marine species.

        Sampling sites have been selected to represent the more heavily
        oiled areas. At each of 20 of the sites, eight one-liter samples
        of surficial sediments will be collected (2 each at the intertidal,
        6-meter, 20-meter, and 100-meter depths) for toxicity testing with
        Mytilus and Ampelisca.   These samples will be stored at 0-4 degree
        celsius and offloaded from the vessel at regular intervals for
        shipment to the testing laboratory. Bioassays will be initiated
        within 10 days of the collection of the samples.

        B.     Oxidation Products of Petroleum

        The fractionation and toxicity testing of polar constituents from
        weathered petroleum will be pursued in a tiered, stepwise fashion.
        A limited pursuit of chemical fractionation and characterization
        will be undertaken in association with the toxicity testing to be
        performed under A/W2.    This preliminary study will include two
        major objectives, and will employ the following approaches:

               1.   Determine whether the toxicity (if any) of organic
               extracts from Exxon Valdez o i 1 -contaminated sediments is
               partitioned between polar and non-polar constituents:

               Approach:

                        perform microtox on unfractionated extracts
                        perform one-step separation on PAC column to
                        fractionate polar from non-polar constituents
                        repeat -microtox on the two separated fractions
                        perform    simple     carcinogenicity/mutagenicity

                                         44









                           bioassay (eg Ames test, S.O.S. Chromotest) on
                           selected fractions

                  2. Determine what types of polar constituents are present in
                  these extracts:


                  Approach:

                           perform HPLC/UV on polar (and non-polar) fractions
                           to identify presence of PNA derivatives
                           perform detailed GC/MS on selected polar fractions
                           to identify and quantify (major)constituents

                  The work should focus (a) on determining whether a
                  significant   fraction   of   the   observed    toxicity    or
                  mutagenicity can be ascribed to polar derivatives in a few
                  of the most biologically active samples, and if so, (b) on
                  a preliminary characterization of the polar constituents
                  that may be involved.    The selection of samples for this
                  chemical fractionation and characterization will be guided
                  by the magnitudes of the Microtox and UVF signals.

                  If this preliminary work suggests that polar constituents
                  could account for significant toxicity in the marine
                  environment, more intensive testing will be performed. At
                  two heavily oiled sites, one untreated and one that has
                  undergone bioremediation (both yet to be selected), and one
                  lightly or unoiled site in PWS, special samples will be
                  taken to assess the concentrations and compositions of
                  petroleum oxidation products in intertidal sediments. These
                  samples will also be taken in conjunction with.A/W Studies
                  2 and 3, probably from the NOAA vessel COBB during the late
                  summer or fall of 1990.      Large quantities of sediments
                  and/or interstitial water will be required to support the
                  necessary development of suitable techniques for bulk
                  fractionation of samples to be tested for toxicity (in C.
                  below) and for chemical characterization and quantification
                  of the polar metabolites.

                  Replicate (3) sediment samples on the order of 10kg (wet
                  weight) each will be collected at each site for exhaustive
                  extraction   with   methylene   chloride.      The    chemical
                  fractionation procedure will include a succession of solvent
                  partitioning, absorption chromatography, HPLC using gel
                  exclusion, and GC-MS. Initial phases of fractionation will
                  follow closely the procedures outlined by MacLeod et al.
                  (1985) for separation and analysis of petroleum compounds,
                  but additional steps will be required for separation and
                  characterization of the more polar fractions.

                  Interstitial water samples should also be examined. Because
                  of uncertainties about the flux of interstitial water in


                                            45









               oiled beaches and the resultant levels of polar metabolites,
               however, it is very difficult to estimate the volume of
               sample that may be required for characterization and
               quantification of polar metabolites. For initial trials, it
               is suggested that "interstitial water" be pumped or siphoned
               from a shallow "well" (i.e., a glass tube inserted to a
               depth of about 30 cm in heavily oiled beach sediments) into
               precleaned glass carboys (18-20 liters) containing acid and
               methylene chloride to carry out the initial extraction and
               to ensure preservation of the samples.        An alternative
               collection technique would be to allow "interstitial water"
               merely to seep into an excavation on the beach and then to
               dip the water into the sample carboys. At each of the sites
               where sediments are collected for analysis, 100 liters of
               "interstitial water" should be collected.           Exhaustive
               extraction with methylene chloride would be followed by
               analytical steps similar to those used for sediments.

               Target compounds of the fractionation and analytical scheme
               will include phenolic, carbonyl, quinone, and carboxylic
               derivatives of polynuclear aromatic hydrocarbons, for
               example:    9-fluorenone, 9-fluorenone carboxylic acids,
               phenanthraquinone as potential derivatives of phenanthrene.
               Analogs related to naphthalene, anthracene, chrysenes,
               benzanthracene, pyrene, and benzpyrene will also be sought
               specifically,    as     Will    oxidation     products      of
               dibenzothiophenes.   GC-MS data will be analyzed also for
               other major constituents in associated polar fractions to
               provide a general characterization of the polar compounds
               found in these samples.

        C.     Toxicity of Oxidized Petroleum Fractions

        Following the initial characterization and quantification of polar
        constituents in oiled sediments and interstitial water,           the
        fractionation process will be scaled up to provide quantities of
        material suitable for toxicity testing.     Based on the results of
        initial fractionation and chemical characterization,         selected
        polar fractions will be assayed for toxicity using the standard
        Microtox bioassay with organic extracts (Schiewe et al. 1985).
        Toxicity of polar fractions will be compared with the better known
        toxicities of aromatic fractions and reference compounds.         The
        composition of all assayed fractions will be checked by GC-MS for
        consistency with previous fractionations of earlier samples.

        D.     Mass Balance Budget for Fate of Spilled Oil (Budget)

        This task is primarily a synthesis function. Information on the
        distributicn and fates of EXXON VALDEZ oil needs to be assembled
        from a number of sources and interpreted in light of existing
        information and models.



                                         46









            The following compartments and processes are proposed for initial
            analysis and inclusion in the Budget. Potential sources of data,
            historical information, and modeling expertise are also noted:

              1.    Floating oil (Distribution in Time & Space)
              2.    Evaporation
              3.    Photooxidation in the atmosphere
              4.    Mousse formation
              5.    Beaching of oil & mousse (T&S)
              6.    Water column accommodation (T&S)
              7.    Photooxidation in water column, in slicks and on beaches
              8.    Biodegradation in water column
              9.    Transport to subtidal sediments
              10.   Biodegradation in sediments


              Representatives of the above noted activities, along with other
              recognized experts on oil weathering and fates, will be
              consulted for recommendations on appropriate approaches to
              synthesis, and for their judgments on the suitability and
              adequacy of existing information for development of the Budget.
              Apart from the information on polar constituents described
              above, no original data is proposed for collection under this
              project.   Timely progress on the Budget will depend on the
              availability of suitable information from other sources and
              projects; chemical data, i.e., from T/S 1, will be of utmost
              importance to the completion of this project. Where existing
              information is found to be deficient, means will be explored
              for gathering of improved information. The reliability of all
              estimates will be assessed and qualified in the final analysis.

              E. Ouality Assurance and Control

              All samples will be taken with careful adherence to QA/QC Plan
              for NRDA.


                                        BIBLIOGRAPHY

              Ahokas, J.T., H. Saarni, D.W. Nebert, and 0. Pelkonen. 1979.
                    The in vitro metabolism and covalent binding of
                    benzo(a)pyrene to DNA catalyzed by trout liver microsomes.
                    Chem. Biol. Interact. 25:103-111.

              Brown, D.A., R.W. Gossett, and S.R. McHugh. 1987. Oxygenated
                    metabolites of DDT and PCBs in marine sediments and
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                    (eds) . Oceanic Processes in Marine Pollution.    Vol. 1.
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              Cabioch, L., J.-C. Dauvin, C. Retiere, V. Rivain, and   D.
                    Archambault. 1982, Les effets des hydrocarbures de  11AMOCO

                                             47










                  CADIZ sur les peuplements benthiques des Baies de Morlaix
                  et de Lannion d'Avril 1978 a Mars 1981. PP. 205-228. In:
                  Ecological Study of the Amoco Cadiz Oil Spill. Rpt of the
                  NOAA-CNEXO Joint Scientific Commission.            U.S. Dept.
                  Commerce, NOAA. Washington, D.C.

             Clark, R.C., and D.W. Brown. 1977. Petroleum: Properties and
                  analyses in biotic and abiotic systems.        PP. 1-89.     In:
                  D.C. Malins (ed.)      Effects of Petroleum on Arctic and
                  Subarctic Marine Environments and organisms.          Vol. I,
                  Biological Effects, Academic Press, New York.

             Dunn, B.P. 1980. Polycyclic aromatic hydrocarbons in marine
                  sediments,    bivalves,    and   seaweeds:       Analysis     by
                  high-pressure liquid chromatography.       PP. 367-377.      In:
                  A. Bjorseth and A.J. Dennis (eds.) Proc. 4th Internatl.
                  Sympos. on Polynuclear Aromatic Hydrocarbons.          Battelle
                  Press, Columbus, Ohio.

             Gundlach, E.R., P.D. Boehm, M. Marchand, R.M. Atlas, D.M. Ward,
                  and D.A. Wolfe.      1983.   The fate of AMOCO CADIZ oil.
                  Science 221: 122-129.

             Jordan, R.R., and J.R. Payne. 1980. Fate and Weathering of
                  Petroleum Spills in the Marine Environment: A Literature
                  Review and Synopsis. Ann Arbor Science Publishers. Ann
                  Arbor, Michigan. 174 pp.

             Karickhoff, S.W. 1981. Semi-empirical estimation.of sorption
                  of hydrophobic pollutants on natural sediments and soils.
                  Chemosphere 10:833-846.

             Krone, C.A., D.G. Burrows, D.W. Brown, P.A. Robisch, A.J.
                  Friedman, and D.C. Malins.       1986.     Nitrogen-containing
                  aromatic compounds in sediments from a polluted harbor in
                  Puget Sound. Environ. Sci. Technol. 20:1144-1150.

             Lech, J.J. and J.R. Bend. 1980. The relationship between
                  biotransformation and the toxicity and fate of xenobiotic
                  chemicals in fish. Environ.. Health Perspectives 35:115.

             Mackay, D. 1981. Fate and behaviour of oil spills. PP. 7-27.
                  In: J.B.Sprague, J.H. Vandermeulen, and P.G. Wells (eds.)
                  Oil Dispersants in Canadian Seas-Research Appraisal and
                  Recommendations. Environment Canada, Toronto.

             MacLeod, W.D., Jr., D.W. Brown, A.S. Friedman, D.G. Burrows, 0.
                  Maynes, R. Pearce, C.A. Wigren, and R.G. Bogar.           1985.
                  Standard analytical procedures of the NOAA National
                  Analytical Facility, 1982-1986: Extractable toxic organic
                  compounds. 2nd Edition. NOAA Technical Memorandum NMFS
                  F/NWC-92. 121 pp.

                                              48










             Means, J.C., J.J. Hassett, S.G. Wood, and W.L. Banwart. 1979.
                    Sorption properties of energy-related pollutants and
                    sediments.   PP. 327-          In: P.W. Jones and P. Leber
                    (eds.)   Polynuclear Aromatic Hydrocarbons.          Ann'Arbor
                    Science Publishers, Ann Arbor, Michigan.

             Means, J.C., S.G. Wood, J.J. Hassett, and W.L. Banwart. 1980.
                    Sorption     of polynuclear aromatic hydrocarbons by
                    sediments and soils.        Environ. Sci. Technol.           14:
                    1524-1528.


             Melancon, M.J.,Jr.,       and J.J. Lech.           1979.       Uptake,
                    biotransformation,     disposition,    and   elimination      of
                    2-methylnaphthalene and naphthalene in several fish
                    species. PP. 5-22. In: L.L. Marking and R.A. Kimerle
                    (eds.)    Aquatic Toxicology.      ASTM STP667.      Am. Soc.
                    Testing and Materials. Philadelphia, Pennsylvania.

             Moore, S.F. and R.L. Dwyer. 1975. Effects of oil on marine
                    organisms: A critical assessment of published data. Water
                    Research 8: 819-

             National Academy of Sciences. 1985. Oil in the Sea. Inputs,
                    Fates, and Effects. National Academy Press, Washington,
                    D.C.

             Neff, J.M., J.W. A   nderson, B.A. Cox, R.B. Laughlin, Jr., S.S.
                    Rossi, and H.E. Tatum.      1976.   Effects of petroleum on
                    survival, respiration and growth of marine animals. PP
                    516-539. In: Sources, Effects & Sinks of Hydrocarbons in
                    the Aquatic Environment.        The American Institute of
                    Biological Sciences, Washington, D.C.

             Overton, E.B., J.R. Patel, and J.L. Laseter. 1979.            Chemical
                    characterization of mousse and selected environmental
                    samples from the AMOCO CADIZ oil spill. PP. 169-174. In:
                    Proceedings, 1979 Oil Spill Conference (Prevention,
                    Behavior, Control, Cleanup),             American Petroleum
                    Institute Publication No. 4308, Washington, D.C.

             Overton,. E.B., J.L. Laseter, W. Mascarella, C. Rashke, I.
                    Noiry, and J.W. Farrington.          1980.       Photochemical
                    oxidation of IXTOC-I oil.        PP. 341-383.      In:    Proc.
                    Sympos. Preliminary Results from the September 1979
                    RESEARCHER/PIERCE IXTOX-I Cruise. NOAA Office of Marine
                    Pollution Assessment, Boulder, Colorado.

             Rice, S.D., A. Moles, J.F. Karinen, S. Korn, M.G. Karls, C.C.
                    Brodersen, J.A. Gharrett, and M.M. Babcock.                1984.
                    Effects of petroleum hydrocarbons on Alaskan aquatic
                    organisms: A comprehensive review of all oil-effects
                    research on Alaskan fish and invertebrates conducted by

                                               49










               the Auke Bay Laboratory, 1970-1981. U.S. Dept. Commerce,
               NOAA Tech. Memo. NMFS/NWC-67. 128 pp.

         Roubal, W.T., T.K. Collier, and D.C. Malins.                    1977.
               Accumulation and metabolism of carbon-14 labeled benzene,
               naphthalene, and anthracene by young coho salmon
               (Oncorhynchus kisutch). Arch. Environ. Contam. Toxicol.
               5:513-529.

         Schiewe, M.H., E.G. Hawk, D.I. Actor, and M.M. Krahn 1985.
               Use of a bacterial bioluminescence assay to assess
               toxicity of contaminated marine sediments.             Canadian
               Journal of Fisheries and Aguatic Sciences 42:      1244-1248.

         Varanasi, U., and J.E. Stein. 1990. Disposition of       xenobiotic
               chemicals    and    metabolites     in   marine    organisms.
               Environmental Health Perspectives (in press).

         Varanasil U., D.J. Gmur, and W.L. Reichert. 1981. Effect of
               environmental temperature on naphthalene metabolism by
               juvenile starry flounder (Platichthys stellatus) and rock
               sole    (Lepidopsetta     bilineata).        Arch.     Environ.
               Contam.Toxicol. 10:203-214.

         Veith, D.G., D.L. Defoe, and B.V. Bergstedt. 1979. Measuring
               and estimating the bioconcentration factor of chemicals in
               fish. J. Fish. Res. Board Canada 36:1040-

         Wolfe, D.A. 1985. Fossil Fuels: Transportation and marine
               pollution. Chapter 2. pp. 45-93. In: Iver W.Duedall,
               Dana R. Kester, P. Kilho Park and Bostwick H. Ketchum
               (eds.), Wastes in the Ocean, Volume 4. Energy Wastes in
               the Ocean. John Wiley & Sons, New York.

         Wolfe, Douglas A. 1987. Interactions of spilled oil with
               suspended materials and sediments in aquatic systems. pp.
               299-316. In: K.L. Dickson, A.W. Maki, and W.A. Brungs
               (eds.), Fate and Effects of Sediment-bound Chemicals in
               Aguatic    Systems.     Proceedings of the 6th         Pellston
               Workshop,     August 12-17, 1984.      Florissant, Colorado.
               Pergamon Press, Oxford, England.

         Wolfe, D.A., R.C. Clark, Jr., C.A. Foster, J.W. Hawkes, and
               W.D. MacLeod, Jr. 1981. Hydrocarbon        accumulation     and
               histopathology in bivalve molluscs transplanted to the
               Baie de Morlaix and the Rade de Brest. PP. 599-616. In:
               AMOCO CADIZ.    Internatl. Symposium on the Amoco Cadiz:
               Fate and Effects of the Oil Spill. Brest, France. 19-22
               Nov 1979. CNEXO, Paris, France.




                                          50














                 BUDGET: NOAA


                 salaries                        $60.0
                 Travel/Shipping                  67.0
                 Contracts                      580* 0
                 Supplies                         13.0
                 Equipment                         0.0
                 Ship Costs                      150.0                                   1

                 Total                          $870.0









































                                                                                        1





                                                51










                           FISH/SHELLFISH INJURY ASSESSMENT


         The grounding of the tanker Exxon Valdez discharged crude oil into
         one of the richest marine ii7sheries communities of the United
         States. Although oil contamination was most severe within PWS, the
         oil spread into large portions of the Gulf of Alaska (GOA), Lower
         Cook Inlet (LCI) , Shelikof Strait, and other North Pacific ocean
         waters off the coasts of Kodiak and the Alaska Peninsula. The fish
         and shellfish populations inhabiting these marine and estuarine
         waters form integral parts of a vast and complex ecosystem which
         also includes various other invertebrate species, birds, and
         mammals (including humans).

         For example, the various life history stages of Pacific herring are
         important forage species for various piscivorous fishes (e.g.
         Pacific salmon, halibut, etc.), birds (gulls, cormorants, eagles,
         loonst   etc.),    mammals    (sea   lions,   seals,   whalest     etc.),
         invertebrates (crabs), and are used for subsistence and commercial
         purposes.    . Regarding Pacific salmon, outmigrating smolts are
         important seasonal prey items for a variety of predatory fish and
         marine birds. Maturing salmon in the high seas and adult salmon
         returning to inland waters are the major portion of the diet of
         marine mammals such as sea lions,'seals, and killer whales. Salmon
         are also the summer mainstay for eagles and many species of gulls.
         Spawning adults in the streams constitute almost 100% of the summer
         diet for bear and some land otter and are a very important link
         between the marine and terrestrial ecosystems. Salmon carcasses in
         streams, estuaries, and lakes are a crucial source of nutrients for
         planktonic communities and benthic organisms which represent the
         bottom rungs of the food chain for a wide variety of animals.

         Various fish and shellfish species are also important components of
         human subsistence, commercial and sport fishery harvests.
         Communities such as Tatitlek, Chenega Bay, and English Bay depend
         upon subsistence fisheries in PWS and LCI for the very existence of
         their residents.      The ex-vessel value of commercial fish and
         shellfish catches within PWS and other affected areas was estimated
         to be $1.3 billion in 1988. The largest recreational fisheries in
         Alaska for salmon, halibut, and rockfish center in Homer and
         Seward; a total of 300,000 angler days was recorded from these
         areas in 1987.     Finally, many non-consumptive users of fish and
         wildlife also utilize the waters affected by the oil spill. Injury
         to fish and shellfish populations and resulting alterations to
         ecological communities would certainly diminish the value of the
         area to this group of people.

         Bioassays prior to EVOS using crude oil from Prudhoe Bay and other
         areas have shown that exposure to concentrations as low as a few
         parts per billion in seawater will cause loss of limbs in Tanner
         crab, immediate death of eggs and larvae of herring, and death of
         Dungeness crab and various shrimp species. To assess the type and

                                            52









           extent of injury done to marine fish and shellfish communities by
           the EVOS, a series of Fish/Shellfish (FIS) studies was developed by
           investigators from various State and Federal agencies.       Species
           were selected for study based on their value as indicators of
           damage, their role as key species within the ecosystem, or their
           direct importance to man as components of subsistence, commercial
           or sport harvests.

           Comparisons of the abundance of larvae, juveniles, or adults
           between oiled and non-oiled waters were chosen as the basic
           experimental units. In some studies, oiled and non-oiled waters
           pertain to different geographic areas; in other studies these terms
           relate to the same area or populations before and after the oil
           spill; in the remaining studies these terms refer to different
           areas and populations before and after the spill. Contamination of
           individual fish and shellfish will be documented by analysis of
           tissue samples, bile samples, and/or testing for induction of
           specific enzymes associated with hydrocarbon exposure. Damages to
           fish and shellfish populations resulting from the oil spill may be
           expressed as lethal (e.g., mortality to specific life history
           stages) or sublethal (e.g., decreased growth, reproduction
           potential, etc.) injuries.    Such injuries to populations could
           cause losses in harvests and use of these species by man, and
           result in undesirable alterations of natural communities which
           might be difficult to restore.

           Project proposals were reviewed and modified through input provided
           by State and Federal agency staff members, State and Federal
           attorneys, various experts retained by the State and Federal
           governments, and many corporate and private individuals. Based on
           these inputs and results from first year studies, a number of
           changes were made for the 1990 fisheries program. salmon studies
           FIS 1, 2, 3, 4, 7, and 8 were continued, as modified, another year
           while F/S 9 (Early Marine Studies outside PWS) which could not be
           initiated in 1989 was not approved for 1990.      Dolly Varden and
           cutthroat trout study FIS 5 which was conducted in 1989 was
           approved for continuation, as modified, in 1990.    The sport fish
           harvest and effort study (F/S 6),conducted in 1989 was not approved
           for continuation in 1990.    A study on Dolly Varden and sockeye
           salmon in LCI (F/S 10) which was approved for 1989 but could not be
           implemented was not approved for 1990. The herring study (F/S 11)
           was expanded and modified considerably from that of 1989 and
           approved for continuation in 1990.    Herring studies outside PWS
           (F/S 12) were completed in 1989 and not proposed for continuation
           in 1990. Clam study F/S 13 was combined with FIS 21 and approved
           for continuation, as modified, in 1990. The crab study within PWS
           (FIS 14) conducted in 1989 was not approved for continuation in
           1990. The crab study outside PWS (F/S 22) was conducted in 1989
           and was approved for continuation in 1990. The spot shrimp study
           (F/S 15) was conducted in 1989 and was approved for continuation in
           1990 with few modifications. The rockfish studies FIS 17 and 23
           were combined and modified for continuation in 1990. Multi-species

                                           53









       trawl surveys, FIS 18 and 24 were conducted in 1989 and modified
       considerably for continuation in 1990. The oyster study (FIS 16),
       larval fish study (F/S 19), underwater observations (FIS 20),
       scallop mariculture study (FIS 25), and the sea urchin study (F/S
       26) were not approved for continuation in 1990. Three new studies
       not conducted in 1989 were approved for implementation in 1990,
       these being sockeye salmon over-escapement (FIS 27), salmon run
       reconstruction (FIS 28), and salmon data base management (FIS 30).












































                                       54












            FISH/SHELLFISH STUDY NUMBER 1

            Study Title:    Injury to Salmon Spawning Areas in PWS

            Lead Agency:   ADF&G

                                       INTRODUCTION

            Wild stock production of pink salmon in PWS has ranged from 10 to
            15 million fish in recent years. Chum salmon returns have ranged
            from 800,000 to 1,500,000. Much of the spawning for pink and chum
            salmon (up to 75% in some years) occurs in intertidal areas.
            Intertidal spawning areas are susceptible to marine contaminants
            and the March 24, 1989, EVOS may adversely affect spawner
            distribution and success in Prince William Sound. To detect injury
            to pink and chum salmon stocks, intertidal contamination will be
            documented and correlated with trends in adult returns.       Return
            estimates are based on accurate appraisals of catch and
            escapements.     This project is designed to document oil
            contamination of intertidal spawning habitat; provide accurate
           .estimates of escapements of wild stocks; and provide estimates of
            the intertidal and upstream area available for spawning. F/S Study
            3 provides estimates of the wild stock component of the commercial
            catch. Results from F/S Study 3 and this study will be combined to
            estimate total return of wild stocks. F/S Study 2 estimates eggs
            and fry per square meter and egg to fry survival by tide zone in a
            subset of the streams in this study.      Egg and f ry density and
            survival data from F/S Study 2 will be combined with stream bed
            area estimates by tide zone from this study to estimate total egg
            deposition and egg to fry survival by tide zone in 138 streams.

            The ADF&G has performed spawning ground surveys of the major salmon
            spawning streams in PWS since the late 1950's. An aerial survey
            program provides weekly estimates of numbers of fish in 218
            spawning streams and a ground survey program on a subset of
            approximately 116 has provided corresponding estimates of numbers
            during the peak of spawning. During 1987 and 1988, funding for the
            ground survey program was severely curtailed and only 58 streams
            were walked. This study includes a thorough and extensive ground
            escapement survey program on salmon spawning streams for which
            there are past ground survey data and includes additional oiled and
            unoiled streams in western PWS. The study also includes ground
            surveys of salmon streams to document the presence of oil in
            intertidal spawning habitat.

            In 1989 a total of 411 streams were surveyed for the presence of
            oil in intertidal spawning areas and 138 streams were included in
            the ground census of pink and chum salmon escapements. In 1990 the
            oil survey will be limited to the 138 streams in the escapement
            censuring portion of the project. The total area of intertidal and

                                            55









        upstream spawning habitat will be estimated for each stream.
        Estimates of stream residence time (stream life) will be made for
        pink and chum salmon in 11 of the 138 streams.

        The results of the study will provide accurate estimates of the
        pink and chum salmon escapement to each stream surveyed; will be
        correlated with escapement estimates based on aerial counts to
        estimate past and current year escapements for 218 streams included
        in the ADF&G aerial survey program; will provide estimates of post
        oil spill distribution of spawning within stream zones and among
        streams; will estimate total available intertidal and upstream
        spawning habitat for each stream; will estimate the average stream
        life for pink and chum salmon in PWS; and will provide an atlas of
        aerial photographs and detailed maps for important spawning sites.


                                    OBJECTIVES

        A.   Determine the presence or absence of oil on intertidal habitat
             used by spawning salmon through visual observation, aerial
             photography, and hydrocarbon analysis of tissue samples from
             intertidal mussels at stream mouth.

        B.   Document the physical extent of oil distribution on intertidal
             spawning areas.

        C.   Estimate the number of spawning salmon, by species, within
             standardized intertidal and upstream zones for 138 streams in
             PWS.

        D.   Enumerate the total intertidal and upstream escapement of pink
             and chum salmon through weirs installed on one or more
             moderately large streams which are representative of streams
             in the aerial and ground escapement survey programs.

        E.   Estimate the accuracy of aerial counts for the 218 aerial
             index streams by comparison of paired ground and aerial counts
             from 138 of the streams on the same or adjacent survey dates
             and by comparison of aerial, ground, and weir counts on one
             stream.

        F.   Estimate the average stream life of pink and chum salmon in at
             least 11 streams in PWS using a variety of techniques.

        G.   Estimate 1961 through 1988 pink and chum salmon escapements to
             the 218 aerial index streams using the average observed error
             in the aerial survey method and on stream life data from 1989
             and 1990.

        H.   Estimate the stream area available for spawning within
             standardized intertidal and upstream zones for the 138 streams
             surveyed.

                                        56









             I.   Produce a catalog of aerial photographs and detailed maps of
                  spawner distribution f or the more important pink and chum
                  salmon streams of Prince William Sound for use in designing
                  sampling transects in the egg deposition and pre-emergent fry
                  studies.

             J.   Identify streams appropriate for enumerating and CWT pink
                  salmon fry.



                                           METHODS



             This project is an integral part of the study of impacts of the
             EVOS on Pacific salmon populations in PWS.     Streams examined by
             this project are a subset of the anadromous salmon streams
             monitored by the ongoing ADF&G aerial survey program.            Two
             additional F/S studies in PWS, pink and chum salmon egg deposition
             and pre-emergent fry studies Study 2 and salmon coded-wire tagging
             studies Study 3, will rely on information about salmon spawning and
             distribution obtained from this project.

             Three crews of two people each will perform foot surveys of
             intertidal and upstream portions of 138 major pink and chum salmon
             spawning streams. Each stream will be visited once prior to the
             salmon returns to measure and mark tide levels and survey
             intertidal areas in and adjacent to the stream for presence of oil.
             Live and dead pink and chum salmon will be enumerated by ground
             survey crews in standardized intertidal and upstream zones in each
             stream. Streams will be enumerated three times at approximately
             two week intervals'during the spawning season.

             Streams to be surveyed will be selected according to the following
             criteria:

               1. Stream is included in the ADF&G aerial survey program.
               2. Stream is included in the pink and chum salmon egg deposition
                  and pre-emergent fry project (F/S Study 2).
               3. Stream was enumerated in prior spawning ground foot survey
                  programs.
               4. Stream is representative of the early, middle, and late run
                  pink and chum salmon stocks in PWS.
               5. Stream is representative of the spatial distribution of pink
                  and chum salmon stocks in PWS and include streams from oiled
                  and unoiled areas.

             Maps of all streams in the program prepared from aerial photographs
             prior to the 1989 field season and modified and corrected during
             the three survey circuits in 1989 will be used and updated during
             the 1990 field season.




                                             57









         The pre-season survey to mark tide zones and document the presence
         of oil in the intertidal area at the stream mouth will be conducted
         in June, prior to the return of the pink and chum salmon.        The
         location of tide levels 1.8f 2.4, 3.0, and 3.7 m above mean low
         water will be measured from sea level using a surveyors's level  and
         stadia rod. Sea level at each site will be referenced to mean    low
         water with site specific, computer generated tide tables which
         predict tides at five minute intervals. Tide zone boundaries will
         be delineated with color coded steel stakes. The linear length of
         the stream within each intertidal zone will be measured with a
         surveyors chain or range finder. The linear length of the stream
         in the upstream zone will be measured similarly on short streams
         and estimated from accurately scaled aerial photos on long streams.
         The average stream width will be determined from systematic width
         measurements taken in each zone. The number of intervals in each
         zone will depend on the length of the zone. Each measurement will
         be recorded at the appropriate location on the stream maps prepared
         in 1989.

         Crews marking, measuring, and mapping tide zones will also conduct
         foot surveys of the intertidal stream bed and adjacent beaches to
         document, map, and classify any oil present.

         During the escapement enumeration portion of the project, streams
         will be surveyed visually from the ground in a systematic order.
         During each stream survey the following data will be recorded:

         - anadromous stream number and name (if available);
         - latitude and longitude of the stream mouth;
         - date and time (24 hour military time);
         - tide stage;
         - observer names;
         - counts of live and dead salmon by species and tide zone (0.0-1.8
           m, 1.8-2.4 m, 2.4-3.0 m, and 3.0-3.7 m above mean low water and
           upstream); and
         - weather and comments on visibility, lighting, and other survey
           conditions.

         All data will be recorded on pre-printed mylar data sheets which
         will overlay a map of the stream.      Maps will be improved and
         modified during the survey to show spawner distribution within each
         zone and the upstream limit of spawning. Particular attention will
         be given to spawner density and distribution observations for the
         48 streams to be sampled during FIS Study 2.

         During the first survey circuit, a composite sample of mussels will
         be collected at the mouth of each stream for hydrocarbon analyses.
         Results of the analyses will be used to document the level of oil
         impact that the stream sustained.     Each sample will consist of
         enough mussels to provide 10 grams of tissue (approximately 30
         mussels) for analysis. The mussels will be collected in the zone
         from 0-2 m above mean low water in the immediate vicinity of each

                                          58










           stream mouth and will be collected above water to avoid
           contamination by hydrocarbons on the water surface. The samples
           from- each stream will be stored in separate, properly cleaned,
           glass jars with teflon lined lids. Appropriate chain of custody
           forms will accompany each sample.

           During all three circuits counts of live and dead salmon will be
           made for the five tide zones (the intertidal zones < 1.8 m, 1.8-2.4
           m, 2.4-3.0 m, 3.0-3.7 m above mean low water and the upstream zone)
           from the 1.8 m tide level to the limit of upstream spawning on all
           138 streams. Tide stage will be monitored continuously and survey
           times and direction will be adjusted accordingly.       If the tide
           stage at the time of the walk is at or below the 1.8 m level the
           stream walk will begin at the mouth of the stream and progress
           upstream. The mouth or downstream limit of the stream will be
           defined as the point where a clearly recognizable stream channel
           disappears or is submerged by salt water.      Fish seen below  the
           downstream limit will be included in an estimate of fish off the
           stream mouth and noted as a comment on the data f orm. If       the
           intertidal portions of the stream above the 1.8 m level         are
           submerged at the time the walk begins, the crew will proceed to the
           upstream limit of the walk, walk downstream, and coincide the end
           of the walk with the time predicted for the tide to be at or below
           the 1.8 m level. The upstream limit of a walk will be determined
           by the presence of natural barriers to fish passage (i.e.
           waterfalls), by the end of the stream, or by the upstream limit of
           spawning. The upstream limit of spawning will be marked on U.S.
           Geological Survey color aerial photos of each stream following each
           survey.

           For counts of live and dead fish on moderate size streams with a
           single channel, crew members will walk together but independently
           count live fish in each intertidal zone.         Crew members will
           individually enter their count on mechanical hand tallies.         A
           maximum of three replicate counts may be made in each zone at the
           request of either observer.   If the two counts differ by more than
           10%, the zone will be recounted until counts differ by 10% or less.
           Upstream counts in a single channel will be similarly conducted at
           convenient stopping points (i.e., log jams or other clear counting
           delineators) . To avoid confusion with counts of live f ish, counts
           of dead fish will be recorded on the return leg of the stream walk.
           For large braided or branched streams, each crew member will count
           separate channels or upstream forks.        To avoid perpetuating
           counting biases within a counting crew, personnel will be rotated
           between crews daily.    When possible, crew members will not be
           assigned to the same streams on succeeding survey circuits.

           Tests for variability among observers and among counting crews
           (observer pairs) will be conducted on 10 streams during each of
           the three enumeration survey circuits.       At test streams, all
           observers will estimate numbers of live and dead pink salmon by
           zone and will record their counts independently. Counts will be

                                            59









         compared after all test streams have been surveyed. Three crews of
         randomly paired observers will also replicate counts on 10 stream
         and results among observed pairs will be compared.

         At 11 of the 138 streams in the ground survey program, fish will be
         include in a stream lif e study.      Stream life studies will be
         modeled in part after previous studies in PWS (   .Helle et al. 1964;
         McCurdy 1984).    For each stream will be captured at the stream
         mouths with beach seines and tagged with individually numbered
         Peterson disk tags color coded for day of capture. Tagging will be
         conducted at weekly intervals at each stream and during each
         tagging episode, 80 fish will be tagged. If fewer than 80 fish
         are available, all fish captured will be tagged. Daily counts of
         live and dead pink and chum salmon will be made by tide zone in
         each of the 11 streams. Live and dead fish bearing tags will be
         enumerated separately by color code and tag n umber. Only fish that
         have died since the previous count will be tallied in the daily
         surveys. To prevent duplicate counts between surveys, tails and
         tags of all dead pink and chum salmon observed will be removed.

         At least one moderately large stream from among the 138 surveyed
         will be weired. The weir will be installed at the six foot tide
         level or the lower level of intertidal spawning. Weir crews will
         be record daily passage through the weir and will tag a portion of
         each day's escapement.     Tags will be numbered sequentially and
         unique color codes will be used for each weekly interval. The weir
         crew will survey the intertidal and upstream portions of the stream
         daily for stream life data. Counts of live and dead pink and chum
         salmon will be made by tide zone.        Fish bearing tags will be
         enumerated by color code and the sequential number on tags from
         dead fish will be recorded.     only f ish that have died since the
         previous count will be tallied in the daily surveys. To prevent
         duplicate counts between surveys, tails and tags of all dead pink
         and chum salmon observed will be removed

         Steams will be divided into categories based on levels of
         hydrocarbon contamination (as determined from visual observations
         species, stream zone, and stream for each will be assigned to one
         of the categories. Categorical data analysis techniques such as
         log linear models using chi-square statistics will be used to
         compare differences in spawning among streams and tide zones, and
         related   these   disruptions    to   the    level  of    hydrocarbon
         contaminations.    Count and spawner distribution data will also be
         compared with historical stream survey data and related to the
         level of hydrocarbon impact.

         Steam life will be estimated using three methods.          The f irst
         estimate is the mean difference between date of tag recovery from
         dead f ish and the tagging date and the estimate will be made of the
         color coded tag lot to examine changes in stream lif e through
         times. The second estimate will use weir data and will be based on
         similar data for numeric tag codes from individual fish. The third

                                           60








            method will be based on the difference in the dates between peak
            life count and the peak dead count.
                                             I

                                          BIBLIOGRAPHY

            Helle, J.H., R.S. Williamson, and J.E. Baily. 1964. Intertidal
                 ecology and lif e history of pink salmon at Olsen Creek, Prince
                 William Sound, Alaska.        U.S. Fish and Wildlife Service,
                 Fisheries No. 483. Washington D.C.

            McCurdy, M.L. 1984. Eshamy District pink salmon streamlife study,
                 1984.    Alaska Department of Fish and Game, Division of
                 Commercial Fisheries. Prince William Sound Data Report No.
                 94-18. Cordova



            BUDGET: ADF&G

            salaries              $ 179.2
            Travel                     2.0
            Contractual              145.3
            Commodities               25.0
            Equipment                 40.0

            Total                 $ 391.5






























                                               61










        FISH/SHELLFISH STUDY NUMBER 2

        Study Title: Injury to Salmon Eggs and Pre-Emergent Fry in PWS

        Lead Agency: ADF&G

                                  INTRODUCTION

        Much of the spawning for pink and chum salmon (up to 75% in some
        years) occurs in intertidal areas. Moles, Babcock, and Rice
        (1987) have shown the adverse effects of oil on pink salmon
        alevins, particularly in saltwater.     The EVOS in PWS occurred
        immediately prior to emergence of pink and chum salmon from
        stream and intertidal spawning areas.      Obviously, these areas
        have the potential to be severely impacted by the oil spill.

        This study along with FIS Studies I and 3 support a comprehensive
        and integrated determination of injury to PWS salmon stocks.
        Results will include documentation of oil in intertidal salmon
        spawning habitat, pre-spill and post-spill estimates of total
        adult returns of wild and hatchery stocks, wild stock spawning
        success, wild stock egg to fry survival, and early marine
        survival of wild and hatchery stocks. Information on the extent
        and persistence of oil in the intertidal zone will be
        supplemented by Coastal Habitat Study 1.       The results of FIS
        Studies 1 through 3 will be used by Economic Uses Study 3 to
        determine the extent of damage to the,Prince William Sound salmon
        resource.


        The ADF&G has sampled pink and chum salmon pre-emergent fry since
        the 1960's in order to predict the magnitude of future salmon
        returns.   The fry dig program has operated at a reduced level
        since 1985.   The oil spill has the potential to cause mortality
        to the critical egg and fry life stages and thus an increased and
        more comprehensive fry dig program is necessary. This project is
        designed to meet this need by assessing the effect of the oil
        spill on egg and fry of wild stock pink and chum salmon.

                                     OBJECTIVES

        1.   Estimate the density of pink and chum salmon eggs (31
             streams) and pre-emergent fry (48 streams) by tide zone in
             study streams.

        2.   Estimate over-winter mortality of pink and chum salmon eggs
             in oiled and control streams based on sampling of 31 natural
             streams.

        3.   Assess reductions in adult returns (if any) associated with
             increased egg to fry over-winter mortality in oiled streams.


                                         62









            4.    Document    hydrocarbon     contamination     using     tissue
                  concentrations of hydrocarbons in alevins and mussels, -and
                  mixed function oxidase (MFO) levels in alevins and eggs from
                  study streams.



                                           METHODS

            There are approximately 900 anadromous fish streams in PWS. Pre-
            emergent fry sampling from some of these streams has historically
            provided an abundance index for pink salmon which is used to
            forecast future pink salmon returns.     In recent years, 25 index
            systems considered representative of pink and chum salmon
            producing streams in PWS have been sampled during the fry dig
            program.   Prior to 1985, sampling had been performed on as many
            as 45 streams.      This study is designed to compare rates of
            mortality and abundance between areas with various levels of oil
            impacts and with data from sampling prior to the oil spill.

            Sampling will consist of egg-digs performed in late September and
            early October, and pre-emergent fry digs conducted in mid-March
            to mid-April.       Preliminary sampling was performed on two
            occasions during the spring of 1989 in an effort to assess fry
            abundance prior to and immediately after oil impact.         on the
            first occasion the 25 streams in the ongoing ADF&G pre-emergent
            index program were sampled along with 14 additional streams.
            During the second event (approximately two weeks after the oil
            spill), 14 of the streams were resampled (representing both oiled
            and non-oiled areas) and an additional 16 streams were surveyed
            to assess their potential as egg and pre-emergent study streams.
            During September and October of 1989 egg digs were conducted on
            31 of these streams.

            spring fry digs in 1990 will be conducted on 48 streams.        These
            will include the 25 streams in the ongoing ADF&G pre-emergent
            index program plus 23 additional streams. The additional streams
            are located in Central to Southwest PWS where the majority of the
            oiling occurred.     New study streams were selected using the
            following criteria:

                       sufficiently large adult salmon returns to indicate a
                       high probability of"success in egg/fry digging;
                       past history of egg/fry digging; and
                       streams which had low to no oil impact in the immediate
                       vicinity of high oil impact streams.      This will help
                       account for possible variability       due to differing
                       climatic/stream conditions.

            The 48 streams span a range of oil impact and include streams in
            the historic sampling program.        Most of the streams with
            suspected or obvious oil impact are new additions.           The 30
            streams in low impact areas include 27 with a history of

                                             63









        sampling, six streams suspected of having received some impact
        including four with a history of sampling, and 12 streams with
        oil visibly present in the intertidal zone, including five with a
        history of sampling.

        As in 1989, egg digs will be conducted in the fall on a subset of
        the 48 streams sampled for pre-emergent fry.       Streams included
        in the fry sampling program, but not the egg sampling, are
        traditional fry sampling streams located on the eastern and
        northern shore of PWS.        These streams are spatially quite
        distinct from the streams studied for oil impact effects. The 13
        streams in low impact areas which were left in the egg dig
        program include four with a history of sampling.        The streams
        suspected of having experienced some impact and the streams where
        impact is visibly obvious are the same as in the fry sampling
        program.

        Sampling methods are identical for the pre-emergent fry and egg
        digs and are modeled in part after procedures described by Pirtle
        and McCurdy (1977).     On each sample stream, four zones, three
        intertidal and one above tidal influence, will be identified and
        marked by crews conducting stream surveys under F/S Number 1
        (PWS). The zones are 1.8-2.4 m, 2.4-3.0 m, 3.0-3.7 m above mean
        low water, and upstream of tidal influence.         Separate linear
        transects 30.5 m in length will be established for the egg and
        pre-emergent fry digs in each zone (one transect for each type
        dig in each zone). The transects will run diagonally across the
        river with the downstream end located against one bank and the
        upstream end against the opposite bank. Overlapping of transects
        will be kept to a minimum to control the influence of fall egg
        digs on perceived abundance of fry during spring         2  sampling.
        Fourteen circular digs (56 per stream), each 0.3 m. will be
        systematically dug along each transect using a high pressure hose
        to flush eggs and fry from the gravel.        Eggs and f ry will be
        caught in a specially designed net.

        Numbers of live and dead fry by species as well as numbers of
        live and dead eggs by species will be collected from each 0.3 m2
        dig.   Additional information such as date, time, zone, and a
        subjective estimate of overall percent absorption of the fry egg
        sacs in the sample will also be collected.

        Tissue samples from pre-emergent pink salmon fry will be
        collected from the intertidal channels of streams. Tissue samples
        will be analyzed for the presence of hydrocarbons characteristic
        of those found in oil from the Exxon Valdez.

        Fry sampled for hydrocarbon analysis will be from the intertidal
        stream bed at a level approximately 2.5 m above mean low water.
        Samples will be collected when the tide stage is below that level
        to avoid contamination from any surface film of oil on salt
        water.   A shovel or clam rake will be used to dislodge the fry

                                         64









             f rom the gravel and a stainless steel strainer which has been
             pre-rinsed in dimethylchloride and dried, will be used to catch
             the fry as they are swept downstream. Captured fry will be placed
             in jars with teflon lined lids and frozen.

             Fry from each tide zone will be collected for MFO analysis and
             these samples will be selected systematically from the digs in
             each transect.       Sampled fry will be preserved in buffered
             formalin solution in glass jars.

             Pre-emergent fry/egg data will be summarized by date, stream,
             level of hydrocarbon impact, stream zone, and number of alive and
             dead, fry and eggs. A mixed effects analysis of covariance will
             be used to test for differences in egg to fry mortality due to
             oiling using the 31 streams sampled for both eggs and pre-
             emergent fry. Degree of oiling and height in the tidal zone will
             be treated as fixed effects.    Height in the tide zone is nested
             within stream, a random effect.       Possible covariates will be
             provided by hydrocarbon analysis of mussel populations in close
             proximity to each stream.

             If no suitable hydrocarbon data are available, analysis of
             variance will be used. Degree of oiling as visually assessed by
             the mapping portion of the assessment of intertidal spawning
             areas will be used to post-stratify streams.       Degree of oiling
             and height in the tidal zone will again be treated as f ixed
             effects.   Height in the tidal zone is nested within streams, a
             random effect.

             Power of the test was estimated for the analysis of variance
             using data from the 1975 and 1976 egg and pre-emergent fry digs
             in PWS.   This study indicated the ability to detect an increase
             of 15% (e.g. 10% mortality to 25% mortality) in egg to fry
             mortality at   = 0.05, 95% of the time.

             These studies will be used to test for 1) differences in egg to
             fry mortality between streams which were oiled and those that
             were not, and 2) increases in fry mortality in 1989 immediately
             after oiling.

             Specific statistics to be estimated are:

                       number of dead and viable eggs per square meter by
                       salmon species, stream, and stream zone;
                       number of dead and live fry per square meter by salmon
                       species, stream, and stream zone; and
                       egg to fry survival by salmon species, stream, and
                       stream zone.







                                              65












                                     BIBLIOGRAPHY



         Moles, A., M.M. Babcock, and S.D. Rice. 1987.         Effects of oil
              exposure on pink salmon, 0. gorbuscha, alevins in a
              simulated   intertidal environment.         Marine    Environment
              Research, 21:49-58.

         Pirtle, R.B. and M.L. McCurdy. 1977.           Prince William Sound
              general districts 1976 pink and chum salmon aerial and
              ground escapement surveys and consequent brood year egg
              deposition and pre-emergent fry index programs.             Alaska
              Department of Fish and Game, Division of commercial
              Fisheries, Technical Data Report 9, Juneau.



         BUDGET: ADF&G


         Salaries                   $ 120.0
         Travel                          4.0
         Contractual                   150.0
         Commodities                    10.0
         Equipment                      18.8

         Total                      $ 302.8





























                                           66












           FISH/SHELLFISH STUDY NUMBER 3

           Study Title: Salmon Coded-Wire Tag Studies In PWS

           Lead Agency: ADF&G

                                    INTRODUCTION


           Two questions must be answered to measure a loss in salmon
           production due to EVOS:        1) which stocks were exposed to
           contaminated waters and 2) to what extent did exposure reduce
           production (catch plus escapement)? This study will contribute to
           estimates of production and survival for hatchery and wild stocks
           in oiled and unoiled areas by quantifying fry outmigration, the
           adult component of the catch, and the escapement to hatcheries.

           Wild stock returns of pink salmon in PWS have ranged from 10 to 15
           million fish in recent years. Chum salmon returns have ranged from
           800 thousand to 1,500,000. Additionally, returns of pink salmon to
           four PWS hatcheries now average more than 20 million fish and
           hatchery chum salmon returns in excess of 1.4 million fish are
           expected.

           Catch and escapement data for wild pink salmon in PWS have been
           collected since 1961.     In 19851 hatchery production became a
           significant part of the total salmon return.     Consequently, pink
           salmon fry tagging was initiated at three area hatcheries in 1986
           to estimate the survival of those stocks and their contribution to
           the 1987 catch. Similar estimates were made for a fourth facility
           based on tagging in 1987 and recoveries in 1988.       FIS Study 3
           estimated catch and survival rates of pink salmon released from
           these four PWS hatcheries based on tags applied in 1988 and
           recoveries of tags in the commercial, cost recovery and hatchery
           brood stocks in 1989.     Tags were also applied to pink, chum,
           sockeye, coho, and chinook salmon releases from PWS area hatcheries
           and to smolts from two wild stocks of sockeye salmon. Tagging in
           1990 will include all the same stocks plus one more wild stock of
           sockeye salmon and six pink salmon wild stocks. Tag recoveries are
           expected for releases at all four pink salmon hatcheries in 1989,
           releases of chum salmon from Main Bay Hatchery in 1986 and from
           Main Bay and Solomon Gulch Hatcheries in 1987, releases of sockeye
           salmon from the Main Bay facility in 1988, and releases of coho
           salmon from Wallace H. Noeremberg (WHN) and Solomon Gulch
           Hatcheries in 1989.









                                            67












                                      OBJECTIVES



         1.   Estimate catch, escapement, and survival rates of pink, chum,
              sockeye, coho, and chinook salmon released from five
              hatcheries in PWS. Outmigrating smolt and returning adults
              from these facilities are exposed to oil in varying degrees.

         2.   Estimate catch of the combined wild stocks of pink salmon in
              PWS and using escapement data from FIS Study 1, estimate
              differences in relative survival rates between pre- and post-
              spill brood years.

         3.   Estimate survival rates of wild pink salmon from three streams
              with contaminated estuaries and three with uncontaminated
              estuaries.

         4.   Provide marked salmon of known origin and oil exposure history
              for recovery by researchers studying early marine migration,
              growth, and survival (FIS Study 4).

         5.   Estimate survival rates of wild stocks of sockeye salmon, two
              from oiled areas, one from an unoiled area.



                                         METHODS

         A subsample of fry or smolt from all hatcheries releasing salmon
         into PWS will be tagged with a coded wire tag (Appendix A). Wild
         stock pink fry and sockeye smolt from both oiled and non-oiled
         areas of the Sound will also be tagged (Appendix B). Tags will be
         applied at rates which will insure that, given a realistic recovery
         effortl sufficient numbers can be recovered in the commercial
         fishery, hatchery cost recovery harvests, and hatchery brood stock
         collections (Appendixes) to allow researchers to estimate the
         contribution of each tag release group by district, week, and
         processor stratum. Release groups represent differences in release
         timing or treatment (i.e. fed vs. unfed fry)

         Tag application will be similar among all hatcheries and among all
         wild stock systems. Fry or smolt will be randomly selected as they
         emerge from incubators or outmigrate from streams, anesthetized in
         a 1 ppm solution of MS-222, adipose f in clipped, and tagged. A
         random sample of 100 fish will be graded for fin clip quality each
         day. The proportion of bad clips in the sample will be used to
         discount the daily release of tagged fish. Clipped fish will be
         tagged and passed through a quality control device to test for tag
         retention. Fish repeatedly rejected will be killed to minimize the
         number of untagged but clipped fish in the release. Fish that
         retain tags will be held for 24 hours to determine short term
         mortality. A sample of tagged fish from each tagger will be taken
         each day and graded for tag placement according to criteria

                                         68









            developed by Peltz and Miller (1988)   Prior to release, a 200 fish
            sample will be randomly sampled to estimate overnight tag
            retention. The proportion of lost tags in the sample will be used
            to estimate tag retention in the daily release.

            At the three Prince William Sound Aquaculture Corporation (PWSAC)
            hatcheries, tagged fish will be released directly into large
            saltwater rearing pens with untagged f ish of the same release
            group. At the Valdez Fisheries Development Association (VFDA)
            Solomon Gulch Hatchery tagged fry will be Placed in small
            enclosures within larger saltwater rearing pens for at least three
            days to allow them to recover from tagging before being mixed with
            unmarked fry from the same release group. At PWSAC hatcheries,
            unmarked f ry entering large pens were counted with Northwest Marine
            Technology counters. At VFDA, unmarked f ry in each pen will be
            estimated from counts of eggs in incubators minus egg mortalities.
            At all facilities, mortalities in the large pens will be estimated
            visually prior to release. Mortality rates based on visual
            estimates will be applied equally to tagged and untagged fish. The
            total number of f ish in group t with valid tags at the time of
            release will be estimated as

                      Tt         (Tt - mt) - (Tt - Mt) Lt,
            where     Tt         total number of fish tagged from group t,
                      Mt         overnight mortality among fish tagged from
                                 treatment group t,
                      Lt         overnight tag loss among fish tagged in
                                 treatment group t.

            The VFDA estimate   includes a term f or short term mortality of
            tagged fish from treatment group t during saltwater rearing (st).
            The number of tagged fish released becomes

                      Tt         (Tt - Mt - St) - (Tt - Mt - St) Lt.

            Hatcheries will release fry when plankton monitoring indices
            indicate peak zooplankton abundance.

            Four hatcheries released 13 groups of pink salmon in 1989. Only one
            of these groups was not tagged. Each of the hatchery pink salmon
            tag groups contained tagged fish at the rate of approximately one
            tag per 570 fish released. The tag rate was held constant across
            release groups to prevent confusion of differential tag mortality
            with variation in survival between release groups (Peltz and
            Geiger, 1988; Geiger and Sharr, 1989). In 1989, chum salmon were
            tagged at the rate of approximately one tag per 60 f ish released at
            the Solomon Gulch Hatchery near Valdez.

            In 1990, hatchery pink and chum salmon tagging will continue at the
            same level of ef fort with the addition of chum salmon at the Esther
            Island Hatchery; approximately 250,000 of these chum salmon will be

                                             69









         tagged in one release group.

         Wild pink salmon will be tagged from six stocks examined in FIS
         Study 2.   Fry will be captured as they emerge using various means.
         The fry will be anesthetized with MS-222 and tagged with Northwest
         Marine Technology tagging equipment and tags. The anesthesia and
         associated trauma will require that the tagged fish be held
         separate from their untagged cohorts, until they appear to have
         fully recovered from the effects of tagging. The extent to which
         the survival and behavior of the tagged fish can be extrapolated to
         other groups of salmon will be assessed at the time of recovery.

         Prior to tagging, hatchery chinook and coho salmon smolt in
         hatcheries will be crowded using seines. A sample of smolt will be
         drawn from each rearing appliance in approximate proportion to the
         number of fish in that appliance. They will be anesthetized with
         MS-222, their adipose fin excised, and a tag applied using
         Northwest Marine Technology equipment and tags. A sample of fish
         from each day's tag production will be retained to estimate short-
         term tag loss and tag induced mortality. Following tagging, the
         tagged fish will be returned to mix with untagged cohorts. All
         mortalities during the first week after tagging will be examined
         and the tag status noted. At the end of a week, the fish will again
         be crowded, and a sample of approximately 2,000-4,000 fish from
         each rearing appliance will be drawn. These fish will be
         anesthetized, and run through a tag detector. Peterson abundance
         estimates for all rearing appliances will be performed and any
         major discrepancies from hatchery inventory records noted. Finally,
         a written description of the tagging will be developed. This will
         include a detailed description of each tag lot, the number of fish
         tagged, the total number of fish in the release lot, the average
         size of the fish at release, a profile of the exposure history of
         the release lot to the oil spill, and all information required by
         the ADF&G Coded-Wire Tag Laboratory which coordinated tagging in
         Alaska.

         In 1989 wild sockeye salmon were tagged at Eshamy and Coghill
         Lakes. Smolt were captured in traps as they migrated to saltwater.
         The smolts were anesthetized with MS-222 and tagged with Northwest
         Marine Technology tagging equipment and tags. The anesthesia and
         associated trauma required that the tagged fish be held separate
         from their untagged cohorts until they appeared to have fully
         recovered from the effects of tagging. As in the wild pink salmon
         tagging, the extent to which the survival and behavior of the
         tagged fish can be extrapolated to other groups of salmon will be
         assessed at the time of recovery. The rate of tag occurrence in the
         stock will be determined from counts at an adult salmon weir in
         each of the systems. All fish passing through the weirs will be
         enumerated and heads from fish with adipose fin marks will be taken
         at the weir for tag removal and decoding. Hatchery produced sockeye
         salmon smolts will be tagged using the methods described for
         chinook salmon above.


                                           70









            The recovery samples are from a stratified sample (Cochran 1977),
            by district and discrete time segments. The recovery will be
            further stratified by processor as described in Peltz and Geiger
            (1988). For each time and area specific stratum, 15% of the pink
            salmon catch and a minimum of 20% of other salmon species catches
            will be scanned for fish with a missing adipose fin. Catch sampling
            will be done in four fish processing facilities in Cordova, one
            facility in Seward, and three facilities in Valdez. When feasible,
            sampling will occur at facilities in Kodiak, Kenai, Anchorage, and
            Whittier and on large floating processors. All deliveries by fish
            tenders to these facilities will be monitored by radio and by daily
            contact with processing plant dispatchers to ensure that the catch
            deliveries being sampled are district specific.

            In addition to catch sampling at the processing facilities,
            approximately 15% of the fish in the hatchery terminal harvest
            areas will be scanned for fish missing adipose fins. There will be
            a brood stock tag recovery effort at each of the three hatchery
            facilities where tags were initially applied. A minimum of 50% of
            the daily brood stock requirements of each facility will be scanned
            for fish with missing adipose fins. Finally, there will be an
            intensive survey of adult pink salmon returning to natural systems
            where tagging was conducted, and a weir will be operated for
            sampling adult sockeye salmon on those systems where sockeye salmon
            were tagged.

            In the catch, terminal harvest, brood stock, and natural system
            surveys, the total number of fish scanned and the total number of
            fish with missing adipose fin will be recorded. The heads will be
            removed from fish with missing adipose fins. Each head will' be
            tagged with uniquely numbered strap tags. Recovered heads will be
            assembled and pre-processed in the Cordova area office. Heads will
            then be sent to the FRED Division Coded-Wire Tag Laboratory in
            Juneau for decoding and data posting.

            A statewide coded-wire tag lab is located in Juneau and operated by
            FRED Division of ADF&G. Coded-wire tag sampling forms will be
            checked for accuracy and completeness. Sampling and biological data
            will first be entered onto the laboratory's data base. Next, the
            heads will be processed. This involves removing and decoding the
            tags, and entering the tag code and the code assigned in the
            recovery survey into the database. Samples will be processed within
            five working days of receipt.

            The first step in the coded-wire tag analysis will be to estimate
            the harvest of salmon from each tag lot, in units of adult salmon.
            Adult salmon from these tagged lots will be recovered in the common
            property fishery, the hatchery cost recovery fishery, and the adult
            brood stock. For the hatchery stock, a modification of the methods
            described in an ADF&G technical report by Clark and Bernard (1987)
            will be used. The specific methods, estimators, and confidence
            interval estimators are described in ADF&G technical reports on two

                                            71









        previous studies of pink salmon in PWS: Peltz and Geiger (1988),
        and Geiger and Sharr (1989). Additional references on methods of
        tagging pink salmon in PWS can be found in Peltz and Miller (1988).
        In the case of the wild stocks, the methods and estimators and
        necessary assumptions are described by Geiger (1988).

        The contribution of a particular tag lot, to a particular fishery
        stratum, is estimated multiplying by the number of tags recovered
        in the structured recovery survey, by both the inverse of the
        proportion of the catch sampled (the inverse sampling rate) , and by
        the inverse of the proportion of the tag lot that was actually
        tagged (the inverse tag rate) . The escapement (brood stock) of each
        tag lot is estimated using methods unique to the particular
        situation. After the contribution to each fishery is estimated for
        the tag lot, the survival is estimated by summing the estimated
        harvest of the tag lot in each fishery, and the estimated
        escapement (brood stock), and dividing by the estimated number of
        fish represented by the tag code.

        Total catches stratified by week, district, and processor were
        obtained f rom summaries of f ish sales receipts (f ish tickets)
        issued to each fisherman. The total hatchery contribution to the
        commercial and hatchery cost recovery harvest is the sum of the
        estimates of contributions in all week, district, and processor
        strata:

                     Ct    zi Xti ( Ni / Si ) pt-
        where:       Ct  = catch of group t fish,
                     Xti = number of group t tags recovered in ith strata,
                     Ni  = number of fish caught in ith strata,
                     Si  = number of fish sampled in ith strata,
                     pt  = proportion of group :t tagged.

        For sampled strata, we used a variance approximation which ignores
        covariance between release groups (Geiger 1988):
                     V (Cd      ZiXtj(Nj/Sipt)2[j - (Ni/Sipt)-'].

        The average tag recovery rate for all processors in a week and
        district will be used to estimate hatchery contribution in catches
        delivered to processors not sampled for that district and week.
        Variances associated with unsampled strata will not be calculated.










                                         72














                                          BIBLIOGRAPHY


            Clark, J.E. and D.R. Bernard. 1987.        A compound multivariate
                    binomial hypergeometric distribution describing coded
                    microwire tag recovery from commercial salmon catches in
                    southeastern Alaska. Alaska Department of Fish and Game,
                    Division of Commercial Fisheries, Informational Leaflet 261.

            Cochran, W. G. 1977. Sampling Techniques, 3rd ed. John Wiley and
                    Sons, New York, New York.

            Geiger, H.J. 1988. Parametric bootstrap confidence intervals for
                    estimates of fisheries contribution in salmon marking
                    studies. Proceedings of the international symposium and
                    educational workshop on fish-marking techniques. University
                    of Washington Press, Seattle. In press.

            Geiger, H.J. and S. Sharr. 1989. A tag study of pink salmon from
                    the Solomon Gulch Hatchery in the Prince William Sound
                    fishery, 1988. Alaska Department of Fish and Game, Division
                    of Commercial Fisheries. In press.

            Peltz, L. and H.J. Geiger. 1988. A study of the effect of
                    hatcheries on the 1987 pink salmon fishery in Prince William
                    Sound, Alaska. Alaska Department of Fish and Game, Division
                    of Commercial Fisheries. In press.

            Peltz,  L. and J. Miller. 1988. Performance of half-length coded-
                    wire tags in a pink salmon hatchery marking program.
                    Proceedings of the international symposium and educational
                    workshop   on   fish-marking   techniques.    University    of
                    Washington Press, Seattle. In press.



            BUDGET: ADF&G


            Salaries                                                  $ 902.0
            Travel                                                         21.0
            Contracts                                                     667.0
            supplies                                                      100.0
            Equipment                                                     300.0

            Total                                                      $1,990.0









                                             73










                Appendix A.                 Coded-wire tagging goals for hatchery
                                            releases of salmon in                    PWS, 1990.



                                                                                     Total
                                                                                     Release
                                                            Valid        Number      /Marked            Number
                                             Projected        Tag        Tags to     Ratio   Number of  Tags       Tag
                    Hatchery        Species   Release         Goal         order     Goal    Tag Codes   \Code       Length


                Armin F. Koernig Pink       120,000,000       200,000      255,000         600        8  30,000       Half
                                                                                                      1  15,000       Half
                Cannery Creek       Pink    150,000,000       250,000      277,000         600        7  37,000       Half
                                                                                                      1  18,000       Half
                Solomon Gulch       Pink    125,000,000       208,333      225,000         600        5  45,000       Half

                Wally Norenburg     Pink    250,000,000       416,667      460,000         600       10  46,000       Half


                GRAND TOTAL         Pink    645,000,000     1,075,000    1,217,000         600       32               Half


                Solomon Gulch       Chum      6,000,000        40,000      40,000          150        1  30,000       Half
                                                                                                      1  10,000       Half
                Wally Norenburg     Chum      50,000,000      100,000      100,000         500        4  25,000       Half


                GRAND TOTAL         Chum      56,000,000      140,000      140,000         400        6               Half


                Ft. Richardson
                    Whittier        Coho         100,000       20,000      20,000          5          1  20,000       Full
                     Cordova        Coho          60,000       10,000      10,000          6          1  10,000       Full

                Solomon Gulch       Coho      1,000,000        30,000      30,000          33         1  30,000       Full

                Wally Norenburg     Coho      2,000,000        70,000      70,000          29         1  50,000       Full


                GRAND TOTAL         Coho      3,160,000       130,000      130,000         24         5               Full


                Main Say            Sockeye   2,500,000       100,000      100,000         25         8  12,500  Order Filled


                GRAND TOTAL         Sockeye   2,500,000       100,000      100,000         25         8  12,500  Order Filled


                Wally Norenburg     King         150,000       30,000      30,000          5          1  30,000       Full


                GRAND TOTAL         King         150,000       30,000      30,000          5          1  30,000       Full


                GRAND TOTAL           ALL   706,810,000      1,475,000   1,617,000         479












                                                                           74









                    Appendix B.                Coded-wire tagging goals f or wild stock of salmon
                                               in PWS, 1990.

                                                                                        Total
                                                                             Valid     Release            Number
                                                              Projected,     Tag       /Marked Number of of Tags       Tag
                    System           Treatment Species        Release        Goat       Ratio  Tag Codes \Code        Length


                    Upper Herring B.   Oiled       Pink       1,000,000      40,000         25         2   25,000      Half

                    Hayden Ck.         Oiled       Pink       1,000,000      40,000         25         2   25,000      Half

                    Loomis Ck.         Oiled       Pink       1,000,000      40,000         25         2   25,000      Half

                    McClure Ck.        Clean       Pink       1,000,000      40,000         25         2   25,000      Half

                    O'Brien Ck.        Clean       Pink       1,000,000      40,000         25         2   25,000      Half

                    Totemoff Ck.       Clean       Pink       1,000,000      40,000         25         2   25,000      Half


                    GRAND TOTAL         ALL        Pink       6,000,000      240,000        25         12  300,000     Half


                    Coghill            Clean     Sockeye      1,000,000      20,000         50         1   20,000      Half

                    Eshamy             Oiled     Sockeye      1,000,000      20,000         50         1   20,000      FuLt

                    Jackpot            Oiled     Sockeye         200,000     20,000         10         1   20,000      Half


                    GRAND TOTAL         ALL      Sockeye      2,200,000      60,000         37         3   60,000      Both


                    GRAND TOTAL         ALL        ALL        8,200,000      300,000        27         15  360,000     Both



































                                                                             75











          FISH/SHELLFISH STUDY NUMBER 4

          Study Title:   Early Marine Salmon Injury Assessment In PWS

          Lead Agencies: ADF&G, NMFS

                                      INTRODUCTION



          The early marine period is a critical one for salmon because it is
          at this time that the greatest mortality is sustained (Parker 1968;
          Bax 1983, Hartt 1980; Foerster 1968; Ricker 1976; Nichelson 1986).
          Mortality is considered to be inversely proportional to the rate of
          growth, since a prolonged juvenile period will result in a pro-
          longed vulnerability to predators (Parker 1971; Healey 1982; Taylor
          1977; Walters et al. 1978). For a possible exception to this, see
          Helle (1980).  Therefore, factors that lower normal growth rates
          during the early marine period, such as toxic effects of exposure
          to hydrocarbons, reduction in prey populations, or increased energy
          expenditures associated with the disruption of normal migratory
          patterns, could have a strong influence on survival.

          Juvenile salmon are especially susceptible to oil toxicity when
          first in seawater (Rice et al. 1975; Rice et al. 1984). Sublethal
          levels of hydrocarbons can affect metabolism and reduce growth of
          juvenile salmon (Rice et al. 1975).    Sublethal levels of water-
          soluble hydrocarbons can also damage olfactory lamellar surfaces,
          conceivably impacting migratory behavior and feeding patterns
          (Babcock 1985).   Oil can also be toxic to littoral and pelagic
          macroinvertebrates (Caldwell et al. 1977; Gundlach et al. 1983).
          Thus, mortality, reduction of reproductive potential, or growth
          inhibition of prey populations could reduce growth rates of
          juvenile salmon, and thus increase their exposure to predation.

          During the past decade, five world-class hatcheries have been
          established within PWS. These facilities, operated by the PWSAC
          and the State of Alaska, produced approximately 535 million
          juvenile salmon in 1989.    The hatchery contribution represents
          roughly half of the total number of juvenile salmon produced in PWS
          this year. CWT program marked roughly 1.3 million juvenile salmon
          this year. Approximately one in every 1,000 juvenile salmon in PWS
          this year was expected to have a CWT. Recoveries of these marked
          fish in PWS will play a major role in our assessment of the impact
          of the oil spill.

          In 1990, the impact assessment will be conducted by the ADF&G and
          the National Marine Fisheries Service (NMFS) . Studies conducted by
          ADF&G will focus on the impact of the oil on growth and migratory
          behavior, and studies conducted by NMFS will focus on pairwise
          comparisons of salmonid growth and behavior in oiled and unoiled
          nearshore rearing habitats.     Sampling will be coordinated to

                                          76









            produce a single cohesive data base of 1) coded-wire tag recoveries
            and 2) zooplankton and epibenthos collections with associated
            temperature data.

            This study emphasizes a coordinated approach to attaining the
            objectives. The studies are mainly complementary. A strong effort
            is required because of 1) the high ecological and economic value of
            the resource and 2) the wide range of habitats utilized by salmon
            during the early marine phase.

                                           OBJECTIVES

            A.   Estimate the effects of oil contamination on abundance,
                 growth, feeding habits, and behavior of juvenile salmon during
                 their early marine residence.

            B.   Describe migration patterns of juvenile salmon relative to
                 oiled and unoiled areas of western PWS.

            C.   Estimate hydrocarbon levels in tissues of j        uvenile salmon
                 collected in oiled and unoiled areas in 1989.

            D.   Determine distribution, abundance, habitat utilization, size
                 and growth, and feeding habits of juvenile pink and chum
                 salmon, in order to compare these parameters with 1989
                 results.


            E.   Determine if sediment contamination has reduced the abundance
                 of primary prey species of harpacticoid copepods.

            F.   Determine if pollution of azoic sediments with hydrocarbons
                 will inf luence meiof auna colonization, especially harpacticoid
                 copepods, in terms of species distribution and abundance.

            PART I: Impacts of Oil Spill on Migratory Behavior and Growth

            The present study is designed to distinguish between the effect of
            oil and other factors on growth and migration by resampling fry in
            a few areas examined in 1989.        It is expected that the major
            difference between these areas in 1990 compared to 1989 will be a
            lower level of oil contamination. In 1990, the study will focus on
            tag lots that will have been released in a period of a week, or
            less.

            Portions of the 1989 sampling program will be discontinued in 1990
            in order to f ocus attention on growth and migrations. Discontinued
            studies will include tow net sampling because of low yields, fry
            stomach analyses because growth can be determined by less expensive
            means, and epibenthic sampling.




                                               77












                                      OBJECTIVES

        A.   Compare the growth of CWT salmon captured in oiled and un-
             oiled areas in 1989 with fry captured in the same areas in
             1990.    Determine at the alpha=.05 level whether size and
             condition factors are different in CWT fry collected in oiled
             and unoiled years.

        B.   Document the impact of oil on the migratory path and speed of
             migration of CWT salmon releases in PWS.        Determine at the
             alpha=.05 level whether migration speeds        and patterns are
             different in oiled and unoiled areas and in     oiled and unoiled
             years.

        C.   Document the hydrocarbon content of CWT fry collected in 1989.
             Determine at the alpha=.05 level whether hydrocarbon content
             differs in CWT fry collected in oiled and unoiled areas in
             1989.


                                          METHODS



        Fry collections will be targeted on tag lots that are released
        during a time of 1 week or less. Recovery of these salmon at later
        times and in different places will allow relatively accurate
        measurements of growth, and reasonable estimates of migration paths
        and migration speeds. Approximately 1.3 million tagged fish will
        be released. The goal will be to recover approximately 30 tagged
        fish in each of several tag lots each sampling time period. Based
        on 1989 recovery data, it is expected that during the proposed 6-
        week field season, sufficient fry should be collected to evaluate
        approximately six tag lots. The recovery effort will be targeted
        on tag lots released from Esther and Armin F. Koernig (AFK)
        hatcheries, because ('I) a good data set is presently available on
        releases from these hatcheries in 1989, (2) the field crew knows
        where fry released from these hatcheries can be collected, and (3)
        the hatcheries are located in an unoiled area (Esther) and an oiled
        area (AFK).

        Most collections, especially early in the season, will be made with
        a beach seine (modified Auke Bay design) and 1811 diameter dip nets
        on two or three beaches in each sampling area.        A 120 ft. purse
        seine (modified Auke Bay design) will be used in nearshore areas
        where the beach seine can not be used.        Water temperatures and
        salinity will be measured with a salinometer at 0 m and 5 m. Tide
        levels and directions will be recorded for each sampling site.

        To avoid excessive mortality when large numbers of fish are caught,
        fish will be placed in a holding tank until processing is
        completed.    Lots of approximately 300 ml of fish (measured by
        displacement in a I-liter beaker) will be put through a 2-inch
        tunnel tag detector (Northwest Marine Technologies) with a small

                                           78









             stream of salt water. When a tag is f ound in the lot, the lot will
             be continuously divided until the tagged fish is found. One 300-ml
             sample of fry will be sorted immediately to determine species
             composition and released. Another sample of approximately 80 fry
             will be preserved in buf f ered f ormalin f or later size measurements.
             This number should be sufficient to identify different size groups
             if they should occur. Remaining fry will be-released.

             All tagged fish will be blot-dried, and measured (snout to fork).
             Tags on these fish will be read later by the FRED Tag Lab. After
             reading the heads will be preserved in 70% ethanol and archived in
             case otolith analyses are desired at a later date.            Chain of
             Custody procedures will be used throughout transfer and storage of
             these samples. Untagged fish will be left in buffered 10% formalin
             for at least 30 days to standardize shrinkage. These fry will be
             rinsed in buffered sea water, blot-dried, weighed and measured.

             Analyses will test the null hypothesis of no difference between CWT
             pink salmon fry collected in oiled and unoiled areas at the alpha
             = 0.05 level. A Chi-square analysis will test the hypothesis of no
             difference in presence/ absence of oil among the different sampling
             areas.


             Analysis-of-variance of CWT fry will help separate the effects of
             oil from other variables contributing to variation in growth rates
             (change in body weight per unit time) and condition factor. other
             variables include year, hatchery of origin, tag code lot, sampling
             time,   and   sampling    area.      Those   variables     contributing
             significantly to differences in growth and condition will be
             further analyzed to assess the relationship.         Where applicable
             analyses will use tag code lot as a blocking variable to evaluate
             effects of oil. In addition, apparent growth rate curves will be
             analyzed using untagged fry caught at the same sample areas during
             the different sample times.

             Migration rates will be calculated using the minimum distance
             between release and recovery sites and the average release date for
             a given tag lot.     Differences in migration rate, distance and
             pattern will be analyzed with ANOVA as described in the above
             section.
















                                               79










        PART II. Impact of Oil Spill on Juvenile Pink and Chum Salmon and
                  Their Habitat



                                    INTRODUCTION

        In 1989, the NMFS component of the early      marine  salmon studies
        focused on pairwise comparisons between oiled and     non-oiled study
        sites in PWS. The objectives were to determine if oil had affected
        distribution, abundance, size and nominal growth ratesi feeding
        habits, and prey abundance.

        Epibenthic harpacticoid copepods produced in the intertidal and
        upper subtidal reaches are an important food resource for juvenile
        pink and chum salmon (e.g., Kaczynski et al. 1973; Healey 1980;
        Godin 1981; Cooney et al. 1981; Landingham 1982; Cordell 1986;
        Taylor et al. 1987; Landingham and Mothershead 1988). The trophic
        link between the benthos and the salmon is the most likely route
        for an impact on juvenile salmon in 1990. The contamination of the
        littoral zone could reduce prey densities by direct toxicity to
        harpacticoid copepods (Bonsdorff 1981; Bodin 1988), or by changing
        harpacticoid species assemblages from those dominated by epibenthic
        species to those dominated by inbenthic species which are not as
        available to visual feeders (Stacey and Marcotte 1987). Uptake of
        hydrocarbons by harpacticoids living in and- on the contaminated
        sediments could also reduce growth.         Contamination from prey
        decreases growth and causes changes in'feeding behavior of juvenile
        pink salmon (Schwartz 1985).      Slower-growing juveniles are more
        susceptible to size selective predation (Parker 1971; Hargreaves
        and LaBrasseur 1985) and thus suffer higher mortality (Healey 1982;
        Taylor et al. 1987; Taylor 1988).

        Proposed research for continuation in 1990 will collect data on
        distribution, abundance, habitat utilization, size and growth, and
        feeding habits of juvenile pink and chum salmon, in order to
        compare these parameters with the 1989 results.        Resolution of
        growth comparisons between oiled and non-oiled locations will be
        increased by using otolith increment analysis (Volk et al. 1984).
        Research will determine if sediment contamination has reduced the
        abundance of primary prey species of harpacticoid copepods and will
        determine if pollution of azoic sediments with hydrocarbons will
        inf luence meiof auna colonization, especially harpacticoid copepods,
        in terms of species distribution and abundance.









                                          so













                                             OBJECTIVES

             (Letters refer to general objectives described above, as well as
             three components listed above.)

             D-1. Test, at alpha = 0.05, if the abundance of juvenile pink
                   and chum salmon does not differ between oiled and
                   non-oiled areas.

             D-2.  Compare distribution and habitat utilization by juvenile
                   salmon between 1989 and 1990.

             D-3.  Test, at alpha       0. 05, if the size and growth r      *ates of
                   juvenile salmon do not differ between oiled and non-oiled
                   areas; to compare growth rates between 1989 and 1990.

             D-4.  Quantify the feeding habits of juvenile pink and chum salmon
                   in terms of fullness, frequency of occurrence, biomass, and
                   index of Relative Importance, and compare oiled and non-oiled
                   areas in 1990 and between 1989 and 1990.

             D-5.  Determine migratory behavior of juvenile salmon based on
                   coded-wire tag recoveries.

             E-1.  Examine if sediment contamination has reduced the abundance of
                   primary prey species (harpacticoid copepods).

             E-2.  Test, at alpha = 0.05, if the abundance of epibenthic prey
                   species for juvenile salmon does not differ between heavily
                   contaminated and lightly contaminated beaches.

             F-1.  Test, at alpha = 0.05, if the colonization of sediments by
                   harpacticoid copepods and other meiofauna is not affected by
                   the presence of oil in the sediments.


                                                METHODS

             Sampling Design

                   Component I (Objective D)

             In order to make direct comparisons between years, the same four
             oiled and four non-oiled locations sampled in 1989 in Western PWS
             will be sampled again in 1990. The locations are categorized as
             bays or migration corridors. At each location, three habitat types
             will be sampled. These habitat types are grossly characterized by
             grade and substrate: low gradient beach (<10% grade, granule-pebble
             substrate) ; medium gradient beach (12-25% grade, pebble-cobble
             substrate) ; and steep gradient beach (>50% grade, bedrock or large
             boulder substrate).      Particular beaches will be selected for

                                                81









         similarity   between    oiled   and   non-oiled    areas   in    such
         characteristics as wave exposure, macrophyte coverage, and
         substrate. Two beaches of each habitat type will be sampled within
         each location in 1990, for a total of 48 sites.         To minimize
         variability due to tide heights, sampling at the sites will be
         restricted to the -1 to +3 tide range. The sites will be sampled
         on each of four sampling cruises from mid-April to early June.

              Component 2 (Objective E)

         Abundance of important harpacticoid prey species will be compared
         between "lightly oiled" and "heavily oiled" beaches within each of
         three oiled embayments.     Comparisons between oil contamination
         levels within an embayment was chosen to minimize the effects of
         geographic variability. Three 40 m transects at the 0 tide level
         will be established for each of the two contamination categories
         within each embayment. Random number series will be used to select
         25 points along each transect for sampling with an epibenthic pump.
         Transects for prey abundance will be sampled during the low-tide
         series encompassed by sampling cruises 1,3, and 4; a different
         embayment will be sampled on each of these cruises.

              Component 3. (Objective F)

         Colonization by meiofauna of azoic sediments will be compared
         between oiled and control sediments. Standard dish pans with holes
         to allow water drainage will be filled with control, low oil (0. 5%)
         and high oil (2.0%) azoic sediiuents.     The pans will be aged in
         running freshwater for 1-2 weeks prior to use. Three pans for each
         level (control, two treatments) will be buried in the lower
         intertidal in two locations (a lightly oiled and a heavily oiled
         location).   The pans will be placed parallel to the water line,
         approximately 5 pan widths apart.      The sediments used in this
         experiment will be collected in Auke Bay, and made azoic by
         freezing. Approximately one*third will be used for the controls.
         The remaining sediments will be divided in half, and mixed with
         Prudhoe Bay crude oil to 0.5% and 2% concentrations. The pans will
         be placed in PWS in late April, and be sampled for meiofauna after
         I day and 4, 6, and 12 weeks.

         Sample Collection

         1. Fish Sampling

         Fish sampling at study sites will be restricted to the -1 to +3
         tide levels to minimize tidal effects between sites. Fish will be
         captured using 37 m beach seines.       Catches will be sorted by
         species and enumerated; all salmon will be checked for the presence
         of CW tags using an OMNI coded-wire tag detector. Each CW tagged
         salmon will be measured, weighed and frozen.       on each sampling
         trip, up to 60 each juvenile pink and 60 juvenile chum salmon from
         each sample site will be preserved in formalin for later length and

                                          82









            weight measurements; 10 of each species of these f ish will be
            randomly selected during processing for diet analysis.                In
            addition, 50 juvenile pink salmon from each embayment site will       be
            retained for otolith analysis, as per standard operating
            procedures. All other fish will be released.

            As time permits, the shoreline within the general vicinity of the
            habitat sites will be surveyed, and additional seine sets made.
            Juvenile salmon collected in these sets will be enumerated, checked
            for coded-wire tags, and used to supplement collections for otolith
            and stomach analyses when insufficient numbers are collected at the
            regularly sampled beaches. All other fishes caught in such sets
            will also be identified and enumerated.

            2. Zooplankton, Epibenthic Harpacticoids, and Meiofauna Sampling

            In the offshore water adjacent to the habitats sampled, triplicate
            samples of pelagic zooplankton will be taken with a 20-m vertical
            haul of a 0.5 m diameter 243 micron net. Epibenthic harpacticoid
            copepods will be sampled using a pump sampler (Cordell and
            Simenstad 1989).

            Meiofauna in the pan experiment (Component 3) will be sampled by
            taking five core samples from each pan, using 50-ml syringes.
            Samples will be f ixed in buf f ered f ormalin in 120-ml glass jars and
            labeled and sealed in the same manner as the other prey samples.

            3. Hydrocarbon Samples

            Mussel samples and sediment samples will be taken for hydrocarbon
            analysis at each location. Mussels will be sampled at or near one
            of the habitats within each site, and frozen.         Three replicate
            samples will be collected in 120 ml glass jars from the top 2 cm of
            sediments from 6-8 spots along the water line adjacent to the beach
            seine site. For the epibenthic transects, the replicate samples
            will be aggregated from within the six random quadrants selected
            for photographing. Two core samples will be taken for hydrocarbon
            analysis from the experimental sediment pans. A blank sample will
            be provided from each site. All sediment samples will be frozen.

            4. Environmental Data

            Water temperature and salinity at 0.5 m depth, wave height, and
            current measurements will be taken at each nearshore site regularly
            sampled, and at each prey transect. Water temperatures at 1 m and
            4 m will be taken in association with each set of zooplankton tows.
            Temperature and salinity will be measured using a Beckman probe
            conductivity-temperature meter. Current will be measured with a
            Marsh-McBirney induction current meter.         Wave height will be
            measured with a meter stick.       Extent of oil deposition and of
            visible oil in the water will also be noted for each habitat. A
            recording temperature/ salinity device will also be deployed at the

                                               83









         two sites where pans of experimentally oiled and control sediments
         are placed, for hourly temperature records. The boundary of the
         oxic-anoxic layer within the sediment pans will.be measured at the
         end of the experiment.

         Sample Processing

         1. Fish Samples

         Coded-wire tagged fish will be stored frozen until processing for
         tags.   The fish will be transported from field collection to the
         Auke Bay Laboratory, then to the ADFG Tag Processing Laboratory in
         Juneau. The tag lab personnel will decode the tags, and transmit
         the information to NMFS and to the ADFG investigator coordinating
         Early Marine Salmon Studies.

         After being weighed and measured, each fish retained for stomach
         analysis will be put into a labeled 20-ml vial filled with 50%
         isopropyl alcohol or 70% ethanol. Subsequent analysis will involve
         excising and weighing the foregut, removing the contents and
         estimating stomach fullness, and reweighing the empty foregut to
         get a measure of total content wet weight. The prey items will be
         identified to a minimum of order level and counted.

         2. Otolith samples

         The sagittal otoliths will be removed from frozen samples of at
         least 50 juvenile pink salmon from each of the four embayment
         locations, for both 1989 and 1990 samples.       Each otolith sample
         will be assigned a sample number corresponding to the original
         sample and the fork length of the individual fish. The otoliths
         will be sent to a qualified contractor, who will process the
         otoliths and determine: number of increments subsequent to the
         hatching and saltwater entry check; width of these increments along
         a standard axis in the posterodorsal quadrat of the otolith; mean
         increment width and associated error term for each 50 fish group.

         3. Zooplankton, Epibenthic Pump, and Meiofauna Samples

         Upon transport to the Auke Bay Laboratory, the samples will be
         logged in by sample number. A total of 96 zooplankton samples, 450
         epibenthic pump samples, and 360 meiofauna core samples will
         require processing.

         4. Hydrocarbon samples

         A total of 32 mussel samples and 472 sediment samples (counting
         triplicates) will be collected. All hydrocarbon samples collected
         in the course of this study will be prioritized by the Hydrocarbon
         Analysis project as to if and when the samples will be processed.
         Procedure for analysis of these samples is detailed in the
         Hydrocarbon Analysis study plan. (Technical Services Study No. 1)

                                           84









           5. Sediment analysis

           A total of 54"sediment samples each from component    2 and 54 from
           component 3 will be collected for organic carbon and nitrogen
           analysis and to quantify the sediment composition.    Processing of
           these samples will also be let to a qualified contractor.

                                      DATA ANALYSIS


           Component 1 (Oblective D)

           Two approaches will be used to compare abundance and size of
           juvenile salmon, and stomach fullness and relative biomass of
           stomach contents: non-parametric comparisons of paired oiled and
           non-oiled locations, and analysis of variance (ANOVA).           The
           Wilcoxin matched-pairs signed-rank test (Daniel 1978) contrasts the
           differences between the a priori pairs of oiled and non-oiled
           locations f or cells that match in terms of time and habitat. In the
           full analysis of variance model, five factors will be considered:
           oil/no-oil (fixed), time (fixed), bay/corridor (fixed), location
           (specific sampling location nested within bay/corridor), and
           habitat type (fixed). To confirm probability levels for the main
           factor of interest (oil) , a randomization procedure will be used to
           generate distribution-free significance levels.

           Nominal growth rates between oiled and non-oiled areas will be
           compared using a exponential growth model, and comparing the
           regression slopes of Ln weight over time with analysis of
           covariance (Zar 1974). ANOVA will be used to compare mean otolith
           increment widths using a partially hierarchal design (Winer 1971)
           involving three factors: year, oil/no-oil, and bays nested within
           oil/no-oil. Condition of juvenile salmon will be compared between
           oiled and non-oiled areas using least squares regression of the
           natural logarithms of weight and length (Cone 1989).         Percent
           similarity indexes (Whittaker 1975) will be calculated for   feeding
           habits between oiled and non-oiled areas, between bays and corridor
           locations, and between 1989 and 1990.

           Component 2 (Oblective E)

           Abundance, percent gravid females, and percent total harpacticoids
           for primary prey species of juvenile salmon will be compared
           between heavily oiled and lightly oiled beaches using ANOVA.
           Because the three embayments will be sampled at different times,
           differences between bays are not of interest as they could be an
           artifact of sampling time. Thus each embayment will be considered
           a separate experiment using a nested ANOVA to compare lightly and
           heavily oiled transects. The transects will be nested within oil
           contamination levels. An alternate analytical approach will be to
           use regression to examine the relationship between abundance of the
           to amount of oil in the sediment, as well as the substrate
           composition, macrophyte coverage, and carbon and nitrogen levels in

                                           85











        the sediments.

        Component 3 (Objec ive F)

        Abundance of total meiof auna and harpacticoid copepods in the
        experimental sediments will be compared using a three f actor,
        fully-crossed ANOVA (Winer 1971). The factors are location of the
        sediments, level of oil contamination in the sediments, and time.

        Data and Sample Archival

        All f ield and laboratory data forms generated through the course of
        this study will be placed in notebooks numbered according to the
        Auke Bay Laboratory Oil Spill Notebook Tracking System (NTS). All
        f ield notes will be similarly cataloged.      Trip reports for each
        sampling cruise and study plans will also be archived within the
        NTS.   Copies of computer data f iles will be maintained on two
        microcomputer hard drives, as well as rotating f loppy disk back-up
        kept in a locked cabinet.


             Table 1. Location of sample sites in Prince
                        William Sound listed as a priori pairs.



        Location                   Type            oil



        Herring Bay                Bay             Yes

        McClure Bay                Bay             No

        Snug Harbor                Bay             Yes


        Long Bay                   Bay             No

        Prince of Wales            Corridor        Yes


        Passage

        Culross Passage            Corridor        No

        Knight Island              Corridor        Yes
        Passage

        Wells Passage              Corridor        No




                                          86












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           Walters, C.J., R. Hilborn, R.M. Peterson, and M.M. Staley. 1978.
                Model for examining early ocean limitation of Pacific salmon
                production. J. Fish. Res. Bd. Can. 35:1303-1315.

           Whittaker, R. H.      1952.   A study of summer foliage insect
                communities in the Great Smoky Mountains, Ecological
                Monographs 2(l):1-44.

           Whittaker, R. H.    1975. Communities and ecosystems.     MacMillan
                Publishing Co., N.Y. 385 pp.

           Winer, B. J. 1971. Statistical principles in experimental design.
                McGraw-Hill, New York. 907 pp.

           Zar, J. H. 1974. Biostatistical analysis. Prentice- Hall, Inc.,
                Englewood Cliffs, NJ.




















                                           91













                                         BUDGET


        BUDGET: ADF&G

        Salaries                                         $82.0
        Travel                                             3.0
        Contracts                                         48.0
        supplies                                           5.0
        Equipment                                         12.0

        Total                                            $150.0


        BUDGET: NOAA                                     Total

        Labor                                          $ 100.0
        Travel                                            15.0
        Contractual Services                             100.0
        Supplies and Materials                            25.0
        Equipment                                         10.0
        Vessel Support:                                  150.0

        Total                                           $ 40d.0


        TOTAL BUDGET

        Category                                         Budget

        Personnel Services                               $182.0
        Travel                                             18.5
        Contractual                                       140.0
        Supplies                                           30.5
        Equipment                                          21.0
        Vessel Support                                    150.0

        Total                                            $550.0













                                          92










             FISH/SHELLFISH STUDY NUMBER S


             Study Title:    Injury to Dolly Varden Char and Cutthroat Trout
                             In PWS



             Lead Agency: ADF&G

                                       INTRODUCTION

             The goal of this study is to compare the survival and growth of
             populations of Dolly Varden Char (char) and cutthroat trout (trout)
             differentially affected by the oil spill in PWS. This project is
             currently in the second year of a    three-year study design. Trout
             and char are estuarine anadromous species that inhabit PWS (Morrow
             1980). Unlike anadromous Pacific salmon, trout and char utilize
             nearshore and estuarine areas for           feeding.     Their marine
             migrations are not as extensive as those of Pacific salmon (Morrow
             1980). Some of the most important stocks of these species inhabit
             areas that have been severely impacted by direct contact with oil
             including Green and Montague Islands and Eshamy Bay (Mills 1988).
             since these species commonly live to age 8 (Morrow 1980), the
             potential exists for both short-term and long-term effects from
             exposure to oil. Study of these species is crucial in that they
             represent finfish species that inhabit the most oil-affected areas
             throughout most of their lives.

             The experimental design for this program is based upon the model
             developed by Armstrong (1970, 1974, 1984) and Armstrong and Morrow
             (1980) to explain the migratory behavior of anadromous char.        This
             model identifies two patterns of life history, fish spawned in lake
             systems and fish spawned in non-lake systems.        For both groups,
             juvenile char remain in freshwater residence in their natal stream
             f or up to f our years.    During their last spring of freshwater
             residence, they smolt to sea. During late summer or early fall,
             f ish that were spawned in lake systems return to their natal stream
             to overwinter in the f reshwater lake.       During the spring, they
             again emigrate into marine waters and annually return to their
             natal lake system during late summer or early fall to spawn and
             overwinter. Fish that were spawned in non-lake systems exhibit a
             more complex migration. Upon smolting, juvenile-char search for a
             lake system to overwinter.      These fish then behave in the same
             manner as do tish that originate in a lake system except that they
             return to their natal stream to spawn and then return to their
             selected lake system to overwinter.

             The migratory habits of anadromous cutthroat trout are less well
             understood than those of anadromous char in Alaska although it
             appears that they exhibit similar migratory habits to char (Jones
             1982).   Trout, however, spawn in the spring as opposed to fall for
             char.


                                               93









         It is hypothesized that two detrimental impacts on these species
         could result from the presence of large amounts of crude oil in
         marine waters: (1) reduced survival; and (2) reduced growth. To
         test whether there was a measurable impact, three stocks of trout
         and char that over winter in watersheds that issue into a marine
         environment which has been directly exposed to oil (treatment
         group) and two stocks of trout and char that over winter in
         watersheds that issue in unoiled areas (control group) were
         selected for study.

         significant changes in stock abundance, composition, or dynamics
         from the initial emigration of stocks within the treatment group as
         compared to stocks from the control group is assumed to be due to
         contact with the oiled marine waters. Evidence from the literature
         indicates that marine migrations can range up to 116 kilometers for
         char (Armstrong 1974) and 80 kilometers for trout (Jones 1982).
         Armstrong's model of migratory behavior provides the basic
         framework for this study.      First, each of the study streams
         represents a stock of fish that annually homes to that specific
         over wintering stream. second, since over winter residency occurs
         entirely in freshwater, fish sampled during the 1989 spring
         emigration had not yet encountered oiled waters. Given this, the
         first sample from each stream (the emigration during 1989) provides
         the baseline data for stocks in control and treatment.


                                  OBJECTIVES


         A.   Test if there is no difference in annual survival rates of
              char and cutthroat trout between treatment and control groups
              during 1989-90 (the test will be done given a level of
              significance of alpha = 0.05.);

         B.   Test if there is no difference in annual growth rates of char
              and cutthroat trout between treatment and control groups
              during 1989-90 (the test will be done given a level of
              significance of alpha = 0.05.);

                                        METHODS


         Trout and char were still in freshwater residence at the time of
         the spill, and the opportunity existed to sample these fish during
         their 1989 emigration prior to any potential exposure to an oiled
         marine environment. Data collected during 1989 became the baseline
         for each system.   Therefore, in addition to comparisons between
         treatment and control, comparisons are also possible for each
         stream within treatment and control between subsequent years' data
         and the 1989 baseline.

         Each study stream consists of a freshwater lake-river system that:
         (1) is a tributary to marine waters that were either impacted by
         large quantities of oil (treatment) or received virtually no oil
         (control) ; and (2) contains stocks of anadromous trout and char.

                                         94









            A weir will be installed and completely block each study stream
            prior to the initiation ofthe 1990 spring emigration.         A smolt
            weir for sockeye salmon will operate at the outlet of Eshamy Lake
            as part of FIS Study #3.       Sampling for char and trout will be
            conducted in conjunction with this project.

            Char greater than 250 mm. in length will be initially clas   sified as
            mature (Blackett 1968). At the conclusion of this year's sampling,
            length frequency data will be analyzed to identify more precise
            classifications for immature and mature fish.

            All emigrating trout and char at each weir site will be counted and
            measured from tip-of-snout to fork-of-tail to the nearest
            millimeter.     Trout and char greater than 199 mm. will be tagged
            with numbered Floy FD-68 anchor tags and fish under 199 mm and
            greater than 149 mm, will be tagged with numbered Floy Fabric anchor
            tags.   All tagged fish will have their adipose fin removed to
            estimate tag loss.

            If all fish can be censused and examined for tags in all years,
            survival will be known for each system. Annual survival will be
            estimated for immature and mature trout and char. The mortality
            rate of spawning char is known to be high, particularly for males
            (Armstrong 1974) and it is probable that the same is true for
            trout. Therefore, the rate of survival estimated for immature char
            and trout will be used to test the hypothesis of equal survival
            between treatment and control groups.

            The hypothesis of equal survival will be tested using a chi-square
            statistic. However, if unknown numbers of fish can be expected to
            be lost past the weir (due to such events as weir washout) , it will
            not be possible to directly estimate survival from the numbers
            released and returned. Instead survival will be estimated using
            mark-recapture methods. Estimates of survival (Seber 1982) from a
            mark-recapture experiment with their 95% confidence intervals at
            three levels of abundance were examined to estimate the sample
            goals required to detect significant differences in survival.

            The hypothesis of equal growth will be tested by analysis of
            individual growth rate. Incremental growth for individuals will be
            computed from recaptured fish.      An Analysis of Variance will be
            conducted with stocks of char or trout serving as replicates within
            the treatment group.     Years, and possibly initial length, will
            serve as f actors in the design.      Differences in average growth
            rates between control and treatment groups will be attributed to
            some external disturbance so long as initial length of f ish is used
            as a covariate.

            During the spring sampling, weirs will be used to count and sample
            the emigration of trout and char from study streams. Weirs will be
            installed approximately 0.5 km upstream from the saltwater terminus
            of the streams. The weirs will be operated by a two-person crew

                                              95









         f rom mid- April to early July.       Downstream live traps will be
         installed.

         All fish captured in the trap will be examined for presence or
         absence of tags, tag scars, and adipose fins. Each fish containing
         a tag from 1989, a tag scar, or missing its adipose fin will be
         considered one recapture event. Recaptured fish with missing tags
         will be retagged.    Fish with no visible tag scar and containing
         their adipose fin (not tagged in 1989) will also be tagged. Each
         fish captured will be identified, counted, and measured (tip-of-
         snout to fork-of-tail to the nearest mm).        Scale smears will be
         collected from the preferred area from all cutthroat trout and
         placed individually on acetate slides in coin envelopes.           Date,
         species,    sex   (if    identifiable    from   external     maturation
         characteristics), and length will be recorded for each fish.
         Recapture events will be recorded separately for fish containing
         tags and fish with missing tags. Tag numbers will be recorded for
         each recapture and each fish tagged.

         All fish found dead impinged on the weir or in the live box will be
         examined for presence of tags and adipose fins, identified, and
         measured as outlined above. Sex and maturity will be determined by
         internal examination, and sagittal otoliths will be collected.
         Datef species, sex, length, maturity, and tag number will be
         recorded. Fish containing tags, tag scars or missing adipose fins
         will be recorded as recaptures.

         Estimates of annual survival will be computed for each study site
         through analysis of tag returns.      If all emigrating fish can be
         examined for marks, the estimates of annual survival (S) can be
         simply computed as:

         S = M2/Rl

         where:

         m2 = number of fish recovered in year y+l
         R1 = number of fish tagged in year y.

         The Jolly-Seber three-sample method (Seber 1982) found in the
         Appendix will be used in the event that each emigrating fish cannot
         be examined at the weirs. Buckland's program RECAP (1980) will be
         used to generate the estimates and variances.

         The sampling event for the purposes of the mark-recapture
         experiment is the emigration of trout and char past the weirs. All
         emigrating fish must cross the weir and therefore are assumed to be
         equally vulnerable to being sampled.         The assumption of equal
         survival of tagged fish will be tested for the different tag groups
         and for the different length classes using chi-square statistics.
         Tag loss will be estimated for fish tagged in 1989, as all tagged
         fish will also have their adipose fin-clipped.

                                            96









            If all weirs hold, the hypothesis of equal survival will be tested
            using a chi-square test for independence. Given a survival rate
            from immature to mature fish of greater than 15% the test will be
            able to detect differences of 7% or more (alpha = .05).      If the
            weirs do not hold and all char are not sampled at the weir, during
            emigration during the second and third year of the experiment; the
            95% confidence intervals of the survival estimated from the multi-
            year mark-recapture experiments will be compared to test for
            significant differences. In order to examine the effect of initial
            length on subsequent survival, the tests and estimates will be
            stratified by tagging length, and if possible a logistic regression
            will be used to estimate this effect.

            Annual individual growth will be calculated from the tag data as
            the difference between length at time of release and length at time
            of recovery.   An Analysis of Variance will be used to test for
            significant differences in growth between fish from control and
            treatment groups.    Variation due to differences in years and
            initial length can be controlled for through the use of a block and
            covariate in the linear model if necessary. The power to detect a
            .5% difference in the growth rate of fish from treatment and control
            areas is estimated to be 90%.

            The assumption of normality will be tested using Kolomogorov's D
            statistic.   In all likelihood the data will not be normally
            distributed and a logarithmic or a rank transformation will be
            necessary.

            The homogeneity of variance assumption will be tested with a
            Bartlett's test.     Again, if the assumption is not valid a
            transformation will be used.


                                      BIBLIOGRAPHY


            Armstrong, R.H. 1970. Age, food, and migration of Dolly Varden
                 smolts in Southeastern Alaska. J. Fish. Res. Bd. Canada
                 27:991-1004.

                 . 1974. Migration of anadromous Dolly Varden (Salvelinus
                 malma) in southeastern Alaska.      J. Fish Res. Board Can.
                 31:435-444.


                 . 1984. Migration of anadromous Dolly Varden char in
                 southeastern Alaska - a manager's nightmare. p. 559-570. In L.
                 Johnson and B.L. Burns [eds.) Biology of the Arctic char,
                 Proceedings of the International Symposium on Arctic Char,
                 Winnipeg, Manitoba, May, 1981.        Univ. Manitoba Press,
                 Winnipeg.

            Armstrong, R.H. and J.E. Morrow. 1980. The Dolly Varden char. p.
                 99-104. In Balon, E.K. [ed.] Chars: salmonid fishes of the
                 genus Salvelinus. Dr. W. Junk b.v., Publisher. The Hague,

                                             97











               Netherlands.

          Blackett' R. F. 1968. Spawning behavior, fecundity and early life
               history of anadromous Dolly Varden Salvelinus malma (Walbaum)
               in southeastern Alaska. ADF&G Research Report. 6:85 p.

          Buckland, S.T. 1980. A modified analysis of the Jolly-Seber
               capture-recapture model. Biometrics 36: 419-435.

          Clutter, R. and L. Whitesel. 1956. Collection and interpretation
               of sockeye salmon scales.         International Pacific Salmon
               Fisheries Commission, Bulletin 9. 159 pp.

          Hepler, K., A. Hoffmann, and P. Hansen. 1989. Injury to Dolly
               Varden char and cutthroat trout in Prince William Sound.
               State/Federal Natural Resource Damage Assessment Preliminary
               Status Report Draft, January 1990.          Fish/Shellfish Study
               Number 5.    Alaska Department of Fish and Game, Sport Fish
               Division, Anchorage.

          Jones, D.E. 1982. Development of techniques for enhancement and
               management of cutthroat trout in southeast Alaska. Alaska
               Department of Fish and Game. Annual Report of Progress,
               Project AFS-42, 23(AFS-42-10-B): np.

          Mills, M.J. 1988. Alaska statewide sport fisheries harvest
               report.    Alaska Department of Fish and Game, Fishery Data
               Series No. 2. 142 pp.

          Morrow, J. E.   1980. The freshwater fishes of Alaska. Alaska
               Northwest  Publishing Company, Anchorage, Alaska. 248 pp.

          Roth, K.J., C.  Whitmore, and P. Hansen. 1990. Prince William
               Sound and Gulf of Alaska sport fishery harvest and effort,
               1989.     State/Federal Natural Resource Damage Assessment
               Preliminary Status Report Draft, January 1990. Fish/Shellfish
               Study Number 6.     Alaska Department of Fish and Game, Sport
               Fish Division, Anchorage.

          Seber, G. A. F. 1982. Estimation of animal abundance and related
               parameters. 2nd edition, Griffin & Company, London. 655 pp.

          BUDGET: ADF&G


          Salaries                   $ 228.0
          Travel                            6.0
          Contracts                        37.3
          Supplies                         18.7
          Equipment                         0.0

          Total                      $ 290.0


                                            98










           FISH/SHELLFISH STUDY NUMBER 7a

           Study Title: Injury to Pink/Chum Salmon Spawning Within
                          Lower Cook Inlet and Kenai Fjords

           Lead Agency: ADF&G

                                      INTRODUCTION

           Wild stocks of pink and chum salmon are a major ecosystem component
           in the outer Kenai Peninsula and Lower Cook'Inlet area, immediately
           "down current" from PWS. Salmon represent a very important food
           source for marine mammals (sea lion, seals), terrestrial mammals
           (bear) , and a wide variety of bird species (eagles, etc.) .      In
           addition, these wild stocks of pink and chum salmon are harvested
           commercially.   In 1988, the year before the oil spill, the ex-
           vessel value of the commercial catch of wild and hatchery stocks of
           salmon from the lower CIK area was more than $8.2 million. Salmon
           are also very important to the sport, subsistence, and personal use
           fisheries. The future abundance of wild stocks of pink and chum
           salmon in the lower CIK areas may be adversely impacted as their
           intertidal spawning areas were affected by the oil spill.* This
           project was designed to evaluate the distribution of pink and chum
           salmon spawning in intertidal and upstream areas as a result of oil
           contamination from the Exxon Valdez oil spill. This project also
           provides spawner distribution data for F/S Study No. 8a).

                                       OBJECTIVES

           A.   Count the numbers of spawning salmon by' species and by
                intertidal and upstream areas for nine streams in the Lower
                Cook Inlet/Kenai Fjords area.

           B.   Produce maps of spawner distribution for each stream sampled.

                                         METHODS

           This project is designed to evaluate changes in numbers and
           .distribution of spawning salmon relative to oil contamination from
           the Exxon Valdez spill of March 1989. Three two-person crews will
           perform foot surveys of intertidal and upstream portions of nine
           major pink and chum salmon spawning streams. Port Dick Creek and
           Island Creek will be surveyed every other day by the first crew,
           Humpy Creek will be surveyed daily by a second crew, and the other
           six streams will be surveyed at least once a week on a rotating
           basis by the third crew. The third crew will be flown in and out
           by aircraft. All surveys will be conducted during low tide between
           July 7 and September 7.

           Streams to be surveyed will be selected using the following
           criteria:



                                            99









              I    The stream must be included in the existing aerial survey
                   program.
              2.   The stream was examined in past spawning ground survey
                   programs.
              3.   A significant fraction of spawning occurs in the
                   intertidal area.

         The nine streams studied in the Lower Cook Inlet area during 1989
         were Windy Creek Left, Port Dick Creek, Windy Creek Right, and
         Island Creek in the Kenai Fjords area and Humpy Creek, China Poot
         Creek, Seldovia River, Tutka Lagoon Creek, and Port Graham Creek.
         All but one historical alevin density index stream, Rocky River,
         was examined.   Rocky River was not included in the 1989 study
         because the effects of logging on that river would have complicated
         analysis.

         Of the streams studied in 1989, Windy Creek Left and Port Dick
         Creek have had oil deposited near the stream mouths, Windy Creek
         Right and Island Creek had oil floating offshore, and the remainder
         had no visible impact.

         Three of the non-oiled streams in Kachemak Bay, China Poot Creek,
         Tutka Lagoon Creek, and Seldovia River, will not be studied during
         1990 because of logistical problems that inhibit sampling efforts
         at these sites.

         Three new non-oiled streams in the Kenai Fjords area, Tonsina Creek
         (in Resurrection Bay) , South Nuka River and James Lagoon Creek,
         will be added to facilitate comparisons with oiled streams. All
         three of these streams have an intermittent history of fry digs.

         During each stream survey the following data will be recorded:

              1.   Stream name;
              2.   Date and time;
              3.   Counts of live and dead salmon by observer, species and
                   location in the stream C(1) 0.0-0.6 m, (2) 0.6-1.2 m, and
                   (3) 1.2-1.8 m below mean high water, (4) the upstream
                   (above tidal inundation) egg-fry dig area, and (5) the
                   upstream area above the egg-fry dig];
              4.   subjective assessment of count quality: (1) the counts
                   are reasonably accurate, (2) the counts are not accurate
                   and a recount may provide a better estimate (e.g. lots of
                   fish in a deep pool, a glare problem, etc.) , (3) the
                   counts are not accurate but a recount will not change the
                   results (e.g. bad viewing conditions due to siltation,
                   wind, rain, etc.).
              5.   Location of tagged fish, tag type, number, and color
                   (Port Dick Creek and Humpy Creek only).
              6.   Observers name(s).

         During foot surveys the numbers of spawning salmon will be

                                         100









           estimated by stream zone f or each study stream.       Stream zones
           represent levels of tidal influence; (1) 0.0-0.6 m below mean high
           tide, (2) 0.6-1.2 m below mean'high tide, (3) 1.2-1.8 m below mean
           high water, (4) the upstream (above mean high tide) egg-fry dig
           area, and (5) the remaining area above the upstream egg-fry dig
           area.


           Stream zones were marked at   each stream during the 1989 season.
           Intertidal zones in the Lower Cook Inlet/Kenai Fiords area were
           measured from the mean high tide level due to large differences in
           mean tidal height between the gulf of Alaska (4 m) and Cook Inlet
           (6 m) sides of the Kenai Peninsula. The stream bed location of the
           tide levels 0.0, 0.6, 1.2, and 1.8 m below mean high water were
           marked with a 0. 3 m 2 fluorescent orange plywood rectangle.      The
           markers were numbered consecutively 1 through 4 with number 1
           furthest downstream at the 1.8 m below mean high water level. A
           fifth marker will be added during the 1990 field season to identify
           the upstream end of the egg-fry dig area (NRDA PIS study 8a). A
           commercial hand held tide computer (Conex Electro-Systems model
           TF0290W TideFinder) with time and location corrections will be used
           to determine tide heights.

           Maps for each stream will be rechecked for boundaries between
           stream zones, distances across the streams within zones, and
           distances between zone boundaries. Areas of spawning concentration
           and preference by species within each stream zone will be recorded
           on the revised maps. These maps will be used later by the Pink and
           Chum Salmon Egg and Pre-emergent Fry Sampling project (NRDA PIS
           8a).

           Surveys will be at low tide and progress upstream from the 1.8 m
           below mean high tide marker (marker number 1) . The upstream limit
           of a survey will be determined by the presence of natural barriers
           to fish passage (e.g. waterfall, log jam, etci), the end of the
           stream, or the absence of spawning salmon.

           Counts from each crew member will be recorded as an independent
           observation. Crew members may either walk together or on opposite
           banks of stream channels depending on terrain and viewing
           conditions. Both crew members will walk up stream forks together
           and not split up. Also, the crew will not divide tasks (e.g. one
           member counts only live salmon while the other counts only
           carcasses, etc.).    survey partners will be rotated on a weekly
           basis to prevent counting bias from being perpetuated. Crews will
           be assigned to a different stream on each succeeding circuit.

           Crews will begin each survey with a "practice count" for a short
           distance.   If their counts differ by more than 10%, they will
           retrace their steps and search for the cause of the difference
           (fish in a deep pool not clearly visible to both crew members, sun
           glare, deep shadow, overhanging vegetation, etc.) and recount as
           many times as necessary until they are satisfied that they can

                                           101









        compensate for visibility problems peculiar to their vantage
        points.    Likewise, crew members may warn each other and are
        encouraged to discuss counting conditions in anticipation of
        problems before they begin counting (e.g. discuss depth and breadth
        of school before counting a deep pool of fish, warn each other of
        difficult viewing conditions, etc.). Thereafter, each member will
        count and record their data independently.

        Both live and carcass counts will be made while walking upstream.
        Carcasses will be marked at Humpy Creek and Port Dick Creek (e.g.
        tail removed) to prevent double counting.           Hand tally counters
        (with 4 banks) will be used when counting. Recounts or stops to
        record counts can be requested at any time and anywhere by either
        crew member.     At a convenient stopping point (e.g. a log jam,
        either end of a deep pool, the start or end of silty water, etc.),
        each crew member will record their counts and rate their counts as
        follows:


              1)   the counts are reasonably accurate and a recount would
                   give very similar results,
              2)   the counts are not accurate and a recount may provide a
                   better estimate (e.g. lots of fish in a deep pool, a
                   glare, wind, or rain problem, etc.),
              3)   the counts are not accurate but a recount will most
                   likely not change the results (e.g. bad viewing
                   conditions due to siltation, wind, rain, etc.).

        If both crew members rate their counts -1 or 3, then proceed to the
        next stream section and count to the next stopping point.               if
        either or both crew members rate their counts 2, then both crew
        members recount that section and record the results of the second
        count. If necessary, both crew members could recount and record
        the results for a third time. After the third trial, proceed and
        count to the next stopping point.

        Pink salmon will be tagged with individually numbered tags to
        determine stream life and movement for Port Dick Creek and Humpy
        creek. These streams will be surveyed on an every other day basis
        with stream location, tag type, color, and number information
        recorded for every tagged salmon observed. Stream life is expected
        to vary over time and between sexes. Fifth (50) fish of each sex
        will be tagged every other day, over an 11 day period.            A beach
        seine will be used to collect fish at the mouth of the streams for
        tagging. The color-number combinations for Port Dick Creek will be
        as follows:


              Day         Male                   Female                  Tag

                1     orange     1-50)    yellow (#51-100)       Peterson disk
                3     orange     1-50)    yellow (#51-100)       adhesive tape
                5     orange     1-50)    yellow (#51-100)       rubber band
                7     red (#101-150)      green (#151-200)       Peterson disk

                                           102









                   9      red (#101-150)     green (#151-200)      adhesive tape
                   11     red (#101-150)     green (1151-200)      rubber band

             The color-number combinations   for Humpy Creek will be as follows:

                  Day         Male                  Female                Tag

                   1      orange (#201-250)  yellow  (#251-300)    Peterson disk
                   3      orange (1201-250)  yellow  (#251-300)    adhesive tape
                   5      orange (#201-250)  yellow  (#251-300)    rubber band
                   7      red (#301-350)     green   (#351-400)    Peterson disk
                   9      red (#301-350)     green   (1351-400)    adhesive tape
                   11     red (#301-350)     green   (#351-400)    rubber band

             The numbered Peterson disk      will be obtained     from commercial
             sources. The second tag type will be self adhesive tape wrapped
             around the caudal peduncle with the two free ends attached together
             and protruding up in to the air with a number written on the tape
             while the third tag type will be a rubber band around the caudal
             peduncle with 15 cm of numbered survey's tape attached.

             A weir will be installed at Humpy Creek to provide known numbers of
             fish in the stream. Foot survey counts will be made on an every
             other day basis while aerial survey counts will be made in
             conjunction with the Commercial Fisheries Division aerial survey
             program. There will be no attempt to adjust foot or aerial survey
             counts to match weir counts as the two are expected to differ.
             Humpy Creek will be mapped and marked for shallow areas with a
             clear view of the sky, deep pools with a clear view of the sky, and
             areas with an overhead canopy.       Counts in these areas will be
             recorded separately as aerial and foot survey counts in these areas
             are also expected to differ.

             Data Analysis

             Total number of salmon by species present at the time of the foot
             survey will be estimated using a simple stratified sampling scheme.
             Each stopping point will be considered the end of a sampling
             strata. Thus, a stream zone could encompass many sampling strata.
             The average and variance of the independent counts will be used to
             estimate the number of fish present within the strata and the
             variance about the estimate. Averages will be summed across stream
             zones to provide estimates of numbers of fish within the zone and
             zones summed to estimate numbers of salmon present during the
             survey. Variances will be weighted by the number of fish estimated
             in the strata and summed across stream zones and stream.

             Stream life and its variance will be the average and corresponding
             variance for the number of days a particular tag lot survives in
             the stream. Changes through time and by sex will be examined.

             Total escapement to each stream will be estimated using the area

                                              103









       under the curve method (similar to that described by Johnson and
       Barrett 1988).   The point estimates and variances from the f oot
       surveys along with stream life estimates and variances will be used
       to estimate total escapement and the corresponding variance.

       Statistics that will be estimated include:

            1.   Number of spawning and dead salmon by species, stream
                 zone, stream, and date and the corresponding variances.
            2.   Stream life and variance  :
            3.   Total escapement and variance.
            4.   Adjustment factors to relate aerial and foot surveys to
                 weir counts.

       A composite sample of mussels (Mytilus sp.) will be collected at
       the mouth of each stream for hydrocarbon analysis. A field blank
       (sample container opened at the collection site, closed and stored
       as if it contained a sample) and two sample replicates will also be
       collected. Results of the analysis will be used to document the
       level of oil impact sustained by the stream.       Each sample will
       consist of enough mussels to provide 10 grams of tissue for
       analysis.    The mussels will be collected from the immediate
       vicinity of all streams.      Collectors will use wooden tongue
       depressors when possible.    All mussels will be above water when
       collected to prevent contamination by surface hydrocarbons. The
       sample containers will be pre-rinsed (with dicloromethane) glass
       jars with teflon lined lids as supplied by I-Chem.      The samples
       will be stored in padlocked containers And kept in a freezer in the
       Homer ADF&G office.     Appropriate chain of custody forms will
       accompany each sample.

       Streams will be divided into 2-3 categories based on levels of
       hydrocarbon contamination (as determined from 1989 visual
       observations and hydrocarbon level in mussel tissues from the 1989
       and 1990 samples) . Counts of salmon by species and stream zone for
       each stream will be assigned to one of the hydrocarbon categories.
       Counts and spawner distribution will be compared with historical
       stream survey data and related to the level of hydrocarbon impact.

                                   BIBLIOGRAPHY

       Johnson, B.A. and B. Barrett. 1988. Estimation of salmon
            escapement based on stream survey data. Alaska Department of
            Fish and Game. Division of Commercial Fisheries. Regional
            Information Report No. 4K88-35. Kodiak.








                                       104













             BUDGET: ADF&G

             Salaries               $ 96.9
             Travel                     7.5
             Contract                   9.6
             Supplies                   3.3
             Equipment                  0.3

             Total                  $ 117.6














































                                              105









         FISH/SHELLFISH STUDY NUMBER 7b


         Study Title:     Injury to Pink Salmon Spawning Areas Within the
                          Kodiak and Chignik Areas

         Lead Agency:     ADF&G
                                     INTRODUCTION



         Large escapements into Kodiak and Chignik streams in 1989 occurred
         as a result of severely limited commercial fishing opportunities
         caused by the EVOS.    Total pink salmon escapements in 1989 were
         20.0 (Kodiak) and 1.4 (Chignik) million fish, whereas target
         escapement goals were 4.0 and 0.7 million pink salmon in the Kodiak
         and Chignik areas, respectively. The magnitude of escapement
         experienced during 1989 is unprecedented and has the potential for
         adversely impacting future returns of pink salmon through density
         related factors such as fungus and disease outbreaks at the egg
         and/or fry stage.

         Pink salmon are a major component of the Kodiak and Chignik area
         ecosystem, providing an important food source for both marine
         mammals, terrestrial mammals, birds, and other fish and shellfish.
         Additionally they annually re-charge fresh water and near shore
         marine environments with nutrients as carcasses decompose after
         spawning.    This species is used for subsistence, sport and
         commercial purposes.    Annual ex-vessel value (1978-1988) of the
         pink salmon harvest is 14.2 and 1.45 million dollars for the Kodiak
         and Chignik areas, respectively (Malloy 1989; Thompson and Fox
         1989).

         Pink salmon commercial fisheries are managed, in part, by
         controlling escapement which is evaluated by aerial and weir
         counting methods. Aerial surveys for pink salmon escapement indices
         using fixed wing aircraft and trained observers have been conducted
         annually for over 30 years in the Kodiak and Chignik management
         areas. Total pink salmon escapement and spawner density estimates
         by index stream, geographical region and management area will
         provide the basis for quantifying the effects of large escapements
         realized during 1989, on forthcoming brood year returns. Estimates
         of total available pink salmon spawning habitat will permit
         assessment of production from empirical escapement densities, and
         allow for determination of optimum spawning density by geographical
         area which will be useful for restoration efforts,.if needed.



                                      OBJECTIVES


         A.   Estimate total pink salmon escapements for streams where
              historic pre-emergent sac fry density data exist.           This
              includes 44 Kodiak and 18 Chignik streams.

                                          106









            B.   Def ine the distribution of spawning pink salmon for index
                 streams within the Kodiak and Chignik management areas. This
                 entails mapping and photographing spawner distribution.

            C.   Estimate total available spawning habitat for index streams
                 within the Kodiak and Chignik management areas.

                                          METHODS

            There are two integral components of this investigation: 1) weir
            counts and repeated aerial and foot surveys for escapement and
            stream life calculations, and 2) stream surveys for collection of
            spawning habitat data necessary for calculating total available
            spawning habitat.

            Trained observers will conduct aerial surveys using fixed wing
            aircraft on the 44 Kodiak and 18 Chignik pre-emergent index
            streams.   Surveys will be conducted weekly on each index stream
            with the program continuing until at least seven surveys over the
            spawning period have been completed or when spawner counts have
            decreased to less than 10 percent of the observed peak count.
            Additional non-index streams (342 Kodiak and 72 Chignik) will be
            surveyed as time and aircraft availability permit. The following
            information for each survey will be collected: 1) stream name and
            statistical number; 2) date, weather conditions, fish visibility
            rating (poor, fair or good) and time; 3) observer, aircraft type
            and pilot; 4) number of live and dead fish of each species (in bay,
            mouth and stream); and 5) general survey comments. Data will be
            recorded on standard forms suitable for data entry into the
            regional survey database. The observer, upon completion of surveys
            for a particular index stream, will map spawner distribution and
            designate the percent of spawning area used. Aerial photographs
            will be taken of spawner distribution for all index streams
            surveyed.

            counting weirs will be located at Akalura, Litnik, Saltery,
            Paramanof, Barling, and Uganik (Pillar Creek will be monitored by
            foot survey).    Pink salmon weir counts will be made daily and
            recorded on standardized forms. once every three days throughout
            the spawning period, foot surveys will be conducted by weir crews
            and additional personnel to enumerate live and dead fish by
            species. Data collected during stream surveys will be recorded on
            standardized forms. Daily weir count and foot survey data will not
            be reported until after the project has been completed. Aerial
            surveys will be completed for all weired index streams with a
            minimum frequency of one survey per week. Data collected during
            these surveys will be kept separate from the routine aerial survey
            data. Information collected for this study component will allow
            for calculation of stream life by system, in-stream population
            estimates, and aerial survey calibration.


                                            107









        At each weir where stream lif e is being estimated, adult pink
        salmon will be tagged with 0. 3m, color coded Floy tags with a
        marking rate of 200 per week.   Tagging will commence approximately
        July 21st and continue for four weeks. Tags will be affixed on a
        single day each week by capturing f ish in a trap located on the
        upstream side of each weir. Each week will have a specific tag
        color for identification of that tagging lot. Recovery effort will
        consist of enumeration of specific color coded fish during foot
        surveys and tags will be recovered and counted from mortalities.

        During the 1989 field season, total available spawning habitat was
        estimated for 31 Kodiak and 14 Chignik preemergent index streams.
        The remaining 17 index streams will be surveyed in 1990 to
        determine total available spawning habitat.

        Relying upon previously constructed maps of Kodiak and Chignik
        index streams with defined limits of historic spawner distribution,
        total and available stream length will be calculated.             The
        available stream length component will be divided into 300 meter
        sections, which will be randomly selected for surveying. Within
        each section, 12 transects spaced 25 meters apart will be run
        perpendicular to the stream bank.

        Pink salmon spawning habitat can be envisioned as a continuum of
        water velocity, depth, substrate size and embededness. According
        to Raleigh and Nelson (1985), substrate size and water velocity
        have the greatest influence on spawning success of pink salmon,
        however, substrate embededness may also have an impact (Platts et.
        al. 1983).     At each transect surveyed, stream width, water
        velocity, depth and substrate embededness will be assessed. Values
        used for these criteria are founded upon averages derived from the
        literature (Andrew and Geen 1960; Chambers 1956; Divinin 1952;
        Krueger 1981; Neave 1966; Wilson et. al. 1981). Available spawning
        habitat will be considered as an area along a transect where depth
        is a minimum of 15.2 cm, substrate size is within 0.6 to 13.8 cm,
        water velocity is 0.3 to 0.9 m/sec and substrate embededness is
        such that gravel is displaced without excessive foot pressure.
        Spawning habitat will be recorded as a percent of the total area
        encompassed by a one meter band along the transect line. Data will
        be entered onto standardized forms and later entered into the
        regional database.     All field personnel responsible for data
        collection will have prior experience with assessing habitat
        variables.

        An additional task of the spawning habitat inventory program will
        be to calculate total spawning area for all of the spawning riffles
        sampled in the pre-emergent fry dig study (F/S Study 8b).
        Estimates will be derived for 44 Kodiak and 18 Chignik Area
        streams. The exact location and approximate dimensions for each
        riffle will be obtained from detailed maps of individual index
        streams and from consultation with pre-emergent fry dig personnel.
        Length of the riffle will be measured on a straight line transect

                                         108









           measure, while width measures will be made one meter apart on both
           sides of the line for the entire length of the riffle.             All
           measurements will be in meters and recorded on standardized forms.
           only substrate size, velocity and substrate embededness variables
           from the above methods will be used to delineate spawning habitat
           in this context.

           Stream life of pink salmon is the length of time an adult is alive
           in the freshwater environment. In 1989, stream life estimates were
           successfully derived from weir and foot survey counts incorporated
           into the Johnson and Barrett (1988) model for two Afognak Island
           systems. Weir and foot survey counts of both live and dead fish
           collected during 1990 will provide for a maximum of 30 data points
           per system to be available for stream life analysis.               Two
           analytical approaches will be used to calculate stream life in
           1990. The first will be identical to that used in 1989, while the
           second will use cumulative and periodic dead fish counts.           The
           first approach relies upon the cumulative weir count and periodic
           foot survey counts being entered into the model and the stream life
           value being iterated until the model output converges upon the
           cumulative weir count. The second approach will involve the
           cumulative dead fish count, periodic foot survey dead counts, and
           the Johnson and Barrett (1988) model, following the steps outlined
           above.   This analysis, in addition to providing a second stream
           life estimate, will also allow for calculating a combined washout
           and predation rate (difference between cumulative dead and
           cumulative weir counts) which will allow for calibrating counts
           derived from stream systems surveyed without weirs.           Computed
           stream life values will be statistically tested and if significant
           differences exist, system specific stream life values will be
           evaluated based upon the variables stream order, orientation,
           geomorphology and stream length.

           Stream life estimated from 'tagging data will consist of determining
           the point at which f if ty percent of the tagged f ish f or a
           particular color code have been recovered. This approach will be
           carried out f or each of the f our weeks f or which tags have been
           affixed. An overall stream 1  -".fe estimate will consist of averaging
           the estimates f or each of -he four weeks and also comparisons
           between systems and weeks following the previously mentioned
           analytical approach for between system tests.

           Instantaneous live pink salmon population size will be estimated
           from cumulative weir counts and periodic (every three days) foot
           survey counts of dead pink salmon, Relying upon either a linear or
           exponential model with in-stream population estimates and aerial
           survey counts as parameters, precision of aerial survey counts by
           geographical location and escapement magnitude can be quantified.
           From this analysis, calibration factors can be determined for use
           in total escapement estimation procedures.

           Temporal aerial survey escapement counts of pink salmon in spawning

                                            109









       streams are, depending upon the frequency and timing of the
       surveys, related to total or cumulative escapement. Defining and
       quantifying variables or relationships that allow transformation of
       aerial survey counts into reliable estimates of total escapement is
       the major task. The Johnson and Barrett (1988) geometric model is
       one such approach to estimating total escapement.          Two data
       components are required for the algorithm, escapement counts over
       time and stream life.     The unit of measurement is area of the
       spawner abundance curve derived from a series of survey counts.
       Two segments comprise the analytical phase of the model, the first
       is calculating number of fish present between survey counts and the
       second is deriving total escapement.

       Total escapement accuracy, according to Johnson and Barrett (1988),
       is related to precision of escapement counts and stream life
       estimates.   The influence that both escapement counts and stream
       life estimates have on the total escapement estimate will be
       evaluated. Total escapement estimates will be calculated for all
       index and non-index streams for both the Kodiak and Chignik
       management areas where aerial and or foot surveys have been
       completed.    Total escapement will also be estimated for index
       streams based upon the historic aerial survey data base (1968-1988)
       so that all data which will be utilized in future analyses will
       have been derived in a similar fashion.

       Available spawning habitat will be determined using equations
       identified in Cochran (1977) and Wolter (1984), (personal
       communication, Alan Johnson Regional Biometrician, ADF&G, Kodiak).
       These equations will allow for calculation of the total available
       spawning habitat and variances associated with the estimates. The
       estimates can then be used for density estimates for spawning pink
       salmon for the 1989 brood year in addition to assessing the
       relationship between density and subsequent returns from previous
       brood year spawning events.

       To derive estimates of potential egg deposition to pre-emergent fry
       survival, an estimate of total area available for pre-emergent fry
       sampling is necessary. The area sampled for an individual fry.dig
       when coupled with the total area estimate will allow for expansion
       of the fry dig data to an estimate of survival. Total area of
       sampling riffles will be estimated by using the habitat measures
       for each riffle and summing over all riffles which are sampled for
       pre-emergent fry. An estimate for each index stream sampled within
       the Kodiak and Chignik management areas will be derived. These
       estimates form the foundation of an analysis component conducted as
       part of FIS Study 8b.

       [email protected] distribution maps will be prepared, in part, by observers
       conducting aerial survey escapement counts as was done for the 1989
       return. In addition, aerial photographs will also be obtained and
       cataloged for reference.


                                        110











                                       BIBLIOGRAPHY

            Andrew, F.J. and G.H. Geen 1960.         Sockeye and Pink salmon
                 production in relation to proposed dams in the Fraser River
                 system. International Pacific Fisheries commission Bull.
                 No.11.

            Chambers, J.S. 1956.     Research relating to study of spawning
                 grounds in natural areas. U.S. Army Corps. Eng. No. Pac. Div.
                 Fish Eng. Res. Prog. 6pp.

            Cochran, W.G. 1977. Sampling Techniques. John Wiley, New York.

            Divinin, P.A. 1952. The Salmon of South Sakhalin. Investia-Tinro.
                 37:69-108.


            Frisell, C.A. and W.J. Liss 1986. Classification of stream habitat
                 and watershed systems in south central Oregon. Unpub. Progress
                 report. Oak Creek Lab. Corvalis Or.

            Hankin, D.G. and G.H. Reeves 1988. Estimating total fish abundance
                 and total habitat area in small streams based on v i s u a 1
                 estimation methods. Canadian Journal of Fisheries and Aquatic
                 Sciences. 45:1413-1424.

            Johnson, B.A. and B.M. Barrett 1988.         Estimation of salmon
                 escapement based on stream survey data: A geometric approach
                 Alaska Department of Fish and Game, Division of Commercial
                 Fisheries, Kodiak. Regional Information Report. No. 4K88-35.
                 8pp-

            Krueger, S.W. 1981. Freshwater habitat relationships, pink salmon
                 (Oncorhynchus gorbuscha). Alaska Department of Fish and Game,
                 Anchorage. 41pp.

            Malloy, L.M. 1989. 1988 Kodiak area salmon management report to
                 the Alaska board of fisheries. Alaska Department of Fish and
                 Game, Division of Commercial Fisheries, Kodiak. Regional
                 Information Report. No. 4K89-5. 171pp.

            Murphy, M.L., J.M. Lorenz, J. Heifetz, J.F. Thedinga, K.V. Koski,
                 and S.W. Johnson 1987.      The relationship between stream
                 classification, fish, and habitat in Southeast Alaska. Tech.
                 Bull. 12., Tongass National Forest. R10-MB-10.

            Neave, F. 1966.   Salmon of the North Pacific Ocean- Part III. A
                 review of the life history of North Pacific pink salmon in
                 British Columbia. International North Pacific salmon
                 fisheries commission Bull. No. 18:71-78.

            Platts, W.S., W.F. Megahan and G.W. Minshall 1983. Methods for
                 evaluating stream, riparian and biotic conditions. U.S. Dept.

                                            III









               of Agriculture, General Tech. Rept. Int-138.

         Raleigh, R.F. and P.C. Nelson 1985.        Habitat suitability index
               models and instream flow suitability curves: Pink salmon.
               U.S. Fish and Wildlife Service. Biol. Rept. 82(10.109). 36pp.

         Thompson, F.M. and J.R. Fox 1989. Chignik management area annual
               f inf ish management report, 1988. Alaska Department of Fish and
               Game, Division of commercial Fisheries, Kodiak.          Regional
               Information Report NO. 4K89-5. 171pp.

         Wilson, W.J., E.W. Trihey, J.E. Baldridge, C.D. Evans, J.G. Thiele,
               and D.E. Trudgen 1981. An assessment of environmental effects
               of construction and operation of the proposed Terror Lake
               hydroelectric facility, Kodiak, Alaska. Arctic Environmental
               Information and Data center, Univ. of Alaska. Anchorage.
               419pp.

         Wolter, K. M. 1894. An investigation of some estimators of variance
               for systematic sampling. Journal of the American Statistical
               Association. 79:781-790.



         BUDGET: ADF&G


         Salaries                    $251.7
         Travel                          5.0
         Contracts                    132.2
         Supplies                      49.3
         Equipment                     22.1

         Total                       $460.3























                                           112










             FISH/SHELLPISH STUDY NUMBER Sa

             Study Title: Injury to Pink and Chum Salmon Eggs and Pre-Emergent
                           Fry Within Lower Cook Inlet and Kenai Fjord

             Lead Agency: ADF&G

                                        INTRODUCTION

             Wild stocks of pink and chum salmon are a major ecosystem component
             in the outer'Kenai Peninsula and Lower Cook Inlet area, immediately
             "down current" from Prince William Sound. Salmon represent a very
             important food source for marine mammals (sea lion, seals),
             terrestrial mammals (bear) , and a wide variety of bird species
             (eagles, etc.) . In addition salmon are harvested commercially. In
             1988, the year before the oil spill, the ex-vessel value of the
             commercial catch of wild and hatchery stocks of salmon from the
             lower Cook Inlet/Kenai Peninsula (CIK) area was more than $8.2
             million. Salmon are also very important to the sport, subsistence,
             and personal use fisheries. The future abundance of wild stocks of
             pink and chum salmon in the lower CIK areas may be adversely
             impacted as their intertidal spawning areas were affected by the
             oil spill. This project continues the evaluation of pink and chum
             salmon egg to fry survival in the intertidal spawning areas
             affected by the EVOS.



                                         OBJECTIVES


             A.   Estimate abundance of pink and chum salmon eggs and pre-
                  emergent fry by intertidal and upstream areas for nine streams
                  in the lower CIK. Six of the streams were studied in 1989.
                  Three unoiled streams in Kachemak Bay were dropped while three
                  Gulf of Alaska streams were added       to provide a better
                  comparison of oiled and unoiled streams in the Gulf of Alaska
                  area.


             B.   Estimate overwinter mortality (egg to pre-emergent fry) of
                  pink and chum salmon eggs.

             C.   Estimate reductions, if any, in pink and chum salmon pre-
                  emergent fry abundance due to oiling.

                                          METHODS

             Sampling will be conducted in two phases: egg-digs performed in
             October and pre-emergent fry digs conducted in March. The number
             of streams to be studied is limited by the number of days in
             October and November with low tides (maximum of +4.0 feet) during
             daylight hours.

             Streams were selected using the following criteria:

                                             113








              1     Sufficiently large adult salmon returns to indicate a
                    high probability of success in egg/fry digging.
              2.    Past history of egg/fry digging.
              3.    Streams covered by FIS Study 7a and aerial escapement
                    survey project.
              4.    Streams can be saf ely studied during the winter and early
                    spring months.

         The nine  streams studied during 1989 were Windy Creek Left, Port
         Dick Creek, Windy Creek Right, and Island Creek in the Kenai Fjords
         area and Humpy Creek, China Poot Creek, Seldovia River, Tutka
         Lagoon Creek, and Port Graham Creek in the Cook Inlet area. All
         but one historical alevin density index stream, Rocky River, was on
         that list. Rocky was not included in the 1989 study because the
         effects of logging would have confused the results.

         of the streams studied in 1989, the first two have had oil
         deposited near the stream mouths, the next two have had oil
         floating offshore, and the remainder had no visible impact.

         Three of the non-oiled streams in Kachemak Bay, China Poot Creek,
         Tutka Lagoon Creek, and Seldovia River, will not be studied during
         1990 because of logistical problems that inhibit sampling efforts
         at these sites.

         Three new non-oiled streams in the Kenai Fjords area, Tonsina Creek
         (in Resurrection Bay), South Nuka River and James Lagoon Creek,
         will be added to facilitate comparisons with oiled streams on the
         Gulf of Alaska. All three were once considered non-index alevin
         density streams with an intermittent history of fry digs.

         Sampling methods are identical for the pre-emergent fry and egg
         digs.   On each sample stream, four zones, 3 intertidal and one
         above tidal inundation, will be identified and marked by crews
         conducting stream surveys during F/S 'Study 7a. The zones are 0.0-
         0.6 m, 0.6-1.2 m, and 1.2-1.8 m below mean high water, and upstream
         of tidal inundation.

         Separate linear transects will be established in each zone (one
         transect for each type dig).      The ttansects will run the entire
         length of the zone.     Overlapping of transects will be kept to a
         minimum to control the influence of fall egg digs on abundance of
         fry during spring   2 sampling.    Fourteen circular digs (56 per
         stream) , each 0. 3 m in size, will be systematically dug along each
         transect using a high pressure hose to flush eggs and fry from the
         gravel. Eggs and fry will be caught in a specially designed net.
         Areas where salmon were not observed spawning during the spawning
         ground surveys (FIS Study 7a) will be avoided. Numbers of live and
         dead fry by species as well as numbers of live and dead eggs by
         species will be collected from each 0.3 m2 dig.              Additional
         information such as date, time, and zone will also be collected.


                                           114








           Eggs and fry will be collected for MFO analysis. A sample of 40
           fish will be preserved in a buffered formalin solution.

           A composite fry sample will be collected from the intertidal area
           for hydrocarbon analysis. A field blank (sample container opened
           at the collection site, closed and stored as if it contained a
           sample) will also be collected. Each sample will consist of enough
           fry to provide 10 grams of tissue (about 110 fry) for analysis.
           The sample containers will be pre-rinsed glass jars with teflon
           lined lids.   The samples will be kept frozen until shipment for
           processing in Auke Bay. Appropriate chain of custody forms will
           accompany each sample.

           A mixed effects analysis of covariance will be used to test for
           differences in egg to fry survival due to oiling.      The level of
           hydrocarbon impact will be determined from hydrocarbon analysis of
           mussels collected in 1989 and 1990 by F/S Study 7a.

           Analysis of variance will be used if no suitable hydrocarbon data
           are available. Degree of oiling as visually assessed by F/S Study
           7a will be used to post-stratify streams.     Degree of oiling and
           height in the tidal zone will be treated as fixed effects. Height
           in the tidal zone is nested within stream, a random effect.

           The number of streams sampled is limited by the window of time
           available for sampling. Power was estimated for the ANOVA using
           data from the 1975 and 1976 egg and pre-emergent fry digs in PWS.
           This analysis indicated the ANOVA could detect an increase of 20%
           (e.g. 10% mortality to 30% mortality) in egg to fry mortality at a
           = 0.05, 90% of the time.

           An assessment of lost fry production will be made if differences in
           egg to fry survival due to oiling are detected. Average survival
           from unoiled areas will be used to estimate potential fry density
           in oiled areas. observed and potential fry densities will then be
           expanded to estimate total observed and potential fry.            The
           difference between the two estimates will be considered lost fry
           production.

           Specific statistics to be estimated are:

                1.    Number of dead and viable eggs per square meter by salmon
                      species, stream, and stream zone.
                2.    Number of dead and live fry per square meter by salmon
                      species, stream, and stream zone.
                3.    Egg to fry survival by salmon species, stream, and stream
                      zone.
                4.    Lost production by salmon species, stream, and stream
                      zone.







                                            115












        BUDGET: ADF&G

        salaries                                               $46.9
        Travel                                                   1.7
        Contracts                                               19.7
        Supplies                                                 1.3
        Equipment                                                1.4

        Total                                                  $71.0
















































                                          116










           FISH/SHELLFISH STUDY NUMBER 8b

           Study Title: Injury to Pink Salmon Egg and Pre-Emergent Fry In the
                          Kodiak And Chignik Management Areas

           Lead Agency: ADF&G

                                       INTRODUCTION

           Large escapements of pink salmon into Kodiak and Chignik streams in
           1989 occurred as a result of severely limited commercial fishing
           opportunities caused by the EVOS. Total pink salmon escapements in
           1989 were 20.0 (Kodiak) and 1.4 (Chignik) million fish, whereas
           target escapement goals are 4.0 and 0.7 million pink salmon in the
           Kodiak and Chignik areas, respectively. The escapement magnitude
           experienced during 1989 is unprecedented and has the potential for
           adversely impacting future returns of pink salmon through density
           related factors such as fungus and disease outbreaks at the egg
           and/or fry stage.

           Pink salmon are a major component of the Kodiak and Chignik area
           ecosystems, providing an important food source for both marine
           mammals, terrestrial mammals, birds, and other fish and shellfish.
           Additionally they annually re-charge fresh water and near shore
           marine environments with nutrients as carcasses decompose after
           spawning.    This species is used for subsistence, sport and
           commercial purposes. Annually, pink salmon comprise 78% and 31%
           (1978-1988) of the Kodiak and Chignik salmon harvest, respectively.
           Ex-vessel value (1978-1988) of the pink salmon harvest is 14.2 and
           1.5 million dollars for the Kodiak and Chignik areas (Malloy 1989;
           Thompson and Fox 1989).

           A total of 386 Kodiak and 90 Chignik streams support populations of
           pink salmon. Pre-emergent sac fry sampling has been conducted in
           44 Kodiak and 18 Chignik streams periodically over the last 20
           years. These streams, referred to as index streams, provide data
           which are utilized for projections of returns and potential
           harvest.

           Potential damage caused by the 1989 brood year escapements upon
           future brood year returns can be quantified by: 1) examination of
           observed versus expected numbers of live fry/dig produced from
           potential egg deposition; 2) comparison of 1989 potential egg
           deposition to pre-emergent fry survival for the odd years 1969 to
           present; 3) evaluation of numbers of live fry/dig for streams with
           optimum spawning density versus streams with spawner densities
           above optimum.


                                        OBJECTIVES

           A.   Estimate potential egg deposition for all Kodiak and

                                            117









            Chignik pre-emergent index streams.

       B.   Estimate pink salmon fry density for Kodiak and Chignik index
            streams.

       C.   Estimate pink salmon survival from potential egg
            deposition to pre-emergent fry.

       D.   Assess changes, if any, of pink salmon pre-emergent fry
            abundance in 1991 due to the oil spill.

       E.   Estimate the 1991 adult pink salmon return by using the 1990
            fry index data.


                                     METHODS

       Potential egg deposition (PED) for each of the 62 Kodiak and
       Chignik Management Area index streams will be determined using
       index stream total escapements and fecundity data collected during
       the 1989 field season.      The PED estimates will be based upon
       average fecundity derived from the relationship of fish length to
       number of eggs carried and the   total escapement estimates derived
       using the Johnson and Barrett (1988) model.

       Pre-emergent sac fry sampling will be conducted on 44 Kodiak and 18
       Chignik index streams.     A majority of these streams. have been
       frequently and consistently sampled each year. Sampling station
       (spawning riffles) selection is founded upon pink salmon spawner
       distribution and specific habitat utilization as recorded from
       aerial surveys. The number of sampling sites per stream is based
       upon escapement magnitude, stream size and observed productivity of
       individual index streams.       Generally, smaller streams where
       escapements average less than 20,000 will have 4-6 sampling
       stations, 6-9 stations for the larger intermediate sized streams
       and the most productive streams, will have 10-15 stations.
       Historically, 10 digs have been completed for each station using a
       pump and associated equipment which hydraulically remove pink
       salmon eggs and fry (both live and dead) from the stream bed. A
       collection frame is used to capture eggs and fry as they are
       displaced from the gravel. Depth of stream bed sampling is 15. 0 to
       46.0 cm with a duration of 1-3 minutes depending on substrate.
       After eggs and fry (both live and dead) are enumerated the
       collection frame is moved to the next dig location and the steps
       repeated.    Sampling is done in an X configuration with equal
       numbers of digs done above and below the center of the X.        Digs
       which are at the extremes of the configuration are those which are
       closest to the stream banks. Ancillary information recorded along
       with egg and fry counts are stream temperature, predator presence,
       stage of fry development, quantities of egg fragments and evidence
       of stream bed scouring or shifts (Brennan 1990).         only minor
       modifications have occasionally beset the above sampling program

                                        118









            and were associated with water conditions, ice coverage or flood
            events which had altered the stream channel. Presently the only
            modification which will be imposed on the sampling program for 1990
            will be that a minimum of 30 digs with at least one live fry be
            obtained for each stream sampled, regardless of the historic number
            of digs done for that system (Johnson 1990). Alternative stations
            for additional digs to meet this constraint will be from
            established sampling sites. Pre-emergent sac fry sampling will be
            conducted in a time frame which will minimize the chances of fry
            emigration prior to sampling.

            Determination of egg to pre-emergent fry survival (1989-1990) will
            be founded upon PED, live fry/dig, and habitat data collected from
            F/S Study 7b.    An estimate of spawning density for all spawning
            riffles sampled for pre-emergent fry will be obtained from the
            detailed spawner distribution maps.

            Utilizing PED and number of live fry/dig data spanning the odd
            years 1969 to 1989, analyses will consist of fitting and assessing
            an empirical relationship. If required for quantifying possible
            outlying data points, climatological variables (precipitation and
            mean monthly temperature) will be assessed as possible causative
            factors.   Damage from this analytical standpoint would be live
            fry/dig values which fall below (descending limb of curve) the
            range of expected PED.     Hypothesis testing using non-parametric
            tests will be used to assess whether observed differences in number
            of live fry/dig are statistically significant.       A signif icance
            level of 0.1 will be used for all statistical analyses.

            The observed difference between potential eggs deposited and
            resultant pre-emergent fry will be designated as fry survival for
            a given year. The equation which will provide the estimate will be
            from Snedecor and Cochran (1967, p.520) with N defined as spawning
            area.


            PED in this framework will be determined from the proportion of the
            total escapement which during odd years utilize this fraction of
            the overall spawning habitat. Again, data for the odd years 1969-
            1989 will be used and comparisons of eggs and fry densities for
            contrasting levels of escapements and years will be made.           if
            needed, climatological conditions will be assessed in relation to
            calculated survival values. Damage, if any, due to escapement
            levels experienced in 1989 will be quantified from this method.

            This analysis component will take into account available spawning
            habitat, total estimated escapement and pre-emergent fry dig data.
            Control streams will be designated based upon a cumulative ranking
            of escapement and total available habitat in which the overall
            density of all streams will be 1.3 fish per   M2 or less. All index
            streams that fall outside of this classification will be designated
            as treatment streams (those with spawner densities above the
            calculated optimum of 1.3 fish/M2).    There are 17 control and 14

                                             119










        treatment streams    within the Kodiak area and 11 control and 3
        treatment streams   in the Chignik area. Analysis will consist of
        comparing the live  fry/dig data in composite for the control versus
        treatment streams. An alternative method will use an independent,
        mutually exclusive classification method for each index stream.
        The above (control    versus treatment) method incorporates streams
        with density values that are above the defined optimum for the
        control group. Index streams (13 Kodiak and 4 Chignik) without
        available spawning habitat estimates will be included in this
        analysis when estimates become available.

                                     BIBLIOGRAPHY

        Brennan, K.R. 1990. History of the pink salmon Pre-emergent fry
              sampling program in the Kodiak and Chignik management areas.
              Alaska Department of Fish and Game. Unpublished manuscript.
              12pp.

        Johnson,    B.A.   1990.      Detecting    changes   in   pink     salmon
              (Onchorhynchus gorbuscha) fry density for Humpy Creek, Alaska,
              and determination of sample size requirements.              Alaska
              Department of Fish and Game, Kodiak. Regional Information
              Report No. 4k9O-3. 6pp.

        Johnson, B.A. and B.M. Barrett 1988. Estimation of salmon
              escapement based on stream survey data: A geometric approach.
              Alaska Department of Fish and Game, Division of commercial
              Fisheries, Kodiak Regional Information Report No. 4K88-35.
              8pp.

        Malloy, L.M. 1989.     1988 Kodiak area salmon management report to
              the Alaska board of fisheries. Alaska Department of Fish and
              Game   Division of Commercial Fisheries, Kodiak. Regional
              Info@mation Report No. 4k89-6. 72pp.

        Snedecor, G.W., and W.G. Cochran 1967. Statistical Methods. Iowa
              State University Press, Ames, Iowa.

        Thompson, F.M. and J.R. Fox 1989. Chignik Management Area Annual
              Finf ish Management Report, 1988. Alaska Department of Fish and
              Game, Division of Commercial Fisheries, Kodiak. Regional
              Information Report No. 4k89-5. 171pp.

        BUDGET: ADF&G


        salaries                 $ 86.4
        Travel                       1.1
        Contracts                    53.0
        Supplies                     8.8
        Equipment                    0.0

        Total                    $ 149.3

                                           120










            FISH/SHELLFISH STUDY NUMBER 11

            Study Title: Injury to PWS Herring

            Lead Agency: ADF&G

                                        INTRODUCTION

            The oil spill in PWS coincided with the annual migration of Pacific
            herring Clupea harengus to near-shore spawning areas. In 1989, a
            significant portion of the spawning area in PWS was located within
            areas contaminated by oil. Additionally, adult spawning herring and
            newly hatched juveniles traversed areas impacted by oil and beach
            cleaning activities.

            It was hypothesized that the oil spill would adversely impact adult
            fish through direct mortality, food shortages, slowed growth, and
            a possible reduction in fecundity. In addition, herring eggs have
            been shown to be particularly susceptible to hydrocarbon
            contamination due to the affinity of hydrocarbon compounds for yolk
            sac material. Although no significant acute mortality was observed
            for adult fish in 1989, significant impacts were measured on egg
            mortality, egg hatching success, and percent viable hatch. Any of
            these adverse effects have the capacity to reduce the abundance and
            availability of herring. Adult and juvenile herring,-as well as
            herring eggs, often form an important item in the diet of marine
            fishes (e.g. salmon and halibut), mammals (e.g. sea lions, seals,
            and whales), and birds (e.g. cormorants, ducks, puffins, gulls).
            Herring also support an important commercial fishery within PWS,
            worth over 12 million dollars in 1988.

            The goal of this project is to determine whether the EVOS will have
            a measurable impact on populations of Pacific herring in PWS.
            Accurate and precise estimates of population abundance, age
            structure, weight, and length composition data are needed to
            accomplish this goal. In addition, the direct effects of oil
            contamination on spawning success and egg survival will be
            determined.



                                         OBJECTIVES

            A.   Expand the normal sampling of herring populations in PWS to
                 increase the precision of herring abundance, age composition,
                 weight, sex ratio, and fecundity estimates. Specifically we
                 intend to:

                 Continue to estimate the biomass of the spawning stock of
                 herring in PWS such that the estimate is within ï¿½ 25% of the
                 true value 95% of the time;


                                            121









               Estimate the age, weight, length (AWL) , and sex composition of
               herring in PWS during 1989 such that age composition estimates
               are within ï¿½ 10% of their true values 95% of the time;

         B.    Continue to document the occurrence of herring spawn in oiled
               and unoiled areas, validating the sites with quantified oil
               level information obtained from shoreline survey maps and
               hydrocarbon analysis of 1989 and 1990 herring eggs and mussel
               tissue.

         C.    Continue to estimate hydrocarbon contamination of, and
               physiological impacts on, adult herring by analyzing tissue
               samples:

               Test the hypothesis that the level of hydrocarbons in herring
               tissues is not related to the level of oil contamination of
               the area from which the herring were sampled. The experiment
               is designed to detect a difference of 1.6 standard deviations
               in hydrocarbon content with the probability of making a type
               I and type II error of 0.05 and 0.1, respectively.

               Estimate the presence and type of damage to tissues and vital
               organs of herring sampled from oil-impacted and un-impacted
               areas.


               Test the hypothesis that the level of hydrocarbons in herring
               eggs is not related to the level of oil contamination of the
               area from which the herring were sampled. The experiment is
               designed to detect a difference of 1.6 standard deviations in
               hydrocarbon content with the probability of making a type I
               and type II error of 0.05 and 0.1, respectively.

         D.    Continue to estimate the proportion of dead herring eggs from
               a subsample of study sites in oiled and un-oiled areas that
               were utilized in the 1989 egg mortality study, expanding the
               data base and providing sample sites for sample collection of
               live and preserved eggs. In addition, add an egg loss study
               at the egg mortality sites to increase the accuracy of the
               spawn deposition biomass estimates.

         E.    Continue to estimate the hatching success, viable hatch,
               occurrence of abnormal larvae, and collect embryonic and
               larval   tissue    for   sublethal    testing    including    MFO,
               cytogenetics, RNA/DNA ration analysis, and others by
               collecting herring eggs from egg mortality sites and control
               sites in Southeast Alaska (Sitka Sound) and rearing them under
               laboratory observation.

                                         METHODS

         This project will be conducted in three parts: (1) herring spawn
         deposition estimation; (2) herring age, weight, length, growth, and

                                           122









            fecundity estimation; and (3) herring egg survival and egg loss
            estimation.

            Herring Spawn Deposition Estimation

            The management of the PWS herring stock is based on a harvest
            policy established by the Alaska Board of Fisheries which specifies
            a maximum 20% exploitation rate for the combined harvest of all
            herring f isheries.   The allowable harvest is based on biomass
            estimates established the previous year modified by the expected
            growth and survival over the year. While aerial surveys were used
            to estimate biomass from 1973-87, spawn deposition surveys were
            performed in 1983 (Jackson and Randall 1983) and 1984 (Jackson and
            Randall 1984), and began to be used as the primary biomass estimate
            in 1988 (Biggs and Funk 1988).       Aerial surveys are easier to
            perform than spawn deposition surveys, but aerial survey biomass
            estimates are not as reliable because of the varying visibility of
            herring schools from the air and because the residence time of
            herring schools on the spawning grounds is unknown. Estimates of
            precision are not available for aerial survey biomass estimates.
            The ADF&G continues to conduct an annual aerial survey of spawning
            biomass to provide in season indicators of run timing and location
            and to collect information on the timing and distribution of
            spawning activity that is used for planning the spawn deposition
            survey.

            This project represents an augmented program to assess the PWS
            herring stock's response to the EVOS.     The original goal of the
            1989 herring spawn deposition survey was to estimate the spawning
            biomass with a precision such that the biomass estimate would be
            within ï¿½ 25% of the true biomass estimate 95% of the time under
            optimal survey conditions. Fishery managers determined that this
            level of precision was acceptable for estimating exploitation rates
            and forecasting future abundance.     If weather or other logistic
            problems hampered the spawn deposition survey sampling effort,
            fishery managers were willing to tolerate reduced precision. The
            EVOS introduced a potentially new and unknown level of mortality on
            herring stocks. The accuracy and precision of estimates of stock
            abundance need to be assured from both oiled and unoiled areas (as
            reflected in objectives 1 and 2).      The opportunity to estimate
            herring biomass with spawn deposition surveys is only available
            during a relatively narrow two week window. After the oil spill,
            the number of divers involved in the survey was increased to assure
            that even if weather problems restricted the available sampling
            time, sufficient numbers of transects could still be performed.
            The number of transects was also increased to provide a level of
            precision such that the biomass estimate would be within ï¿½ 25% of
            the true biomass 95% of the time. The amount of time devoted to
            skiff surveys of spawning areas was also increased. Skiff survey
            delineation of spawning area boundaries should help to increase the
            level of precision of spawn deposition surveys and provides
            important documentation of the occurrence of herring spawn in oiled

                                            123











         and unoiled areas.

         The aerial survey project will provide a map indicating the general
         location of herring spawning areas.       A skif f survey will then
         delineate the boundaries of each spawning area in more detail.
         Transects will be placed perpendicular to the shoreline at
         locations selected randomly from the shoreline maps of spawning
         areas.   Divers will swim along the transects and systematically
         place 0.1 m2 quadrants at 5 m intervals. Divers will estimate the
         total number of eggs in each quadrant.            All egg-containing
         vegetation will be removed from a subset of the quadrants for later
         enumeration of the number of eggs in a laboratory procedure. These
         enumerated egg counts will be used to correct bias in diver-
         estimated egg counts and estimate the precision of the diver
         estimates. The survey design is described in detail by Biggs and
         Funk (1988), and follows closely the two-stage sampling design of
         similar surveys in British Columbia (Schwiegert et al. 1985), and
         in Southeast Alaska (Blankenbeckler and Larson 1982, 1987).         The
         surveys use random sampling at the first stage (transects) , and
         systematic sampling at the second stage (quadrants within
         transects). Random sampling in the second stage is not feasible
         because of underwater logistical constraints (Schwiegert et al.
         1985).    In addition to the two-stage design, the survey is
         stratified by five areas within PWS (Southeast, Northeast, North
         Shore, Naked Island and Montague), because of the geographic
         separation of these areas and the potential for herring in these
         areas to be discrete stocks.

         mean egg densities along each transect will be combined to estimate
         an overall average egg density.        The observed widths of the
         spawning bed along each of the transects will be used to estimate
         the average spawning bed width.         The average width, average
         density, and total spawning bed shoreline length (verified from the
         skiff survey) will be used to estimate the total number of eggs
         deposited in each of five area strata established within PWS.
         Using the average fecundity and sex ratio derived from the AWL
         sampling portion of this project, the total number of eggs
         deposited will be converted into population numbers and biomass.
         Based on the variances obtained during the 1989 survey, 160
         transects would be needed to insure that the estimated biomass
         would have a 95% chance of being within 25% of the true biomass
         (161 transects were conducted in 1989 with a 95% chance of being
         within 19% of the true biomass).

         Sampling Procedure:

         The general locations of spawning activity will be derived from
         visible milt observed in the water column during scheduled aerial
         surveys. This information will be compiled and summarized on maps
         showing spawning locations and the number of days on which milt was
         observed.



                                          124









            Using this information, skiff surveys will be conducted in season,
            by members of the spawn deposition dive team, to verify the
            accuracy of spawning area maps derived from aerial survey data.
            Diving where herring have spawned is not recommended for at least
            5 days after spawning activity has ceased because of water
            visibility problems caused by milt and because large numbers of sea
            lions are usually present.

            The shoreline area containing herring spawn on the map verified by
            skiff survey will be divided into the smallest segments resolvable
            on the scale of the map (0. 1 mile or less). A total of 160 of the
            shoreline segments will be selected at random from all of the
            spawn-containing shoreline segments.         Each transect will be
            assigned a number and its location drawn     on waterproof field maps
            that can be taken out in the dive skif f .  The dive team leader will
            determine the exact transect location within the randomly selected
            shoreline segment by identifying a shoreline feature (tree, rock,
            cliff, etc.) located above the high tide line as the dive skiff
            approaches the shore, but before bottom profiles, bottom
            vegetation, or herring spawn are visible from the skiff.
            A 0.1 m 2 quadrant constructed of PVC pipe will be used for the
            sampling frame. A depth gauge and compass will be fastened to the
            quadrant. Data will be recorded on pre-printed single matte mylar
            forms attached to PVC clipboards, using a large weighted
            carpenter's pencil attached to the clipboard. Normally the dive
            team leader will make egg density estimates and record data while
            the assistant diver sets and follows the compass course, measures
            distancest and carries and places the quadrant.

            Sampling along the transects will occur in the following manner:

                 1.    A compass course perpendicular to the shoreline at the
                       transect location will be set on the compass attached to
                       the sampling quadrant.

                 2.    The first quadrant will be placed within the first 5
                       meters of spawn by tossing the quadrant.

                 3.    The lead diver will estimate and record the number of
                       eggs in the quadrant.     The number of eggs is normally
                       recorded in units of thousands.       The vegetation type,
                       percent cover, substrate, and depth are also recorded.

                 4.    The assistant diver will measure four complete 1 m hand-
                       spans offshore, along the compass course.            Halfway
                       through the fifth hand-span, the assistant diver will
                       gently toss the quadrant ahead approximately one-half
                       meter and allow it to come to rest. The lead diver then
                       makes another estimate at the new quadrant location.

                 5.    This process continues every 5 meters until the apparent

                                              125










                 end of the spawn is found. Divers will verify the end of
                 the spawn by swimming at least an additional 20 m past
                 the end of the spawn, unless a steep drop-off is
                 encountered.

      Data codes have been developed for the vegetation types and species
      that are encountered in PWS. If more than one is present in the
      quadrant sampled, the three most common are recorded on the data
      forms.   Percent cover is a simple estimate of the percentage of
      plant cover that exists within the quadrant sampled (e.g., if half
      the area is covered, the cover is 50%).

      Approximately every fifth quadrant will be used as a special diver
      calibration sample. Both divers will estimate the number of eggs
      in the quadrant in a manner such that neither can see the other's
      estimate.     Divers will attempt to remove all egg-containing
      vegetation and scrape eggs off rock substrate, placing the material
      in numbered mesh bags.      A sample size goal of 80 calibration
      samples per diver was established, including 20 in each of four
      vegetation categories (eelgrass, fucus, large brown kelp, hair
      kelp), based on 1988 and 1989 survey results. Calibration samples
      should also be spread over a wide range of egg densities.           The
      spawn deposition project leader will track the number of samples
      collected by each diver by vegetation group and density to ensure
      that sufficient calibration samples are taken in each category.
      Upon completing a dive shift, calibration sample material will be
      removed from the numbered mesh bags and placed in nalgene ziploc
      bags. Gilson's solution will be poured over the sample so that all
      material is completely immersed.      A label will be made for each
      sample (preferably in pencil on mylar) containing the transect
      number, both diver's estimates, date, and vegetation type. Five or
      6 calibration sample bags can be stored in a 5 gallon plastic
      bucket.   Samples should not be stacked over one another to prevent
      spilling and mixing. Procedures for the enumeration of the number
      of eggs  in each calibration sample are described, including the
      formulas  used to prepare Gilson's solution and the other chemicals
      used for sample processing.

       Data Analysis:

       Biomass Estimation

       The 1990 spawn deposition survey was patterned after the 1988 and
       1989 spawn deposition survey in Prince William Sound (Biggs and Funk
       1988, Biggs In Press). The overall biomass estimator is:


                                   (T   BI)
                             B                                  (1)
                                        R)


     where:


                                        126









              B =   estimated spawning biomass in tonnes,
              T =   estimated total number of eggs (billions) deposited in an
                    area,
              BI =  estimated tonnes of spawning biomass required to produce one
                    billion eggs, and
              R     estimated proportion of eggs disappearing from the study
                    area from the time of spawning to the time of the survey
                    (egg loss).

              The estimates for T and BI are derived from separate sampling
              programs and are thus independent.         Ignoring the unknown
              variability in R, the estimated variance for the product of
              the independent random variables T and B1, conditioned on R
              is:

                             [T'Var(BI) + B"Var(T) - Var(T)-Var(B')]
                 Var(BIR) =                     (1-R)2                 ; where   (2)

              Var(BI) = an  unbiased estimate of the variance of BI; and
              Var(T) = an   unbiased estimate   of the variance of T
                             (Goodman 1960).

                                  Total Number  of Eggs (T)

              The total number of eggs deposited in an area is estimated
              from a two-stage sampling program with random sampling at the
              primary stage, followed by systematic sampling at the
              secondary stage, using a sampling design similar to that
              described by Schwiegert et al. (1985). In computing variances
              based on the systematic second stage samples it is assumed
              that eggs are randomly distributed in spawning beds with
              respect to the 0.1 m   2 sampling unit.   While this assumption
              was not examined, in practice the variance component
              contributed by the second sampling stage was much smaller than
              that contributed by the f irst stage, so that violations of
              this assumption would have little effect on the overall
              variance. The total number of eggs (T), in billions, in an
              area is estimated as:

                                         T=N- 91- 10-6, where                  (3)

                N     = LIVO.1 = the total number of possible transects;
                L     = the shoreline length of the spawn-containing
                        stratum in meters;
                VO.1    0.3162 m = width of transect strip;
                        average estimated total number of eggs (thousands)
                   -6   per transect; and
                10      conversion from thousands to billions of eggs.

                The average total number of eggs per transect strip (in
                thousands) is estimated as the mean of the total eggs (in

                                             127


~0







              thousands) for each transect strip using:

                                     n

                                            ....ere          (4)
                                      n

              ~q9~1i = M~qi ~- ~2qY~qi; and
              y~q,= average quadrant egg count in transect i (in thousands
                    of eggs);

              i    transect number;

             ~4qM~qi    ~4qw~qi~ql~4qVO.1 = number of possible quadrants in transect i;
              ~0qw~qi   transect length in meters; and

              ~'h  number of transects actually sampled.

              The average quadrant egg count within a transect, yi~,~-is
              computed as:

                                ~M~_
                                .=I y~qij F where               (5)
                                ~0qm~qi

                    quadrant number within transect i,
              ~0qm~i    number of quadrants actually sampled in transect i, and
              y~qi~qj  adjusted diver-estimated egg count (in thousands of eggs)
                     from the diver calibration model for quadrant j in
                     transect i.

              The variance of T is similar to that given by Cochran
              (1963) for three stage sampling with primary units of
              equal size, although in this case the expression is
              modified because the primary units (transects) do not
              contain equal numbers of secondary units (quadrants), and
              the variance term for the third stage comes from the
              general linear model used in the diver        calibration
              samples:
                    2~q(~ql~qo-6~q)2~q[ (1~-f ~q1)  f 1 (1~-f 2)    f~q1f2
         Var(T) = N       _ -    S~q12  + ~_~_~qF~q_ ~- S2 2 + ~q_~6qF~_~q_ ~* ~S3~q1~q], (6)
                             n          ~4qZ m~qi            ~0qm~qi
                                        ~qi~q=~q1

                   n      ~4q9~q,~0q)2
                   ~24qZ (~8q9~q, i ~8q-
              ~qs~0qi2                variance among transects,
                        n-1






                                        128
 





                 2    n  2    (Yij Y-,)2
               s      71M                 variance among quadrants,
                 2    j=1 i j=1

                 2    n
               s      z  Var(y,,)  sum of the variances of the
                 3                    individual predicted quadrant egg counts
                                            from the diver calibration model,

                      n
               f,     -  = proportion of possible transects sampled, and
                      N


                      m
               f2     -  = proportion of quadrants sampled within transects
                      Mi   (same for all transects).


               Diver Calibration

               Diver observations of vegetation species will be
               aggregated into four vegetation categories based on
               structural and phylogenetic similarities of plants in the
               quadrant: eelgrass, fucus, hair kelp, and large brown
               kelp.   Diver estimates of egg numbers are approximately
               proportional    to  laboratory-enumerated   counts!    but
               systematic biases in the diver estimates can be accounted
               for by vegetation type and density (Biggs and Funk 1988,
               Biggs In Press).     Individual diver effects were not
               significant in the 1988 and 1989 survey, but potential
               differences among individual divers will be examined. The
               basic form of models used to account for biases in diver
               observations is:


                              a   Dj  Vk    Bik
                      Yijk  e * e    e    Xijk -e    where         (7)


               a      a constant;
               Dj     parameters representing the effect of j1h diver;
               V k    parameters representing the effect of the k"
                      vegetation type;
               8 jk   parameters controlling the functional form of the
                      relationship between the diver estimate and laboratory-
                      enumerated egg count for diver j in vegetation type k;
               Yijk = the ith laboratory egg count in the vegetation-
                      diver stratum jk;
               X ijk =the ith diver estimate in vegetation-diver stratum jk;
                      and
                 e    a normally distributed random variable with mean 0 and
                      variance a2.


                                          129









         A multiplicative-effect model is chosen because relative
         estimation errors are expected to change with egg density.
         The distribution of laboratory-enumerated egg counts for
         a given diver estimate was positively skewed in the 1988
         and 1989 surveys (Biggs and Funk 1988, Biggs In Press), so
         that the logarithmic transformation used to estimate the
         parameters of the multiplicative-effect model also
         stabilized the variance and corrected the skewness of the
         egg density estimates. After a logarithmic transformation
         model 7 becomes:


            loge (Yijk) = a + Di + Vk + Bik. loge (Xijk) + 6     (8)
         Bik = the slope of the relationship between the logarithm
              of the diver estimate and the logarithm of the
               laboratory-enumerated egg count.

         In logarithmic form, the model comprises a linear analysis
         of covariance problem with two factor effects (vegetation
         and diver) and 1 covariate (diver-estimated egg number) .
         The SAS Institute Inc. (1987) procedure for general linear
         models will be used to obtain least squares estimates of
         parameters and evaluate variance components. In addition
         to the two factor effects and one covariate, terms for
         diver-vegetation group interactions, density-vegetation
         group interactions and density-diver interactions will be
         considered in the analysis of covariance. Three-way and
         higher level interaction effects will not be considered
         because the objective is to derive a simple model with a
         relatively small number of parameters. Backward stepwise
         procedures will be used to determine subsets of the six
         effects that explain the maximum amount of variability in
         the data with the smallest number of parameters. During
         the backward stepwise procedures, effects will be included
         or eliminated from the model based on the probability
         level of F ratios for partial sums of squares.

         Translation of the predicted values from the logarithmic
         model, equation (8), back to the original scale, equation
         (7), requires a correction for bias.     The bias in the
         expected value of Yijk is eXp(12a2) when the true variance
         of Y k    a2, is known. Laurent (1963) gives an exact
         expression for the bias correction that incorporates
         additional terms when a2 is estimated from a sample. For
         the diver calibration data, the biases in estimating a2
         from a sample were less than 0.05% (Biggs and Funk 1988),
         so expected values for Yijk are estimated from:

                        a   Di  Vk   Bik    12S2
           E (Yijk)   e * e    e    Xijk - e   where           (9)

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                   s    the mean squared error from the general linear model.
                   The variance of individual predicted Y    is estimated
                   from:

                     Var(Y  ) = [e (2Y  + a2)   [e    - 1      (10)

                   Although the above expression is appropriate when a is
                   known (Laurent 1963), s will assumed to be an unbiased
                   estimate of a for the 1990 study since only a small bias
                   was introduced into estimates of the mean when s was used
                   to estimate a in past years (Biggs and Funk 1988).

                   Spawning Biomass per Billion Eggs (B')

                   Catch sampling programs will be used to estimate the
                   relationship between spawning biomass and egg deposition.
                   The tonnes of spawning biomass required to produce one
                   billion eggs (B') will be estimated as:


                                     W    S
                           B'                 10   where                 (11)
                                     F(Wf)
              W      = estimated average weight in grams of all herring (male
                        and female) in the spawning population in an area;
              S      = estimated ratio of total spawning biomass (male and
                        female) to female spawning biomass;
              F(W)   = estimated fecundity at the average weight of females in
                       the spawning population in an area, in numbers of eggs;
                        and
                                                    10-6  conversion from grams to tonnes
             103        units conversion factor = - =
                                                    10-9  conversion from eggs to billions

                  Estimates of average weight, sex ratios, and fecundies are not
                  independent. The variance of B' is approximately:


               Var(B') = (10  ){ [S/F(W)] Var(W)
                                 + [W/F(W)] Var(S)
                                 + [WS/F(W) ] Var(F(W ))
                                 + 2Cov(W,S) [S/F(W )] [W/F(W )]

                                               131








                           2COV[@R,F(@q-f)] [SIF(Rf)] [@@SIF(Rf)']

                           2Cov[S,FFWf)]-[W/FFWf)] -CWS/F(Wf)']        (12)

         The covariance terms containing S, Cov(W,S) and Cov[S,F(Wf)lf
         will not be included in the estimate for 1990. These terms
         were not included in the estimate of Var(B') in 1988 and 1989
         because S was estimated from either the same pooled AWL
         samples or from a single AWL sample. However, Cov(9,S) and
         Cov[S,F(wf)] probably contribute a small amount to Var(B')
         since the term involving Cov['W F('Wf)) was very small in 1988
         and 1989.

         Correction for Egg Loss

         The only component needed for the biomass estimate that has
         not been estimated within the present study is egg loss (the
         proportion of eggs disappearing from spawning areas between
         the time of spawning and the time of surveys).        Before the
         extensive use of SCUBA diving to survey herring egg
         deposition, estimates of egg loss were relatively high.
         Montgomery (1958) estimated that egg loss was 25 to 40% for
         Southeast Alaska, and Blankenbeckler and Larson (1987) used
         similar estimates in their early egg deposition surveys in
         Southeast Alaska. However, Haegele et al. (1981), counducting
         diving surveys in British Columbia, argued that egg loss was
         only about 10%. They based this assumption on the fact that
         most spawn was deposited in the subtidal zone where egg loss,
         primarily due to predation and wave loss, was probably less
         than had been observed in the intertidal zone. Presently, egg
         loss is assumed to be 10% in British Columbia, Southeast
         Alaska and PWS since the timing of diving surveys in relation
         to spawning has been standardized among these areas (W.
         Blankbeckler, ADF&G, Ketchican, personal communication; Biggs
         and Funk 1988). To test this assumption, an initial study of
         actual egg loss within PWS will be conducted in conjunction
         with the egg survival study during 1990.

         Herring Age, Weight, Length, Growth and Fecundity Estimation

         Mean Weight and Sex Ratio

         Mean weight and sex ratio will be estimated from AWL samples
         collected from the commercial catch and ADF&G test fishing
         conducted before or after commercial openings. AWL samples
         will be collected from the spawning population in each of the
         spawn deposition summary areas (Southeast, Valdez Arm, North
         Shore, Naked Island, and Montague Island). The approximate
         timing of peak herring spawning in each summary area will be

                                        132









                 determined from aerial survey sightings of milt and herring
                 schools.   All herring AWL samples taken during the time of
                 peak spawning in each area will be pooled to obtain estimates
                 of mean weight and sex ratio for each summary area. Average
                 weights and sex ratios for all of PWS will be estimated as the
                 average of the estimates from each of the areas weighing by
                 the spawn deposition biomass estimate in each area.

                 The estimated sex ratio, S, is expressed as the ratio of the
                 number of herring of both sexes in the AWL samples to the
                 number of females. The binomial distribution will be used to
                 estimate the proportion of females, p, in samples, where S
                 1/p. The variance of S.is then given by:
                                     S'(S-1)
                            Var(S) =        1                        (13)
                                        n

                 where n is the number of herring in the AWL sample.

                                            EGG LOSS

                 Commercial and test fishing catches will be sampled for AWL,
                 fecundity, and roe maturity information. These data are used
                 to estimate spawning biomass and spawn deposition, forecast
                 herring returns, and evaluate effects of the oil spill on
                 survival. Information on fecundity, average weight of females,
                 and sex ratio are also important components of the spawn
                 deposition biomass estimator. AWL sampling will be intensified
                 in 1990 to increase the precision of biomass estimates and,
                 therefore, enhance the possibility of detecting oil spill
                 impacts upon herring stocks.

                 Sampling will begin as soon as concentrations of herring
                 appear in near shore areas that can be sampled with purse
                 seine gear.      Efforts will be made to sample major
                 concentrations of herring throughout PWS at periodic intervals
                 throughout the spawning period. The major objective of this
                 portion of the study will be to determine the age, sex, and
                 size composition of all major herring concentrations in the
                 general areas of Valdez Arm and the Eastern District, the
                 North Shore, Naked Island, and Montague Island. Results of
                 the aerial survey program will be used to direct test f ishing
                 efforts within each area.

                 Each week during the sampling period, early April through
                 early May, six to eight samples of herring will be collected
                 through test fishing or from the commercial catch. A sample
                 of 403 herring is needed to simultaneously estimate the
                 proportion of at age of a multinomial population such that 95%
                 of the time the estimated proportions will be within ï¿½10% of
                 the true proportions (Thompson 1987). Therefore, efforts will

                                               133









          be made to obtain samples consisting of approximately 450
          herring to allow for the occurrence of unreadable scales
          (usually less than 5% of the sample). Herring samples will be
          flown from the fishing grounds each day to Cordova for
          processing. Augmentation of the standard AWL sampling program
          will be needed to collect sufficient samples for hydrocarbon
          analyses, fecundity estimates, and oocyte loss measurements.
          All AWL data will be collected using personnel and funding
          from the standard (i.e. non-oil spill related) AWL sampling
          program conducted by ADF&G within PWS.

          The following data will be collected for each herring sampled:

          1. sex (determined by examination of gonads);
          2. standard length (in mm);
          3. weight (in grams);
          4. age (determined by examination of scales);
          5. capture information (date of capture, fishing district,
             subdistrict,local name for the location, fishing vessel
             name, gear type);
          6. herring number on data form; and
          7. data form number.

          Fecundity

          Additionally, a subsample of herring will be collected to
          estimate f ecundity.   The average fecundity at the average
          female weight (F(Wf)) from expression (11) is a component of
          the spawn deposition survey biomass estimator.       The spawn
          deposition survey attempts to estimate spawning biomass so
          that the 95% confidence interval is within ï¿½ 25% of the actual
          biomass estimate. If fecundity sampling is to contribute no
          more than 1% to the confidence interval width, a sample of 85
          females of exactly the average weight of females in the
          spawning population is needed. Since average female weight is
          unknown at the time of sampling, more herring must be sampled
          over a range of sizes.       Based on the precision of 1989
          fecundity sampling, a sample size of 130 herring would be
          needed to provide the desired level of precision.             An
          additional 100 samples clustered around the average size of
          females in 1989 will be taken to compare with the past year's
          data. The average weight of a female in the fecundity sample
          in 1989 was 119 grams. The predicted average weight for the
          population in 1990 is 142 grams that translates to an average
          predicted length of 215 to 225 mm. Therefore, sampling should
          be clustered about the 210 mm to 230 mm length classes is
          desirable.

          Effects of the oil spill on fecundity will also be examined by
          testing for differences in fecundity among five areas: (1)
          Southeast Shore including Simpson and Sheep Bays, Port
          Gravina, and Port Fidalgo; (2) Northeast Shore including

                                        134









               Valdez Arm and Tatitlek Narrows; (3) North Shore; (4) Naked
               Island; and (5) Montague Island. While extensive mortality of
               adult herring from the oil spill has not been documented, it
               is possible that sublethal stresses could result in reduced
               fecundity.

               Herring fecundity samples will be collected concurrently with
               AWL samples.    To accomplish this, at least five individual
               test purse samples will be subsampled. Females within these
               purse seine samples will be randomly selected within 10 mm
               length classes until stratum goals are reached. The roe sacs
               from each selected females herring will be removed and placed
               in a ziploc bag labeled with the AWL number corresponding to
               that female. Each individually packaged roe sample will then
               be placed in a larger plastic bag labeled with the sample date
               and location. Standard laboratory procedures have been
               developed to process fecundity samples.

               Samples for hydrocarbon analyses will also be obtained from
               herring collected at each of the four locations (Naked Island,
               Galena Bay, Cedar Bay, and Stockdale Harbor):

               1. three gut samples for hydrocarbons;
               2. three viscera samples for hydrocarbons;
               3. three muscle samples for hydrocarbons; and
               4. three gonad samples for hydrocarbons.

               General observations on the prevalence of nematodes, liver and
               gall bladder condition, and fullness of gut will also be made
               for each herring collected for hydrocarbon analyses. Standard
               protocol, including sample sizes and collection strata, for
               collecting herring eggs for hydrocarbon analyses will be
               followed.

               In addition to the 500 ovaries collected for fecundity
               analysis, 50 ovaries will be collected and preserved in a
               buffered formalin solution for oocyte loss measurements. An
               additional 25 preserved ovaries will be obtained from Sitka
               Sound, Southeastern Alaska, for use as a control.         Atretic
               eggs and histopathological damage in the sac roe of the adult
               herring will be recorded during oocyte loss observations.

               A linear relationship was found between fecundity and weight
               for herring samples collected in 1988 and 1989 (Biggs and Funk
               1988). In 1990, the fecundity-weight relationship will again
               be examined using data pooled across all areas. Average
               fecundity for each area will be estimated from the fecundity-
               weight relationship using the average female weight from each
               area.   The average fecundity for each area will then be
               applied to the spawn deposition biomass estimator (F(Wf) in
               expression   (11).     The variance of estimated average
               fecundities will be approximated using the variance of

                                              135








              predicted means from the fecundity-weight linear regression
              (Draper and Smith 1981):
                                  I          (Wf  WF7 I
               Var[F(i@f)    S2 1-n   +  q  + z (Wi - WF)      where      (14)
                2
               s    =  residual mean square from the fecundity-weight linear
                       regression;
               Wf   =  average weight of female fish in the spawning
                      population;
                      average weight of females in the fecundity sample;
               Wi     weight of individual females in the fecundity sample;
                n     total number of females in the fecundity sample; and
                q     total number of females in the AWL sample.

              General Linear Model (GLM) extensions of linear ANOVA
              techniques will be used to test for year and area effects in
              growth and fecundity.

              Herring Egg Survival and Egg Loss Estimation

              oil contamination of herring spawning sites and exposure of
              spawning herring to oil may cause mortality to herring eggs,
              decrease hatching success, reduce larval viability, and impair
              larval growth.    The major objective of this portion of the
              study will be to measure immediate, easily observable
              mortality of herring eggs in a subsample of the sites used in
              1989.   In 1990, nine sites will be used to conduct the egg
              loss study, collect hydrocarbon samples, collect live eggs for
              the laboratory portion of the study, and to gather samples for
              sublethal impact testing.

              Three study transects will be re-established in each of three
              areas used during 1989 (assuming those areas receive spawn in
              1990): Naked Island, Fairmont Bay, and Rocky Bay on North
              Montague Island.     The ratio of live to dead eggs will be
              determined along each transect from subsamples of 100 eggs.
              Dead eggs turn an opaque white color and are easily identified
              with low power magnification under a binocular microscope.
              Mussel tissue samples will also be collected for hydrocarbon
              analysis.

              A 1990 laboratory egg incubation experiment, similar to the
              one conducted in 1989, will be carried out by a private
              consultant contracted by ADF&G.           This experiment will
              determine the survival of herring eggs and larvae collected
              from the nine study sites in PWS and three control sites in
              Sitka Sound, Southeast Alaska.

              Divers will establish the location of mean low water (MLLW) at
              the start of each dive. Each dive team will attempt to sample

                                              136









             three transects each day. Each transect will be sampled every
             two days until most herring eggs have hatched (about 20 May) .
             A total of twelve to sixteen dives will be made along each
             transect over the course of egg development.

             The location of each transect will be marked.     Divers will
             work along transects by following a compass course set
             perpendicular to shore.   During the first dive, five sample
             stations at the +1, 0, -5, -15, and -30 foot depths will be
             marked underwater with weighted floats anchored by a spike.
             Station depths, corrected for tide stage, will be determined
             using diver's depth gauges.     Three samples of vegetation
             containing at least 100 eggs will be collected at each depth
             along the transect whenever possible.

             The following data will be recorded the first time each
             transect is sampled:

             1. transect number;
             2. site description (location, exposure, plant community);
             3. number of depth strata from which herring eggs were
                obtained; and,
             4. original treatment category (high, medium, low, or no
                oil-impact).

             The following data will be recorded every time each transect
             is sampled:

             1. transect number and location
             2. date;
             3. dive time;
             4. treatment level;
             5. air and water temperature;
             6. maximum depth; and,
             7. number of live, dead, and other eggs per sample.

             Herring eggs and mussels will be collected at each site for
             hydrocarbon analysis on the first day. Three samples each of
             eggs and mussels (six per transect) will be collected from
             each sampling location, including the three control sites in
             Sitka Sound, at the lowest tide stage at which mussels occur
             (usually about 5 ft below MLLW) .    Collection methods will
             follow established protocol, including chain of custody forms.

             During one of the sampling trips to each transect, herring
             eggs and associated vegetation will be collected for the
             laboratory incubation project. Herring eggs will be collected
             at nine sites within Prince William Sound and three sites
             within Sitka Sound. At each site, three samples of vegetation
             containing at least 300 eggs will be collected at three depths
             (MLLW, -5 ft, and -15).


                                          137









          Herring eggs will also be collected and preserved in a
          phosphate buffered formalin solution, using sea water, for
          biochemical analysis.    Results of these analyses may help
          determine the extent of oil exposure from determination of
          sublethal effects.

          Finally, herring egg samples will be collected from each of
          the 12 study sites for cytogenetic analysis. Ten egg patches
          consisting of approximately 1000 eggs each (5 ml) will be
          preserved in a buffered formalin solution from each study site
          (i.e. a total of 120 samples). A subsample of eggs will be
          taken from each sample jar and analyzed for mitotic
          aberrations in the embryonic and yolk cells.           Detailed
          methodology will be provided by the lab contracted to perform
          the service.

          Egg survival data will be summarized by level of hydrocarbon
          impact, transect, depth, date of sample collection, and
          proportion of live eggs. Several different analyses will be
          conducted to test for differences in egg survival due to the
          level or amount of oil. The first analysis will be a nested
          mixed factor ANOVA incorporating all possible factors and
          interaction effects like:

       Yijkl = u + Ai + Bj (Ai) + Ck + Dt + ACtk + ADil + CDk1 + ACDikt +6ijkL' (15)

         where,
          Yijkt = the arc sin transformed proportion of live eggs;
          U     = grand mean;
          Ai    = oil impact level (treatment; fixed effect);
          Bi    = transect (random effect; nested within treatment);
          Ck    = depth (fixed effect);
          Dt    = time interval (days) between spawning and sample
                  collection (random effect);
          AC1k + ADR + CD kt +ACD ikt = interaction terms; and
          fijkt = error terms, which, after arsine transformation are
                  assumed to be normally distributed with mean 0 and
                  variance o.2

          The second analysis will be an analysis of covariance (ANCOVA)
          where both treatment (Ai) and time (D,) will be treated as
          covariates.   Treatment and depth will be treated as f ixed
          effects, while transect (nested within treatments) and time
          will be treated as random effects. This model will describe
          the decrease in the proportion of live eggs over time, using
          time as a covariate, and will reduce the number of parameters
          that must estimated for the model.

          Egg loss is the only component of the spawn deposition biomass
          estimator that has not been measured. In the past, a 10% egg
          loss factor was applied to all transect data to adjust the

                                        138









              total spawned biomass estimate.     In 1990 a preliminary egg
              loss study will be conducted in conjunction with the egg
              survival study to determine whether the 10% egg loss factor is
              appropriate for use at PWS study locations.

              The same three transects used in each of three areas for the
              egg survival study will be used in the egg loss study: Naked
              Island, Fairmont Bay, and Rocky Bay on North Montague Island.
              Egg loss will be estimated by observing changes in egg density
              over time at these locations.

              To avoid sampler bias in selecting samples, as was done for
              the egg survival study, a marked leadline, 20 m or less in
              length, will be used to select samples. The leadline will be
              placed parallel to shore and to the left of each transect
              station. Egg density estimates will be taken within 0.1 M2
              sample quadrants using the same procedures described for spawn
              deposition diver transects. For each transect, five egg
              density estimates will be made at each of five depths (+l, 0,
              -5,-15,-30) ft depths). Divers making egg density estimates
              for the egg loss study will be calibrated in a similar manner
              used for divers assisting in spawn deposition surveys. one
              egg count calibration sample will be collected at each
              transect and at each depth level. For the calibration sample,
              all herring eggs and vegetation will be removed from a 0.1 M2
              sample quadrant. Counts of eggs within the calibration sample
              will be made in the laboratory at a later time. Egg density
              estimates and egg counts will be conducted every other day
              f rom the time of spawning in each area until the time of
              hatching (a period of approximately 20-25 days). It should be
              possible to obtain egg density estimates and egg counts for
              about eight days during the study. This would result in a
              total of approximately 1,800 egg density estimates (three
              areas; 3 transects per area; five depths per transect; five
              egg density estimates per depth; eight days) and 540 egg
              counts (three areas; three transects per area; f ive depths per
              transect; one egg count per depth; eight days) f or the season.

              Egg loss data will be summarized by area, transect, depth,
              date of sample collection, and estimated egg density.        Egg
              density estimates will be adjusted for observer (diver)
              biases, following procedures set forth for diver calibration
              in the spawn deposition survey, prior to analyses. The change
              in egg density over time for each transect and depth will be
              examined.


                                        BIBLIOGRAPHY

            Biggs, E.D., and F. Funk. 1988. Pacific herring spawning ground
                 surveys for Prince William Soundl 1988, with historic overview.
                 Regional Information Report 2C88-07, Alaska Department of Fish and
                 Game, Anchorage, 73 p.

                                            139










        Blankenbeckler, W.D. and R. Larson. 1982. Pacific herring (Clupea
              harengus pallasi) spawning ground research in Southeastern Alaska,
              1978, 1979, and 1980. Alaska Department of Fish and Game Technical
              Report No. 69. 51 P.


        Blankenbeckler, W.D. and R. Larson. 1987. Pacific herring (Clupea
              harengus vallasi) harvest statistics, hydroacoustical surveys, aget
              weight, and length analysis, and spawning ground surveys for
              Southeastern Alaska, 1980-1983. Alaska Department of Fish and Game
              Technical Data Report No. 202. 121 p.

        Cochran, W.G. 1963. Sampling techniques. John Wiley and sons,
              New York.

        Draper, N.R. and H. Smith. 1981. Applied regression analysis.
              John Wiley and Sons, New York.

        Goodman, L.A. 1960. On the exact variance of products. Journal
              of the American Statistical Association 55:708-713.


        Haegele, C.W., R.D. Humphreys, and A.S. Hourston. 1981.
              Distribution of eggs by depth and vegetation type in Pacific
              herring (Clupea harengus pallasi) spawnings in Southern British
              Columbia. Canadian Journal of Fisheries Aquatic Sciences 38:381-
              386.

        Hourston, A.S., H. Rosenthal, and H. von Westernhagen. 1984. Viable
              hatch from eggs of Pacific herring (Clupea harengus pallasi)
              deposited at different intensities on a variety of substrates.
              Canadian Technical Report of Fisheries and Aquatic Sciences 1274.

        Jackson, M. and R.C. Randall. 1983. Herring spawn deposition
              surveys in Prince William Sound, 1983. Alaska Department of Fish
              and Game, Prince William Sound Data Report No. 83-6. 15 p.

        Jackson, M. and R.C. Randall. 1984. Herring spawn deposition surveys,
              Prince William Sound, 1984.    Alaska Department of Fish and Game,
              Prince William Sound Data Report 84-16. 15 P.

        Laurent, A.G. 1963. Lognormal distribution and the translation
              method: description and estimation problems.        Journal of the
              American Statistical Association 58:231-235.

        Montgomery, D.T. 1958. Herring spawning surveys if Southeastern
              Alaska.    United States Fish and Wildlife Service, Bureau of
              Commercial Fisheries, Marine Fisheries Investigations Field Opera-
              tions Report. 22 p.

        Palsson, W.A. 1984. Egg mortality upon natural and artificial
              substrata within Washington State spawning grounds of Pacific
              herring (Clupea harencfus nallasi) , M.S. Thesis, University of

                                         140









                 Washington, Seattle.

            SAS Institute Inc. 1987. SAS/STAT Guide for personal computers,
                 version 6 edition. SAS Institute, Cary, North Carolina.


            Schweigert, J.F., C.W. Haegele, and M. Stocker. 1985. Optimizing
                 sampling design for herring spawn surveys on the Strait of Georgia,
                 B.C. Canadian Journal of Fisheries and Aquatic Sciences 42:1806-
                 1814.


            Thompson, S.K. 1987. Sample size for estimating multinomial
                 proportions. The American Statistician 41:42-46.



            BUDGET: ADF&G


            salaries                   $ 121.7
            Travel                          6.9
            Contracts                     398.0
            supplies                       16.8
            Equipment                      15.0

            Total                      $ 558.4
































                                             141











      FISH/SHELLFISH STUDY NUMBER 13

      Study Title: Effects of Hydrocarbons on Bivalves


      Lead Agency: ADF&G

                                   INTRODUCTION



      Bivalve mollusks are an important component of the f ood chain,
      existing as prey f or bear and sea otters, and bivalves support
      subsistence and sport fisheries in PWS. Because they are relatively
      sedentary and occupy nearshore areas, bivalves may be particularly
      susceptible to contamination by oil. In contrast to finfish species
      which metabolize hydrocarbons at a much higher rate, bivalves
      metabolize hydrocarbons at a reduced rate and are therefore much more
      likely to bioaccumulate hydrocarbons.       It is hypothesized that
      increased hydrocarbons in nearshore sediments could affect bivalves
      for a long period of time by increasing mortality, decreasing growth,
      or causing sublethal injuries. The effects of oil on the growth and
      survival of littleneck clam (Protothaca staminea) in particular and
      other bivalves in general have been well documented (Anderson et al.
      1982, Anderson et al. 1983, Augenfeld et al. 1980, Dow 1975, Dow
      1978, Keck et al. 1978).

      This study seeks to continue to evaluate the potential effects caused
      by the oil spill by comparing data obtained from several beaches
      representing different levels of oil contamination. The effects of
      the mechanical cleaning of beaches following the spill will also be
      evaluated.   Documenting effects on littleneck clams, butter clams
      (Saxidomus giganteus), and razor clams (Siliqua patula) is required
      to determine the scope of impact by the oil spill on these species,
      associated ecosystem elements, and current and future employment,
      recreation, and lifestyles of coastal communities on PWS.

                                    OBJECTIVES

      A.   Test if the level of hydrocarbons in bivalves and in sediments
           is not related to the level of oil contamination of a beach.

      B.   Document the presence and type of damage to tissues and vital
           organs of bivalves sampled from beaches such that differences of
           ï¿½5% can be determined between impact levels 95% of the time.

      C.   Test if the growth rate of littleneck, butter and razor clams is
           the same at beaches of no oil impact, intermediate or high
           levels of oil impact and intermediate or high levels of oil
           impact in areas which had been treated.

      D.   Test if the proportion of dead clams is not related to the level

                                       142











                  of oil contamination or treatment at a beach.

            E.    Document numbers of young-of -the-year clams and test if the
                  proportion of young-of -the-year clams is not related to the
                  level of oil contamination or treatment at a beach.


                                            METHODS

            This study will be conducted by the ADF&G and represents a
            consolidation of the former FIS Studies 13 and 21.        During April
            through June, 1990, beaches will be selected which, if possible,
            coincide with those sampled during 1989. An emphasis will be placed
            on beaches that are known to be important habitat for bear or sea
            otters.   It is possible that the baseline beaches sampled in 1989
            will not be resampled because they do not constitute bear and/or
            otter habitat. Sites will be chosen inside PWS which have not been
            impacted by oil, which have received moderate to heavy oiling, and
            which have been cleaned of oil by mechanical means. Sites outside of
            PWS will be chosen which have not been impacted by oil and which have
            received heavy to moderate oiling. Because razor clams are not found
            in the same habitat as butter or littleneck clams, razor clam beaches
            along the Kenai and Alaska peninsulas will be located which were
            affected and unaffected by oil.

            Beaches of known bear and/or sea otter habitat and known to contain
            clams will be classified by oil contamination levels.       Nine study
            sites for littleneck or butter clams in PWS representing three levels
            of oil contamination (subjectively rated as no contamination,
            intermediate or high contamination and intermediate or high
            contamination which had been treated by mechanical means) will be
            sampled. Beaches with no oil contamination are Hell's Hole, Double
            Bay, and Simpson Bay.         Beaches with moderate or heavy oil
            contamination which have been treated or untreated include Gibbon
            Anchorage, Snug Harbor, Wilson Bay, North Chenega Island, Horseshoe
            Bay and Green Island.      Sites not sampled in 1989 are selected
            contingent upon habitat suitability.

            Since cleaning efforts were  not as intense outside of PWS, and since
            a smaller number of sites have been chosen theref the additional
            sample level based on mechanical treatment will not be investigated
            outside of PWS.    Eight study sites for littleneck or butter clams
            representing two levels of oil contamination (subjectively rated as
            no contamination or moderate to high contamination) were chosen to be
            sampled in the Lower Cook Inlet and Kodiak areas. Beaches with no
            oil contamination are Jakalof Bay, Kachemak Bay and Seldovia Bay in
            lower Cook Inlet and Port Bailey on Kodiak Island.        Beaches with
            moderate to heavy oil contamination are Windy Bay, Tonsina Bay and
            Port Dick in Lower Cook Inlet and Kupreanof Strait on Kodiak Island.

            Six sites have been chosen for sampling razor clam habitat
            representing two levels of oil contamination (subjectively rated as
            no or high contamination) .    Beaches with no oil contamination are

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      Halibut Bay, Polly Creek and Augustine. Beaches with moderate or
      heavy oil contamination are Swishak, Alinchak and South Nuka Island.

      For each sample site, the following site description information will
      be recorded:   site orientation (N-NW etc.), latitude, longitude,
      beach slope, low tide height, percent dominant substrate composition,
      temperature and salinity of the water, weather and wave action.
      Temperature and salinity of the water will be measured at a distance
      of approximately 5 meters offshore from the sampled beach at the
      daily low slack tide.

      Beaches will be sampled for littleneck and butter clams at maximum
      low tides for a monthly tidal cycle.     For beaches which had been
      sampled in 1989, 1990 tidal heights and time of year will be matched
      with the 1989 values as closely as possible. At each beach, three
      sampling transects will be run to insure complete coverage of the
      beaches as distribution of oil on the beaches is unknown. Transects
      will be perpendicular to the water's edge and parallel to each other
      with a total distance between each transect of 15 meters. Transects
      are perpendicular to the water to insure complete sampling of clam
      habitat. The top of each transect is placed at the +1.6 meter tide
      level and the bottom of the transect at the lowest tide level.

      Prior to sampling, the upper distribution of clams will be determined
      by removing sediment to a depth of 30 cm (12 in) along a trench
      adjacent to the proposed transect. The trench is dug starting from
      the top of the transect and continuing until clams are encountered.

      A total of eight quadrants will be sampled from each transect to
      obtain2hydrocarbon and necropsy specimens. Sample quadrants are each
      0.25 m (0.5 m by 0.5 m). Additional sampling or complete sampling
      of each transect (all possible sampling quadrants) may be necessary
      if insufficient numbers of clams are recovered within the eight
      sampling quadrants to meet project objectives.     Quadrants will be
      sampled from the top to the bottom of each transect as the tide
      recedes. The distribution of clams will extend below the low tide
      levels encountered during each sampling event. However, the bottom
      of each transect and the bottom sampling quadrant will occur at the
      daily low tide level. The upper layer of sediment will be removed
      and washed through a 1 mm mesh screen to retain small young-of-the-
      year clams. The remainder of the sediment is washed through a larger
      3 mm mesh screen.

      Razor clam habitat tends to be comprised of long and broad sandy
      beaches. Because of the size of the area inhabited by razor clams,
      and due to the time and manpower required for a full scale study of
      this species, there will be no attempt made to estimate the abundance
      of razor clams on the beaches being surveyed. The primary objectives
      of this portion of the study are to obtain hydrocarbon and necropsy
      samples, and to collect a sufficient number of razor clams for age
      and growth determination.


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            Razor clams inhabitat the 0.91 m to -1.22 m tidal range (Quinn and
            Jones ' 1989). In order to minimize sampling effort, a tidal height
            known to contain a large number of clams will be established at
            approximately 0 m to -0.33 m on each beach. A transect along this
            tidal height will be dug with a high pressure pump (pre-emergent fry
            pump) until the desired sample size has been collected.

            A total of nine sediment samples will be collected from each beach
            site (triplicates from each transect)..   All sediment samples will be
            collected before bivalve sampling is performed.         The triplicate
            hydrocarbon samples from each transect will be composite sediment
            samples which will be collected by scooping one tablespoon (15 cc) of
            sediment to a depth of 2 to 3 cm from each of the eight sample
            quadrants on a transect.

            All samples from each transect will be placed in 8 oz glass jars
            rinsed with methylene chloride. Each-jar will be labelled with the
            site name, latitude, longitude, date, "SEDIMENT", transect number,
            sample number, names   of the sampling team members, "BIVALVE", and
            11ADF&G11. Data will be recorded on the appropriate form.

            Triplicate composite sediment samples will be taken from the razor
            clam beach transect. This will provide 3 samples per beach and 9
            samples per treatment level.

            The small sub-samples of sediment taken from each sampling quadrant
            will provide a representative mixture of sediment composition and
            contamination throughout the transect.       Three composite sediment
            samples for each transect at each site provides 27 composite samples
            for each impact level (no, intermediate or high, and intermediate or
            high with treatment). The industry standard is 8 samples for each
            treatment level.     A sample size of nine composite samples is
            considered an adequate number of samples to detect a difference in
            sediment contamination between impact levels at the desired a and 8
            levels. This coverage level is being tripled.

            Three common species of clam, littleneck clam Protothaca staminea
            butter clam Saxidomus giganteus., and razor clam Siligua patula wili
            be sampled for hydrocarbon analysis, necropsy, and age and growth
            statistics.

            Specimens for hydrocarbon analysis will be taken from each sampling
            quadrant before any other specimen sampling is conducted. Bivalves
            of each species will be randomly selected for hydrocarbon analysis
            from sampling quadrants at each site.

            0ne hydrocarbon sample for each species will be obtained from each
            transect.   For littleneck clams and butter clams, each hydrocarbon
            sample will be composed of 14 specimens. The 14 specimens from each
            transect (1 hydrocarbon sample) will be selected by randomly picking
            two clams with a shell length of 2-5 cm from each of the eight
            sampling quadrants and discarding two clams selected at random.

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         Each hydrocarbon sample for razor clams will optimally be composed of
         six to eight individuals.    Six to eight razor clams with a shell
         length of 2-5 cm will be randomly collected at the beginning, middle
         and end of the collection transect, for a total of three samples per
         site.

         Bivalve samples are being limited to a particular size range because
         rates of uptake, metabolism, and depuration by clams probably change
         with size. If specimens of the desired size are not found in each of
         the sampling quadrants, then the desired number of additional
         specimens will be collected from the other sample quadrants.

         Combined tissue samples from each sampling quadrant will provide a
         representative   mixture    of   bivalve   tissue   composition    and
         contamination throughout the transect.     The desired size of each
         composite tissue sample is 15 qm. The number of bivalves to provide
         this sample from each transect was estimated based on the average
         size of individuals of each species.          An estimate of three
         hydrocarbon samples from each site is needed for detecting
         contamination between impact levels. A sample size of nine composite
         samples per three impact levels within the sound will allow the
         detection of differences in hydrocarbon content of 1.9 standard
         deviations with a and B levels of 0.05 and 0.1, respectively. A
         sample size of 12 composite samples per two impact levels outside of
         the sound will allow the detection of differences in hydrocarbon
         content of 1.4 standard deviations with a and B levels of 0.05 and
         0.1, respectively.

         Collection of specimens for necropsy will begin only after all
         hydrocarbon samples have been taken. Total sample size is 20 live or
         moribund specimens of each species taken at random from each beach
         site. Noticeable numbers of moribund animals will be documented and
         sampled separately. With 20 bivalves sampled from each beach, the
         total sample for each treatment (no, intermediate or high oil
         contamination, and intermediate or high oil contamination which has
         been treated) will be 60 within the sound and 80 for each of two
         treatment levels (oiled and not oiled) outside the sound.         This
         sample size will allow detection of differences in presence of tissue
         damage of ï¿½5% with 95% confidence between samples obtained from
         beaches with different levels of oil impact. This sample size will
         allow detection of gross differences between beaches with no, medium
         or high oil impact and medium or high oil impact which have been
         treated by mechanical means.

         one specimen of each species will be randomly selected from each
         sampling quadrant. This will yield a total of 24 specimens. Four
         specimens from the 24 collected will be randomly selected and
         discarded from the sample to achieve a sample size of 20 specimens.
         Twenty razor clams will be collected at random along the beach
         sampling transect.

         For littleneck and butter clams, a total of 100 specimens will be

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         collected from each transect at each site.    From.each transect six
         sampling quadrants will be selected at random. From each of these,
         12 specimens will be randomly sampled from the quadrant containers.
         Fourteen specimens will be randomly sampled from the remaining two
         quadrant containers.

         The sample of 100 specimens per species from each transect will
         provide 300 samples from each beach or 900 and 1,200 clams per
         species for each level of beach impact inside and outside of PWS,
         respectively.   Sample size for growth is based on the difference
         between mean shell height (width) for age i and age i+l clams,
         variance in shell height for age i+l clams, probability of making a
         type I error equal to .01 and probability of making a type II error
         equal to .05 (Netter and Wasserman 1985). Data for mean shell height
         and variance in shell height was taken from Paul and Feder (1973) for
         littleneck clams and Nickerson (1977) for butter clams. Sample size
         for detecting difference in growth at age of clams between impact
         levels was estimated at 261-275 littleneck clams for each impact
         level. This sample size was rounded up to 300 clams. The purpose of
         3 sites for each impact level is to provide replicates at each impact
         level. The sample size required for detecting difference in growth
         at age was somewhat smaller for butter clams, however because not all
         size ranges were represented in the available data, the larger sample
         size of 300 clams was recommended for this species as well.

         All shells will be collected from each quadrant and the number of
         live clams, the number of dead hinged shells, and the number of half
         shells will be recorded. One hundred hinged shells from dead clams
         taken in the sampling quadrant located at the median tidal height
         (quadrant 4) will be retained for age analysis.     If possible, some
         will have the microstructure analyzed to determine the year of death.
         .If less than 100 hinged shells are found in the three mid-tidal
         height sampling quadrants, additional shells will be collected at
         random at this approximate tidal height until the sampling objective
         of 100 dead shells is obtained.

         The sample size for determining the age composition of razor clams is
         based on data taken from Clam Gulch (Quinn and Jones, 1989). Quinn
         and Jones recommend a sample size for determining age composition of
         between 300 and 400 clams per stratum of interest. A minimum of 300
         razor clams per beach will. be collected for size and age
         determination.

         A total of 600 clams will be submitted for microstructure analysis.
         A random sample of 200 clams collected from each of three
         representative beaches (no contamination, intermediate or high oil
         contamination, and intermediate or high oil contamination which has
         been treated) located within PWS will be analyzed.     In particular,
         this analysis will look for the presence of a "check" in the shell
         material which has been laid down by clams as a possible response to
         the oil spill. Growth which has occurred since the "check" will be
         examined. Growth rates will be reported as well as estimated ages.

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         To further quantify oil impacts on clam growth and to discount site
         effects, littleneck clams will be transplanted from oiled to non-
         oiled areas and from non-oiled to oiled areas. Three oiled beaches
         and three non-oiled beaches will be chosen for this purpose.
         Criteria for selecting paired oiled/non-oiled beaches, to the extent
         possible, will include similarity in profile, drainage and length-
         frequency distribution of bivalves.

         Two tidal heights will be selected, each of which has an adequate
         number of specimens at paired beaches. Clams will be transplanted to
         the same tidal height from which they originated.      At each tidal
         height, three locations will be established creating triplicate
         sampling stations at each height.  2  Each location will consist of
         three adjacent clearly marked 0.25 m plots. one plot will be marked,
         but will not be disturbed until clams are sampled for growth.
         Another plot will be dug to a depth of 0.3 m and all of the removed
         clams and sediment will be replaced in the plot.     Clams from this
         plot will have a small notch filed into the ventral edge of the
         valves to mark the time of disturbance. All clams will be removed
         from the third plot which will be dug to a depth of 0.3 m and the
         transplanted clams will be placed in this plot along with the
         original sediment.    The clams which have been removed will be
         collected for comparison with the clams in the undisturbed plots at
         the end of the experiment.

         Clams to be transplanted will be obtained by digging a trench along
         the prescribed tidal height of the donor beach until 150 clams
         between 15  mm and 35 mm. in length have been collected.       Fifteen
         millimeters is considered to be the smallest size which can
         effectively be tagged. Clams less than 35 mm are selected to narrow
         the range   of ages for which differences in growth are being
         determined  and because the maximum growth rate appears to occur
         within this size range. A sample of 50 specimens from each of three
         plots will provide 150 samples from each tidal height at each beach
         and 450 clams for each tidal height and level of beach impact.
         Sample size for growth is based on the difference between mean shell
         height for age i and age i+1 clams, variance in shell height for age
         i+1 clams, probability of making a type I error equal to . 01 and
         probability of making a type II error equal to .05 (Netter and
         Wasserman 1985). The sample size was determined after comparing data
         for mean shell height and variance in shell height taken from Paul
         and Feder (1973) and Nickerson (1977). The sample size for detecting
         between impact level differences in growth at age of clams in the
         size range of 15 mm to 35 mm was estimated at 133 clams from the Paul
         and Feder data and at 85 clams from the Nickerson data for each
         impact level.   The higher estimate was rounded up to 150 clams by
         including the next smaller size group (age 5-6).    The purpose of 3
         sites for each impact level is to provide replicates at each impact
         level.

         Transplanted clams will be identified by marking each clam with a
         numbered floy tag secured with a quick-drying adhesive. All marked

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         clams will have a small notch f iled into the ventral edge of the
         valves to mark the time of transplantation. Individual clams will be
         measured at the beginning and end of the experiment. At the end of
         the growing season (October 1990), clams will be removed from each of
         the plots described above and analyzed f or growth.      Wet and dry
         weights of clams will also be recorded so that clam condition can be
         compared in terms of a weight to height ratio. Hydrocarbon samples
         will be taken during the experiment.

         To address objective A (hydrocarbons in sediments and bivalve
         tissues), an ANOVA will be used to test for differences in
         hydrocarbon content in sediment between sites.        Differences in
         sediment hydrocarbon content will verify that control sites (areas of
         no oil impact) are in fact "controls". These differences will also
         permit post-stratification of sample sites according to level of
         impact. An analysis of variance will be performed on the hydrocarbon
         content of clam samples among sites. The results of this test will
         be related to the level of sediment impact.

         Objective B will be met through ANOVA contingent upon the processing
         of necropsy samples. These samples will be processed if hydrocarbon
         analysis is positive.

         To provide baseline (pre-impact) information on variance in growth at
         age among sites, an analysis of variance on growth parameters from
         clams taken during 1989 between areas will be conducted.        Growth
         parameters will be determined for various growth curves, such as
         Gompertz, von Bertalanffy, or polynomial equations.             Growth
         parameters will be presented for the most appropriate growth models
         only. A similar ANOVA will be conducted on growth parameters from
         clams taken during 1990 between areas. Those beach sites which are
         resampled in 1990 will be subjected to an analysis of variance on
         growth parameters obtained from fitting algorithms for clam growth
         after impact (1990 and beyond) and will be compared to growth
         parameters for clam growth prior to impact (approximately 1979-1989)
         to resolve impact of oil contamination on growth (Objective C).
         Graphics will be used to display differences in growth among areas
         over time, including growth curves (size at age) and growth increment
         at age by year for each beach.

         To meet objectives D and E, a chi-square or an appropriate
         nonparametric test will be used to test for significant differences
         in proportions of dead clams (objective D) and young-of-the-year
         clams (objective E) between treatment levels.      Appropriate tests
         involving relative abundance measures may also be used to meet
         Objective E.

                                     BIBLIOGRAPHY

         Anderson, J.W., J.R. Vanderhorst, S.L. Kiesser, M.L. Fleishmann, and
              G.W. Fellingham. 1982. Recommended Methods for Testing the Fate
              and Effects of Dispersed Oil in Marine Sediments. In Oil Spill

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           Chemical     Dispersants:          Research,     Experience,       and
           Recommendations. ASTM Special Technical Publication
           840. Tom E. Allen Ed. Philadelphia, PA 19103. pp. 224-238.

     Anderson, J.W., R.G. Riley, S.L. Kiesser, B.L. Thomas, and G.W.
           Fellingham. 1983.       Natural Weathering of Oil in Marine
           Sediments: Tissue Contamination and Growth of the Littleneck
           Clam, Prototheca staminea. Canadian Journal of Fisheries and
           Aquatic Sciences. 40(Suppl. 2):70-77.

     Augenfeld, J.M., J.W. Anderson, D.L. Woodruff, and J.L. Webster.
           1980. Effects of Prudhoe Bay Crude 0 i 1 -Contaminated Sediments on
           Protothaca staminea (Mollusca: Pelecypoda) : Hydrocarbon Content,
           Condition Indexf Free Amino Acid Level. Marine Environmental
           Research. 4(1980-81):135-143.

     Dow,  R.L. 1975. Reduced Growth and Survival of Clams Transplanted to
           and Oil Spill Site. Marine Pollution Bulletin. 6(8):124-125.

     Dow,  R.L. 1978. Size-Selective Mortalities of Clams in an oil Spill
           Site. Marine Pollution Bulletin. 9(2):45-48.

     Keck, R.T., R.C. Heess, J. Wehmiller, and D. Maurer. 1978. Sublethal
           Effects of the Water-soluble Fraction of Nigerian Crude Oil on
           the Juvenile Hard Clams, Mercenaria (Linne). Environmental
           Pollution. 15:109-119.

     Neter, J., W. Wasserman, and M. Kutner. 1985. Applied Linear
           Statistical Models. Richard D. Irwin, Homewood Illinois.

     Nickerson, R.B. 1977. A Study of the Littleneck Clam (Prototheca
           staminea Conrad) and the butter clam (Saxidomus giganteus
           Deshayes) in a habitat permitting coexistence, Prince William
           Sound, Alaska. Proceedings of the National Shellfisheries
           Association. 67:85-102.

     Paul, A.J. and H.M. Feder. 1973. Growth, recruitment, and
           distribution of the littleneck clam, Protothaca staminea in
           Galena Bay, Prince William Sound, Alaska.          Fishery Bulletin
           71 (3) :665-677.

     Quinn, II, T.J., and N.F. Jones. 1989. Razor Clam (Siliqua patula,
           Dixon) Investigations on the Eastside Cook Inlet Beaches. Juneau
           Center for Fisheries and Ocean Sciences, University of Alaska
           Fairbanks, Project Report UAF-JCFOS-8902. 165 p.








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             BUDGET: ADF&G

             Salaries             $ 121.4
             Travel                    5.0
             Contracts               100.8
             Supplies                  0.0
             Equipment                 2.0

             Total                $ 229.2















































                                               151










     FISH/SHELLFISH STUDY NUMBER 15


     Study Title:    Injury to PWS Spot Shrimp

     Lead Agency: ADF&G


                                  INTRODUCTION



     This project will continue to determine possible damage to spot
     shrimp, Pandalus platyceros, due to the EVOS.      Spot shrimp are a
     representative species of the deep water near shore benthic
     ecosystem, serving as a food source for a variety of fish. They are
     a commercially important species and also support subsistence and
     personal use fisheries in PWS. This project is a continuation of FIS
     Study 15 which was conducted during 1989-90.

     Spot shrimp are known to be sensitive to oil contamination in both
     the larval and adult phase, and the effects of oil on spot shrimp in
     particular and shrimp in general are well documented (Anderson et al
     1981, Brodersen et al 1977, Brodersen 1987, Mecklenburg, Rice and
     Karinen 1977,     Sanborn and Malins 1980, Stickle et al 1987,
     Vanderhorst 1976). To determine the impacts that hydrocarbons from
     the spill may have had on spot shrimp, samples will again be
     collected from the three oiled and three non-oiled sites in western
     PWS which had been surveyed in 1989.     The data collected from the
     samples will be analyzed to determine tissue hydrocarbon levels and
     tissue damage. The collected data will also be tested to confirm or
     reject the hypothesis that there is no significant difference in
     hydrocarbon levels between the oiled and non-oiled areas. Relative
     abundance, in terms of catch per unit effort, at each study site and
     changes in relative abundance over time will be tested to determine
     possible relationships with the level of oiling. A comparison with
     historical records will also be made. The size composition of the
     stock at each site will be estimated and, dependent upon recruitment
     to the fishing gear, analyzed to determine whether the 1989 year
     class suffered a high mortality rate in areas of high oil impact
     relative to other year classes in non-oiled areas.        Spot shrimp
     fecundity will also be determined and tested for significant annual
     and interannual differences between oiled and non-oiled sites.


                                   OBJECTIVES

     A.   Estimate the relative abundance by weight and sex of spot
          shrimp and the relative abundance by weight of incidentally
          caught pink and coonstripe shrimp in oiled and unoiled areas and
          compare these values to those obtained during the first
          assessment survey in 1989.

     B.   Compare size and age frequencies (by sex and depth stratum)
          between sites using mixture model analysis.

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              C.   Estimate fecundity, egg mortality, and other sublethal effects
                   between oiled and non-oiled areas over time, and            determine
                   whether those effects result in adverse changes in reproductive
                   viability.

              D.   Analyze tissue and egg samples for presence of hydrocarbons and
                   compare differences between oiled and non-oiled sites. Test the
                   hypothesis that the level of hydrocarbons is not related to the
                   level of oil contamination present at a site.

              E.   Document injury to tissues and compare differences between
                   oiled and non-oiled sites if warranted by results from tissue
                   hydrocarbon analysis.

                                               METHODS

              This project uses commercial spot shrimp pots of a standardized size
              to catch spot shrimp in oiled and unoiled areas. Shrimp specimens
              will be analyzed for Prudhoe Bay crude oil levels and necropsied to
              determine if damage has occurred to tissues as a result of oil
              contamination. As in the 1989 study plan, oiled and unoiled areas
              will be sampled in two phases which correspond with two stages of egg
              development.    The first phase will occur in early November (1990)
              following the fall molt and egg extrusion.        The second phase will
              occur in early March (1991) just prior to egg hatching. The sampling
              strategy will   be identical during both phases. Relative abundance
              estimates of spot shrimp will be made using a stratified pot
              deployment based on depth and location. Size distribution, species
              composition, and reproductive data will also be collected. Previous
              spot shrimp research in PWS is documented by Kimker and Donaldson
              (1987), Donaldson (1989), Donaldson and Trowbridge (1989), and Kruse
              and Murphy (1989).

              This project will be carried out in two general areas. One will be
              an area of little apparent impact, the northwestern portion of PWS.
              This area includes Unakwik Inlet, the site of previous ADF&G research
              on abundance and growth of spot shrimp.        The second area will be
              central and southwestern PWS, an area of generally high oil impact.
              This area includes Green Island where ADF&G test fishing occurred in
              1981.   Within each of these two areas, fishing will take place at
              three sites. In the northwestern sound, test fishing will occur in
              Unakwik Inlet, Port Wells, and Culross Passage. In the central and
              southwestern sound, test fishing will take place near Herring Bay,
              Chenega Island, and Green Island. Shrimp distribution in these areas
              has been established by surveying the commercial fleet.

              Fishing will take place at six sites - three in oiled areas and three
              in non-oiled areas. Each site will be stratified by depth. Stratum
              1 will be shallow waters - 20 to 70 fathoms. Stratum 2 will be deep
              waters - 70 to 120 fathoms. Based on past research, spot shrimp are
              not abundant below those depth ranges. Because of the difficulty of
              placing the gear at precise depths, it is impractical to divide the

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          depth into more than two strata. Strata span 50 fathoms in depth or
          approximately 65 to 85 fathoms in width along the bottom at slopes of
          75 to 100 percent. Fishing a 100 fathom string will span the width
          of each strata and allow for a complete placement of gear over the
          strata.

          Eleven pots spaced 10 fathoms apart will be fished on a long line so
          that each string of pots is 100 fathoms long. One 100 fathom string
          of gear constitutes a sampling station. Two stations will be fished
          in each stratum at each site for a total of 22 pots per stratum per
          site, or 44 pots per site. Forty-four pots is the most that can be
          fished in a day while collecting all of the various samples and data.
          If necessary, pots will be redeployed an additional day at each site
          and at each depth until a minimum of 500 shrimp are captured per
          depth stratum. A total of 264 pots will be fished during each time
          period.

          Water temperature, salinity, and dissolved oxygen concentration by
          depth will be recorded using a CTD, transferred from the CTD to a
          micro-computer and stored on diskette.      CTD casts will be at one
          station in the deep stratum every day. The CTD will be lowered at a
          rate of 60 meters per minute. Because of the configuration of the
          CTD, only readings from the downcast will be used.

          Total weight of catch, sub-sample weight, and the weight of each
          species in a sub-sample will be recorded for each pot on a paper form
          at the time the pot is retrieved. The total weight of shrimp per pot
          will be determined by weighing the contents of each pot on an
          electronic scale. The average number of shrimp per kilogram will be
          determined. If less than 500 shrimp are estimated to be contained in
          all of the pots, all of the shrimp will be sampled. If the pots are
          estimated to contain more than 500 shrimp, a constant proportion by
          weight of each pot will be sampled for a total sample of 500 shrimp.

          Each sub-sample will be sorted by species.       Weight and number of
          animals will be recorded for each species. Only spot shrimp will be
          retained for further data collection. All spot shrimp in the sub-
          sample will be measured for carapace length to the nearest 0.1
          millimeters using a digital caliper and sex will be determined as
          male, transitional, or female. For female spot shrimp, egg color and
          stage of development (eyed or uneyed) ; relative clutch size; presence
          of breeding dress and egg parasites or parasitic externa will be
          noted.    Each female retained for fecundity analysis will be
          identified with a code number to allow cross reference of fecundity
          and other data.

          Specimens for necropsy analysis will be taken after the catch is
          weighed and processed. Twenty shrimp from a single station in each
          stratum will be selected randomly to make up a necropsy sample.
          Necropsy samples will be labeled with the date, station number,
          latitude and longitude, sample number, project leader's name,
          species, and agency.

                                            154









         To prevent contamination, specimens for hydrocarbon testing will be
         taken from the pot immediately after removal from water and before
         contents are weighed.     Three spot shrimp will form one composite
         sample.   Each composite will be taken from a different pot.         Two
         replicates of the composite will be taken randomly from one station
         in the stratum and the third replicate will come from the other
         station.   Three samples per site per depth stratum result in nine
         samples per depth stratum (three sites X three samples) per impact
         level and 18 samples per oil impact level (nine samples X two depth
         strata). This will allow hypothesis testing to detect differences in
         hydrocarbon levels of 1.2 standard deviations with the probability of
         a type I or type II.error being 0.05 and 0.10, respectively.

         The number of specimens for one hydrocarbon analysis is dependent on
         the size of the specimens collected.      Tissue volume based on the
         average size of the species was estimated and the number of specimens
         needed to provide 15 gm. of tissue was calculated to be three spot
         shrimp. An estimate of three hydrocarbon samples from each treatment
         level is needed for detecting contamination between levels.

         Twenty five egg bearing females will be taken at random from each
         station to estimate fecundity and egg mortality.         A total of 24
         stations will yield a total sample size of 600 females. Specimens
         from each station will be individually labeled. Each sample bag will
         be labeled with project leader's name, species name, "eggs", date,
         station, and agency name.

         Fecundity will be determined by removing the eggs from the pleopods,
         drying each egg mass to a constant weight, weighing a sub-sample of
         a known number of eggs, and expanding the sub-sample weight to the
         weight of the entire clutch. Carapace length will be taken for each
         specimen at the time the eggs are removed and recorded on the
         fecundity form.

         A minimum number of five shrimp from each station will be sampled for
         fecundity which will allow an adequate sample (30 per depth strata
         per oil impact level) to test for differences in fecundity between
         depth strata and oil impact level.

         Objective A will be addressed by estimating the average catch per pot
         by weight, sex, and species.        ANOVA will be used to test for
         significant differences in each of these categories between strata
         (depth) , sites, and oiled versus non-oiled areas.       To def ine the
         relationship between hydrocarbon levels and changes in relative
         abundance, statistics for analysis of covariance or an appropriate
         multivariate technique will be calculated to contrast differences in
         hydrocarbon content and relative abundance in- oiled and non-oiled
         areas.   Changes in average catch per pot over time will also be
         analyzed between different depth strata, sites, and oiled and non-
         oiled areas.

         A size frequency distribution will be made by species and sex to

                                           155









         address objective B.    The hypothesis that there is no significant
         difference between strata, and oil impact levels for size frequency
         distribution will by tested using quantile-quantile plots, Chi-square
         tests or other appropriate methods.      A t test or a similar non-
         parametric test will be used to test for similarity in means.
         changes in size frequency distribution over time will be examined by
         comparing data collected during, phase one and phase, two. A. t test
         for means and an,appropriate method for comparing distributions will
         be used to look for significant differences between time.periods as
         well.

         To meet objective C, the relationship between size and fecundity will
         be examined. The percentage of spot shrimp females bearing eggs; the
         stage of spot shrimp egg development (color and,presence or absence
         of eyes); the percentage of spot shrimp egg fouling and egg
         mortality; the fecundity by size; and the relative clutch size will
         be determined for each station and each phase. Chi-square tests @will
         be used to test for differences in stratal sites.and levels in data
         which involve percentages and proportions. Differences between
         strata, sites, and impact,levels:for fecundity and relative size of
         clutch will be tested for using analysis of variance.       ANOVA will
         also be used to test for a significant difference in the above
         measures between phase one and* phase two which may provide an
         estimate of the number of eggs -dying over the course of the brood
         period or estimates of differences in egg viability.

         To address objectives D and   E, the average levels of   oil present in
         spot shrimp tissue by strata  and site will be estimated. Significant
         differences in hydrocarbon concentrations between oiled and unoiled
         sites will be tested by analysis of variance. To further define the
         impact of hydrocarbon -levels on the stock, the percentage of animals
         with abnormal tissues in oiled and unoiled areas*will be determined.
         A chi-square test will be utilized to test for significant
         differences in percentage of animals with abnormal tissues between
         strata, sites, and impact levels.

                                      BIBLIOGRAPHY

         Anderson, J.W., S.L. Kiesser, R.M. Bean, R.G. Riley    and B.L. Thomas.
              1981. Toxicity of 'chemically dispersed oil to shrimp exposed to
              constant and decreasing concentrations in a flowing system. In:
              .1981 Oil Spill Conference (Prevention, . Behavior, Control,
              Cleanup), Proceedings. Washington D.C. American Petroleum
              Institute. Pp. 69-75.

         Brodersen, C.C., S.D.  Ricef, J.W. Short, T.A. Mecklenburg,   and J.F.
              Karinen. 1977.    Sensitivity of larval and adult Alaskan shrimp
              and crabs to acute exposures of the water-soluble fraction of
              Cook Inlet crude oil. In: 1977 Oil Spill Conference (Prevention,
              Behavior,, Control, Cleanup) , Proceedings.      Washington, D.C.
              American Petroleum Institute. pp. 575-578.


                                           156









         Brodersen, C.C. 1987. Rapid narcosis and delayed mortality in larvae
              of king crabs and kelp shrimp exposed to the water-soluble
              fraction of crude oil. Marine           Environmental Research.
              22(1987):233-239.

         Donaldson, W. 1989. Synopsis of the Montague Strait experimental
              harvest area 1985 - 1988. Alaska Department of Fish and Gamef
              Division of Commercial Fisheries, Regional Information Report
              No. 2C89-04. 21 pp.

         Donaldson, W. and C. Trowbridge. 1989. Effects of rigid mesh panels
              on escapement of spot shrimp (Pandalus I?latyceros) from pot
              gear.    Alaska Department of Fish and Game, Division of
              Commercial Fisheries, Regional Information Report No. 2C89-05.
              22 pp.

         Kimker, A. and W. Donaldson. 1987. Summary of 1986 streamer tag
              application and overview of the tagging project for spot shrimp
              in Prince William Sound. Alaska Department of Fish and Game,
              Division of Commercial Fisheries, Prince William Sound
              Management Area Data Report 1987-07.

         Kruse, G. and P. Murphy. 1989. Summary of statewide shrimp workshop
              held in      Anchorage during October 24-26, 1988.           Alaska
              Department of Fish and Game, Division of Commercial Fisheries,
              Regional Information Report No. 5J89-##.

         Mecklenburg, T.A., S.D. Rice, and J.F. Karinen. 1977. Molting and
              survival of king crab (Paralithodes camtschatica) and coonstripe
              shrimp (Pandalus hypsinotus) larvae exposed to Cook Inlet crude
              oil water-soluble fraction. In: D.A. Wolfe (ed.). Fate and
              Effects of Petroleum Hydrocarbons in Marine Ecosystems and
              Organisms. Pergamon Press, New York, NY. pp. 221-228.

         Sanborn, H.R. and D.C. Malins. 1980. The disposition of aromatic
              hydrocarbons in adult spot shrimp (Pandalus platyceros) and the
              formation of metabolites of naphthalene in adult and larval spot
              shrimp. Xenobiotica. 10(3):193-200.

         Stickle, W.B., M.A. Kapper, T.C. Shirley, M.G. Carls, and S.D. Rice.
              1987. Bioenergetics and tolerance of the pink shrimp (Pandalus
              borealis) during long-term exposure to the water-soluble
              fraction and oiled sediment from Cook Inlet crude oil. In: W.B.
              Vernberg, A. Calabrese, F.P. Thurberg, and       F.J.     Vernberg
              (eds.). Pollution Physiology of Estuarine Organisms. Belle W.
              Baruch Libr. Mar. Sci. 17, Univ. S. C. Press, Columbia. pp. 87-
              106.


         Vanderhorst, J.R., C. I. Gibson, and L J. Moore. 1976. Toxicity of No.
              2 fuel oil to coonstripe shrimp. Marine Pollution Bulletin.
              7(6):106-108.


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     BUDGET: ADF&G


     Personal Services          $ 44.0
     Travel                         1.4
     Contractual                   15.0
     Supplies                       4.6
     Equipment                      0.0

     Total                      $ 65.0















































                                        158











              FISH/SHELLFISH STUDY NUMBER 17

              Study Title:    Injury to Demersal Rockfish and Shallow
                              Reef Habitats in PWS and Along the Lower KP

              Lead Agency: ADF&G

                                          INTRODUCTION

              In light of the findings of the first year of study of potential
              impacts on rockfish populations conducted in 1989, continued study
              of demersal rockf ish populations and shallow reef habitats is
              warranted for 1990. Unlike many species of marine fish, demersal
              rockfish complexes are relatively sedentary, residing near rocky
              reefs and boulder fields. The potential impact of the oil spill on
              various nearshore assemblages is dependent upon location of various
              rockpiles. The potential uptake of various contaminants will be
              related to the level of oil contamination and food web
              characteristics of these reefs.         of primary importance are
              questions of transport of oil to subsurface habitats and the
              RPtential f or residual persistence of this contamination.        Khan
              (1987) reports that crude oil can contaminate sediments and persist
              for long periods of time in the environment.

              Under these conditions, the petroleum hydrocarbons can exert a
              broad range of effects on animals, from impaired feeding, growth,
              reproduction, and changes in behavior; to tissue and organ damage,
              damage to blood cells, changes in enzyme activity and changes in
              parasite densities (Khan 1986; Khan 1987; Kiceniuk and Khan 1986;
              Rice 1985; Wennekens et al. 1975; Malins et al. 1977; Rice et al.
              1977; Gundlach et al. 1983; Hose et al. 1987; Spies et al. 1982).
              These possible affects are especially critical to demersal rockfish
              since they are long-lived, recruitment is low, and the potential
              for long-term stock decline due to chronic exposure to crude oil is
              high.  Continuation of this study will help determine long term
              histopathological effects on the fish and will quantify the extent
              to which hydrocarbons persist in the environment.

              Only limited baseline data are available for rockfish populations
              in PWS and along the lower Kenai Peninsula (LKP). Rockfish were
              studied as part of a study of nearshore f ish assemblages during the
              years 1977-1979 in PWS (Rosenthal, 1980) and Morrison studied
              select reefs along the LKP during 1980 through 1984.             These
              investigations   provided    descriptions   of   selected     rockfish
              populations including estimates of species and prey composition,
              density, length and age composition.



                                           OBJECTIVES


              A.   Determine the presence or absence of hydrocarbons in demersal

                                               159









             rockf ish, benthic prey species, benthic suspension feeders,
             and sediments from two control and two treatment sites in PWS
             and two control and two treatment sites along the LKP.

        B.   Determine the physiological effects resulting from oil
             contamination through histopathological examination of five
             organs, enzyme activity, examination of red blood cells for
             circulating micronuclei; and the examination of developing
             embryos.

        C.   Determine the feasibility of using toxicological analysis of
             gonads and pituitary glands to ascertain effects of oil
             contamination on growth and reproduction.

        D.   Determine the feasibility of using otolith microstructure to
             evaluate depressed growth as a result of oil contamination.


                                       METHODS

        Eight sites (four treatment and four control) in PWS and along the
        LKP will be sampled in 1990. Demersal species of rockfish, benthic
        and   epibenthic   invertebrates,    and   finfish    prey    species,
        unconsolidated benthic sediments and sessile suspension feeders
        will be collected at each sample location for analysis of
        hydrocarbons. From the results of these analyses the mechanism of
        hydrocarbon uptake in demersal rockfish and the extent to which
        hydrocarbons persist in reef ecosystems may be determined.         The
        effects of sublethal hydrocarbon contamination in demersal rockfish
        may be determined through histopathological examination of five
        organs; evaluation of enzyme activity; examination of red blood
        cells for circulating micronuclei; and, the examination of
        developing embryos.     The feasibility of evaluating affects on
        growth and fecundity through toxicological and biochemical analysis
        of gonad and pituitary tissues, as well as determining depressed
        growth through examinations of otolith microstructures, will be
        explored. Results will be compared between control and treatment
        sites.   A pilot sampling trip will be made to determine what
        species are present and to evaluate sampling techniques and site
        selection.

        Criteria for choosing sample reefs were based on:                   (1)
        accessibility to boat and diving operations (ocean floor
        surrounding the reefs were approximately 20 fathoms or less in
        depth) ; (2) exposure of surface waters to oil; (3) location of
        reported kills and/or sublethal contamination of demersal rockfish;
        (4) occurrence of sampling by other oil spill assessment studies
        relative to this study and (5) previous study sites of Rosenthal or
        Morrison.

        A systematic sampling design will be used to identify sampling
        sites within each reef. Transects will be established at discrete


                                         160









             depths by deploying an anchor line along specific contours of the
             reef and each end will be marked by anchored flag pole assemblies.
             Coordinates, length, depth, and orientation of the transect will be
             recorded. The actual number of sample sites will be depend on the
             length of the transect and the orientation of the reef in the ocean
             currents. During the pilot sampling trip prey and benthic species
             that are  common to all reefs and that are not transient will be
             listed as  target species. These species will then be collected at
             each reef during the sampling trip.      Sampling will be conducted
             during late July and early August, the time frame that Rosenthal
             (1980) identified as near the peak abundance of rockfish in
             nearshore areas.    Collection methods for finfish, prey species,
             sediment, and sessile invertebrates are outlined below.

             Twenty adult demersal rockf ish (target primarily yelloweye rockf ish
             Sebastes ruberrimus) will be collected at each sample site using
             hook and line jigging techniques. The sample size is based on the
             number required for histological evaluation as determined by the
             Histopathology Technical Group (Meyers, 1989). Baited lures will
             be lowered to the substrate and raised enough to allow for adequate
             jigging action. When a fish is on the line it will be retrieved
             slowly in order to allow the air bladder to equilibrate and prevent
             extrusion of the stomach and regurgitation of its contents. Where
             excessive depths make this impractical, divers will enclose the
             fish in a dive net to retain the stomach contents upon
             regurgitation. Where hook and line techniques do not yield results
             divers will verify the presence or absence of demersal rockfish
             assemblages and if, present, collect them using spear guns.
             Stomach contents will be collected to determine composition of the
             prey species.    Species identification of adult rockfish will be
             accomplished using the methods of Kramer and O'Connell (1988) and
             Hart (1973).

             Fifty juvenile demersal rockfish will be collected using variable
             mesh, monofilament gillnets set in the shallow areas of the reef
             and in intertidal zones adjacent to the reefs.       Given estimated
             proportions of 0.6 and 0.2 respectively, sample size was determined
             (Zar 1984) to be 50, where a =.05.        Species identification of
             juvenile rockf ish species will be accomplished using the methods of
             Matarese et al. (1989).

             Ten samples of prey species (Rice, 1990) at each reef will be
             collected for hydrocarbon analysis. The sample size is based on
             the number required for hydrocarbon analysis as established by the
             Analytical Chemistry Group (Manen, 1989).        The species to be
             collected will be determined during the pilot sampling trip.
             Additional information used to select prey species will be based on
             the analysis of stomach content samples and previous food ecology
             studies (Rosenthal et al., 1988; Rosenthal, 1980).              Divers
             outfitted with SCUBA gear will use an air-lift sampler to collect
             benthic prey species at each site (Chess, 1978).        The air-lift
             sampler uses suction to collect all organisms from a square meter

                                              161









         area and deposits them into a sampling container. Additional food
         organisms may be collected using a variety of other techniques
         depending on the target species. crab pots and shrimp pots will be
         used to collect crustacean species. Trammel nets, plankton tows,
         and diver controlled nets will be used to capture appropriate
         target species.

         .Nine sediment samples (Rice, 1990) will be collected at each sample
         site by divers outfitted with SCUBA equipment prior to the
         collection of air-lift samples outlined above. Each sample will
         consist of ten 2 cc scoops taken from the top 2 cm of the substrate
         along a 10 m long transect. Excess water will be poured of f at the
         surface and the sample will be frozen. Three sediment samples will
         be collected at each reef.

         Three samples of sessile      filter feeders (Rice, 1990) will be
         collected from each reef by   divers outfitted with SCUBA equipment.
         Each sample will consist of pieces of two or three sessile filter
         feeders. Enough samples will be collected to at least half fill a
         4 oz. hydrocarbon sampling jar.

         Samples collected will be handled differently depending upon the
         data required and type of analysis being conducted. The following
         sections explain each type of preparation that will be used. Most
         samples collected will be used for only one type of analysis,
         however, each rockfish captured will be used or prepared for a
         variety of purposes. Rockfish will be processed in the following
         specific order: 1) immediately after capture blood samples will be
         drawn and slides prepared; 2) rockfish will be measured to the
         nearest millimeter (fork length) and weighed to the nearest gram
         for calculation of condition factor; 3) tissue will be sampled for
         hydrocarbon analysis and histopathological evaluation according
         procedures outlined in proceeding sections; and, 4) otoliths will
         be removed for later age determination.

         Length (fork length) , to nearest millimeter, and weight, to the
         nearest gram, will be used to calculate a relative condition
         factor.    Condition factors will be calculated for all rockfish
         captured.

         Ten of the 20 rockfish (Rice, 1990) collected at each reef will be
         prepared for hydrocarbon analysis. All samples will be collected
         from live fish. Bile samples will be collected first by removing
         the whole gall bladder and emptying the bile into 0.5 oz. amber
         sampling jars. Ten grams each of stomach, pyloric caeca, liver,
         and muscle tissue will be collected from each rockfish.            Each
         tissue type will be stored in separate 4 oz. sampling jars.

         Ten samples of prey species (Rice, 1990) will be collected at each
         reef. Different preparation methods will be conducted depending
         upon the prey species being collected. Larger fish will be handled
         in the same manner as the rockfish. Smaller fish, and other small

                                           162









           organisms where dissection is not practical, will be collected
           whole in sufficient numbers to fill a 4 oz. sampling jar half to
           three-quarters full.

           Twenty live demersal rockfish, including the ten sampled for
           hydrocarbons, will be collected at each reef for histopathological
           analysis and processed under the guidelines outlined by the
           Histopathology Technical Group (Meyers, 1989). Blood samples will
           be collected using a heparinized syringe inserted between the
           vertebrae in the caudal peduncle and smears made and fixed for
           later staining with May-Grunwald-Giesma stain. The liver will be
           visually examined for discoloration, blotchiness, and firmness and
           its condition recorded. One centimeter sections of tissue will be
           removed from the following organs: liver-pancreas, kidney, gills,
           gonads, and eyes.    All developing embryos will be collected and
           preserved in a neutral formalin solution.

           Sagittal otolith pairs will be collected from fifty juvenile
           yelloweye rockfish (measuring less than 200 mm) from each reef.
           Age validation studies involving daily growth increments, such as
           Boehlert and Yoklavich (1987), typically utilize otoliths from
           juveniles because growth is deposited more rapidly, and
           physiological checks and daily growth increments are more visible.
           Upon collection, otoliths will be rinsed and stored dry in pairs in
           coin envelopes.

           Juvenile otoliths will be prepared for examination following
           methods outlined by Boehlert and Yoklavich (1987). Otoliths will
           be viewed under transmitted light with a compound microscope at
           40OX magnification.     Presence and location of hyaline zones
           comprising annuli, daily growth increments, and checks resulting
           from physiological factors including a reduction in growth will be
           examined.   The feasibility of distinguishing differences in the
           type of zones will be explored by measuring the width of growth
           zones deposited over consecutive periods of time (days and years).
           Where physiological checks are clearly discernible from annuli, the
           presence of checks will be determined with respect to annuli.
           Checks deposited within the growth zone of the previous year will
           be noted. The proportion of otoliths containing checks within this
           growth zone will be compared between control and treatment groups.

                                      DATA ANALYSIS

           Data analysis will consist primarily of the comparison of results
           between control and treatment groups for each of the following:

           LeCren's relative condition factor (Kn) (Anderson and Gutreuter,
           1983) will be calculated for each adult and juvenile rockfish.
           The mean condition factor for adult and juvenile rockfish for each
           reef will be calculated and differences between control and
           treatment groups will be tested using ANOVA.


                                           163









         Rockfish tissues, prey species and sessile filter feeders will be
         analyzed f or presence of hydrocarbons. Proportions of contaminated
         samples in each category will be compared between control and
         treatment groups.

         For each species the proportion of treatment sites containing
         contaminated samples will be compared to the proportion of control
         sites with contaminated samples using a two-sampled z-test from Zar
         (1984).

         Tissues will be examined for histopathological abnormalities and
         enzyme activity, and blood will be examined for circulating
         micronuclei by the Histopathological Technical Group.           The
         proportion of samples showing evidence of histopathological
         abnormalities will be compared between control and treatment groups
         for each tissue type using the z-test from Zar (1984).

         Otoliths from juvenile demersal rockfish will be examined as
         described in the methods section.         Proportion of otoliths
         containing checks between the last two annuli will be compared
         between control and treatment groups using the z-test from Zar
         (1984). Age composition and mean length-at-age will be calculated
         for each species of rockfish.



                              BIBLIOGRAPHY


         Anderson, R.O., and S.J. Gutreuter. 1983. Length,
              weight, and associated structural indices.    Chapter 15 IN:
              Fisheries Techniques, L.A. Neilson and D.L. Johnson eds.,
              American Fisheries Society, Bethesda, Maryland.

         Boehlert, G.W. and M.M. Yoklavich. 1987. Daily growth increments
              in otoliths of juvenile black rockfish, Sebastes melanops: An
              evaluation of autoradiography as a new method of validation.
              Fishery Bulletin: Vol. 85, No. 4. pp. 826-832.

         Chess, J.R. 1978, An airlift sampling device for in situ
              collecting of biota from rocky substrate. Marine Technology
              Society Journal, 12:20-23.

         Gundlach, E.R., P.D. Boehm, M. Marchand, R.M. Atlas, D.M. Ward, and
              Douglas A. Wolfe. 1983. The fate Amoco Cadiz oil. Science
              221:122-129.


         Hart, J.L. 1973.     Pacific Fishes of Canada.      Bulletin 180,
              Fisheries Research Board of Canada. Ottawa, Ontario, Canada.
              pp. 388-453.

         Hepler, K., A. Hoffmann, and T. Brookover. 1989. Injury to
              rockfish in Prince William Sound.     State/Federal resource
              damage assessment data summary report. Fish/Shellfish Study

                                        164









                 Number 17. ADF&G Sport Fish. Anchorage, Alaska.

           Hose  J.E., J.N Cross, S.G. Smith and D. Deihl. 1987. Elevated
                 circulating     erythrocyte    micronuclei     in    fishes     from
                 contaminated sites in California.           Marine Environmental
                 Research, 22:167-176.

           Khan  R.A. 1986. Effects of chronic exposure to petroleum
                 hydrocarbons on two species of marine fish infected with
                 hemoprotozoan, Trypanosoma muranensis. Can. J. Zool. 65:2703-
                 2709.

           Khan R.A. 1987. Crude oil and parasites in fish. Parasitology
                 Today, 3:99-102.

           Kiceniuk J.W. and R.A. Khan.           1986.     Effects of petroleum
                 hydrocarbons on Atlantic cod, Gadus Morhua, following chronic
                 exposure. Can. J. Zool. 65:490-494.

           Kramer, D.E. and V.M. O'Connell. 1988. Guide to Northeast Pacific
                 Rockfishes Genera Sebastes and Sebastolobus. University of
                 Alaska Marine Advisory Bulletin No. 25.

           Malins, D.C., E.H. Gruger, Jr., H.O. Hodgins, N.L. Karrick, and
                 D.D. Weber.        1977.     Sublethal effects of petroleum
                 hydrocarbons and trace metals, including biotransformations,
                 as reflected by morphological, chemical, physiological,
                 pathological, and behavioral indices. OCS Energy Assessment
                 Program. Seattle, Washington.

           Manen, C. A., Chairperson. 1989. State/federal damage assessment
                 plan, Analytical Chemistry Group, National Marine Fisheries
                 Service, Auke Bay, Alaska.

           Matarese A.C., A.W. Kendall Jr., D.M. Blood, and B.V. Vinter.
                 1989. Laboratory guide to early life history stages of
                 northeast Pacific fishes. NOAA Tech. Rep. NMFS 80. National
                 Oceanic and Atmospheric Adm., National Marine Fisheries
                 Service. Seattle, Washington 98115. 625 pp.

           Mey ers, T. R., Chairperson. 1989. State/federal damage assessment
                 plan, Histopathology Technical Group, Alaska Department of
                 Fish and Gamel Fisheries Rehabilitation, Enhancement, and
                 Development Division, Juneau, Alaska.

           Ricef S.D., J.W. Short, and J.P. Karinen. 1977. Comparative oil
                 toxicity and comparative animal sensitivity.         In: "Fate and
                 effects of petroleum hydrocarbons in marine organisms and,
                 ecosystems, Proceedings", Wolfe, Douglas A., ed., Pergamon
                 Press, New York. pp. 78-94.

           Rice, S.D. 1985. Effects of oil on fish. Chapter 5 IN:

                                              165









             Petroleum effect in the Arctic environment. F.R. Engelhardt
             ed. pages 157-182.       Elsevier Applied Science Publishers,
             London.

       Rice, S.D. 1990. Personal communication. Analytical Chemistry
             Group, National Marine Fisheries Service, Auke Bay, Alaska.

       Rosenthal, R.J., Victoria Moran-O'Connell and Margaret C. Murphy.
             1988.         Feeding     ecology     of    ten     species      of
             rockf ishes (Scorpaenidae) f rom the Gulf of Alaska. Calif . Fish
             and Game 74:16-37.

       Rosenthal, R.J.        1980. Shallow water fish assemblages in
             northeastern gulf of Alaska: habitat evaluation, species
             composition,abundance, spatial distribution and trophic
             interaction. Prepared for NOAA, OCSEAP Program. 84 pp.

       Rubin, J. 1988. A review of petroleum toxicity and fate in the
             marine environment, with implications for the development of
             a penalty table for spilled oil.           Institute for Marine
             Studies, University of Washington. Seattle, Washington.

       Spies, R.B., J.S. Felton, and L. Dillard. 1982. Hepatic mixed-
             function oxidases in California flatfishes are increased in
             contaminated environments by oil and PCB ingestion. Marine
             Biology 70:117-127.

       Wennekens, M. P., L. B. Flagg, L. Trasky, D. C. Burbank,
             R. Rosenthal, and F. F. Wright. 1975. Anatomy and potential
             costs of an oil spill upon Kachemak Bay. Alaska Department of
             Fish and Game, Habitat Protection Section Anchorage, Alaska.

       Zar, J.H. 1984. Biostatistical Analysis. Prentice Hall, Inc.,
             Edgewood Cliffs, New Jersey.



       BUDGET: ADF&G


       Personnel              $    40.9
       Travel                       2.7
       services                    63.6
       supplies                     1.2
       Equipment                    1.0

       Total                   $ 109.4








                                          166









            FISH/SHELLFISH STUDY NUMBER 18

            Study Title:    Prince William Sound Trawl Assessment

            Lead Agency:    NOAA



                                        INTRODUCTION

            This project is a continuation of a multispecies trawl survey to
            collect samples from bottomfish for    'hydrocarbon analyses.    Its
            purpose is to determine if bottomfish in PWS are still exposed to
            oil or oil components from the EVOS and, if so, the geographical
            extent of the exposure. The study will be conducted for 12 days
            during June 1990.

                                         OBJECTIVES

            A.   Collect bile and tissue samples, and stomach contents from
                 bottomfish, especially the utilized species.

            B.   Use CTD instrument to profile water characteristics throughout
                 the sampling area.

            C.   Preserve any fish observed with abnormalities of any type for
                 subsequent analysis.

                                          METHODS

            Sample collection will be done from the RV John N. Cobb using 400-
            mesh Eastern otter trawls at known trawl locations in the Sound.
            CTD profiles will be taken at each trawl station to provide
            information on the structure of water masses.

            Because the samples must be obtained from live fish, the hauls will
            be of short duration (probably 10 minutes or less) to avoid death
            from capture. The fish will be placed in live tanks and samples
            taken immediately after capture.     Procedures for taking samples
            will be the same as in the 1989 survey, with the exceptions that
            the samples will be obtained only from live fish and that samples
            will be frozen immediately after being taken.

            Six tows per day are planned for each of the 10 days. The 60 tows
            will be distributed among the areas and depth strata used for the
            1989 summer survey. The 1989 summer survey sampled six areas but
            the 1990 survey will sample only five of the six areas; one area,
            Port Wells, had negligible catches in 1989 and this area will not
            be sampled in 1990.     The same depth strata used f or the 1989
            sampling will be used for the 1990 sampling:




                                            167











                          Stratum               Depth (fm)

                               1                  10-50


                               2                   51-100


                               3                  101-200


                               4                  201-400



       Eleven depth strata occur in the five areas:


                      Area                 Depth strata to be sampled

         Hinchenbrook                                 2,3

         Orca/Fidalgo                                 1,2

         Central Basin                                3,4

         Knight Island                               2,3,4

         outside                                      2,3



       The tows (60) will duplicate the 1989 stations to maximize the
       number of successful tows and provide coverage throughout the
       Sound. The stations to be sampled in 1990 include both oiled and
       unoiled 1989 areas and extend from the central basin south to
       Montague Strait.    Species that will be sampled will include
       halibut, walleye pollock, flathead sole, and Pacific cod.        The
       maximum number of live individuals that can be handled will be
       processed prior to retrieving the next tow. Halibut and pacific
       cod are not anticipated to be as common in the hauls as walleye
       pollock and flathead sole.     If time permits at the end of the
       cruise, stations where halibut and walleye pollock were few in
       number will be resampled to increase the number of hydrocarbon
       samples for these species.

       Exposure of fish to oil will be determined by measuring
       concentrations of metabolites of aromatic petroleum compounds in
       bile. Analytical procedures used for the bile metabolite assays
       will   likely   include   excitations/emission    measurements    at
       wavelengths where naphthalene and phenanthrene fluoresce.         if
       exposure is documented through bile analysis, analysis of tissue
       and stomach content samples will occur.     Estimated exposures to
       petroleum hydrocarbons will be available to other investigations
       (particularly Study Number 24) to assess environmental damage using

                                       168










              statistical and simulation models. These other studies will meld
              bile and tissue chemistry to establish relationships between
              biological damage and estimated exposures to hydrocarbons

              BUDGET:   NOAA

              salaries             $    65.4
              Travel                    10.7
              Ship Coast               100.0
              Supplies                  10.0
              Equipment                  0.0

              Total                $ 186.1












































                                               169









         FISH/SHELLFISH STUDY NUMBER 22

         Study Title: Injury to Crabs outside PWS

         Lead Agency: NOAA

                                    INTRODUCTION



         Dungeness crabs in Alaska occupy nearshore areas in protected bays
         and estuaries. These habitats are usually characterized by fine
         benthic sediments and minimal wave action.         If oil becomes
         incorporated in shallow subtidal sediments it persists and can
         affect crab populations for several years after an oil spill (Krebs
         and Burns 1977, Boehm et al. 1987).        Dungeness crab may be
         especially susceptible to contamination by petroleum hydrocarbons
         because they often burrow into benthic sediments; ovigerous female
         Dungeness crab, in particular, are known to spend long periods (up
         to 10 months) burrowed into sediments while brooding their eggs.

         Several studies have documented deleterious effects on crabs
         exposed to petroleum hydrocarbons.    Sublethal concentrations can
         result in early post-molt autotomy of limbs, behavioral disorders
         and reduced reproductive capacity (Karinen and Rice 1974, Krebs and
         Burns 1977, Karinen et al. 1985 and Malan 1988).           Sex and
         reproductive state may determine responses of crabs to oil
         pollution.    Krebs and Burns (1977) noted a greatly reduced
         proportion of females in populations of the fiddler crab, Uca
         pugnax, at oil contaminated sites in Buzzards Bay, Massachusetts.
         Reproductively active ghost crabs, Ocypode quadrata, are more
         sensitive to the water soluble fraction of crude oil than are crabs
         not in reproductive condition (Jackson et al. 1981).

         This project is a continuation of work begun in 1989 and will
         provide quantitative data regarding adverse impacts on populations
         of Dungeness crab outside PWS as a result of the EVOS. The project
         will provide information on hydrocarbon levels in benthic sediments
         occupied by crabs as well as hydrocarbon levels in the tissues of
         crabs in contaminated and uncontaminated areas near Kodiak Island
         and the eastern Alaska Peninsula. It will also provide biological
         data on fecundity, reproductive capacity, and distribution and
         relative abundance of the crabs.      These data will permit the
         assessment of short-term losses caused by contamination of
         harvestable crabs and long-term impacts owing to adverse effects on
         crab reproduction. The data will also contribute to the long-term
         data base for management of fisheries and assessment of future oil
         spills.

         Products will include estimates of the amount of petroleum
         hydrocarbons taken up by the tissues of crabs inhabiting areas with
         contaminated sediments, estimates of the impact of hydrocarbons
         taken up by crab reproductive tissues on crab fecundity and

                                         170








            reproductive capacity and identification of possible contamination
            pathways from sediments to crab reproductive tissues and developing
            eggs.


                                         OBJECTIVES



            A.   Determine the levels of hydrocarbons, if present, in Dungeness
                 crabs in oiled and unoiled sites along the eastern Alaska
                 Peninsula and/or near Kodiak Island.

            B.   Assess reproductive condition of crabs in oiled and unoiled
                 areas by measuring such variables as percentage of ovigerous
                 crabs, fecundity and egg loss, condition and development.

            C.   Determine the incidence of limb loss and of abnormalities in
                 newly formed crab exoskeletons in oiled and unoiled areas.

            D.   Compare the strength of larval settlement in oiled and unoiled
                 areas using artificial substrates.

            E.   Identify potential methods and strategies for restoration of
                 lost use, populations, or habitat where injury is identified.



                                           METHODS



            The study will be conducted at eight sites with populations of
            Dungeness crab near Kodiak Island and the eastern Alaska Peninsula.
            Five sites will be in oiled areas and three will be reference sites
            in unoiled areas. Final site selection will, of necessity, be made
            in the field and will depend on the presence of Dungeness crab
            populations as determined by diver observations.           A list of
            candidate sites will be compiled prior to departure.         Dungeness
            crab will be sampled by diving. sex, carapace width, presence or
            absence of an egg clutch and external physical condition will be
            recorded for each crab.

            A total of 30 live female crab' will be sampled from each site
            during each sampling period. Divers will swim three transects to
            collect female crabs. Three randomly-selected subsamples of ten
            female crab each will be taken from the divers' total catch. The
            specimen number, carapace width, fresh weight, clutch size and a


                 1    Determination of sample sizes for all variables covered
            by this project depends on estimates of sample size for
            comparable variables by Margaret C. Murphy in the Project
            operational Plan on "The effects of hydrocarbons on reproduction
            inlDungeness crab."


                                             171









         description of the physical condition of each female crab in the
         samples will be recorded. The left fifth pleopod will be removed
         from each crab and fixed in 5% formalin for subsequent processing
         to estimate egg development, egg mortality and egg fouling. Three
         crab will be randomly selected from the ten crab in each sample,
         measured and sacrificed for ovaries and hepatopancreas.         Three
         ovaries will equal one composite hydrocarbon sample.            Three
         hepatopancreas will equal one composite hydrocarbon sample. One
         composite hydrocarbon sample of eggs will be taken by clipping a
         small portion of the egg clutch (4 g = 1/3 of pleopod) from the
         right fifth pleopod of each of the three crab. The remaining seven
         crab from the subsample of ten will be returned to the sea.

         Artificial substrates will be used to assess the intensity of the
         settlement of larval Dungeness crab at oiled and unoiled sites.
         Ten artificial substrates will be installed at about 0.5 m above
         the bottom at each site.    At those sites with eelgrass beds the
         collectors will be placed about 3 m apart along an isobath just
         below the lower limit of the eelgrass; at those sites lacking
         eelgrass beds the substrates will be placed 3 m apart along the 6
         m isobath. The substrates will be put in place in mid-May and will
         be sampled for the megalopae of Dungeness crab in July and August.
         The substrates will be retrieved in August at the time of the
         second sampling.

         Sediment samples will be collected at all sites.         Divers will
         collect three composite sediment samples along a 30 m transect laid
         parallel to shore in the area where divers collected the crabs.
         Each composite sample will consist of eight subsamples collected
         randomly along the 30 m transect. All sediment samples collected
         by divers will be taken from the top 2 cm of the sediment column.

         Physical oceanographic data will be collected at each site during
         each sampling period using an instrument that measures CTD. CTD
         will be recorded every 2 seconds as it is lowered to the bottom and
         raised to the surface.      The CTD measuring instrument will be
         deployed once at each site during each sampling period.

         Definitive analysis of the chemical composition of petroleum
         hydrocarbons in the sediments, tissues and eggs will be
         accomplished in the laboratory with gas chromatography/mass
         spectrometry as directed by the Analytical Chemistry Control Group.
         The types of analyses to be performed on the samples will be
         determined by the Analytical Chemistry Group and will include 1)
         characterization of oil in marine sediments and cra    *b tissues, 2)
         total organic carbon on selected samples, and 3) size fraction
         analysis on representative sediment samples. Prescreening analyses
         of   collected    samples   will    occur   prior    to   full     gas
         chromatography/mass spectrometry analysis in areas of low
         likelihood of oiling. Details of the methods used    in the chemical
         analyses are recorded under the Quality Assurance    Program.


                                          172









           The number of specimens required for one hydrocarbon analysis
           depends on the amount of tissue available in a crab and the need
           for a composite sample. Three Dungeness crab are enough to provide
           15 g of ovarian tissue. One pleopod from an average clutch would
           provide 15 g of crab eggs, but a sample representative of more than
           one crab is desirable. Therefore egg clips from the clutches of
           three crab will be combined to form a composite sample for
           hydrocarbon analysis. Three hydrocarbon samples from each site are
           the minimum needed to detect contamination between oiled and non-
           oiled sites.   A sample size of 30 crab was estimated to be an
           adequate number to determine differences in reproductive output
           between impact levels.

           All data will be tested for heteroscedasticity with Bartlett's test
          ,or equivalent. Data will be reported as means and 95% confidence
           intervals calculated according to a standard formula (Sokal and
           Rohlf 1981).   Parametric statistics (analysis of variance and
           Scheffe's a posteriori test) will be used to test for differences
           in means between oiled and non-oiled sites if underlying
           assumptions of the parametric procedures are met, otherwise
           nonparametric tests (eg. the Kruskal-Wallis test) will be employed.
           Variables to be tested will include hydrocarbon concentrations in
           Dungeness crab tissues, the reproductive parameters of Dungeness
           crabs, crab larval production and viability and hydrocarbon content
           of sediments in crab habitat.

           Further multivariate statistics (eg. analysis of covariance, rank
           correlation coefficients, discriminate analysis) will be computed
           if the above summary statistics indicate relationships may exist
           between Dungeness crab hydrocarbon content, reproductive capacity,
           sediment hydrocarbon content, and physical oceanographic factors.


                                      BIBLIOGRAPHY



           Boehm, P. D., M. S. Steinhauer, D. R. Green, B. Fowler, B.
                Humphrey, D. L. Fiest, and W. J. Cretney. 1987. Comparative
                fate of chemically dispersed and beached crude oil in in
                subtidal sediments of the arctic nearshore. Arctic 40, supp.
                1: 133-148.


           Jackson, L., T. Bidleman" and W. Vernberg. 1981. Influence of
                reproductive activity on toxicity of petroleum hydrocarbons to
                ghost crabs. Mar. Pollut. Bull. 12: 63-65.

           Karinen, J. F. and S. D. Rice. 1974. Effects of Prudhoe Bay crude
                oil on molting tanner crabs, Chionoecetes bairdi. Mar. Fish.
                Rev. 36: 31-37.






                                           173









       Karinen, J. F., S. D. Rice, and M. M. Babcock. 1985. Reproductive
            success in Dungeness (Cancer magister) during long-term
            exposures to o i 1 -contaminated sediments. Final Report-OCSEAP
            P-Unit 3008, Anchorage, Alaska, 28 pp.

       Krebs, C. T. and K. A. Burns. 1977. Long-term effects of an oil
            spill on populations of the salt-marsh crab Uca pugnax.
            Science 197: 484-487.

       Malan, D. E. 1988. The effects of Qatar light crude oil on the
            saltmarsh crab Sesarma catenata and its implications in the
            field: toxicity to adults and larvae. S. Afr. J. mar. Sci. 7:
            37-44.

       Neter, J., W. Wasserman, and M. Kutner. 1985. Applied Linear
            Statistical Models. Richard D. Irwin, Homewood, Illinois.

       O'Clair, C. E. and J. L. Freese. 1988. Reproductive condition of
            Dungeness crabs, Cancer magister, at or near log transfer
            facilities in southeastern Alaska. Mar. Env. Res. 26: 57-81.

       Sokal, R. R. and F. J. Rohlf 1981. Biometry. W. H. Freeman and
            Company, San Francisco. 859pp.


       BUDGET: NOAA


       salaries             $ 28.0
       Travel                     8.0
       Ship Costs               50.0
       Contracts                12.0
       supplies                   6.0
       Equipment                  6.0

       Total                   110.0

























                                        174









             FISH/SHELLFISH STUDY NUMBER 24

             Study Title:    Assessment of Oil Spill Impacts on Fishery
                             Resources: Measurement of hydrocarbons and their
                             metabolites, and their effects, in important species.

             Lead Agency: NOAA

                                         INTRODUCTION



             Preliminary analyses of data collected in 1989 indicates that
             several nearshore fish species were exposed to petroleum or
             petroleum derivatives in and around PWS, subsequent to the EVOS
             (Varanasi et al., 1990). Because petroleum and its components can
             cause severe damage to fishery resources, this study provides for
             the continued monitoring of the nearshore fisheries resources of
             PWS and adjacent areas. Such monitoring will include measurement
             of petroleum exposure and short-term effects, as was done in the
             summer and fall of 1989, but will also encompass an assessment of
             long-term    biological    effects,    including    measurements     of
             reproductive dysfunction and histopathological lesions of liver,
             gill, kidney, and gonad.

             Certain petroleum components C e.g. aromatic hydrocarbons (Ahs) ) can
             cause reproductive toxicity and teratogenicity in rodents (Shum et
             al., 1979; Gulyas and Mattison 1979, Mattison and Nightingale,
             1980).    Similarly, reproductive impairment has been noted in
             benthic fish residing in contaminated areas of San Francisco Bay
             (Spies and Rice, 1988) and southern    *California (Cross and Hose,
             1988). Moreover, English sole from areas of Puget Sound having
             high sediment concentrations of Ahs showed inhibited ovarian
             maturation (Johnson et al., 1988), and fish from these areas that
             did mature often failed to spawn after hormonal treatment to induce
             spawning (Casillas et al., 1990).

             In general, reproductive impairment (including reduced plasma
             levels of the sex steroid hormone, estradiol) was found in English
             sole which showed evidence of exposure to aromatic compounds.
             Moreover, our laboratory studies have shown that plasma levels of
             estradiol are reduced in gravid female English sole exposed to
             chemical contaminants extracted from urban sediments (Stein et al.,
             1990), and, more importantly, our preliminary studies have also
             shown that exposure to Prudhoe Bay crude oil reduced plasma levels
             of estradiol in gravid female rock sole.

             In  view of our findings last year that several nearshore fish
             species in and around PWS have been exposed to crude oil
             components, including Ahs, the assessment of possible reproductive
             dysfunction in animals from impacted areas will be very important
             in determining biological damage to living marine resources as a
             result of the oil spill.      ovaries of selected species will be

                                              175









       histologically examined to determine if ovarian maturation is being
       affected in animals from oil-impacted areas, and to determine
       fecundity and levels of plasma estradiol in these same animals.
       Combined with measurements of metabolites in bile and enzyme
       activities in liver, these studies will enable us to estimate the
       degree of reproductive dysfunction which may be occurring in oil-
       exposed fish.

       Exposure of animals to crude oil can also result in changes at the
       tissue and cellular levels (National Academy of Sciences, 1985).
       Examples of such changes after exposure of fish to o i 1 -contaminated
       sediments include liver hypertrophy and fatty liver in winter
       flounder (Payne et al., 1988) and the occurrence of hepatocellular
       lipid vacuolization in English sole (McCain et al., 1978) . Certain
       Ahs (e.g., benzo(a]pyrene) are known carcinogens in rodents (Lutz,
       1979), and studies with several bottomfish species show that, of
       the xenobiotic chemicals in sediments, Ahs are most strongly
       associated with high prevalences of liver lesions, including
       neoplasms (Malins et al., 1984; Myers et al., 1987; Black et al.,
       1983; Varanasi et al., 1987) . Generally, histopathological lesions
       of the types noted above do not become manifest until at least
       several months after exposure.     However, by the summer of 1990,
       fish in and around oil impacted sites will have potentially been
       exposed to petroleum components for        more than one year, and
       juveniles of some species of salmon will have potentially been
       exposed during most of their developmental period.        Accordingly,
       assessment of histopathological effects in selected species is
       strongly warranted, and is included in this proposal.

       This study will continue to measure exposure to oil and oil
       components in the biota of PWS and other areas affected by the oil
       spill, by determining levels of hydrocarbon metabolites in bile and
       by measuring hepatic enzyme activities.       A range of biological
       effects, especially indicators of reproductive dysfunction and
       histopathological effects will be measured. By employing such a
       broad spectrum of state-of-the art chemical, biochemical and
       biological methods, analytical data will be obtained to document
       the degree of exposure and resultant biological effects of
       petroleum hydrocarbons on economically and ecologically important
       fish species.       This information for important Alaskan fish
       species, will be incorporated into models for use in estimating oil
       spill impacts on fishery resources.

                                     OBJECTIVES

       A. Sample selected nearshore fish species from 14 sites inside
       and outside PWS, with emphasis on sites outside PWS.               Site
       selection is primarily based on data from last year's sampling and
       analyses. Representative sediment samples will also be taken from
       each sampling site for subsequent chemical analysis.

       B. Estimate the exposure to petroleum hydrocarbons by measuring

                                        176









              levels of hydrocarbon metabolites in bile of the above species from
              oiled and nonoiled habitats to detect significant differences in
              bile concentrations with a = 0. 05 and b = 0. 10.         Additionally,
              stomach contents of fish showing high levels of hydrocarbon
              metabolites in bile will be analyzed for hydrocarbons, such to
              detect signif icant dif f erences in concentrations with a = 0. 05 and
              b = 0.10

              C. Estimate the induction of hepatic aryl hydrocarbon hydroxylase
              (AHs) activity or increased levels of cytochrome P-45OIAl in the
              above species from oiled and nonoiled habitats such to detect
              statistical differences in levels of effects with a = 0.05 and b
              0.10.

              D. Estimate the prevalence of pathological conditions in the above
              species from oiled and nonoiled habitats such to detect statistical
              differences in levels of effects with a = 0.05 and b = 0.10.

              E. Estimate the levels of plasma estradiol, the degree of ovarian
              maturation, and fecundity in adult females of two of the above
              species (Dolly Varden char and yellowfin sole) from oiled and
              nonoiled habitats such to detect statistically significant
              differences with a = 0.05 and b = 0.10.

              F.   Estimate temporal changes in the parameters described in
              Objectives B&C, by comparing data obtained in 1990 to data obtained
              in 1989. In order to assess either recovery or increased damage of
              habitats from the oil spill, trends in these parameters must be
              statistically significant at a = 0.05 and b = 0.10.

              G. Construct simulation models similar to those of Schaaf et. al.
              (1987) for important Alaskan fish species for use in estimating oil
              spill impacts on fishery resources. These models will incorporate
              pre-spill information from the fisheries literature on mortality
              and fecundity together with information on reproductive impairment,
              pathological conditions, and biochemical effects in fish exposed to
              petroleum hydrocarbons as a result of the spill.

                                              METHODS


              General Strategy and Approach

              Samples of biota will be collected from approximately 14 sites
              during 1990, from mid-May to mid-June. Sites will generally be the
              same sites occupied last year, and will be located in potentially
              oil-impacted and unimpacted areas in PWS, in CI, and in embayments
              on the KP, Alaska Peninsula, and Kodiak Island. As feasible, the
              sample locations will be coordinated with A/W Study 2.             Dolly
              Varden char and juvenile salmon will be sampled in intertidal
              areas, whereas f latf ish (e. g. Pacif ic halibut, yellowf in sole, rock
              sole and flathead sole) will be sampled in subtidal areas. Salmon
              and halibut were selected primarily because of their economic

                                                177









          importance, and f lathead sole, yellowf in sole, rock sole, and Dolly
          Varden     were selected because of their wide geographical
          distribution and year-round residency in the sampling areas.
          surf icial sediment samples for establishing levels of petroleum
          hydrocarbon residues will be collected at all sites, with analyses
          projected to be done under A/W Study 2.

          Petroleum exposure of fish will primarily be assessed by measuring
          (a) concentrations of metabolites of aromatic petroleum compounds
          in bile, and (b) AHs activities in liver.               These types of
          measurements are necessary because petroleum hydrocarbons in fish
          are rapidly metabolized to compounds that are not detectable by
          routine chemical analyses. AHs activity in fish is due primarily
          to a single cytochrome P-450, apparently cytochrome P-4501Al
          (Varanasi et al., 1986, Buhler and Williams 1989). Measurement of
          hepatic AHs activity will provide a very sensitive indicator of
          contaminant exposure of sampled animals (Collier and Varanasi,
          1987). Moreover, the induction of AHs activity indicates not only
          that contaminant exposure has occurred, but also that biological
          changes have occurred as a result of the exposure. In addition to
          measuring AHs activity, cytochrome P-4501Al will be directly
          quantitated    in   selected    liver   or   tissue    samples    by    an
          immunochemical method recently developed at the University of
          Bergen (Collier et al., 1989; Goksoyr, 1990).

          Other biological effects in fish will be estimated by examining
          selected species for pathological conditions and by assessing
          reproductive    impairment     in   suitably    mature    female     fish.
          Pathological conditions will include grossly visible abnormalities
          (e.g., fin erosion) and other lesions diagnosed by histological
          procedures    (e.g-,    gill    necrosis,    liver    cell     necrosis).
          Reproductive capacity will be estimated by examining the
          developmental stages of ovaries and by measuring plasma levels of
          certain reproductive hormones (Johnson et al., 1988), in addition
          to measuring fecundity (Cross and Hose, 1988).          The two primary
          species for assessing reproductive impairment are Dolly Varden char
          and yellowfin sole. It is anticipated that, during the proposed
          sampling period (May/June), these two species will be at an
          appropriate stage in their reproductive cycle for such assessments
          to be done.    Concurrent laboratory studies will be conducted to
          determine the effects of known doses of oil and oil components on
          reproductive processes in these or related species.

          Samples of sediment, and selected stomach contents of fish (from
          fish whose bile had evidence of oil exposure) will be analyzed
          (sediment under A/W Study 2) for hydrocarbons by recently
          developed, scientifically sound and cost-effective analytical
          procedures involving high-performance liquid chromatography, gas
          chromatography and mass spectroscopy (Krahn et al., 1988).

          Environmental injury will be determined using statistical and
          simulation models, which will be developed as part of these

                                             178









           proposed studies, as well as from other investigations with related
           fish species. The bile and tissue chemistry data will be used to
           establish relationships between biological damage and estimated
           exposures to petroleum hydrocarbons.

           Sampling Methods

           sampling activities will be conducted at several sites in PWS and
           the GOA. Sample collection will be performed from a NOAA vessel
           (and its launches) at water depths of approximately 0 to 320
           meters. At each site, sediment samples will be collected with a
           box corer, VanVeen or Smith-McIntyre grab.       Sediments will be
           stored at - 200 C. The coordinates and depths of each station will
           be recorded.

           Fish will be collected with a bottom trawl, long-line gear, gill
           nets! or beach seines.    Bottom trawls will be performed with an
           otter trawl.   Tows will be of 5 to 15 minutes duration. In order
           to reduce contamination of the catch by free oil, trawling will
           avoid areas of surface films or slicks. Individuals of selected
           target fish species will be sorted and examined for externally
           visible lesions; up to 30 fish of selected species will be
           measured, weighed, and necropsied; and tissue samples will be
           excised and preserved in fixative for histopathological examination
           or frozen for chemical analyses.

           Laboratory Analyses

           1. Bile Metabolite Assay (analyses done under Technical Services-
           il

           Samples of bile will be injected directly into a liquid
           chromatograph and a gradient elution conducted (Krahn et al., 1984,
           1986a, b, c) . Two fluorescence detectors are used in series. The
           excitation/ emission wavelengths of one detector are set to 290/335
           nm,   where   metabolites    of   naphthalene    (NPH)    fluoresce.
           Excitation/emission wavelengths of the other detector are set to
           260/380 nm, where metabolites of phenanthrene (PHN) fluoresce. The
           total integrated area for each detector is then converted
           (normalized) to units of either NPH or PHN that would be necessary
           to give that into-grated area.

           2. Liver Aryl Hydrocarbon Hydroxylase (AHs) Activity and Cytochrome
           P-4501AI Analysis

           Hepatic microsomes will be prepared essentially as described by
           Collier et al. (1986) and microsomal protein will be measured by
           the method of Lowry et al. (1951), using bovine serum albumin as
           the standard. AHs activity will be assayed by a modification of
           the method of Van Cantfort et al. (1977) as described by Collier et
           al. (1986), using 14C-labeled benzo(a]pyrene as the primary
           substrate. All enzyme assays will be run under conditions in which

                                           179










         the reaction rates are in the linear range for both time and
         protein.    Cytochrome P-4501Al will be measured by an ELISA
         utilizing rabbit antibodies to cytochrome P-450c isolated from
         Atlantic cod (Goksoyr,1990).

         3. Histopathology

         Histopathological procedures to be followed are described in the
         report from the Histopathology Technical Group for Oil Spill
         Assessment Studies in PWS, Alaska. Briefly, the procedures will
         involve the following: (a) tissues preserved in the field will be
         routinely embedded in paraffin and sectioned at five microns
         (Preece, 1972) ; and (b) paraffin sections will be routinely stained
         with   Mayer's    hematoxylin    and   eosin,    and    for    further
         characterization of specific lesions, additional sections     will be
         stained using standard special staining methods (Thompson, 1966;
         Preece, 1972; and Armed forces Institute of Pathology, 1968). All
         slides will be examined microscopically without knowledge of where
         the fish were captured.       Hepatic lesions will be classified
         according to the previously described diagnostic criteria of Myers
         et al. (1987). Ovarian lesions will be classified as described in
         Johnson et al. (1988).

         4. Reproductive Indicators

         Reproductive activity will be assessed by examining the ovaries of
         the sampled fish histologically to determine their developmental
         stage, and for the presence of ovarian lesions that would be
         indicative of oocyte resorption (Johnson et al., 1988).          other
         parameters associated with reproductive activity will also be
         measured, including fecundity (Bagenal and Braum, 1971), plasma
         vitellogenin (Gamst and Try, 1980; DeVlaming et al., 1984) and
         estradiol (Sower and Schreck, 1982) levels, and gonadosomatic index
         (ovary wt/gutted body wt x 100) .     Relationships between ovarian
         maturation, fecundity, plasma estradiol, plasma vitellogenin, and
         petroleum hydrocarbon exposure will then be evaluated.

                                     DATA ANALYSIS

         Where possible, non-parametric statistical tests will be employed
         to avoid assumptions that the data are normally distributed. The
         principal non-parametric tests that will be used are Spearman rank
         correlation, which has about 0.91% of the power of product-moment
         correlation when the parametric assumptions are met (Zar, 1984),
         and the heterogeneity-G statistic. Spearman rank correlation will
         be used for estimating uptake and metabolism of petroleum
         hydrocarbons from oiled and non-oiled habitats when an independent
         measure of contamination (e.g. , levels of Ahs in sediment) is
         available.

         The heterogeneity-G statistic (Sokal and Rohlf, 1981) will be used
         to study prevalence of pathological conditions at oiled and non-

                                          180









           oiled habitats.    In addition, logistic regression (appropriate
           where the outcome variable is binomial) will be used to model the
           prevalences   of    pathological   conditions    in   relation     to
           contamination.

           The Kruskal-Wallis test (a non-parametric form of ANOVA) will be
           used for supporting statistical analyses of variation in sediment
           and fish hydrocarbon levels at sites sampled.          If the null
           hypothesis of no differences among sites is rejected at a = 0.05,
           a non-parametric multiple comparison test (Dunn, 1964; Hollander
           and Wolfe, 1973; Zar, 1984) will be used to determine differences
           between sites at a = 0. 05.     Principal components analysis and
           LOWESS (Chambers et al., 1983) will also be employed for this
           purpose; both are methods of exploratory data analysis rather than
           inferential statistical methods.    Cohen (1977) will be used for
           computations of statistical power.

           Products will include information on the distribution and
           concentrations of petroleum hydrocarbons and their metabolites in
           fish tissues and in sediments obtained from sites in Alaska; the
           hepatic activities of AHs and levels of cytochrome P-45OIAl in fish
           from sites in Alaska;     and the distribution and prevalence of
           histopathological disorders and reproductive impairment among
           selected species from those sites.        Chemistry data will be
           submitted in the f orm of data tables and distribution maps, and all
           data will be stored in computerized data management programs. Fish
           pathology data will be reported in the form of distribution maps,
           tables describing disease frequencies of each species examined,
           photographs of gross and microscopic properties of abnormalities,
           figures representing various types of biological data (e.g.,
           length-weight, age-weight) and discussions of the relative
           importance of the types of abnormalities found. comparisons of the
           characteristics of these abnormalities will be made with similar
           conditions previously reported in other marine areas of the world.
           The data management formats were designed in cooperation with the
           National Oceanographic Data Center.

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                   waters. International Biology Programme Handbook 3. (W.E.
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           Black, J.J. 1983. Field and laboratory studies of environmental
                   carcinogenesis in Niagara River Fish. J. Great Lakes Res.
                   9:326-334.


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       Buhler,   D.R. and D.E. Williams. 1989. Enzymes involved in
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       Casillas, E., D. Misitano, L.J. Johnson, L.D. Rhodes, T.K. Collier,
                 J.E. Stein, B.B. McCain, and U. Varanasi.                  1990.
                 Inducibility of spawning and reproductive success of female
                 English sole (Parophrys vetulus) from urban and nonurban
                 areas. Submitted to Mar. Environ. Res.

       Chambers, J. M., W. S. Cleveland, B. Kleiner, and P. A. Tukey.
                 1983. Graphical methods for data analysis. Belmont, CA:
                 Wadsworth International Group. 395 p.

       Cohen, Jacob. 1977. Statistical power analysis for the
                 behaviorial sciences. New York: Academic Press. 474 pp.

       Collier, T.K., J.E. Stein, R.J. Wallace, and U. Varanasi. 1986.
                 Xenobiotic metabolizing enzymes in spawning English sole
                 (ParoRhrys vetulus) exposed to organic-solvent extracts of
                 sediments from contaminated and reference areas.           Comp.
                 Biochem. and Physiol. 84C:291-298.

       Collier, T.K. and U. Varanasi. 1987. Biochemical indicators of
                 contaminant exposure in flatfish from Puget Sound, Wa. pp
                 1544-1549. In: Proceedings Oceans 187 IEEE, Washington,
                 D.C.


       Collier, T.K., B.-T. L. Eberhart, and A. Goksoyr. 1989.
                 Immunochemical quantitation of cytochrome P450 IA1 in
                 benthic fish from coastal U.S. waters. Proc. Pac. NW Assoc.
                 Toxicol. 6:9. (Abstract).

       Cross, J.N. and J. Hose. 1988. Evidence for impaired reproduction
                 in white croaker (Genyonemus lineatus) from contaminated
                 areas off Southern California. Mar. Environ. Res.
                 24:185-188.


       DeVlaming, V., R. Fitzgerald, G. Delahunty, J. J. Cech, Jr., K.
                 Selman, and M. Barkley. 1984. Dynamics of oocyte
                 development and related changes in serum estradiol 17-8,
                 yolk precursor, and lipid levels in the teleostean fish,
                 Leptocottus armatus. Comp. Biochem. Physiol. 77A:599-
                 610.

       Dunn, 0. J. 1964. Multiple contrasts using rank sums.
                 Technometrics 6: 241-252.

       Gamst, 0. and K. Try. 1980. Determination of serum-phosphate

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                    without deproteinization by ultraviolet spectrophotometry
                    of the phosphomolybdic acid complex. Scand. J. Clin. Lab
                    Invest. 40:483-486.

           Goksoyr, A. 1990. An ELISA for monitoring induction of cytochrome
                    P-4501Al in fish liver samples. manuscript in
                    preparation.

           Gulyas, B.J. and D.R. Mattison. 1979. Degeneration of mouse
                    oocytes in response to polycyclic aromatic hydrocarbons.
                    Anat. Rec. 193:863-869.

           Hollander, M. and D. A. Wolfe. 1973. Nonparametric statistical
                    methods. New York: John Wiley. 503 p.

           Johnson, L.J., E. Casillas, T.K. Collier, B.B. McCain, and U.
                    Varanasi. 1988. Contaminant effects on ovarian
                    development in English sole (Parophrys vetulus) from
                    Puget Sound, Washington. Can. J. Fish. Aquat Sci.
                    45:2133-2146.


           Krahn, M.M., M.S. Myers, D.G. Burrows, and D.C. Malins. 1984.
                    Determination of metabolites of xenobiotics in bile of
                    fish from polluted waterways. Xenobiotica. 14:633-646.

           Krahn, M.M., L.J. Kittle, Jr., and W.D. MacLeod, Jr. 1986a.
                    Evidence for oil spilled into the Columbia River. Mar.
                    Environ. Res. 20:291-298.


           Krahn, M.M., L.D. Rhodes, M.S. Myers, L.K. Moore, W.D. MacLeod,
                    Jr., and D.C. Malins. 1986b. Associations between
                    metabolites of aromatic compounds in bile and occurrence
                    of hepatic lesions in English sole (Parophrys vetulus)
                    from Puget Sound, Washington. Arch. Environ. Contam.
                    Toxicol. 15:61-67.

           Krahn, M.M., L.K. Moore, and W.D. MacLeod, Jr. 1986c. Standard
                    Analytical Procedures of the NOAA National Analytical
                    Facility, 1986: Metabolites of Aromatic Compounds in Fish
                    Bile. Technical Memorandum NMFS/F/NWC-102, 25 pp.
                    (Available from the National Technical Information
                    Service of the U.S. Department of Commerce, 5285 Port
                    Royal Road, Springfield, VA 22161).

           Krahn, M.M., C.A. Wigren, R.W. Pierce, L.K. Moore, R.G. Bogar,
                    W.D. MacLeod, Jr., S.-L. Chan, and D.W. Brown. 1988.
                    Standard Analytical Procedures of the NOAA National
                    Analytical Facility, 1988: New HPLC Cleanup and Revised
                    Extraction Procedures for Organic Contaminants.
                    Technical Memorandum NMFS/F/NWC-153, 52 pp. (Available
                    from the National Technical Information Service of the
                    U.S. Department of Commerce, 5285 Port Royal Road,

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                Springfield, VA 22161).

       Lowry, O.H., N.J. Rosebrough, A.L. Farr, and R.J. Randall. 1951.
                Protein measurement with the Folin phenol reagent, J.
                Biol. Chem. 193:265-275.

       Lutz, W.K. 1979. In vivo covalent binding of organic chemicals
                to DNA as a quantitative indicator in the process of
                chemical carcinogenesis. Mutat. Res. 65:289-356.

       MacLeod, W.D., Jr., D.W. Brown, A.J. Friedman, D.G. Burrows, 0.
                Maynes, R.W. Pearce, C.A. Wigren, and R.G. Bogar. 1985.
                Standard Analytical Procedures of the NOAA National
                Analytical Facility, 1985-1986: Extractable Toxic
                Organic Compounds, 2nd Ed. NOAA Technical Memorandum
                NMFS F/NWC-92, 121 pp. (Available from the National
                Technical Information Service of the U.S. Department of
                Commerce, 5285 Port Royal Rd., Springfield, VA 22161;
                PB86-147873).

       Malins,  D.C., B.B. McCain, D.W. Brown, S-L. Chan, M.S. Myers,
                J.T. Landahl, P.G. Prohaska, A.J. Friedman, L.D. Rhodes,
                D.G. Burrows, W.D. Gronlund, and H.O. Hodgins. 1984.
                Chemical pollutants in sediments and diseases in bottom-
                dwelling fish in Puget Sound, Washington. Environ. Sci.
                Technol. 18:705-713.

       Mattison, D.R. and M.S. Nightingale. 1980. The biochemical and
                genetic characteristics of murine ovarian aryl
                hydrocarbon (benzo(a]pyrene) hydroxylase activity and its
                relationship to primordal oocyte destruction by
                polycyclic aromatic hydrocarbons..Toxicol. appl.
                Pharmacol. 56:399-408.

       McCain,  B.B., H.O. Hodgins, W.D. Gronlund, J.W. Hawkes, D.W.
                Brown, M.S. Myers, and J.H. Vandermeulen. 1978.
                Bioavailability of crude oil from experimentally oiled
                sediments to English sole (Parophrys vetulus) and
                pathological consequences. J. Fish. Res. Board Can.
                35:657-664.


       Myers, M.S., L.D. Rhodes, and B.B. McCain. 1987. Pathologic
                anatomy and patterns of occurrence of hepatic neoplasms,
                putative preneoplastic lesions and other idiopathic
                hepatic conditions in English sole (Parophrys vetulus)
                from Puget Sound, Washington, U.S.A. J. Natl. Cancer
                Inst. 78:333-363.

       National Academy of Sciences. 1985. Oil in the Sea; Inputs,
                fates and effects. National Academic Press, Washington,
                D. C. 601pp.


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             Preece,  A. 1972. A Manual for Histologic Technicians. 3rd
                      edition. Little, Brown and Co., Boston, 428 pp.

             Schaaf,  W.E., D.S. Peters, D.S. Vaughan, L. Coston-Clements, and
                      C.W. Krouse. 1987. Fish population responses to chronic
                      and acute pollution: the influence of life history
                      strategies. Estuaries 10: 267-275.

             Shum, S., N.M. Jensen, and D.W. Nebert. 1979. The murine Ah locus:
                      in utero toxicity and teratogenesis associated with genetic
                      differences in benzo[a]pyrene metabolism. Teratology
                      20:365-376.


             Sokal, R. and F. Rohlf. 1981. Biometry. (Second Ed.)            W.H.
                      Freeman and Co.: San Francisco, CA, 859 pp.

             Sower, S. A. and C. B. Schreck. 1982. Steroid and thyroid
                      hormones during sexual maturation of coho salmon
                      (Oncorhynchus kisutch) in seawater or freshwater. Gen.
                      Comp Endocrine. 47:42-53.

             Spies, R.B. and D.W. Rice, Jr. 1988. Effects of organic
                      contaminants on reproduction of the starry flounder
                      Platichthys stellatus in San Francisco Bay. II.
                      Reproductive success of fish captured in San Francisco
                      Bay and spawned in the laboratory. Mar. Biol. 98:191-200.

             Stein, J.E., T. Hom, H.R. Sanborn, and U. Varanasi. 1990. Effects
                      of exposure to a contaminated-sediment extract on the
                      metabolism and disposition of 173-estradiol in English
                      sole (Parophrys vetulus). Manuscript in preparation.

             Van Cantfort, J., J De Graeve, and J.E. Gielen. 1977. Radioactive
                      assay for aryl hydrocarbon hydroxylase. Improved method
                      and biological importance. Biochem. Biophys. Res. Commun.
                      79:505-511.

             Varanasi, T.K. Collier, D.E. Williams, and D.R. Buhler. 1986.
                      Hepatic cytochrome P-450 isozymes and aryl hydrocarbon
                      hydroxylase in English sole (Parophrys vetulus).
                      Biochem. Pharmacol. 35:2967-2971.


             Varanasi, U., D.W. Brown, S-L. Chan, J.T. Landahl, B.B. McCain,
                      M.S. Myers, M.H. Schiewe, J.E. Stein, and D.D. Weber.
                      1987. Etiology of tumors in bottom-dwelling marine fish.
                      Final Report to the National Cancer Institute under
                      Interagency Agreement Y01 CP 40507.

             Varanasi, U., S-L. Chan, R.C. Clark, Jr., T.K. Collier, W.D.
                      Gronlund, M.M. Krahn, J.T. Landahl, and J.E. Stein. 1990.
                      Oil Spill Progress Report. Shellfish and Groundfish Trawl
                      Assessment Outside Prince William Sound. 30 p.

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        Zar, J.H. 1984. Biostatistical Analysis. Prentice-Hall:
                 Eaglewood Cliffs, NJ, 620 pp.


        BUDGET: NOAA

        Salaries                                 $230.0
        Supplies                                   35.4
        Travel                                     19.7
        Equipment (disposable)                     14.9
        Vessel support                            150.0

                                                 $450.0











































                                          186









            FISH/SHELLFISH STUDY NUMBER 27


            Study Title: Sockeye Salmon Overescapement

            Lead Agency: ADF&G

                                        INTRODUCTION



            commercial fishing for sockeye salmon in 1989, was curtailed in
            upper CI, the outer Chignik districts, and the Kodiak areas due to
            presence of oil in the fishing areas from the EVOS. As a result,
            the number of sockeye salmon entering four important sockeye
            producing systems (Kenai/Skilak, Chignik/Black, Red, and Frazer
            Lakes) and two less important lake systems (Akalura and Af ognak or
            Litnik lakes) greatly exceeded levels that are thought to be most
            productive. Sockeye salmon spawn in lake associated river systems.
            Adult salmon serve an extremely important role in the ecosystem,
            providing food for marine mammals, terrestrial mammals, and birds.
            Additionally, carcass decomposition serves to charge f resh water
            lake systems with important nutrients. Juvenile salmon which rear
            in lakes for one or two years serve as a food source for a variety
            of fish and mammals.       Sockeye salmon are also an important
            subsistence, sport, and commercial species. The ex-vessel value of
            the commercial catch of sockeye from these lake systems has
            averaged about $42 million per year since 1979, with the 1988 catch
            worth $115 million.     Sockeye salmon returns to the Kenai River
            system support some of the largest recreational fisheries in the
            State.


            Overly large spawning escapements may result in poor returns by
            producing more rearing juvenile sockeye than can be supported by
            the nursery lake's productivity (Kyle et al. 1988).       In general,
            when rearing f ish abundance greatly exceeds the lake I s carrying
            capacity, prey resources are altered by changes in species and size
            composition (Mills and Schiavone 1982, Koenings and Burkett 1987,
            Kyle et al. 1988) with concomitant effects on all trophic levels
            (Carpenter et al. 1985). Because of such changes, juvenile sockeye
            growth is reduced, mortality increases, larger percentages holdover
            for another year of rearing; and the poor quality of smolts
            increases marine mortality.     Where escapements are two to three
            times normal levels, the resulting high juvenile densities crop the
            prey resources to the extent that more than one year is required to
            return to normal productivity. Rearing juveniles from subsequent
            brood-years suffer from both the poor quality of forage and from
            the increased competition for food by holdover juveniles (Townsend
            1989).   This is the brood-year interaction underlying cyclic
            variation in the year class strength of anadromous fish.

            This project will examine the effects of large 1989 spawning
            escapements on the resulting progeny for a select subset of the

                                             187










         above mentioned sockeye nursery lakes. Three impacted lake systems
         where the 1989 escapements were more than twice the desired levels
         (Kenai/Skilak in upper CI; Red and Akalura lakes on Kodiak Island)
         were selected. Upper Station Lake which is near the two impacted
         lakes on Kodiak did not receive a large escapement and will be
         examined as a control.

         This study is necessary to obtain a more timely assessment of
         impact as adult sockeye, produced from the 1989 escapement, will
         not return until the 1994/1995 season. Further, total return data
         are not available for individual Kodiak sockeye systems due to the
         complex mixed-stock nature of the commercial fisheries and the
         inability to estimate stock-specific catches.


                                     OBJECTIVES

         A.       Estimate the number, age, and size of sockeye
                  salmon juveniles rearing in selected freshwater
                  systems.

         B.       Estimate the number, age, and size of sockeye
                  salmon smolts migrating from selected freshwater
                  systems.

         C.       Determine ef f ects of large escapements resulting
                  from fishery closures caused by the EVOS on the
                  rearing capacity of selected nursery lakes
                  through:

                  a.   analysis of age and growth of juveniles and smolts
                  b.   examination of nursery area nutrient budgets and
                       plankton populations.



                                       METHODS


         Numbers of adult sockeye salmon that entered selected spawning
         systems outside PWS prior to and during 1989 have been estimated at
         weir stations or by sonar. This information was collected during
         projects routinely conducted by the ADF&G as part of their resource
         management program.     Optimal escapement levels, which on the
         average should produce maximum sustained yield, have been based on
         either past relationships between spawners and returning progeny or
         the extent of available spawning and rearing habitat. The baseline
         program will continue at each site including but not limited to
         estimates of adult sockeye escapement and collection of scales for
         age analysis.

         For each of the 4 lake systems identified, the response (abundance,
         growth, and freshwater age) of rearing juveniles from the 1989
         escapement will be studied through its likely period of freshwater

                                         188









             residence, early summer 1990 to spring 1992.

             The total number of juvenile sockeye in each lake will be estimated
             through hydroacoustic surveys conducted during the summer (late
             June) and fall (September-October) of 1990, 1991, and 1992.         Age
             and size information as well as diet items will be obtained from
             samples of juvenile sockeye collected from concurrent mid-water
             trawl netting surveys. Survey transect designs for hydroacoustic
             sampling and tow-netting have been established for Kenai and Skilak
             lakes (Tarbox and King 1989), and will be developed for each
             additional lake in the study. The basic survey design will be a
             stratified random sample where each lake is subdivided into areas
             and survey transects randomly selected in each area.               Such
             programs, funded through other studies, are already in place for
             Tustumena and Afognak lakes.     Depending on densities of rearing
             juvenile sockeye, estimates of fish densities will be made for each
             transect either by echo integration or by echo counting.          Total
             f ish population estimates will be computed, by summing transect
             populations, along with 95% confidence intervals (Kyle 1989).

             Freshwater growth and age of sockeyp salmon rearing juveniles from
             all study systems will be determined from scale and otolith
             measurements made either by direct visual analysis of scales or on
             an Optical Pattern Recognition system.      In cases where data are
             available (e.g., Kenai and Skilak Lakes) , growth of progeny from
             the 1989 spawning escapements will be compared with growth (size)
             of progeny produced from spawnings within these systems during
             prior years.

             The total number of smolt migrating from each system will be
             estimated with a mark-recapture study during 1990, 1991 and 1992
             using inclined plane traps after Kyle (1983), and Tarbox and King
             (1989). Smolt will be captured in traps, sampled for age and size
             information, marked with Bismark Brown Y (a biological dye), and
             transported upstream of -the traps and released for subsequent
             recapture (Rawson 1984).     Periodic retesting will determine the
             capture efficiency of the traps under changing river conditions
             during the spring. Total population estimates (with 95% confidence
             intervals) will be made using catch efficiencies, and weekly number
             weighted smolt size and age information will be calculated using a
             computer spreadsheet developed by Rawson (personnel communication,
             1985).   Size and ages of sockeye smolts from the 1989 spawning
             escapements will be compared with smolt information from spawnings
             within these systems during prior years. Finally, smolt programs
             consistent to those for the study lakes are planned, under separate
             funding, for Tustumena and Afognak Lakes.

             Limnological studies will monitor the response of the lakes to the
             high juvenile rearing densities and to estimate the carrying
             capacity parameters of euphotic volume, nutrient budgets (carcass
             enrichment) , and zooplankton biomass, body-sizes, and population
             shifts. Approximately six limnology surveys will be conducted at

                                              189









        two stations, during 1990, 1991, and 1992, to determine zooplankton
        species abundance and body-sizes, nutrient chemistry, and
        phytoplankton abundance for Kenai/Skilak, Red, Akalura, and Upper
        Station lakes. Carrying capacity parameters exist for Afognak and
        Tustumena lakes based on ongoing studies by FRED and Commercial
        Fish Divisions.

        In cases where seasonal data are available (e.g., Akalura, Kenai,
        and Skilak lakes), limnological parameters taken during residence
        of the juveniles from the 1989 spawning escapements will be
        compared to parameters within these systems during prior years. In
        addition, randomized intervention analysis (RIA) will be used to
        detect changes in the systems with large escapements relative to a
        control or reference system (Carpenter et al. 1989).

        In addition to RIA, the holistic approach proposed here involves
        several evaluation procedures to assess the effects of sockeye
        salmon overescapement.

        First, fresh-water production from the 1989 escapements will be
        assessed in Kenai/Skilak, Red, Akalura, and Upper Station lakes.
        This will be accomplished through analysis of growth, freshwater
        survival (in particular over winter survival), and freshwater age
        of sockeye smolt populations. Also planktonic food sources will be
        assessed through estimation of abundance of zooplankton prey
        biomass and numbers of species. Any anomalies will be determined
        by analysis of freshwater growth recorded on archived scales,
        historical freshwater age composition, and modelled freshwater
        survivals; and from results of previous studies as well  as the 1990
        smolt characteristics from each of the study systems.

        Second, future sockeye salmon production from the 1989   parent year
        and subsequent parent years will be estimated based on
        spawner/recruit    relationships     incorporating    a    brood-year
        interaction term.      Losses of adult sockeye production from
        subsequent parent years may result from negative effects of progeny
        of the 1989 escapement on the lake's carrying capacity and/or from
        continued high escapements due to the inability to harvest the runs
        because of oil in the fishing area.            The spawner/recruit
        relationships will be estimated from historical stock specific
        return data (where available) , and generalized spawner/recruit data
        scaled to the carrying capacity parameters (i.e., euphotic volume
        and zooplankton biomass) of the nursery lakes where stock specific
        return data are not available (Geiger and Koenings 1990).

        Third, experimental and empirical sockeye life history/production
        models (Koenings and Burkett 1987, Koenings et al 1989) will be
        used to compare salmon production by life-stage at escapement
        levels consistent with management goals to the 1989 escapements.




                                         190












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             Geiger, H. J., and J. P. Koenings. 1990. Escapement goals for
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             Koenings, J. P., J. E. Edmundson, G. B. Kyle, and J. M. Edmundson.
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             Koenings, J.  P., R. D. Burkett, M. Haddix, G. B. Kyle, and D. L.
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             Kyle,  G. B.  1983. Cresent Lake sockeye salmon smolt enumeration
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             Kyle,  G. B., J. P. Koenings, and B. M. Barrett. 1988. Density
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                                              191










        Mills, E. L., and A. Schiavone, Jr. 1982. Evaluation of fish
              communities through trophic assessment of zooplankton
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        Rawson, Kit. 1984. An estimate of the size of a migrating
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        Tarbox, K.E., and B.E. King. 1989. An estimate of
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              through the use of dual beam hydroacoustic techniques in
              1989. Alaska Department of Fish and Game, Commercial Fish
              Division Regional Information Report No. 2S90-1.

        Townsend, C.R. 1989. Population cycles in freshwater fish.
              Journal of Fish Biology 35(Supplement A):125-131.


        BUDGET: ADF&G


        Personnel Services              $168.2
        Travel                             4.9
        Contractual                      88.7
        Supplies                         52.7
        Equipment                        77.5

        Total                           $392.0

























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            FISH/SHELLFISH STUDY NUMBER 28


            Study Title: Salmon Oil Spill Injury Model and Run
                          Reconstruction

            Lead Agency: ADF&G

                                     INTRODUCTION



            There are at least two approaches to the determination of damage
            to PWS fishery resources. The first approach is the "bottom up"
            view. Here one begins with escapements in the oiled and unoiled
            areas and projects return based on the various life history
            parameters observed for the two adult return areas. The damages
            are the dif f erence in adult production between the oiled and
            unoiled areas. The second approach is the "top down" view. Here
            one begins with the returns (district catches plus escapements)
            and estimates the stock specific return and return per specimen
            for oiled and unoiled areas based on a reconstruction of the run.
            Damages are defined as lost production of adults and are
            estimated from the differences in return per spawner applied to
            the parent escapements.

            This study will estimate damages to PWS fishery resources based
            on both the "bottom up" (i.e. life history modeling) and "top
            down" (i.e. run reconstruction) views.

            In the life history modeling approach, it is necessary to add
            together the factors at various life history stages over several
            individual river systems and salmon species.          Examples of
            potential factors include: reduced growth of fry, increased
            mortality of eggs and fry, loss of spawning habitat, increased
            early marine mortality, and overescapement. The magnitude of the
            overall loss in productivity for an individual salmon stock can
            best be understood by considering the survival at each life
            history stage (egg, fry, smolt, subadult, adult) over the life
            span of all fish of the same age class, and over all age classes
            present in the population.     Note that survival of future age
            classes must be considered if the detrimental effects of oiling
            are persistent.    A bookkeeping program is necessary to take
            advantage of the data already being collected, and to integrate
            existing historical data into documentation of the actual and
            potential damages due to oiling.

            In the "top down" view, it is necessary to estimate the stock
            specific return (i.e. catch plus escapement) so that return per
            spawner in oiled and unoiled areas can be estimated. Because the
            fisheries in PWS harvest mixed stocks of fish, it will be
            necessary to reconstruct the stock specific abundance over time

                                           193









        in each of the fishing districts to estimate stock specific
        catches.


                                   OBJECTIVES




        Life  History Modeling.

        A.    Develop a computational framework to account for specific
              effects of oiling on species, stock, and life history stages
              of salmon populations in PWS, Cook Inlet, Kodiak, and the
              Chignik areas.

        B.    Estimate the "status quo or no-oiling"        values f or all
              parameters implicit in the computational framework that are
              most consistent with the scientific literature and give the
              best description of the aggregate of stocks' historical
              population dynamics.

        C.    Estimate the "oiling" values for all parameters implicit in
              the computational framework that are most consistent with
              the synthesis of the individual stocks responses as
              identified in the NRDA studies and/or responses deduced from
              the available scientific literature.

        D.    Develop estimates of salmon injury by comparison of future
              simulations of salmon production under the "oiling" and "no-
              oiling" model parameter values.

        Run Reconstruction.

        A.    Develop a computational framework for estimating stock
              specific abundance over time in the 8 fishing districts in
              Prince William Sound.       The approach will be a two
              dimensional (multi-stock and multi-district) generalization
              of the comprehensive timing model of Schnute and Sibert
              (1983).

        B.    Analyze the historical timing data and tagging data
              necessary to develop simplifying assumptions to derive
              estimable parameters.   Test the run reconstruction approach
              by reconstructing the 1988 pink salmon run, develop
              estimates of hatchery contribution, and compare those to the
              hatchery contributions observed in the coded-wire tag (CWT)
              study.

        C.    Reconstruct the 1990 pink salmon run to Prince William Sound
              and develop estimates of return per spawner for oiled and
              unoiled areas.




                                        194













                                         METHODS

           The life history model and run reconstruction model will be
           developed by a select group of experts under contract to ADF&G.

           The life history model will have the following properties:

                1.   The salmon stocks and areas included in this computer
                     based mathematical model are those included in the
                     portions of F/S studies 1-10 as approved by the
                     trustees as well as any stocks that were observed to
                     have suffered overescapement in 1989 as a result of the
                     presence of oil.

                2.   The model will have sufficient temporal or life history
                     structure to account for all of the potential oil
                     related    injuries  that   might   affect   the    future
                     production of salmon.

                3.   The model will have stochastic elements to account for
                     natural variation.

                4.   The model will project future abundance of salmon by
                     individual stocks and will enable the comparison of
                     future scenarios of salmon abundance with and without
                     oiling.

           With regard to the run reconstruction model, the following
           relates the multi-stock and multi-district generalization of
           Schnute and Sibert (1990) for PWS Pink Salmon.     There are eight
           fishing districts in Prince William Sound and eight stocks
           consisting of the aggregate of spawning streams within the
           respective fishing district.

           Define the following:

                ii(t) = cumulative entry to District j
                Ej(t) =   cumulative escapement in District j
                Xij(t)    cumulative entry to District j from District i
                Pj(t)     total number of fish in District j
                Cj(t)     cumulative catch in District j
                Cij(t)    cumulative catch of stock i in District j

                            8
                Cj(t)    E Ci.                                    (1)
                           J=i

                                           195









         P j (t)   I j (t) + E Xkj M   - Cj(t) - E Xjt(t) - Ej(t)         (2)
                             k                        1


         The timing functions I., Xi1j,       and P.   will be estimated by
         reconstructing back from the cumulative catches (C,(t)) and
         cumulative escapements (E,(t)). Several assumptions must be made
         in order to solve the above generalization of            Schnute and
         Sibert's model.       To do so requires extensive        analysis of
         historical timing and tagging information.               Examples of
         assumptions are: 1. Entry of pink salmon into Prince William
         Sound occurs in Districts 6, 7, 8 (i.e. Southwestern, Montague,
         and Southeastern, respectively). 2. Rate of exploitation is the
         same for all stocks within a given District.

                                   DISCUSSION

         Note that the life history and run reconstruction models will
         accommodate harvest in existing mixed stocks fisheries and will
         enable the comparison of alternative commercial fisheries harvest
         policies.     This will facilitate the evaluation of fisheries
         restoration strategies that attempt to rebuild damaged stocks by
         reducing catch in fisheries that exploit stocks damaged and
         stocks not damaged by the oil spill.

         Activities during the first year include: analyses of historical
         data, developing efficient software for computer simulation, and
         synthesis of model parameters for the no-oiling scenario.
         Activities during the second year include analysis of NRDA
         results, including run reconstruction, to developing the best
         estimate of the various salmon stocks response to oiling;
         synthesis of these results into altered parameters in the model;
         develop the best scientific. estimates of future salmon stocks
         production under the no-oiling and oiling scenarios.

                                      BIBLIOGRAPHY

         Schnute, J and J. Sibert. 1983. The salmon terminal fishery: a
              practical, comprehensive timing model. Can. J. Fish. Aquat.
               Sci. 40:835-853.


         BUDGET: ADF&G

         Personnel                         $58.9
         Travel                              5.2
         Contractual                       100.0
         Supplies                            1.0
         Equipment                          10.0

         Total                            $175.1


                                           196











           FISH/SHELLFISH STUDY NUMBER 30

           Study Title:    Data Base Management

           Lead Agency:    ADF&G

                                       INTRODUCTION

           Large quantities of data are being analyzed in order to demonstrate
           the fact and extent of injury to natural resources due to oiling.
           The purpose of this study is to make original data readily
           available to agency and non-agency personnel so that data analysis
           can be conducted, and so that all analyses can be accomplished in
           an efficient and cost effective manner.      The data to be placed
           under the database management system (DBMS) will be drawn from two
           categories; 1) historical data necessary to the interpretation and
           implementation of the results of NRDA studies, and 2) data
           resulting from NRDA studies.

                                        OBJECTIVES

           A.   To construct a cost effective DBMS to readily retrieve and
                order data from original data in electronic form according to
                user specified criteria of time, space, and selection of
                variables.     The DBMS should be constructed to meet the
                following criteria, in order of priority: 1) completeness of
                contentst 2) speed of retrieval, and 3) ease of use in
                assembling primary data into data sets for further analysis by
                other software. Furthermore, the DBMS will take advantage of
                existing DBMS applications currently available in the ADF&G.

           B.   To develop the structural facilities for individuals to access
                data that is physically located at different sites.          To
                accomplish this, a LAN (local area network) facility must be
                developed in the Cordova and Anchorage ADF&G offices, as well
                as to develop a system for linking these with existing LAN's
                in Juneau and Kodiak.     Note that objective B, although a
                necessity for. this project, will be met by a concurrent and
                separately funded project "statewide data base system"
                currently being implemented by ADF&G.

                                         METHODS

           A distributional data base management system, using SQL software,
           will be developed.   The system will be flexible to accommodate the
           data physically located in Kodiak, Anchorage, Cordova, and Juneau.

           The DBMS system will be accessed through a linked system of LAN's,
           (Juneau, Anchorage, Kodiak, and Cordova) . The DBMS can be accessed
           by any user with an IBM compatible PC that has access to the
           Anchorage LAN.     Interface software using "WINDOWS" will be

                                           197









        developed and made available to individuals to facilitate non-
        programmer access to the DBMS systems.

        The following data, for all species and from Prince William Sound,
        Cook Inlet, Kodiak, and Chignik areas, will be incorporated into
        the DBMS:

             1.   All NRDA project data.
             2.   Salmon escapement data, including weir counts, stream
                  counts, aerial survey counts, and sonar counts.
             3.   Biological data including age composition, size, sex,
                  growth, and stock composition.
             4.   Pre-emergent and egg density.
             5.   Groundfish and shellfish survey data.

        In addition, the DBMS will have access to statewide fish ticket
        system data which includes commercial fisheries catch and effort
        data by area, species, and gear type.

        This project will be developed concurrently with the development of
        the ADF&G statewide data base system which is being funded with
        State of Alaska general funds. It is the intent to develop LANS in
        the Anchorage and Cordova ADF&G offices. These new LAN's will be
        linked with existing Kodiak and Juneau LAN's to facilitate
        statewide access to the above DBMS as well as to accommodate the
        need to access data, currently in electronic form, located in
        Kodiak and Juneau.      For example the catch data cited above is
        currently in the statewide f ish ticket system data base which
        resides in Juneau.    The network will accommodate all Commercial
        Fisheries Division personnel and have the potential capacity to be
        expanded to all departmental personnel.



        BUDGET:   ADF&G


        Personnel Services            $ 80.0
        Travel                           5.0
        Contractual                      0.0
        Supplies                         1.0
        Equipment                        34.0

        Total                          $120.0












                                         198











                                  MARINE MAMMAL ASSESSMENT



            Although the most visible impact of the EVOS on marine mammals was
            the large number of dead sea otters, other marine mammal species
            were potentially injured by the spill, including Steller sea lions,
            harbor seals, killer whales, and endangered humpback whales.

            In 1989, seven studies were assembled and implemented to gather
            information on injury to marine mammals.        Aerial surveys for
            stranded cetaceans were also conducted.         Additional data on
            injuries to sea otters were gathered at the sea otter
            rehabilitation centers.

            All of these studies, except one, will be continued in 1990.
            Marine Mammals Study Number 3, Cetacean Necropsies to Determine
            Injury from the EVOS, is discontinued.        No oil spill related
            cetacean strandings are expected in the second year. Cancellation
            of this study does not exclude the possibility of collecting
            samples opportunistically, should fresh carcasses be encountered.

            In many cases, the 1990 studies have been expanded and modified in
            response to knowledge gained during the f irst year and comments
            from reviewers and the public.     The sea otter study is far more
            extensive than last year and will look at possible physiological
            and toxicological impacts that could result in long-term, sublethal
            injuries.   The assessment of population effects is also greatly
            expanded and will look closely at pre and post spill populations,
            population dynamics, and reproductive biology. Data from studies
            on Steller sea lions and harbor seals will provide information on
            toxicological effects of the EVOS. The ongoing cetacean studies
            are intended to provide information on changes in cetacean use of
            the spill zone, to assess impacts that may not become apparent
            until the second year, and to corroborate information on injury to
            killer whales gathered during the 1989 studies.

















                                             199











                      MARINE MAMMAL STUDY NUMBER 1

        Study Title:   Effects of the EVOS on the Distribution and
                       Abundance of Humpback Whales in PWS, Southeast
                       Alaska, and the Kodiak Archipelago

        Lead Agency: NOAA

                                    INTRODUCTION

        During the f irst year of the humpback whale damage assessment,
        photographs of individual humpback whales occurring in PWS and
        Southeast Alaska were collected from May to September 1989 to
        assist in determining the impact of the EVOS on humpback whale lif e
        history and ecology.     In PWS, f our dedicated research vessels
        traversed 9,623 nautical miles to search for and photograph whales,
        reflecting 260 days of field research.        In Southeast Alaska,
        researchers working from five different vessels spent 1,011 hours
        searching for whales for a total of 230 days of field research. An
        additional 155 hours were spent off Kodiak conducting marine mammal
        sighting surveys.

        Concerns about the North Pacific humpback whale stock encountering
        or being exposed to oil is well founded based on evidence in the
        literature.    The humpback whale is currently listed as an
        endangered species. Changes in abundance of humpback whales (after
        being exposed for one season to the EVOS) would more likely occur
        in the second year.

        This study will obtain photographs of individual humpback whales
        occurring in PWS from early June to late September 1990. Calves of
        the year will be documented.       Photographs collected will be
        compared to the Alaskan photographic database for the years 1977 to
        1989 to determine if changes have occurred in whale abundance,
        seasonal distribution, continuity of habitat usage, and mortality
        and natality rates.       Results of this research will allow
        determination of the extent of injury (displacement) or loss
        (reduction in numbers) to humpback whale populations as a result of
        the EVOS.


                                     OBJECTIVES

        A.   Count and individually identify humpback whales entering PWS.

        B.   Test the hypothesis that humpback whale distribution and
             abundance within PWS is similar to that reported for 1989 and
             previous years.

        C.   Test the hypothesis that humpback whale natality has not
             changed since the EVOS.

        D.   Test the hypothesis that humpback whale mortality rates have

                                        200









                  not changed since the EVOS.



                                             METHODS


             Shore-based camps (shared with personnel from the killer whale
             project) will be established in PWS to conduct photo-identif ication
             studies on humpback whales from small boats (June through
             September 1990). Camp locations will be sinilar to those set up in
             1989.    Early in the season, camps will be located in the
             northwestern area of PWS (Naked Island); the southwestern region
             at Squire Island (off the southwest side of Knight Island); and
             either on Hinchinbrook Island or off the northern side of Montague
             Island. Camps may be moved during the field season based on whale
             distributional data collected during the study.          Each humpback
             whale camp will be staf fed by at least two biologists equipped with
             one small boat. For consistency in data collection, key personnel
             will remain in the field throughout the 4-month period.

             Weather permitting, field personnel will spend an average of 8 to
             10 hours per day conducting boat surveys searching for whales.
             Effort will be comparable to the 1989 season.          Specific areas,
             known for whale concentrations, will be investigated first.
             However, if reports of whales are received from other sources (e.g,
             sighting network described below) these areas are examined.            if
             whales are not located in "known" areas and opportunistic sighting
             reports are not available; a general search pattern will be
             developed and implemented. Travel routes taken by whales will be
             surveyed. When whales are sighted, researchers end their general
             search effort and approach the whales to collect photo-
             identification information.       A humpback whale survey form is
             completed for each encounter.

             To obtain a high-quality photograph, an approach within 30-60
             meters is required. Photographs are taken of the ventral surface
             of the fluke and left side of the dorsal fin.

             Daily effort logs are maintained each day which will permit 1)
             quantification of the amount of time searching for whales versus
             photographing whales,     2) quantification of search effort under
             different weather conditions; 3) daily vessel trackline, and 4) an
             estimation of the number of vessels/ aircraft encountered in the
             study area.

             To increase the sighting effort within PWS to ensure that all
             whales are being seen and photographed, a marine mammal sighting
             network will be organized throughout the PWS area. This network
             will record all sightings of whales collected opportunistically
             from Alaskan State Ferries and private aircraft and boaters. Whale
             sightings are reported directly to the whale research vessels.
             Field teams respond by searching out the area where whales were
             reported to collect photographic data.

                                               201









         All photographs of humpback whales will be analyzed for individual
         identification. An individual whale's ventral aspect of the fluke
         is recorded (notes and sketches) . Photographs are then grouped by
         individual.    Each individual whale identified is then visually
         compared to the historical photographic database.             A second,
         independent matching analysis will be performed to ensure accuracy.
         Considerable expertise exists in recognizing individual whales
         through computer matching of color patterns. Once all photographs
         are properly cataloged and evaluated, it is then possible to
         determine 1) the identification of individual whales and 2) if the
         individual whales have altered their distributional patterns.

         To avoid biases in data interpretation, it is important that the
         amount of effort in searching for and photographing whales in 1990
         is at least equal to (but not less than) that completed in previous
         years. When comparing differences in sightings per unit effort,
         either the Kolmogorov-Smirnov or Mann-Whitney test will be used.

         Calves of the year will be noted and their mothers identified.
         Natality (number of calves per adult female) will be calculated for
         each area. Comparisons of natality among years will be made using
         either Chi-square tests or Z tests for comparing differences
         between two proportions (selection of test based on sample size).
         Stranded animals found during the 1990 season will be reported.
         Distributional comparisons will be made on a qualitative basis.

                                       BIBLIOGRAPHY

         The following humpback whale articles are pertinent to the studies
         being conducted in Alaska.

         Baker, C. S.        1985.    The Population Structure and Social
               organization of Humpback Whales (Megaptera novaeangliae) in
               the Central and Eastern-North Pacific. Ph.D. Dissertation.
               University of Hawaii. 306 pp.

         Baker, C. S. and L. Herman.           1987.    Alternative population
               estimates of Humpback Whales (Megaptera novaeangliae) in
               Hawaiian Waters. Canadian Journal of Zoology, 65: 2818-2821.

         Hall, J. S. 1979. A Survey of Cetaceans of Prince William Sound
               and Adjacent Waters--their Numbers and Seasonal Movements.
               In:   Environmental Assessment of the Alaskan Continental
               Shelf. NOAA OCSEAP Contract No. 01-6-022-15670. 72 pp.

         Hall, J. D. 1981. Aspects of the Natural History of Cetaceans of
               Prince William Sound, Alaska. Ph.D. Dissertation. University
               of California     Santa Cruz. 148 pp.

         Hall, J. D.       i982.    Prince William Sound - Humpback Whale
               Population and Vessel Traffic Study. Final Contract Report
               No. 81-ABG-00265 to National Marine Fisheries Service, 20 pp.

                                            202










           Johnson, J. J. and A. A. Wolman. 1984. The Humpback Whale
                  (Megaptera novaeangliae). Marine Fisheries Review 46(4):30-
                  .37.

           Jurasz, C. M. and V. P. Jurasz. 1979. Feeding Modes of the
                  Humpback Whale (Megaptera novaeanaliae) in Southeast Alaska.
                  Sci. Rep. Whales Res. Inst. No. 31: 69-83.

           Katona, S., B. Baxter, 0. Brazier, S. Kraus, J. Perkins, and H.
                  Whitehead. 1979. Identification of Humpback Whales by Fluke
                  Photographs. In: H. E. Winn and B. L. Olla (eds). Behavior
                  of Marine Animals - Current Perspectives in Research, Vol. 3:
                  Cetaceans: pp. 33-44. Plenum Press, New York.

           Rice, D. W. 1978. The Humpback Whale in the North Pacific:
                  Distribution, Exploitation, and Numbers.       In:   Report on a
                  Workshop on Problems Related to Humpback Whales (Megaptera
                  novaeangliae) in Hawaii. NTIS Report PB-280 794. pp. 29-44.

           Watkins, W. A., K. E. Moore, D. Wartzok, and J. H. Johnson.
                  1981. Radio Tracking of Finback (Balaenoptera physalus) and
                  Humpback (Megaptera novaeangliae) Whales in Prince William
                  Sound, Alaska. Deep-Sea Research 78: 577-588.

           Wing, B. L. and K. Krieger. 1983. Humpback Whale Prey Studies
                  in Southeastern Alaska, Summer 1982. Report by Northwest and
                  Alaska Fisheries Center Auke Bay Laboratory, 60 pp. National
                  Marine Fisheries Service, NOAA, P. 0. Box 155, Auke Bay,
                  Alaska   99821.

           von  Ziegesar, 0. 1984. A Survey of the Humpback Whales in
                  Southwestern Prince William Sound, Alaska 1980, 1981, and
                  1983.  A Report to the State of Alaska, Alaska Council on
                  Science and Technology, 68 pp.

           von Ziegesar, 0. and C. 0. Matkin. 1989. A Catalogue of Prince
                  William Sound Humpback Whales Identified by Fluke Photographs
                  Between the Years 1977 and 1988.        2 8 Pages.    North Gulf
                  Oceanic Society, P. 0. Box 15244, Homer, Alaska




           BUDGET: NOAA


           Salaries                      $   0.0
           Travel                            6.0
           Contracts                        80.0
           Supplies                          2.0
           Equipment                         4.0

           Total                         $ 92.0


                                              203












         MARINE MAMMAL STUDY NUMBER 2

         Study Title:   Assessment of Injuries to Killer Whales in PWS,
                        Kodiak Archipelago, and Southeast Alaska


         Lead Agency: NOAA

                                    INTRODUCTION

         During the first year photographs of individual killer whales
         occurring in PWS, Southeast Alaska, and the Kodiak Archipelago were
         collected from May to September 1989 to assess the impact of the
         EVOS on killer whale life history and ecology.        In PWS, f our
         dedicated research vessels traversed 9,623 nautical miles searching
         and photographing whales, reflecting 260 days of field research.

         This year's study will obtain photographs of individual killer
         whales occurring in PWS and adjacent waters from early June to late
         September 1990.      Calves of the year will be documented.
         Photographs collected will be compared to the Alaskan photographic
         database for the years 1977 to 1989 to determine if changes have
         occurred in whale abundance, seasonal distribution, continuity of
         habitat usage, pod integrity, and mortality or natality rates.
         Results of this research will allow determination of the extent of
         injury (displacement) or loss (reduction in numbers) to killer
         whale populations as a result of the EVOS.

                                     OBJECTIVES

         A.   Count the number and individually identify killer whales
              within PWS and adjacent waters.

         B.   Test the hypothesis that killer whale distribution within
              PWS and adjacent waters is similar to that reported for
              previous years (1984-1989).

         C.   Test the hypothesis that pre- and post-spill killer whale
              pod structure and integrity have remained constant.

         D.   Test the hypothesis that killer whale natality rates
              within PWS have not changed since the EVOS.

         E.   Test the hypothesis that killer whale  mortality rates
              within PWS have not changed since the  EVOS.

                                      METHODS

         Shore-based camps will be established in PWS to conduct photo-
         identification studies on killer whales from small boats (May
         through September 1990). Camp locations will be similar to those
         set up in 1989. Early in the season camps will be located in the

                                         204









            northwestern area of PWS (Naked Island) , the southwestern region at
            Squire Island (off the southwest side of Knight Island); and either
            on Hinchinbrook Island or off the northern side of Montague Island.
            Camps may be    moved during the field season based on whale
            distributional  data collected during the study.        Each camp is
            staf f ed by at  least two biologists and one small boat.           For
            consistency in  data collection, key personnel remain in the field
            throughout the  4-month period.

            Weather permitting, field personnel will spend an average of 8 to
            10 hours per day conducting boat surveys searching f or whales.
            Effort will be comparable to the 1989 season.        Specific areas,
            known for whale concentrations, are investigated first. However,
            if reports of whales are received from other sources (e.g, sighting
            network described below) , those areas are examined. If whales are
            not located in known areas and opportunistic sighting reports are
            not available, a general search pattern will be developed and
            implemented. Travel routes typically taken by whales are surveyed.
            When whales are sighted, researchers stop further search efforts
            and    approach  the   whales    to   collect    photo-identification
            information.   A killer whale survey form is completed for each
            encounter.    When whales are encountered, researchers select a
            vessel course and speed to approximate the animals' course and
            speed to facilitate optimal photographic positioning.

            To obtain a high-quality photograph, an approach within 30-60
            meters is required. Photographs are taken of the left side of the
            whale's dorsal fin and saddle patch. Any high-performance camera
            system can be used to collect the data.

            Daily effort logs are maintained each day which will permit 1)
            quantification of the amount of time searching for whales vs
            photographing whales,    2) quantification of search effort under
            different weather conditions; 3) daily vessel trackline, and 4) an
            estimation of number of vessels/aircraft encountered in the study
            area.


            To increase the sighting effort within PWS to ensure that all
            whales are being seen and photographed, a marine mammal sighting
            network will be organized throughout the PWS area. This network
            will record all sightings of whales collected opportunistically
            from Alaskan State Ferries and private aircraft and boaters. Whale
            sightings are reported directly to the whale research vessels.
            Field teams respond by searching out the area where whales were
            reported to collect photographic data.

            To account for the possible displacement of killer whales to areas
            outside PWS and to confirm that the missing whales are not
            elsewhere (e.g., particularly the absence of the 22 individuals of
            AN pod), photographic studies will be conducted off Kodiak Island.



                                             205









        A marine mammal sighting network will be organized throughout
        Alaska which includes sightings collected opportunistically from
        Alaskan State Ferries and private aircraft and boaters. To provide
        extended coverage throughout the GOA, marine mammal sighting
        information collected by NOAA ships and other research vessels that
        have been working areas of interest will be examined. All killer
        whale data will be extracted and summarized. If photographs were
        collected; an attempt will be made to obtain them.

        All photographs of killer whales will be analyzed for individual
        identification.   Each negative (or prints as needed) is placed
        under a dissection microscope for identification purposes and notes
        and sketches made. Photographs are then grouped by individuals.
        Each identified whale is then visually compared to the historical
        photographic database available at the Pacific Biological Station,
        Nanaimo, British Columbia, Canada.      Once all photographs are
        properly entered and evaluated, it is then possible to determine 1)
        if all members of the pod were present, and 2) if pod
        structure/ integrity is similar to previous years. Missing animals
        are noted. The stability of resident pods over time is such that
        if an individual is listed as missing for at least one year, that
        missing whale is considered dead.

        To avoid biases in data interpretation, effort in searching for and
        photographing whales in 1990 will at least be equal to (but not be
        less than) that completed in previous years. For a large pod ( >12
        animals), the liklihood of obtaining photographs of all individuals
        are increased as the number of encounters are increased.         Some
        individuals, and certain pods, are more likely to approach vessels
        making photographic documentation easier; while others keep a
        considerable distance away making for more difficult conditions.
        Whale behavior also plays a role when attempting to obtain
        photographs of individual whales. If the pod is resting (typically
        grouped together) , it is easier to obtain photographs of all whales
        than when the pod is travelling (spread out through an area) .
        Researchers with prior killer whale experience in a particular area
        who are capable of recognizing individuals, will also enhance the
        likelihood of accounting for all whales within a pod.

        Calves of the year will be noted and their mothers identified.
        Natality (number of calves per adult female) will be calculated for
        each pod for each year and comparisons made between resident and
        transient groups using descriptive statistics.      Mortality rates
        through 1989 will also be calculated for resident groups.
        Mortality for transient pods will be calculated when necessary data
        are available.

        General location of whales will be recorded each time photographs
        are taken, allowing comparisons of pod distributions among years.
        Changes in normal distribution patterns will be reported.



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                                           BIBLIOGRAPHY

             The following killer whale articles are pertinent to the studies
             being conducted in Alaska.

             Anon. 1982. Report on the workshop on identity, structure, and
                   vital rates of killer whale populations.        Rept. Int. Whal.
                   Commn, 32: 617-631..

             Balcomb, K. C. 1978. Orca Survey 1977. Final Report of a Field
                   Photographic Study Conducted by the Moclips Cetological
                   Society in Collaboration with the U. S. National Marine
                   Fisheries Service on Killer Whales (Orcinus orca) in Puget
                   Sound. Unpub. Report to the Marine Mammal Division, National
                   marine Fisheries Service, Seattle, Washington, 10 pages.

             Bigg, M. A. 1982. An Assessment of Killer Whale (Orcinus orca)
                   Stocks off Vancouver Island, British Columbia.         Rept. Int.
                   Whal. Commn., 32: 655-666.

             Braham, H. W. and M. E. Dahlheim. 1982. Killer Whales in Alaska
                   Documented in the Platforms of Opportunity Program.           Rept.
                   Int. Whal. Commn. 32: 643-646.

             Calambokidis, J., J. Peard, G. H. Steiger, J. C. Cubbage, and R.
                   L. DeLong.    1984.   Chemical contaminants in marine mammals
                   from Washington State.      Natl. Oceanic Atmospheric Admin.,
                   Tech. Memo, NOS OMS, 6: 1-167.

             Ellis, G. 1987. Killer Whales of Prince William Sound and
                   Southeast Alaska. A Catalogue of Individuals Photoidentif ied,
                   1976-1986. Sea World Research Institute/Hubbs, Marine Research
                   Center, Technical Report No. 87-200. April 1987. ,

             Fowler, C. W. 1984. Density Dependence in Cetacean Populations.
                   In "Reproduction in Whales, Dolphins, and Porpoises". Eds. W.
                   F. Perrin, R. L. Brownell, and D. P. DeMaster.         Rept. Int.
                   Whal. Commn., Spec. Issue 6: 373-380.

             Hall, J. D. 1981. Aspects of the Natural History of Cetaceans
                   of Prince William Sound. Ph.D. Dissertation. University of
                   California - Santa Cruz. 148 pp.

             Heyning, J. E. and M. E. Dahlheim.             1988.     Orcinus orca.
                   Mammalian Species Account, No. 304, pp. 1-9, 4 figs.

             Leatherwood, S., K. C. Balcomb, C. 0. Matkin, and G. Ellis.
                   1984.   Killer Whales (Orcinus orca) of Southern Alaska -
                   Results of Field Research 1984 Preliminary Report. Hubbs Sea
                   World Research Institute Tech. Report No. 84-175, 59 pp.

             Leatherwood, S., A. Bowles, E. Krygier, J. D. Hall, and S.

                                               207









             Ignell.    1985.   Killer Whales (Orcinus orca) in Southeast
             Alaska, Prince William sound, and Shelikof Strait; A Review of
             Available Information. Rept. Int. Whal. Commn., SC/35/SM 7.,
             10 pp.

        Perrin, W. F. and *S. B. Reilly.      1984. Reproductive Parameters
             of Dolphins and Small Whales of the family Delphinidae. In
             "Reproduction in Whales, Dolphins, and Porpoises". Eds. W. F.
             Perrin, R. L. Brownell, and D. P. DeMaster. Rept. Int. Whal.
             Commn., Spec. Issue 6: 97-134.

        von Ziegesar, 0., G. Ellis, C. Matkin, and B. Goodwin. 1986.
             Repeated Sightings of Identifiable Killer Whales (Orcinus
             orca) in Prince William Sound, Alaska 1977-1983. Cetus, Vol.
             6, No. 2, 5 pp.




        BUDGET: NOAA

        Salaries                    $    45.0
        Travel                           10.0
        Contracts                        180.0
        Supplies                         10.0
        Equipment                        10.8

        Total                       $    255.8




























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            MARINE MAM14AL STUDY NUMBER 4

            Study Title:    Assessment of Injury to Steller Sea Lions in PWS
                            and the GOA


            Lead Agency:    NOAA

            Cooperating Agency:    ADF&G


                                        INTRODUCTION

            Steller sea lions (Eumetopias Jubatus) are the largest and one of
            the most conspicuous pinnipeds inhabiting the GOA. The north GOA
            contains a major portion of the worldwide habitat of this
            species. Regularly used haulouts are located throughout PWS and
            along the Gulf coast.      Major breeding rookeries occur at the
            entrance to PWS, along the eastern Kenai Coast, in the Barren
            Islands, in the northern Kodiak area, at Chirikof Island south of
            Kodiak, and in the Semidi Islands, south of Shelikof Strait
            (Calkins and Pitcher 1982; Loughlin et al. 1984; Merrick et al.
            1987).

            Steller sea lions were present in large numbers in PWS during the
            oil spill, were exposed to oil immediately after the spill, and
            may continue to be exposed for several more years.            Initial
            observations indicated that sea lions did not attempt to avoid
            the oil.   Oiled sea lions were reported at haulouts by several
            observers.

            Steller sea lion populations have declined substantially in much
            of their range since at least 1970 (Braham et al. 1980; Calkins
            1985; and Loughlin et al. 1984).       This decline appears to be
            accelerating in the northern Gulf of Alaska (Loughlin et al.
            1990; Calkins and Goodwin 1988; Merrick et al. 1987 Loughlin et
            al. 1984).    The NMFS has listed this population as threatened
            under terms of the Endangered Species Act. Further reductions of
            this species in this area could result in adverse ecological
            impacts on the marine ecosystem.

            Various studies and observations suggest that several thousand
            sea lions move across the northern GOA in the spring; probably
            most return to the large rookeries along the Kenai coast and
            northern Kodiak to pup and breed (Calkins and Pitcher 1982).
            Many of these animals use PWS during the period of March through
            May (Calkins and Pitcher 1982).

            This study addresses the impacts of the EVOS on the Steller sea
            lion population in PWS and the GOA.        Sea lion pups will be
            counted at rookeries from Chowiet Island to Seal Rocks.        These
            counts will be compared between years for 1989 and 1990 as well
            as compared to historical data of a similar nature.      The counts
            will be used to monitor relatively large changes which may occur

                                            209








        in the population.    Premature pupping will be investigated by
        comparing premature pupping rates at an area close to the oil
        spill (Cape St. Elias) to an area a substantial distance from the
        spill (Chirikof Island).        It is assumed that, because of
        proximity, the sea lions at Cape St. Elias had a higher exposure
        rate to the oil than those at Chirikof Island. Premature pupping
        has been suggested as a possible toxicological consequence of
        exposure to hydrocarbons during gestation.     This would not have
        been a likely effect in 1989 because the spill occurred late in
        the gestation period. Toxicological and histological examination
        of tissues from sea lions will provide information on absorption
        and possible damage caused by hydrocarbons including the pups
        born prematurely.    Tissues have been taken from both animals
        collected specifically for that purpose and from animals found
        dead in oiled areas.    All animals collected for tissue analysis
        were examined by a certified veterinary pathologist.          Tissue
        analysis may show if injury occurred to sea lions.

                                    OBJECTIVES

        A.   Test the hypothesis that premature pupping occurs at a
             higher rate at a hauling area nearer the oil spill.

        B.   Test the hypothesis that pup production is lower in the
             vicinity of the oil spill.

        C.   Estimate hydrocarbon levels in sea lion tissues to within
             10% of the actual value 95% of the time.

        D.   Test the hypothesis that tissue damage has occurred.

                                      METHODS

        Premature pupping has occurred historically in the GOA sea lion
        population (Calkins and Goodwin 1988) and may be accelerated by
        toxic effects of oil.      observations and searches for aborted
        fetuses will be made at all hauling areas and rookeries visited
        after March 1989. Premature pupping will be measured at Cape St.
        Elias and at Chirikof Island by stationing observers at these
        locations for a 4 week period during April and May.             Each
        premature birth will be recorded and each fetus will be examined.
        Tissues will be preserved from each animal examined for
        hydrocarbon and histological analysis.     Adults will be counted
        daily at each location and a rate of premature births to adults
        present will be determined. Daily observations will be conducted
        using spotting scopes and binoculars.

        Pup production will be measured by counting pups directly at the
        six rookeries within the oil spill area from Chowiet Island to
        Seal Rocks (Calkins and Pitcher 1982).      Sea lions from all of
        these rookeries could be assumed to be impacted. This count has
        been conducted in June/July 1989 and will be conducted again in
        1990.


                                        210








            In order to insure accurate hydrocarbon analysis of tissues, it
            is important to preserve tissues within six hours after death.
            Accordingly, sea lions will be collected under terms of a permit
            issued by the NMFS, upon consultation with NMFS, if available
            tissue analyses indicate further collections are warranted.

            In accordance with established criteria of the histopathology
            technical group, a Board certified veterinary pathologist will
            perform histopathological analysis of all sea lion tissues and a
            second board certified pathologist will perform an independent,
            blind reading of a subsample of histology slides.         Reference
            histology slides will be retained and archived toxicological
            samples will be frozen and stored.

            Data analysis for comparing premature pupping between two areas
            to determine if the proportion of premature pups born to adults
            in an area close to the oil spill is higher than an area further
            away from the oil spill will be tested with a two sample t-
            statistic on rate (Snedcor and Cochran 1980) at alpha=0.05 in the
            lower tail.  The normality assumption will be examined with Q-Q
            plots (Hoaglin, Mosteller and Tukey 1985) and if necessary, the
            data will either be transformed to meet this assumption or a
            Mann-Whitney, nonparametric statistic will be used (Conover
            1980).

            Analysis of pup counts will utilize a regression model to predict
            expected numbers of sea lion pups in the absence of the EVOS.
            Because sea lion pup numbers have been declining since 1979, the
            1990 pup count will be compared to the 1989 count and historical
            data to determine if it is lower than what the regression model
            suggests-. A Hotelling's T2 statistic will be used to test if the
            observed 1990 count is significantly lower than the predicted
            value from the regression equation modeling of the pre-1989 sea
            lion decline (Neter and Wasserman 1974).     The validity of the
            model will be tested using data from counts from unoiled areas.

            It is assumed that the distribution of pup counts is normal. The
            regression model would accurately predict pup numbers in the Gulf
            of Alaska in the absence of the oil spill. The regression model
            is correctly specified, and has constant variance.              The
            proportion of sea lions exhibiting hydrocarbon uptake will be
            estimated and an exact 95% confidence interval determined using
            the binomial distribution (Ostle and Mensing 1982).

                                      BIBLIOGRAPHY

            Braham, H. W., R. D. Everitt, and D. H. Rugh. 1980. Northern sea
                lion population decline in the. eastern Aleutian Islands.
                Fish. Bull. 44:25-33.

            Calkins, D. G. 1985. Sea lion pup counts in and adjacent to
                Shelikof Strait.    Final report submitted to North Pacific
                Fisheries Management Council, contract 84-1.             Alaska

                                           211








              Department of Fish and Game, Anchorage Alaska. 13pp.

        Calkins D. G. and E. Goodwin. 1988. Investigation of the
              declining sea lion population of the Gulf of Alaska.
              National Marine Mammal Laboratory contract NA-ABH-00029.
              Alaska Department of Fish and Game, Anchorage, Alaska. 76pp.

        Calkins, D. G. and K. W. Pitcher. 1982. Population
              assessment,ecology, and trophic relationships of Steller sea
              lions in the Gulf of Alaska.     In: Environ. Assess. of the
              Alaskan Cont. shelf. Final Reports.    19:445-546.

        Conover, W. J. 1980. Practical nonparametric statistics
              2nd ed. John Wiley and Sons, New York. 493 pp.

        Hoaglin, D. C., F. Moesteller, and J.W. Tukey. 1985. Exploring
              data tables, trends, and shapes.    John Wiley and Sons, New
              York. 527 pp.

        Johnson, R. A. and D. W. Wichern. 1988. Applied multivariate
              analysis.   2nd ed.    Prentice Hall, Englewood Cliffs, New
              Jersey. 607 pp.

        Loughlin, T. R., D. J. Rugh, and C. H. Fiscus. 1984.
              Northern sea lion distribution and abundance: 1956-80.       J.
              Wildl. Manage. 48:729-740.

        Loughlin, T. R., A. S. Perlov, and V. A. Vladimirov. 1990.
              Survey of northern sea lions (Eumetopias jubatus) in
              the Gulf of Alaska and Aleutian Islands. NOAA
              technical memorandum NMFS F/NWFC- 176. 26 pp.

        Merrick, R. L., T. R. Loughlin, and D. G. Calkins. 1987.
              Decline in abundance of the northern sea lion Eumetopias
              jubatus in Alaska, 1956-86. Fish. Bull. 85:351-365.

        Mendenhall, W., R. L. Scheaffer, and D. D. Wackerly. 1981.
                Mathematical statistics with applications. Second
                edition. Duxbury Press, Boston, Mass. 686pp.

        Neter, J. and W. Wasserman. 1974. Applied linear statistical
              models: regression, analysis of variance, and      experimental
              designs. Richard D Irwin, Inc. Homewood, Illinois. 842pp.

        Ostle, B. and R. W. Mensing. 1982. Statistics in research,
              3rd ed. The Iowa State Univ. Press. Ames, Iowa. 595 pp.

        Snedecor, G. W. and W. G. Cochran. 1980. Statistical
              methods. Seventh edition. The Iowa State Univ. Press, Ames
              Iowa. 507pp.





                                         212











            BUDGET: NOAA

            Personnel                    $ 107.4
            Travel and per them              6.0
            Services                         45.5
            Commodities                      6.8                                         1
            Equipment                        5.5

            TOTAL                          $171.2




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                                             213














        MARINE MAMMAL STUDY NUMBER 5

        StudyTitle:     Assessment of Injury to Harbor Seals in PWS and
                        Adjacent Areas

        Lead Agency:    NOAA

        Cooperating Agency:      ADF&G


                                     INTRODUCTION

        Harbor seals (Phoca vitulina richardsi) are one of the most
        abundant species of marine mammals in PWS and adjacent areas.
        They are resident throughout the year, occurring primarily in the
        coastal zone where they feed and haul out to rest, bear and care
        for their young, and molt (Hoover 1988).          They are used for
        subsistence purposes by Native residents in the area. Unlike fur
        seals (Callorhinus ursinus) and sea lions (Eumetopias Jubatus),
        harbor seals do not form distinct rookeries during the pupping
        and breeding season.      Pups are born at the same locations as
        those used as haulouts at other times of year.           Some of the
        largest haulout sites in PWS, and adjacent waters to those
        haulouts, were directly impacted by substantial amounts of oil
        during the EVOS.     oil that moved into the GOA impacted harbor
        seal habitat at least as far to the southwest as Tugidak Island.
        Harbor seals swam through oil and breathed at the air/water
        interface.    On haulouts they crawled through and rested on oiled
        rocks and algae. Pups were born on the haulouts in May and June
        while some of the sites still had oil on them, resulting in pups
        becoming oiled.     The same locations were also used during the
        molt in August and September.

        Trend count surveys indicate that the number of harbor seals in
        PWS declined by 40% from 1984 to 1988, and similar declines have
        been noted in other parts of the northern GOA (Pitcher 1989).
        Additional impacts on harbor seal populations are therefore of
        particular concern.

        Three ringed seals (Phoca hispida) exposed in the laboratory to
        fresh Norman Wells crude oil all died within 71 minutes; six
        seals exposed for 24 hours at a field site showed minor damage to
        the   eyes,   kidneys,   and    liver   (Geraci   and   Smith    1976).
        Hydrocarbons were rapidly absorbed into the body fluids and
        tissues when ringed seals were exposed to oil by either immersion
        or ingestion (Engelhardt et al. 1977).         In 1974, oil from an
        unknown source came ashore at a grey seal (Halichoerus gryRus)
        pupping beach in Wales.      Two pups drowned when they became so
        encased in oil that they were unable to swim, and oiled pups
        reached a lower peak weight at weaning than did unoiled pups
        (Davis and Anderson 1976).

                                          214










            Following the spill, f ield observations were made of seals in
            oiled and unoiled areas of PWS.        Carcasses of 39 seals were
            necropsied and sampled;      19 that were f ound dead or died in
            captivity, and 20 that were collected specifically for sampling.
            Histopathological and toxicological analyses are in progress.

            Last year aerial surveys were conducted during June to count the
            number of harbor seal pups and non-pups on 25 oiled and uno      'iled
            haulouts in PWS. Aerial surveys were also conducted at the same
            25 haulouts'during the fall molt.      Results of the fall surveys
            have bee 'n compared to results,of surveys flown in 1984 and 1988
            to determine whether trends in numbers are similar in oiled and
            unoiled areas.

            This    project   proposes   to   complete   histopathological     and
            toxicological analyses of harbor seal tissues and to provide
            .counts of harbor seals on haulouts in oiled and unoiled parts of
            PWS and during pupping and molting in two additional years (1990
            and 1991). . Data from aerial surveys will be used to evaluate
            whether changes occurred in the     'distribution and abundance of
            harbor seals following the, EVOS, and whether such changes
            coincided with the presence or absence of oil in the Area or on
            the haulouts.    Toxicological analyses of tissues from oiled and
            unoiled seals will allow an assessment of whether hydrocarbons
            were assimilated by the seals and how contaminant levels changed
            over time.     Histopathological examinations will determine the
            types and degrees of toxic damage. to tissues. Survey and
            laboratory data, in combination with historical data for PWS,
            will be used to evaluate whether the EVOS caused a reduction in
            pup productivity at oiled sites in 1989 and 1990, and whether
            changes in abundance during the 1989 fall molt were due to the
            EVOS. This information can then be used to make recommendations
            regarding restoration of lost use, populations, or habitat where
            injury is identified.

                                         OBJECTIVES

            A.      Test the hypothesis that harbor seals found dead in        the
                    area affected by the EVOS died due to oil toxicity.

            B.      Test the hypothesis that seals exposed to oil from         the
                    EVOS assimilated hydrocarbons to the extent that harmful
                    pathological conditions resulted.

            C.      Test the hypothesis that the abundance of harbor seals on
                    the trend count route during pupping and molting decreased
                    in oiled areas of PWS as compared to unoiled areas.

            D.      Test the hypothesis that pup production was lower in oiled
                    than in unoiled areas, or than in years not affected by
                    the EVOS.


                                              215













                                     METHODS

       For one week during pupping in June 1990, small boats will be
       used to observe seals and seal haulouts in oiled areas. Haulout
       sites will be inspected for the presence of oil or dead animals.
       Seals will be examined using 7 to 10-power binoculars and a 25-
       power spotting scope to determine whether any have oiled pelage.
       Seals observed will be classified as to the degree of pelage
       oiling (heavy, moderate, light, or none).     If any carcasses are
       found that are in suitable condition, they will be necropsied by
       trained biologists, veterinarians, or pathologists, and samples
       will   be   obtained    and   preserved   for   toxicological     and
       histopathological examination.

       A maximum of 12 additional harbor seals will be collected, under
       a permit f rom NMFS.    Most will be collected at or adjacent to
       sites impacted by the EVOS. One or more seals will be collected
       from an area not impacted by the spill, such as southeast Alaska.
       Each animal will be necropsied as soon as possible after death by
       qualified personnel.

       Collected animals will be measured, weighed, and photographed;
       time, date, location, and circumstances of collection will be
       noted; and any gross abnormalities will be recorded.            Blood
       samples for serum, plasma, and whole blood analyses will be
       taken. Samples will be taken for histopathology and toxicology.
       Chain of custody will be maintained for all samples. Samples for
       histopathology will be stored in formalin until they are
       analyzed.    Reference histology slides will be retained and
       archived.   Toxicology samples will be frozen and stored until
       they are sent to an approved laboratory for analysis.

       Aerial surveys will be conducted during pupping in June and
       molting in September along a previously established trend count
       route (Calkins and Pitcher 1984; Pitcher 1986, 1989) that covers
       25 haulout sites and includes 6 sites impacted by the EVOS
       (Agnes, Little Smith, Big Smith, Seal and Green islands, and
       Applegate Rocks), 16 unoiled sites, and 3 intermediate sites that
       were not physically oiled but were adjacent to oiled areas.
       Visual counts will be made of seals at each site and photographs
       taken of large groups for later verification.

       During June, separate counts will be made of pups and non-pups.
       Pupping surveys are needed in 1990 and should be done in 1991
       since there are no historical data available from PWS during the
       pupping season with which to compare the 1989 results. Breeding
       and embryo implantation for 1990 pups occurred while seals were
       still exposed to oil on haulouts and while hydrocarbon levels in
       tissues may have been abnormally high.

       Surveys during the molt in 1990 and possibly 1991 are necessary

                                       216










            to determine whether observed changes in the number of seals on
            oiled sites between 1988 and 1989 persist.

            All statistical tests for significance will use alpha = 0.05.
            Statistical testing is not appropriate for all objectives.       The
            assessment of cause of death of animals found in areas impacted
            by the EVOS (objective A) will require expert evaluation of
            limited and varying toxicology and histopathology data sets.

            Toxicological results for each seal collected will be entered
            into a data base along with information on date and location of
            collection; presence of oil in the area; degree of external
            oiling of the seal; age, sex, size, and reproductive condition.
            Hydrocarbon levels in the tissues will be tabulated by individual
            and by groups based on age, sex, collection location, and degree
            of oiling.     Differences between groups will be tested where
            possible using ANOVA (Neter and Wasserman 1974).

            Types of pathology detected will be listed for each specimen and
            will be grouped into tables by sex, age, collection location, and
            degree of oiling.    Incidence of pathology will be expressed as
            the percentage of the total number of animals in the group that
            exhibited a particular type of anomaly.     Incidence of pathology
            will be evaluated in light of toxicological results for each
            specimen.

            Harbor seal surveys must be conducted within biological time
            windows imposed by the pupping and molting periods.            While
            results of previous harbor seal trend counts have indicated that
            it is desirable to obtain 7-10 counts during a survey period
            (Pitcher 1986, 1989), the actual number of counts is frequently
            limited by the number of days suitable for flying.            During
            pupping, the survey window cannot be extended to accommodate
            sample size needs since, as pups grow and are weaned, they become
            increasingly difficult to differentiate from adults when observed
            f rom the air.   Similarly, during the molt it is necessary to
            confine surveys to the period when maximum numbers are thought to
            haul out.

            Aerial surveys of harbor seals do not estimate the total number
            of seals present since they do not account for seals that are in
            the water or seals hauled out at locations not on the trend count
            route. Surveys provide indices of abundance based on the number
            of hauled out seals counted on the trend count route.
            Interpretation of trend count surveys relies on the assumption
            that counts of harbor seals on select haulout sites are valid
            linear indices of local abundance. We assume that within a given
            biological window, such as the pupping or molting period, haul
            out behavior remains the same f rom one year to the next, and
            counts can thus be compared.        Standardization of procedures
            minimizes the affects of variables such as tide and weather that


                                            217










         could influence the number of seals hauled out on a given day.

         The trend count route includes haulouts impacted by the EVOS, as
         well as haulouts that are north, east, and south of the primary
         area impacted by oil. There is an adequate sample of both oiled
         and unoiled areas.

         There are no historical data on the distribution of harbor seals
         in PWS during the pupping period.         The first surveys during
         pupping were conducted in June 1989 after the EVOS. In order to
         gather these data it will be necessary to conduct surveys in at
         least 1990 and 1991. These data will be used in a retrospective
         analysis comparing counts of seals in oiled and unoiled sites
         between years and using the same statistical techniques employed
         for fall molting surveys (Frost 1990).

         Fall molting surveys of the trend count route were conducted in
         1983, 1984, 1988, and 1989.    The 1984, 1988, and 1989 counts are
         considered reliable and will be used for comparisons with data
         collected in 1990 and possibly 1991. Analysis of count data and
         comparisons   to   other   years   will    be  conducted     following
         statistical    methodology    used   for    1989   molting     surveys
         (Frost 1990).

         A repeated measures ANOVA (Winer 1971) will be conducted on the
         trimean  (Hoaglin et al. 1985) of the site count data in order to
         examine  trends in abundance at oiled versus unoiled sites.        The
         trimean  statistic will be used as the measure of central tendency
         because sets of counts at a single location sometimes show
         bimodal distributions or extreme variations.           This analysis
         assumes  random samples, constant variance, and normality of the
         differences. If necessary, transformations (Snedecor and Cochran
         1980) will be used to ensure constant variance and normality.
         The test assumes that the mean proportion of the population
         hauled out on the trend count route is constant over years.
         Hypotheses addressing Objective C will be tested using orthogonal
         contrasts derived from the ANOVA.

         In order to compare pup production at oiled and unoiled sites, a
         one-way analysis of co-variance (Neter and Wassermann 1974) will
         be performed on the square roots of the trimeans (Hoaglin et al.
         1985) of pup counts, using the square roots of non-pup counts as
         a covariate.    The square root transformation will be used to
         correct for non-constant variation of the count data (Snedecor
         and Cochran 1980). Linear contrasts (Neter and Wasserman 1974),
         where the average number of pups is adjusted to a common number
         of adults, will be used to test working hypotheses.





                                          218













                                         BIBLIOGRAPHY

             Calkins, D. and K. Pitcher. 1984. Pinniped investigations in
                    southern Alaska:1983:84. Unpubl. Rep. ADF&G, Anchorage,
                    AK 16pp.

             Davis, J. E. and S. S. Anderson. 1976. Effects of oil pollution
                    on breeding gray seals. Mar. Poll. Bull. 7:115-118.

             Engelhardt, F. R., J. R. Geraci, and T. G. Smith. 1977. Uptake
                    and clearance of petroleum hydrocarbons in the ringed
                    seal, Phoca hispida. J. Fish Res. Board Canada 34:1143-
                    1147

             Frost, K. J. 1990. Marine Mammals Study Number 5: Assessment
                    of injury to harbor seals in Prince William Sound,
                    Alaska, and adjacent areas. State-Federal Natural
                    Resource Damage Assessment for April-December 1989.
                    Unpubl. Prelim. Status Rep. ADF&G, Fairbanks, AK. 27pp.

             Geraci, J. R., and T. G. Smith. 1976. Direct and indirect
                    effects of oil on ringed seals (Phoca hispida) of the
                    Beaufort Sea. J. Fish. Res. Board Canada 33:1976-1984.

             Hoaglin, D. C., F. Mosteller, and J. W. Tukey. 1985. Exploring
                    data tables, trends, and shapes. John Wiley & Sons. New
                    York. 527pp.

             Hoover, A. A. 1988. Pacific harbor seal. Pages 125-157 in J.
                    W. Lentfer (ed). Selected Marine Mammals of Alaska:
                    Species Accounts with Research and Management
                    Recommendations. U.S. Marine Mammal Commission,
                    Washington, D.C.

             Neter, J., and W. Wasserman 1974. Applied linear statistical
                    models. Richard D. Irwin, Inc., Homewood, Illinois.
                    842pp.

             Pitcher, K. W. 1986. Harbor seal trend count surveys in
                    southern Alaska, 1984. Unpubl. Rep. ADF&G, Anchorage,
                    AK. 10pp.

             Pitcher, K. W. 1989. Harbor seal trend count surveys in southern
                    Alaska, 1988. Final Rep. Contract MM4465852-1 to U.S.
                    Marine Mammal Commission, Washington, D.C. 15pp.

             Snedecor, G. W. and W. G. Cochran. 1980. Statistical Methods.
                    Iowa State University Press, Ames, Iowa. 507pp.




                                              219










         Winer, B. J. 1971. Statistical principles in experimental
                 design. 2nd Ed. Mcgraw-Hill, New York, New York.
                 907pp.




         BUDGET: NOAA


         salaries                      $    82.7
         Travel                             15.1
         Contracts                          42.1
         Supplies                             5.4
         Equipment                          14.0

         Total                         $    159.3








































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           MARINE MAMMAL STUDY NUMBER 6A

           Study Title:    Assessment of the Magnitude, Extent, and Duration
                           of Oil Spill Impacts on Sea Otter Populations in
                           Alaska.


           Lead Agency:    FWS

                                       INTRODUCTIOX

           In the first year following the EVOS, several hundred sea otters
           are known to have died as a result of contamination by oil. Death
           occurred from hypothermia and from severe liver, kidney, and lung
           damage as a result of ingestion of oil and inhalation of toxic
           aromatic compounds present during the early period of the spill.
           Long-term or chronic effects of oil on sea otters are not known,
           but initial results from the first year's studies indicate sea
           otter populations have been detrimentally affected.         Potential
           effects may occur as the result of debilitating or sublethal
           injury, accumulation of toxins, and loss or contamination of the
           food supply.   The capacity of the population to recover to pre-
           spill levels is not known. This study will assess the impacts of
           the oil spill on Alaska sea otter populations through (1) surveys
           of wild populations living in oiled and unoiled areas, (2) genetic,
           hematological, histopathological and toxicological analysis of
           tissues collected from live and dead sea otters, (3) analysis of
           survival, reproduction and movements of adult females and pups
           living in oiled and non-oiled areas and (4) analysis of population
           dynamics of dead and living sea otters recovered from or living in
           oiled and non-oiled areas.


                                        OBJECTIVES


           A.   BOAT SURVEYS


                1.    Test that differences in sea otter densities are not
                      significantly different between oiled and unoiled areas.

                2.    Test for differences in sea otter densities between pre-
                      and post-event surveys in oiled and unoiled areas.

                3.    Estimate the magnitude of any change between pre- and
                      post-event sea otter population estimates in PWS.

                4.    Estimate post-event sea otter population size and monitor
                      population trends of sea otters in PWS.

                5.    Estimate winter 1990 offshore densities of sea otters in
                      oiled and unoiled areas.





                                            221











       B.   HISTOPATHOLOGY AND TOXICOLOGY

            1.    Test the hypothesis that sea otters residing in regions
                  that were not affected by the oil spill have lower levels
                  of hydrocarbons in their visceral f at and whole blood
                  than sea otters residing in areas that were affected by
                  oil.

            2.    Test the hypothesis that sea otter carcasses f ound in
                  oiled portions of the Alaska coastline subsequent to the
                  oil spill contain levels of hydrocarbon contamination
                  similar to those in sea otters killed immediately as a
                  result of the spill.

            3.    Test the hypothesis that sea otter carcasses f ound in
                  oiled areas subsequent to the spill contain higher
                  burdens of hydrocarbon contaminants than sea otter
                  carcasses found in non-oiled areas or those analyzed
                  before the spill.

            4.    Evaluate the nature and cause of death of sea otters that
                  died subsequent to the oil spill by performing complete
                  gross and histopathological examinations of carcasses
                  recovered after September 1, 1989.

       C.   CAPTURE OF ADULT FEMALE AND JUVENILE SEA OTTERS

            1.    Test the hypothesis that pup -survival pre-weaning is not
                  different between oiled and non-oiled areas.

            2.    Test the hypothesis that weanling survival at various age
                  intervals is not different between oiled and non-oiled
                  areas.


            3.    Test the hypothesis that survival of adult female sea
                  otters is not different in oiled and non-oiled areas.

            4.    Test the hypothesis that pupping rates of adult female
                  sea otters are not different between oiled and non-oiled
                  areas.


            5.    Evaluate the movements of weanling and adult female sea
                  otters with respect to areas in PWS that have been
                  affected by the oil spill.

            6.    Test the hypothesis that blood values (obtained from
                  complete blood counts and blood panel) do not differ
                  between samples collected from otters from oiled and non-
                  oiled areas.






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             D.   CAPTURE OF ADULT MALE SEA OTTERS

                  1.    Test the hypothesis that blood values (hematogram and
                        chemistry) do not differ among male sea otters living in
                        the oil spill zone and males living in non-oiled areas.

                  2.    Test the hypothesis that variation of DNA content in
                        lymphocytes does not differ among male sea otters living
                        in the oil spill zone and males living in non-oiled
                        areas.


                  3.    Test the hypothesis that DNA structure in sperm cells
                        (measured by the stability of nuclear chromatin) does not
                        dif f er among male sea otters living in the oil spill zone
                        and males living in non-oiled areas.

                  4.    Test the hypothesis that spermatogenic function, measured
                        by a DNA profile of the testicular cells, does not differ
                        among male sea otters living in the oil spill zone and
                        males living in non-oiled areas.

                  5.    Test the hypothesis that proportion of morphologically
                        normal sperm cells, measured by light microscopy, does
                        not differ among male sea otters living, in the oil spill
                        zone and males living in non-oiled areas.

                  6.    Test the hypothesis that levels of hemoglobin adducts,
                        measured   by   isoelectric    focusing    and    capillary
                        electrophoresis of hemoglobin, do not differ among male
                        sea otters living in the oil spill zone and males living
                       An non-oiled areas.

                  7.    Test the hypothesis that levels of plasma proteins,
                        including haptoglobin, quantified by gel electrophoresis,
                        do not differ among male sea otters living in the oil
                        spill zone and males living in non-oiled areas.

             E.   ANALYSIS OF POPULATION DYNAMICS BASED ON CARCASSES IN MORGUE

                  1.    Test the hypothesis that the sex and' age structure of
                        dead otters recovered during the 5-month period after the
                        oil spill did not dif f er among various geographic regions
                        and, hence, can be pooled for demographic analysis.

                  2.    Test the hypothesis that age structure of dead otters
                        collected after the spill does not differ from the age
                        structure of otters which died of natural causes before
                        the spill.

                  3.    Assess potential biases in the sample of dead otters
                        collected caused by differential mortality and/or
                        differential probability of carcass recovery.

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             4.   Develop sex and age specific survival schedules that
                  ref lect natural survival in the populations prior to the
                  spill.

             5.   Develop age specific fecundity schedules that reflect
                  natural reproductive rates in the populations prior to
                  the spill.

             6.   Construct a population model for assessing population
                  recovery in areas affected by the oil spill.

                                       METHODS


        BOAT SURVEYS

        Surveys will be conducted from small boats manned by an operator
        and two observers.

        A stratified random sampling design, including shoreline,
        coastal/pelagic and pelagic strata, will be used to meet Objectives
        A1-5.     Approximately 29% of the shoreline and 25% of
        coastal/pelagic and pelagic transects will be surveyed once in
        March 1990 and three times (one survey each in June, July and
        August) during summer 1990 jointly with Bird Study Number 2. All
        sea otters within transect boundaries will be recorded.            The
        shoreline stratum includes all water within 200 m of any shoreline,
        and will be surveyed by traveling 100 m offshore, parallel to the
        coast, at 5-10 knots.      The shoreline stratum is divided into
        transects consistent with those used during 1984-1985 surveys.
        (Irons et. al., 1988).     Sampling strip width and protocols are
        similar for pelagic surveys.

        Strip transect sampling is conducted under the assumption that all
        sea otters located within the transect are sighted.          If this
        assumption is not met, then population estimates are biased low.
        If sufficient time and resources are available, an attempt will be
        made to verify the boat-based observations with concurrent land-
        based observations during the summer 1990 field season.            The
        sightability assumption is not critical to this study however,
        since pre-spill observations were not corrected for this factor.
        Results produced by this study should be considered "estimates of
        surface abundance" or "population indices" rather than simply
        "population estimates".

        Abundance estimates will be calculated independently for shoreline,
        coastal and pelagic environments using ratio estimation techniques
        (Cochran, 1977).    Estimates calculated from second-year surveys
        will be compared to earlier estimates for the determination of
        injury to the sea otter population within PWS.        Differences in
        otter densities will be tested using two sample t-tests and/or
        ANOVA, dependent upon post-stratification of oil condition.


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             HISTOPATHOLOGY AND TOXICOLOGY

             Tissue samples for histology will be collected from dead sea otters
             recovered in or adjacent to habitats affected by oil. Histology
             samples will be sent to the Armed Forces* Institute of Pathology for
             processing and analysis.      For toxicology, duplicate samples of
             liver, kidney, skeletal muscle, bile and fat will be collected from
             each sea otter carcass that is recovered from the oil spill zone or
             areas outside of the oil spill zone that could serve as controls.
             Collection procedures will follow strict guidelines outlined by the
             Analytical Chemistry Working Group.

             Tissues from carcasses will be graded for degree of necrosis. Mean
             values for the degree of necrosis will be computed for tissues of
             sea otters of various age, sex, and location parameters provided
             sufficient sample sizes exist. Contaminant data from tissues will
             be stratified by degree of necrosis, and the effect of necrosis
             will be tested.    A comparison of contaminants will also be made
             between sea otter carcasses found in the oil spill zone and 4
             control animals examined in 1986. Data will be checked for
             normality; if needed, appropriate transformations will be made.
             Blood values and contaminant values will be compared between
             treatment and control groups using t-tests or ANOVAs, at a = 0.05.

             CAPTURE OF ADULT FEMALE AND JUVENILE SEA OTTERS

             In addition to sampling dead sea otters, fat and blood will be
             sampled from free-ranging sea otters residing in areas affected by
             the oil spill as well as from otters living in non-oiled control
             areas.   The sampling design calls for sampling of blood and fat
             from a total of 100 reproductively mature females and 100 pups and
             a total of 100 reproductively mature males. Up to 36cc of blood
             will be collected from captured animals.       At least 4cc of whole
             blood will be frozen for toxicology, and the remaining blood will
             be processed as needed for additional assays.         From sea otters
             which are implanted with transmitters, a small (1/2 inch diameter)
             piece of visceral fat will be removed prior to closing the incision
             and frozen for toxicology.

             The experimental design for the capture and telemetry study takes
             advantage of the fact that the oil from the EVOS directly covered
             less than one-half of PWS. That situation has been used to develop
             a treatment/ control study where the portion of PWS that was covered
             by the spill is the treatment area, and the unaffected portion of
             eastern PWS, specifically Port Gravina, Port Fidalgo, and Sheep
             Bay, is the control area.

             Intensive studies of sea otters using radio telemetry will
             concentrate on reproductively mature females and large pups in each
             area.   The pup portion of the study will be initiated in late
             summer, 1990. The female study was initiated in October 1989 but
             only 23 females in non-oiled habitat and 9 females in oiled habitat

                                               225










        were instrumented.   Additional radios will be put on females in
        spring 1990. Up to 50 reproductively mature females and pups will
        be instrumented in both the treatment and control areas.

        Sea otters will be caught primarily in unweighted tangle nets or
        dip nets. Tangle nets will be set in areas used by sea otters and
        anchored at one end. The nets will be monitored closely to prevent
        captured sea otters from fighting.       Captured animals will be
        removed from the nets and placed in holding cages and transported
        to a temporary holding cage.    Captive sea otters will be fed ad
        libitum a combination of fresh frozen dungeness crabs and razor
        clams.

        The transmitters will be implanted into the body cavity by a
        qualified veterinarian. Surgical procedures will follow Williams
        and Siniff (1983) and Garshelis and Siniff (1983).      Transmitters
        measure 311 x 211 x 111, weigh 120 g, and are coated with an inert
        material suitable for implantation in sea otters. The transmitters
        contain a coiled antenna and are powered by batteries that provide
        an operating life of up to 1,000 days. Following immobilization
        with a combination of fentanyl citrate and azaperone (Kreeger et
        al. 1989), abdominal surgery will be performed. During surgeryl
        the animal's status will be monitored by observation of capillary
        perfusion, color of mucous membranes, respiration rate and depth,
        and heart rate. While the sea otters are still anesthetized they
        will be marked with one Temple Tag in each of the flippers and
        implanted with a passive glass transponder chip (10 mm x 2 mm)
        injected under the skin in the gluteal area (Thomas et al. 1987).
        A 30cc blood sample will be taken from each sea otter for blood
        hemotograms and chemical analyses. A premolar will be removed from
        each adult for age determination.

        After release, attempts will be made to relocate each animal at
        least bi-weekly from either a boat or airplane. Attribute data
        for each relocation, including group size, whether or not the focal
        animals have pups, behavior of focal animal, sea condition, and
        presence or absence of tags, will be collected. During the pupping
        season and shortly following that period, efforts will be made to
        locate reproductively mature females at least weekly.

        Following instrumentation, efforts will also be made to locate pups
        at least weekly.     Previous studies have shown relatively high
        mortality at weaning in normal populations (Monnett, unpublished
        data). Therefore, frequent relocations of weanlings will increase
        the chances of recovery of carcasses as soon after death as
        possible.

        All fresh-dead sea otters found in either the treatment or control
        areas will be sent immediately for necropsy. Samples of tissues
        for contaminant and histology analysis will be collected.

        Alternatives to the implanting of transmitters were considered,

                                         226









           including 1) radio tracking devices attached to the outside of the
           animal, 2) dyes, 3) visual tags attached to flippers, and 4) no
           marking. External telemetry devices have been tried in the past
           but are easily damaged by the animal and the environment and have
           only an average of about 60 days operational time compared to up to
           3 years for internal implants. Dyes are not feasible in the marine
           environment and would adversely affect the animals fur.       Temple
           Tags,and a glass transponder chip will be used in conjunction with
           each implant but by themselves would not allow for the tracking of
           the animals.

           Using standard sample size calculations for testing the difference
           between two proportions (Snedecor and Cochran, 1967; p. 221), it
           was determined that a sample size of 50 gives a 79% chance of
           finding a significant difference at - = .2, given that the
           population proportion changes by .2 from an initial value of .5.
           A sample of 50 represents a minimum number at which significant
           differences between groups might be detected, and the maximum
           number that can be realistically instrumented and radio tracked.

           Reproductive rates are estimated by counting the number of females
           observed with pups divided by the total number of females.
           Estimates of survival and reproduction can be calculated over
           various time intervals.

           Reproductive data will be compared between treatment and control
           areas using contingency tables analysis.       Two-way contingency
           tables will be used except when interactions among age, sex, or
           location are of interest.    In that case three-way or multi-way
           contingency tables based on log-linear models will be used (Sokal
           and Rohlf, 1981).    Survival estimates will be obtained by the
           product limit method and differences in survival patterns will be
           tested with log-rank tests (Pollock et al., 1989).

           Data on movements and dispersal will be compared between treatment
           and control areas. Distance between successive locations, distance
           between extreme locations and the minimum convex polygon will be
           calculated for each radio-marked sea otter and stratified by sex,
           age and reproductive status (Garshelis and Garshelis, 1984; Ralls
           et al. 1988).   Dispersal distance, here defined as the shortest
           distance between the site of weaning (or the location of the last
           sighting of females with their pups) and the midpoint of their
           first established activity center will be compared for sea otter
           pups.


           CAPTURE OF ADULT MALE SEA OTTERS

           Proposed approaches to damage evaluation in male sea otters include
           analysis of blood panels, blood proteins, blood toxin levels, DNA
           content and structure (in blood lymphocytes, sperm and testis
           cells), and sperm morphology.


                                           227










    Blood panels (hematograms and chemistry) are a standard diagnostic
    procedure that will be used. Information on chemical damage will
    be obtained by examination of blood proteins.         Specifically,
    increased levels of hemoglobin adducts are indicative of chemical
    exposure (Sabbioni and Neumann, 1990; Tornqvist et al., 1988) and
    analysis of plasma proteins, with specific examination of
    haptoglobin binding, can also be of diagnostic value in assessing
    the health of an individual (Van Pilsum et al., 1986).

    Nuclear DNA content of blood lymphocytes is a sensitive indicator
    of damage to developing cells from clastogenic contaminants in the
    environment.   Cells can be measured by flow cytometry and, for
    normal samples, the resulting frequency histogram of DNA content
    should have a very low coefficient of variation. Deviations from
    the normal DNA content are detected as an increase in the
    coefficient of variation, reflecting damage to the chromosomes.

    Spermatozoa are another cell type in which damage to DNA is readily
    assayed by flow cytometry. (Evenson, 1986).         The structural
    stability of sperm nuclear DNA decreases after exposure to toxic
    compounds (Evenson, et al., 1985, 1989). The stability of the DNA
    is inversely related to male fertility (Ballachey et al., 1987;
    Evenson et al., 1980).    Morphology is an alternate indicator of
    genotoxic damage to sperm cells (Wyrobeck et al., 1983), and thus
    proportion of normal sperm in samples from otters living in oiled
    versus non-oiled regions will be compared. Testicular cells will
    also be collected by fine needle aspiration and examined by flow
    cytometry to determine proportions of germ cells.

    Males will initially be caught in two areas: 1) Western PWS; and
    2) Eastern PWS. The first area was directly affected by spilled
    oil and thus is the treatment area. The latter area will serve as
    a control.    Blood samples, testicular fine needle aspirations
    (Hendriks et al., 1969; Thorud et@al., 1978; Nseys et al., 1984;
    Sandqvist et al. , 1986; B. Purscell, pers. comm.) and electro-
    ejaculated sperm cells (Salisbury et al., 1978; Howard et al.,
    1986; Wildt et al., 1989) will be collected from each otter.
    Following analysis of these samples a decision will be made whether
    males should be caught in the following three areas: 1) The KP
    (treatment) ; 2) Kodiak Island (treatment)      and 3) Sitka, in
    southeast Alaska (control).

    It is estimated that a minimum sample size of 18 otters for each
    control area and 18 otters for each treatment area will be required
    to give an 80% chance of detecting a significant difference of .10
    in the proportion of damaged sperm cells between the groups at a =
    .05. Twenty animals from each treatment and control area will be
    sampled.

    Blood will be obtained by jugular venipuncture and'samples will be
    handled according to established protocols for the given tests.
    Complete blood counts and veterinary panels will be done on the

                                    228









                  blood samples.     A subsample of the blood will be allocated f or
                  measurement of DNA content of lymphocytes.

                  An additional subsample of blood will undergo assays f or hemoglobin
                  adducts. Plasma protein levels will be quantified.         DNA in testis
                  and sperm cells will also be measured. For flow cytometry of sperm
                  cells, samples will be prepared as described for the SCSA by
                  Ballachey et al. (1988).       For flow cytometry of the testicular
                  cells, the samples will be prepared as described by Thorud et al.
                  (1980). , A premolar will be taken from each otter for age
                  determination.      Otters will be tagged and implanted with a
                  transponder chip prior to release.

                  A one-way MANOVA will be used to test for differences among the
                  geographic groups, using a significance level of a = .05. Linear
                  contrasts will be used to make specific comparisons of the groups.
                  Analyses on various subsets of variables will be handled separately
                  (i.e., 1) blood panels, 2) blood    DNA/ lymphocytes, 3) blood proteins
                  and 4) sperm and testis cells). Toxicology data, when available,
                  will be analyzed in a similar manner.          Prior to analysis, the
                  variables to be tested will         be examined and transformed as
                  necessary to see that they meet     the assumptions of the MANOVA.

                  ANALYSIS OF POPULATION DYNAMICS     BASED ON CARCASSES IN MORGUE

                  All sea otter carcasses found in the spill zone have been kept in
                  frozen storage.     All carcasses not yet examined will be removed
                  from the freezer and thawed. Degree of decomposition and amount of
                  oil on the carcass will be used to subjectively place each animal
                  into one of three categories, killed during the spill due to
                  exposure to oil, died before the spill, and died during the spill
                  but unrelated to oil exposure. Standard body measurements (total
                  length, weight, bacula length) will be recorded for complete
                  carcasses and sex will be determined based on external genitalia or
                  tooth measurements if the carcass is not intact. Sections from a
                  premolar and canine tooth extracted from the skulls of each carcass
                  will be stained and mounted on slides with the age of each dead sea
                  otter estimated to the nearest year by counting the number of
                  cementum,   lines present      (Schneider    1973,    Garshelis     1984).
                  Reproductive tracts of all adult females which are not badly
                  decomposed will be examined for implanted fetuses, placental scars,
                  and corpora albicans. Where possible the approximate age and sex
                  of fetuses will also be determined.        All data collected on each
                  carcass as well as information available on recovery date and
                  location and comments will be incorporated into a database which
                  will be used for analysis.

                  The sex and age data will be summarized using a 2 x 3 x 4
                  contingency table, representing 3 geographic areas (PWS, KP, AP)
                  and 4 age classes (pup, immature, mature, and old) .           Log-linear
                  analysis will be used to test for differences between area, sex,
                  and age.

                                                     229









             The age structure of dead otters collected after the spill in the
             various geographic areas will be compared to the age structure of
             otters collected on beaches in PWS prior to the spill (Johnson
             1987). Significant post spill increases in prime age animals will
             be indicative of a major mortality event unrelated to normal
             mortality processes.

             Results from the analysis of age structure will determine if
             segments of the age structure data should be eliminated from
             survival rate estimation because of possible sampling biases. From
             these results the age structures will be constructed for survival
             estimation using techniques described by Chapman and Robson (1960)
             and Robson and Chapman (1961).      An initial analysis will be
             performed using all age classes and the model for constant
             survival. Chi-square tests will be used to test if the model of
             constant survival adequately fits the data.   If constant survival
             does not appear appropriate, the contribution of each age class to
             the chi-square statistic will be examined to determine which age
             classes the assumption of constant survival appears appropriate.
             The "segment" method (Chapman and Robson 1960) will then be used to
             estimate annual survival for these age classes. If data from other
             sources indicates that the assumption of a "stationary" population
             is not met the survival estimates will be adjusted using estimates
             of rate of change in the population (Eberhardt 1988). Estimates of
             the impact of senescence on the survival rates of the oldest age
             classes will be obtained by using minimum chi-square or nonlinear
             least square techniques to fit age structure data to the 3-
             component survivorship model developed by Siler (1979) and modified
             by Eberhardt (1985).      Estimates of the parameters in the
             survivorship model will then be used to construct an age-specific
             survival schedule for incorporation into a population model.

             Results of the examination of female reproductive tracts and the
             variability in sizes of fetuses will be used to construct a
             fecundity schedule.   These data will then be fit to Eberhart's
             (1985) fecundity model using minimum chi-square or nonlinear least
             square techniques. Estimates of the parameters in the fecundity
             model will then be used to construct an age-specific fecundity
             schedule for incorporation into a population model.

             A Leslie matrix (Leslie 1945, 1948) type population model will be
             constructed using the survivorship and fecundity schedules
             developed from the analysis described in objectives 4 and 5. The
             stable age distribution will be calculated using Lotka's (1907)
             equation as modified by Cole (1954) for populations where births
             are concentrated in a short time interval each year (Eberhardt and
             Siniff 1988).    This stable age distribution will be used to
             construct an initial population.   Population projections will be
             simulated using a commercial spreadsheet and the fecundity and
             survival schedules developed from the carcass data.             The
             performance of the simulated population will be compared to data on
             the general demographic characteristics of the PWS population

                                             230









        available from past and current telemetry studies.                  These
        comparisons will suggest adjustments to the fecundity and survival
        schedules and possibly incorporation of density dependent
        mechanisms into the model. A series of simulation experiments will
        then be conducted to explore possible recovery patterns of the PWS
        population following the mortality event caused by the oil spill.

                                     BIBLIOGRAPHY


        Ballachey, B.E., D.P. Evenson, and R.G. Saacke. 1988. The Sperm
              Chromatin Structure Assay: Relationship with alternate tests
              of semen quality and heterospermic performance of bulls. Jnl.
              Androl. 9(2):109-115.

        Ballachey, B.E., W.D. Hohenboken, and D.P. Evenson.                 1987.
              Heterogeneity of sperm nuclear chromatin structure and its
              relationship to bull fertility. Biol. Reprod. 36:915-925.

        Ballachey, B.E., H.L. Miller, L.K. Jost, and D.P. Evenson. 1986.
              Flow cytometry evaluation of testicular and sperm cells
              obtained from bulls implanted with zeranol. Jnl. Anim. Sci.
              63:995-1004.

        Bickham, J.W., B.G. Hanks, M.J. Smolen, T. Lamb, and J.W. Gibbons.
              1988. Flow cytometric analysis of the effects of low-level
              radiation exposure on natural populations of slider turtles
              (Pseudemys scrinta). Arch. Environ. Contam. Toxicol. 17:837-
              841.


        Chapman, D. G. and D. S. Robson.     1960.    The analysis of a catch
              curve. Biometrics 16:354-368.

        Cochran, W.G. 1977. Sampling Techniques. John Wiley and Sons, Inc.
              New York, New York. 428pp.

        Cole, L.C. 1954. The population consequences of life history
              phenomena. Quarterly Review of Biology. 29:103-137.

        Eberhardt, L.L. 1985. Assessing the dynamics of wild populations.
              J. Wildl. Manage. 49:997-1012.

        Eberhardt, L.L. 1988.      Using age structure data from changing
              populations. J. Applied Ecology 25:373-378.

        Eberhardt, L.L. and D.B. Siniff. 1988. Population model for Alaska
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              Evenson, D.P., P.H. Higgins, D. Grueneberg, and B.E. Ballachey.
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              Evenson, D.P. and M.R. Melamed. 1983. Rapid analysis of normal
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              Garshelis, D.L. and D.B. Siniff.              1983.     Evaluation of
                   radiotransmitter attachments for sea otters.          Wildl. Soc.
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              Garshelis, D.L. and J.A. Garshelis.            1984.     Movements and
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              Garshelis, D.L., J.A. Garshelis, and A.T. Kimker. 1986. Sea otter
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        Jameson, R.J. and A.M. Johnson. 1987., Reproductive characteristics
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        Johnson, A.M. 1987. Sea Otters of Prince William Sound, Alaska.
             Unpubl. Report, U.S. Fish and Wildlife Service, Alaska Fish
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        Kenyon, K.W. 1969. The sea otter in the eastern Pacific Ocean.
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        Kreeger, T.J., C; Monnett, L.M. Rotterman, and A.R. DeGange. 1989.
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        Nseyo, U.0., L.S. Englander, R.P. Huben, and J.E. Pontes. 1984.
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        Pollock, K.H., S.R. Winterstein, C.M. Bunch, and P.D. Curtis. 1989.
             Survival analysis in telemetry studies: the staggered entry
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        Ralls, K., T. Eagle, and D.B. Siniff. 1988. Movement patterns and
             spatial use of California Sea otters. In D.B. Siniff and K.
             Ralls (eds.) , Population Status of California Sea Otters. OCS
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         Schneider, K.B.    1973.   Age determination of sea otters.         Final
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              loss in California Sea otters. In D.B. Siniff and K. Ralls
              (eds.) , Population Status of California Sea Otters. OCS Study
              MMS 88-0021, USDI, Minerals Manage. Serv. pp. 13-32.

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              Freeman & Co., San Francisco, CA. 859 pp.

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                                            234









           Wyrobek, A.J., L.A. Gordon, J.G. Burkart, M.W. Francis, R.W. Kapp
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                                           235













        MARINE MAMMAL STUDY 6B

        Study Title:    Pre to Post Spill Comparisons of Sea Otter
                        Mortality in PWS Following the EVOS

                                    INTRODUCTION

        Much of the initial work to determine injury to sea otters caused
        by the EVOS focused on readily observable signs of acute injury.
        Efforts have since shifted toward studies to identify possible long
        term effects due to acute or chronic exposure to hydrocarbons in
        the environment. Changes in the characteristics of mortality (ie.
        mortality rates, age and sex composition of mortality) from pre to
        post spill time periods may be indicative of reduced viability of
        sea otters exposed to oil or hydrocarbon residues in the
        environment.

        Work by Kenyon (1969) and Johnson (1987) documented patterns of
        mortality for sea otter populations within areas at various stages
        of reoccupation. Their findings indicate extremely low levels of
        mortality for prime age otters.

        The Green Island area in southwestern PWS has a well established
        otter population and is within the oil spill zone. Johnson's study
        provides 10 years of base line mortality data for this area as well
        as 10 years of mortality data for the more recently established
        populations in the non-oiled, northeastern portion of PWS. Using
        the same beach survey methods as Johnson (1987) this study will
        seek to determine if the overall characteristics of mortality have
        changed from pre spill levels for both oiled and non-oiled
        habitats.


                                     OBJECTIVES

        A.    Test the hypothesis that post-spill levels of mortality
              (number of carcasses per linear kilometer of beach surveyed)
              are not different from pre-spill levels of mortality in PWS.

        B.    Test the hypothesis that the proportion of prime age carcasses
              found on beaches in post-spill surveys is not significantly
              different from proportions found in pre-spill beach surveys in
              PWS.

        C.    Test the hypothesis that the proportion of female carcasses
              found on beaches in post-spill surveys is not different from
              proportions found in pre-spill beach surveys in PWS.






                                         236












                                         METHODS


            Sampling Methods

            For valid comparisons, beaches surveyed and methods used will be
            the same as those used by Johnson (1987). Treatment beaches to be
            surveyed will include those on Green Island, Little Green Island,
            Channel Island, and the barrier islands northwest of Gibbon
            Anchorage on Green Island. Control beaches will include'those in
            the Hell's hole, Olsen Bay area of Port Gravina in the northeast
            portion of PWS. These beaches will be walked once during April or
            May before summer revegetation occurs which may hide old carcasses
            washed high on the beach.

            Skulls will be taken from carcasses and a tooth extracted for aging
            (Garshelis 1984). Any fresh carcasses collected will be necropsied
            as soon as possible and tissue samples for toxicology and
            histopathology will be collected. Badly decomposed carcasses or
            partial remains may have no evidence indicating the sex of the
            individual. In these cases, if a canine tooth is present and the
            carcass is that of an adult, sex may be determined by canine
            diameter (Johnson 1987, Lensink 1962).

            All teeth will be sectioned and prepared according to standard
            procedures.    Teeth will be read (aged) separately by two
            experienced readers with no knowledge of where the tooth was
            collected or other information on the carcass. Necropsies will be
            performed and histopathology samples will be prepared and analyzed
            according to standard protocols.

            DATA ANALYSIS

            Levels of mortality for a given year using beach survey data are
            influenced by a number of variables (ie. weather and current
            patterns, yearly changes in otter distribution and abundance) and
            are variable (2 to 34 carcasses found in any one year on Green
            Island area beaches). However multiple years data with associated
            variance will provide a basis for comparing pre and post spill
            mortality levels. To do so, only those beaches providing at least
            five years of pre spill data will be resurveyed for comparisons.
            A mean number of carcasses per kilometer of beach will be
            calculated for each pre spill year in both areas of PWS and for
            post spill data as they are collected.

            The proportion of prime age otter carcasses will be calculated for
            each year. Prime age in this study refers to those age groups with
            uniformly high survival rates as measured by pre spill data. Based
            on Johnson (1987), prime age are animals between 2 and 8 years old
            in the Green Island area and those between 2 and 10 years in the
            Port Gravina area.

            The proportion of female otters will be calculated for each year.

                                            237









        Changes in these proportions could reflect changes in the
        proportions   of  males   and   females   in  the   area due      to
        immigration/emigration or initially high mortality of one group at
        the time of the spill.     Changes may also reflect differential
        levels of chronic mortality between sexes due to unequal levels of
        susceptibility to hydrocarbon toxins or unequal levels of exposure
        to toxins because of spatial segregation.

        These three variables will be analyzed separately for the two
        areas. Pre spill data represent the control in this study and post
        spill data represent the treatment observations. Analysis will be
        a t-test using years as replicates for each dependent variable. In
        the case of the first post spill year's analysis the variance will
        be estimated entirely from pre-spill data.

        The most sensitive indicator of abnormal change in mortality will
        be the proportion of prime age carcasses found. This variable is
        not influenced by many of the confounding variables associated with
        the other two and a significant change in this parameter is the
        most meaningful biologically.

        A summary of Johnson's pre spill mortality data for the Green
        Island area shows the proportion of prime age carcasses relative to
        total carcasses found on beaches to range from 0.0 to 0.28 with a
        9 year average of 0.12 (n = 163 carcasses, SD = 0.094). Assuming
        post spill variability to be the same, a proportion of 0.32 or
        greater in the first post spill year would represent a significant
        increase in post spill, prime age mortality      at a = 0.20.      A
        proportion of 0.51 would represent a significant increase at a
        0.05.


                                   BIBLIOGRAPHY

        Garshelis, D. L. 1983. Age estimation of living otters. J. Wildlife
             Manage. 48(2):456-463.

        Johnson, A. M. 1987. Sea ot ters of Prince William Sound, Alaska
             Unpublished Report, U.S. Fish and Wildlife Service, Alaska
             Fish and Wildlife Research Center, Anchorage, AK.

        Kenyon, K. W. 1969. The sea otter in the eastern Pacific ocean.
             North Amer. Fauna 68. 352 pp.

        Lensink, C. J. 1962. The history and status of sea otters in
             Alaska. Ph.D. Thesis, Purdue Univ. 188 pp.








                                        238










            MARINE MAMM.AL STUDY NUMBER 6C


            Study Title:    A Drif t Study To Assess The Fate and Recovery of
                            Sea Otter Carcasses In PWS.


                                        INTRODUCTION

            Four hundred and ninety sea otter carcasses were recovered f rom PWS
            during the EVOS response.         Based on information from the
            rehabilitation ef fort and the recovery of carcasses throughout the
            spill zone, it is likely that more sea otters were killed in PWS
            than in any other area affected by the oil spill.      There are no
            data to indicate what proportion of the sea otter carcasses from
            PWS stayed within or drifted out of PWS and were lost or recovered
            elsewhere.
                                          OBJECTIVE

            Determine whether simulated sea otter carcasses (floats) deployed
            in PWS remain in or drift out of the Sound.


                                           METHODS


            Thirty simulated sea otter carcasses (floats) will be designed for
            the study. Design of the floats is crucial because the float must
            have drift characteristics similar to a sea otter carcass. Each
            float will be marked with a visible tag containing the address and
            phone number of the FWS should one of the floats be recovered. In
            addition, each float will contain a small radio transmitter with an
            external whip antenna that has an operating life 40-50 days.
            Floats will be deployed by boat in sea otter habitat affected by
            the oil spill in PWS. Ten floats will be deployed in PWS in three
            consecutive releases.    If feasible, deployment of ten simulated
            otter carcasses will be concurrent with the ongoing drift study,
            "An Assessment of Damage to Seabirds in PWS and the Western GOA
            Resulting from the EVOS.11

            Following release, the simulated carcasses will be relocated using
            fixed-wing aircraft outfitted with 2 4-element yagi antennas and a
            telemetry receiver.    Up to 8 hours per day for 25 days will be
            devoted to tracking the floats during the drift experiment. Within
            PWS fixed-wing aircraft will fly parallel to the shoreline at an
            elevation of 1500 ft.       A systematic search pattern will be
            developed for offshore areas within the Sound. Outside of the PWS
            the aircraft will fly at 3,000 ft over open water following a
            systematic search pattern. The aircraft periodically will search
            the coastline of the KP. Relocations will be marked directly on
            detailed charts or topographic maps of the study area and entered
            into the computer as UTM coordinates.

            Information on the recovery locations of sea otter carcasses from
            Prince William Sound and the Kenai Peninsula during the oil spill

                                             239










        response will be used to estimate recovery rates.

        Recovery locations f or simulated sea otter carcasses deployed in
        PWS will be stratified into two groups: recoverable (on shore or
        within 200 m of shore) or nonrecoverable (> 200 m off shore in the
        Sound or outside of PWS) . Floats that remain offshore or that are
        not found will be considered unrecoverable. The overall recovery
        rate of simulated carcasses in PWS will be estimated as the
        proportion of carcasses that drift into habitat in which they could
        have been recovered.


        BUDGET: FWS


                                   6A         6B        6C        Total

        Salaries             $     389.8      5.0         0     $    394.8
        Travel                      41.0      2.6         0.8          44.4
        Contractual                422.3      1.0        24.7        448.0
        Commodities                153.6      1.6         0.6        155.8
        Equipment                   53.8      0.8         7.4          62.0

        Total                $ 1,060.5        11.0       33.5     $1,105.0





























                                         240













             MARINE MAMMAL STUDY NUMBER 7

             Study Title:    Assess the Fate of Sea Otters Oiled and
                I            Rehabilitated as a Result of the EVOS

             Lead Agency:    FWS

                                         INTRODUCTION

             The capturej cleaning, and care of sea otters contaminated with oil
             during the EVOS oil spill has been the focus of considerable
             attention and effort. During the initial weeks of the spill, the
             health of many of the sea otters brought to the cleaning center in
             Valdez was severely compromised by exposure to the oil, and many
             died.   The chronic effects of exposure to oil on otters which
             survived and were released into the wild are unknown. Given that
             the underlying goal of the rehabilitation program was to release
             sea otters back into the wild as functioning members of their
             environment, it was important that a long-term evaluation of the
             process be undertaken.    This information will assist in guiding
             future cleaning operations for sea otters as well as aiding our
             understanding of how exposure to crude oil from the EVOS affected
             sea otters.   Forty-five rehabilitated sea otters were implanted
             with radio transmitters in summer 1989 and released shortly
             thereafter in eastern PWS. Radio tracking of those individuals is
             ongoing.   Twelve of the instrumented sea otters are known dead;
             several others are missing.     Preliminary evidence suggests that
             mortality of rehabilitated sea otters is higher than a sample of
             animals instrumented in eastern PWS as a control.


                                          OBJECTIVES


             A.   Test the hypothesis that survival of sea otters that underwent
                  oiling, cleaning, rehabilitation and release is not different
                  from that of sea otters that were not affected by the oil
                  spill.

             B.   Test the hypothesis that survival of rehabilitated sea otters
                  that re-enter oiled areas does not differ significantly from
                  that of rehabilitated sea otters that remain in oil free

                  areas.


             C.   Test the hypothesis that reproductive rate of female sea
                  otters that underwent oiling, cleaning and rehabilitation does
                  not differ significantly from that of female sea otters that
                  were not affected by the oil spill.

             D.   Document the movements of rehabilitated sea otters relative to
                  impacted habitat in western PWS and the KP.



                                              241













                                      .METHODS

        Sampling Methods

        Thirty-six of the instrumented sea otters were from the KP, either
        from the Valdez, Seward, or Homer sea otter facilities.           The
        remaining nine implanted otters were from PWS.        comparisons of
        effects of severity of oiling as well as the effects of fresh crude
        oil vs. weathered crude oil on survival of sea otters released back
        into the wild were intended; however, because only nine sea otters
        oiled in PWS with fresh crude oil were suitable for implantation,
        analyses comparing the effects of oil type and degree will be
        limited.

        Forty-five rehabilitated' sea otters were instrumented prior to
        release.   A sample of 50 female sea otters from non-oiled areas
        instrumented as part of Marine Mammal Study Number 6 (M/M) will
        serve as control. Twenty-three control animals were instrumented
        in eastern PWS during fall 1989.       Additional animals will be
        instrumented this spring as part of the control group. An existing
        population of 58 radio-marked sea otters in the vicinity of the
        release sites for the rehabilitated sea otters is also available
        for comparison.    For specifications on the transmitters and the
        implant protocol see the study proposal for M/M Study 6.

        Using standard sample size calculations   for testing the difference
        between two proportions (Snedecor and C   'ochran, 1967; p. 221), a
        sample size of 45 gives a 75% chance of finding a significant
        difference at alpha = 0.20, given that the population proportion
        changes by .2 from an initial value of .5.

        All sea otters used in this study were released back into the wild
        in eastern PWS as recommended in the FWS Draft Release Strategy
        Plan for Rehabilitated Sea Otters.      The release sites were not
        directly affected by oil from the spill and were occupied by sea
        otters prior to the release. Male sea otters were released in a
        male area in Nelson Bay. Females were released in a female area in
        Sheep and Simpson bays.    The release sites represent a short to
        moderately long translocation for sea otters captured in western
        PWS and along the KP.

        After the initial 20 day monitoring period, the frequency of
        relocation has depended upon weather, and the sex, age,
        reproductive status, and whereabouts of the marked animals.
        Relocations of all animals were intended to be made at least
        biweekly but for many of the animals, movements have been erratic
        and unpredictable, therefore they have been difficult to relocate.
        During the pupping season and shortly following that period
        attempts will be made to locate reproductively mature females at
        least weekly although this will be impossible for some animals.
        Those animals captured on the KP and that have returned to the KP

                                         242









           have only been relocated occasionally.

           Attribute data for each relocation, including group size, number of
           pups in the group, whether or not the focal animals had a pup, and
           behavior of focal animal, are also collected. At the end of each
           workday, locations of each otter are entered directly into a
           computer along with the attribute data. Periodically, those data
           are transferred to Anchorage, where they will be analyzed using
           geoprocessing software and statistical software, including SAS. In
           addition to location fixes, qualitative assessments of the health
           status of each rehabilitated sea otter are being made.

           Marked sea otters that have died following release are collected as
           soon as possible.    Carcasses that are in suitable condition are
           necropsied.    Tissue samples will be taken for histology and
           toxicology.

           Additionally, histopathological samples taken from sea otters that
           died in the rehabilitation centers will be analyzed. Results will
           be important for analyzing the effectiveness of current
           rehabilitation techniques, for assessing the need for changes in
           these techniques and for providing other information important for
           future sea otter restoration efforts.


           Estimates of survival rate of the rehabilitated sea otters will be
           calculated for comparison with those of control animals and other
           populations of sea otters within and outside of Alaska.

           Survival estimates will be obtained by the product limit method and
           differences in survival patterns will be tested with log-rank tests
           (Pollock et al., 1989).

           Reproduction and movements of rehabilitated, implanted and control
           sea otters will also be examined in the proposed study.           The
           reproductive rate will be estimated for each population by counting
           the number of females in each group (rehabilitated and controls)
           observed with a pup, and dividing this value by the total number of
           females observed.     However, given the small sample sizes of
           females, sufficient data to examine reproductive rates in a
           rigorous statistical sense may not be available. Several measures
           will be used in the analysis of movements including distance
           between successive locations, minimum convex polygon, and distance
           between extreme locations (Garshelis and Garshelis 1984, Ralls et
           al. 1988).   Since many of the sea otters that make up the radio
           telemetry portion of this study were originally captured on the KP
           and in western PWS, the release sites represented a short to
           moderate translocation. The influence of translocation distance on
           movements of sea otters will be examined by regressing
           translocation distance on the daily rate of movement or on
           dispersal distance. Dispersal distance is defined as the distance
           from point of release to the location of the translocated sea
           otter's first activity center at which it becomes sedentary.

                                           243












                                     BIBLIOGRAPHY


         Garshelis, D.L. and J.A. Garshelis. 1984. Movements and
                    management of sea otters in Alaska.     J. Wildl. Manage.
                    48:665-678.

         Kleinbaum, D.G. and L.L. Kupper. 1978. Applied Regression
                    Analysis and Other Multivariable Methods.           Duxbury
                    Press/Wadsworth Publishing Co., Belmont, California.

         Pollock, K.H., S.R. Winterstein, C.M. Bunch, and P.D. Curtis. 1989.
                    Survival analysis in telemetry studies: the staggered
                    entry design. J. Wildl. Manage. 53: 7-15.

         Ralls, K., T. Eagle, and D.B. Siniff. 1988. Movement
                    patterns and spatial use of California Sea otters. In
                    D.B. Siniff and K. Ralls (eds.), Population Status of
                    California Sea Otters.     OCS Study MMS 88-00211 USDI,
                    Minerals Manage. Serv. pp. 33-63.

         Siniff, D.B. and K. Ralls. 1988. Reproduction, survival and
                    tag loss in California Sea otters. In D.B. Siniff and K.
                    Ralls (eds.) , Population Status of C@lifornia sea otters.
                    OCS Study MMS 88-0021, USDI, Minerals Manage. Serv. PP.
                    13-32.

         Snedecor, G.W. and W.G. Cochran. 1967. Statistical Methods. 6th
                    Edition. The Iowa State University Press, Ames, IA.

         Sokal, R.R. and F.J. Rohlf. 1981. Biometry. 2nd Edition. W.H.
                    Freeman and Co., San Francisco.




         BUDGET: FWS


         Personnel                       $     42.5
         Travel                                  5.0
         Contractual                           87.6
         Commodities                           11.0
         Equipment                         -     0.9

         Total                           $     147.0










                                           244












                          TERRESTRIAL MAMMAL INJURY ASSESSMENT



           Terrestrial mammals are an important part of the ecosystem in the
           area affected by the EVOS. A wide variety of species are present,
           many of which use intertidal habitats that were heavily impacted by
           oil. They are important to humans for recreational viewing, sport
           and subsistence hunting, and commercial and subsistence trapping.

           In the 1989 damage assessment plan, 19 terrestrial mammal species
           were identified as potentially being impacted by oil. Of those,
           five were selected for intensive field study and nine were chosen
           for general assessment only. In 1990, intensive damage assessment
           studies in the field will be continued for three species: deer,
           river otter, and brown bear.     A literature review will also be
           completed to gather information on the importance of intertidal
           habitat use by black bear.

           The deer study will focus on detection of lethal injury during the
           spring of 1990 when deer are concentrated on beaches and,
           therefore, most likely to come in contact with oil.          If no
           mortality attributed to oil is detected, this project will be
           discontinued. The river otter and brown bear studies will explore
           both lethal and sublethal injury. The river otter study includes
           examination of animals found dead and assessment of oil impacts on
           populations, food habits, and habitat use. The brown bear project
           will examine mortalities and assess impact on reproduction and
           population density

           A laboratory study to determine the influence of hydrocarbons on
           reproduction in ranched mink will also be conducted. It commenced
           in 1989 and will end in July 1990.         The work will provide
           information on whether sublethal doses of hydrocarbons will
           influence reproduction in mammals. Mink will provide a model for
           other related terrestrial and marine mammal species.


















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       TERRESTRIAL MAMMAL STUDY NUMBER I

       Study Title:    Assessment of the Effect of the EVOS on the Sitka
                       Black-tailed   Deer   in   PWS   and    the    Kodiak
                       Archipelago

       Lead Agency:    ADF&G

                                   INTRODUCTION

       Sitka black-tailed deer (Odocoileus, hemionus sitkensis) are the
       most abundant large mammal on the islands of PWS and the Kodiak
       Archipelago.   ADF&G wildlife biologists estimate that there are
       15,000 to 20,000 deer in PWS and up to 100,000 deer on the Kodiak
       Archipelago.     In addition to the intrinsic values of this
       resource, it also has a substantial economic value to residents
       of Alaska.

       During late winter and early spring, deer in PWS and Kodiak
       usually concentrate on beaches and along a relatively narrow
       fringe near the coast (ADF&G 1986). Groups of over 500 deer have
       been observed on some beaches.       These areas commonly have a
       reduced snow depth or are snow-free, and deer forage on
       intertidal marine vegetation, coastal sedges, grasses, shrubs,
       and herbaceous vegetation in the forest understory (ADF&G 1986).

       Hinchinbrook, Montague, and Hawkins Islands contain most of the
       deer habitat in PWS. Beaches on Hinchinbrook and Hawkins Islands
       generally were not effected by the EVOS, whereas the northern
       portion of Montague Island was lightly oiled. Deer also occur in
       relatively high densities on some of the other islands in PWS
       that were heavily impacted by oil. Deer are abundant throughout
       the Kodiak Archipelago.     Light to very light EVOS impacts were
       reported along most Kodiak beaches, with heaviest concentrations
       occurring on the east side of Shuyak Island and along portions of
       Uyak Bay on Kodiak island.

       oil has affected several types of coastal deer forage, and deer
       have been observed feeding on oiled kelp. It is anticipated that
       deer will be adversely affected if they consume vegetation that
       has been contaminated by oil. Small to moderate amounts of crude
       oil consumed by deer and other ruminants may cause, direct
       mortality due to disruption of the rumen fermentation process and
       aspiration of rumen fluid into the lungs (Rowe et al. 1972).
       Sublethal injury also could occur, reducing animal health and
       affecting reproduction.

       When oil reached beaches where deer were concentrated in late
       March/April 1989, snow was already melting in upland areas. Some
       deer had begun their annual spring movements away from the coast
       and into higher elevations.         This fact, coupled with the
       substantial increase in human activity on beaches soon after the

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             spill undoubtedly reduced the potential for deer exposure to oil.
             However, the increased human activity probably pushed some deer
             away from preferred beach feeding areas prematurely, forcing them
             into areas with deeper snow.           This would cause accelerated
             mortality because the energy reserves of deer are at an annual
             low state during late winter/early spring.                Unfortunately,
             quantification of such additional       indirect "natural" mortality
             was not possible.     The winter and spring of 1989-90 may be the
             best time to investigate potential impacts of oil on wintering
             deer.    If winter temperatures and snowpacks are within normal
             limits, deer will concentrate along      beaches sometime in the mid-
             December to mid-February period and human activity will be far
             less than it was from late March through late fall 1989.

                                            OBJECTIVES

             A.    Test the hypothesis that deer on heavily oiled islands have
                   tissues and rumen contents that have been contaminated by
                   oil.

             B.    Test the hypothesis that deer found dead have rumen contents
                   in their lungs.

             C.    Estimate the number of dead deer per unit area on both a
                   heavily oiled and a non-oiled island in the Sound, if
                   substantial numbers of deer concentrate on oiled beaches in
                   the late winter of 1989-90, and there is evidence to suggest
                   that some of these deer are dying from oil contamination.



                                             METHODS

             A sample of live deer has been collected and examined for
             hydrocarbon contamination.       Deer were collected in areas near
             beaches in PWS and the Kodiak Archipelago that had been affected
             by oil.     These collections       occurred during various periods
             throughout  the year.

             Deer were   collected on Afognak Island on 7 April 1989, prior to
             any reported EVOS impacts in the area.            Tissue samples were
             collected, wrapped in Reynolds aluminum foil and frozen for
             histopathological analysis.

             Deer near oiled beaches on Shuyak Island were taken on 4 May
             1@89.    Necropsies were conducted by a pathologist immediately
             after    collection    and   tissue   samples    were   collected     for
             histopathology and hydrocarbon analysis.           Additional deer on
             Shuyak and from PWS were taken near oiled beaches from-* 31 May
             through 15 June 1989.       Gross necropsies were performed in the
             field,. by wildlife biologists and tissue samples were collected
             for histopathology and hydrocarbon analysis.


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       Live deer were collected in oiled areas or areas that are known
       to be heavily used by deer hunters in PWS and the Kodiak
       Archipelago during August and September 1989.      Gross necropsies
       were performed in the field by wildlife technicians and tissue
       samples were collected for future histo3athology and hydrocarbon
       analysis.   Small amounts (approx. 2 cm ) of liver and skeletal
       muscle from each animal were boiled, smelled and tasted in an
       attempt to detect obvious evidence of oil contamination.

       Additionally, several samples were made available for analysis
       from dead deer collected by ADF&G staff on or near oiled beaches
       in PWS in April, and from deer found dead and turned in by
       various workers associated with the EVOS throughout the spring
       and summer of 1989.

       Flights will be made over selected beaches in PWS as often as
       possible, but not more frequently than every two weeks during the
       winter of 1989-90. If information obtained during these flights,
       or observations from individuals in the field indicate that deer
       are concentrating on oiled beaches, additional deer collections
       will be made and searches conducted for dead deer in those areas.
       If deer behavior, gross necropsy, and examination of lungs
       suggest that deer are dying from oil, systematic surveys will be
       conducted on a heavily oiled island and a control island of
       similar size, topography, and deer density. The carcass of each
       dead deer that is found will be examined in the field by a
       biologist and recent mortalities will be examined by a
       pathologist. Pellet group counts on each island will be done to
       correct for different deer densities (Kirchhoff and Pitcher
       1988a, Kirchhoff and Pitcher 1988b). If it is assumed that deer
       carcasses are distributed in a "patchy fashion", a systematic
       sampling scheme should be close to optimal (Snedecor and Cochran
       1980) and this procedure will provide an estimate of deer
       mortality per unit area on each island.

       Throughout all phases of this study we will attempt to identify
       potential alternative methods and strategies for restoration of
       lost use, populations, or habitat if injury is identified.        The
       final report will include a listing of suggested ways to address
       long-term restoration projects.

       Tissues which were collected will be analyzed as outlined by the
       EVOS Histopathological Technical Group and the Hydrocarbon
       Technical Group.    All statistical analyses will be performed at
       an alpha level of 0. 05.      Sample sizes will be used that are
       adequate to detect at least 1 deer affected by oil contamination
       with a given percentage of certainty, for varying proportions of
       the population contaminated by hydrocarbons.      These sample size
       calculations are based on a binomial distribution (Mendenhall,
       Schaffer and Wackerly 1981), and assume that the sample size is
       very small compared to the population total.        This study will
       assume that at least 10% of the deer population was affected by

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            EVOS; therefore, a total sample of at least 29 deer will be
            collected to be 95% certain of collecting at least one deer
            affected by hydrocarbons.



                                        BIBLIOGRAPHY

            Alaska Department of Fish and Game. 1986. Alaska Habitat
                   Management Guide - Life Histories and Habitat
                   Requirements of Fish and Wildlife. Ak. Dept. Fish and
                   Game.' 763pp.

            Kirchhoff, M.D. and K.W. Pitcher. 1988a. Evaluation of
                   methods for assessing deer population trends in
                   southeast Alaska. Fed. Aid Wildl. Res. Prog. Rep. W
                   -22-6. Job 2.9. 13pp.

            Kirchhoff, M.D. and K.W. Pitcher. 1988b. Deer pellet-group
                   surveys in southeast Alaska, 1981-1987.     Fed. Aid
                   Wildl. Res. Prog. Rep. W-22-6. Job 2.9. Objective 1.
                   13pp.

            Mendenhall, W., R.L. Schaffer, and D.D. Wackerly. 1981.
                   Mathematical Statistics with Applications. Duxbury
                   Press. Boston, Mass. 686 pp.

            Rowe, L.D., J.W. Dollahite, and B.J. Camp. 1972. Toxicity
                   of two crude oils and of kerosine to cattle. J. Amer.
                   Vet. Med. Asso. 162(2):61-66.

            Snedecor, G.W. and W.G. Cochran. 1980. statistical
                   Methods; Seventh Edition. Iowa St. Univ. Press.
                   Ames, Iowa. 507 pp.



            BUDGET: ADF&G


            Personnel                     $ 60.6
            Travel & per them                  4.0
            Contracts & Services              52.0
            Supplies                           8.0
            Equipment                          0.0

            TOTAL                         $ 124.6










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        TERRESTRIAL MAMMAL STUDY NUMBER 2

        Study Title:    Review of Literature on Intertidal Habitat Use by
                        Black Bear


        Lead Agency:    ADF&G

                                    INTRODUCTION

        There is a dense population of black bear (Evarctos americanus) in
        PWS. They are omnivorous, opportunistic feeders near the top of
        the food chain. Black bears are known to feed in intertidal areas
        and, therefore, have the potential to contact oil directly by
        eating sludge washed ashore, grooming oiled hair, eating
        contaminated intertidal organisms, or scavenging carcasses of
        mammals and birds killed by oil offshore and deposited on beaches.


        A study of the impact of the EVOS on black bear populations was
        proposed in t 'he 1989 damage assessment plan.   That effort proved
        not feasible, given the logistical difficulties of bear capture in
        the densely forested habitat of PWS.        The literature search
        proposed for 1990 will provide helpful background information for
        evaluating the need for a revised detailed population study.

                                     OBJECTIVE


        Determine importance of intertidal habitat use by black bear to
        establish the likelihood of significant impact due to beached oil.

                                      METHODS

        Black bear literature will be searched to identify and retrieve any
        information on the importance of intertidal habitat use.. The final
        product will include a list of citations accompanied by abstracts
        of each paper and a summary that includes relevant information from
        all sources.


        BUDGET: ADF&G

        This study will be a contract for a period March 1, 1990 - February
        28, 1991 and will not exceed $10,000.

        Contract                  $ 10.0

        Total                     $ 10.0








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           TERRESTRIAL MAMMAL STUDY WUMBER 3


           Study Title:   Assessment Of The Effect Of The EVOS On River
                          Otters In PWS


           Lead Agency:   ADF&G

                                      INTRODUCTION

           River otter (Lutra canadensis) populations in PWS rely on
           intertidal and subtidal environments for food.          Studies of
           similar coastal populations in southeastern Alaska documented
           that marine fishes, crabs, and other invertebrates dominated food
           habits (Larson 1983, Woolington 1984).    Because critical habitat
           for this species was heavily contaminated by oil, otter
           populations are at risk by direct contact with oil or by
           environmental changes to other habitat components.         Data on
           density prior to the oil spill are lacking, but river otters were
           probably abundant. The goal of this study is to determine if the
           EVOS will have measurable effects on these populations.          The
           approach is to 1) examine carcasses to determine direct effects
           of oil, 2) compare pre- and post-spill river otter dietary
           information from scats, 3) validate the use of a control area and
           then, 4) compare population density and various biological
           aspects between oiled and control study areas.

           Necropsy and tissue samples obtained from otter carcasses
           recovered from oiled beaches will provide information on possible
           short-term impacts.    Magnitude of short-term loses cannot be
           measured directly because the proportion of recovered carcasses
           is unknown.

           This study will use parallel data collected in a control area
           (Esther Passage) and an area heavily contaminated by oil (Knight
           Island) to test for impacts on river otters.      Radio telemetry,
           rates of fecal deposition, food habit analysis, home range
           determinations, and analyses of habitat selection by otters will
           provide population characteristics, trends, and indexes for
           comparing the two areas.       Additionally, necropsy and tissue
           analyses of animals collected outside of the study areas will
           provide data on presence of hydrocarbons and their long-term
           effects on individual animals.     Results f rom the study on the
           effects of hydrocarbons on captive mink (Terrestrial Mammal Study
           Number 6) will provide the context for interpreting hydrocarbon
           levels in river otters.










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                                    OBJECTIVES
        Direct Effects

        Al -       Determine cause of death for river otter recovered from
                   oiled  areas   via   necropsy   and    histopathological
                   procedures.
        A2 -       Test (a = 0. 05) for higher hydrocarbon levels in river
                   otter in oiled versus unoiled areas.

        Population Change

        Bi -       Estimate population sizes of river otter within 10% of
                   the true value 95% of the time, on representative oiled
                   and control study areas using mark-recapture methods
                   and test (a = 0.05) for lower population levels in
                   oiled versus control areas.
        B2         Estimate the rate of fecal deposition within 10% of the
                   true value 95% of the time for river otter. This rate
                   will be used as an index to population size to test (a
                   =' 0.05) for lower rate of deposition in oiled versus
                   control study areas.
        B3         Test  (a = 0.05) for lower survivorship of river otter
                   in oiled versus control study areas.

        Food Habits

        B4 -       Test (a = 0.05) for differences in food habits of river
                   otters before and after the oil spill on the oiled
                   study area.
        B5 -       Test (a = 0.05) for differences in food habits of river
                   otters on oiled and control study areas.

        Habitat Use

        B6 -       Test (a = 0.05) for differences in activity patterns
                   (foraging) of river otters between oiled and control
                   study areas. (limited funding and man power may not
                   allow data collection for this objective)
        B7 -       Use homerange size and use patterns to test (a      0.05)
                   for differences in habitat selection in river otters
                   between oiled and control study areas'.

                                      METHODS

        The initial impact assessment concentrated on locating two study
        areas (control vs. oil impacted) with comparable numbers of
        active latrine sites for mink and river otters.      Each site was
        given a unique name, plotted on a map and field marked for future
        relocation, and a site d rawing with a rough description made in a
        field notebook.     Sites were cleaned of all scats and then
        revisited five times between June and September 1989, to obtain

                                        252










            data on continued use.     After the initial visit the number of
            scats present were recorded in addition to scat collection for
            later analyses.

            information obtained during the 1989 initial study for impact
            response was used in developing the study design for this
            project.   The 59 latrine sites in the control area and 57 sites
            in the oiled area will be the focus of efforts to live capture
            otters.   Most of these sites will also be utilized to provide
            scat samples for the study.       With qualifications, information
            obtained on otter densities, habitat selection, and population
            response to oil will be available for extrapolation to other
            areas of PWS.    Standard operating procedures will be developed
            for each segment of the long range study to insure data validity.

            The following are methods for collecting data by objective.

            Direct Effects.

            Al-  Necropsy and histopathology will be performed according to
                 standard procedures.
            A2   Up to 20 additional animals may be collected outside the
                 study area to provide hydrocarbon and histological samples.
                 Necropsy and similar tissue analysis will continue to be
                 made on dead otters found throughout the entire area
                 impacted by the oil.

            Population Change

            Bi-  River otters will be live trapped at latrine sites in the
                 control and oiled study areas. Modified Hancock live traps
                 and drugging boxes to hold otters, as described by Me1quist
                 and Hornocker (1979), will be used. Weather permitting, all
                 traps will be monitored daily.     All traps will be equipped
                 with a transmitter that signals a sprung trap. Animals will
                 be held only as long as necessary to complete the marking
                 process and provide for their recovery from surgery.
                 Animals will then be. released at their original capture
                 site.

                 Techniques for implantation of radio transmitters will be as
                 described by Woolington (1984).     Surgery will be done by a
                 licensed    veterinary/biologist     or    project     personnel
                 specifically trained in the technique.     Each transmitter is
                 equipped with a "mortality model' so the fate of individual
                 animals can be determined.

                 Radioisotope implants in otters will be used to estimate
                 population density in the oiled and control study areas
                 using a mark-recapture method.           Marking will be by
                 implantation of radio-labeled, polylactic acid (PLA) tablets
                 to provide a long lasting tracer that can be detected in
                 feces (scats) of river otter (Crabtree et al. 1989).
                 Recoveries of scats from latrine sites will provide the
                 "recaptures" for analysis.      This mark-recapture technique

                                             253









             has been employed in carnivore studies (Kruuk et al. 1980),
             including river otters (Knaus et al. 1983, Shirley et al.
             1988).

             Animals instrumented with VHF transmitters will have radio
             labeled PLA tablets implanted intra-peritoneally. This
             method allowed detection for over 10 months in the scats of
             coyotes (Canis latrans) (Crabtree et al. 1989).       A gamma
             spectrometer will be used to detect and identify radio
             labeled scats.

             Sampling of latrine sites will provide the "recaptures" for
             simple mark-recapture analysis (Seber 1982).     Twenty river
             otters in each study area will be uniquely marked. A closed
             population model will be used, employing radio transmitters
             to determine exactly how many marked animals are resident in
             the study area while scats are being sampled.             Mark-
             recapture models for closed populations are well established
             (Dennis et al., In Press; Seber 1982).     Latrine sites will
             be cleared of scats at the start of a sampling period, and
             visited every one to two days until a predetermined number
             of scats has been collected.

             The distribution of marked animals is likely not to be
             random, due to the necessity of focussing our capture effort
             in locations of high animal abundance. Biases can result if
             the recovery of scats is uneven across low and high density
             areas within each main study area. A special effort will be
             made to randomize the recovery of otter scats to ensure
             every scat is equally likely to be collected.

        B2_  Rates of fecal deposition will be used as an index to
             population size in oiled and control areas.          The same
             latrine sites used for mark-recapture population      estimates
             will be used for estimating fecal deposition rates.

        B3_  Estimates of survival will depend on data obtained from
             otters instrumented with radio transmitters. Data will be
             obtained coincidental to data gathered for objectives B1.
             B6, and B7-

        Food Habits

        B4 & B5_  Food habits of river otter will be described from prey
             remains in their feces.      Such procedures have been used
             successfully in past studies (Gilbert and Nancekivell 1982).
             A preliminary survey of latrine sites conducted in late
             April and early May 1989, located 59 latrines in the control
             area and 55 latrines in the oiled area.            Feces were
             collected at each site and resampled four times.

             Scats from river otter will be distinguished from those of
             other mammals by their characteristic morphologies (Murie
             1954).


                                        254









                  Latrine sites will be resampled when snow free in late
                  spring 1990 in the same manner as those collected following
                  the oil spill.    Thereafter, we tentatively plan to collect
                  feces from latrines 1-2 times/week from June through mid-,
                  September 1990 on both control and oiled areas.

                  Laboratory analysis of prey remains in feces of river otter
                  will follow standard procedures (Bowyer et al. 1983).

                  Because of differential digestibility of prey and variable
                  rates of passage through the gut, volumetric measures of
                  prey    remains    in   mustelid    feces    are     meaningless.
                  Consequently, the analysis will be confined to the
                  occurrence of prey items in latrines and will be expressed
                  in terms of percent of latrines with food items, and percent
                  of total food item (Bowyer et al. 1983).         To assure that
                  subsamples from a latrine are representative of that site,
                  all feces from that site will be mixed and a series of
                  subsamples (about the volume of an individual scat) will be
                  drawn and analyzed separately. Sampling will continue until
                  the function between number of prey items and number of
                  samples becomes asymptotic.      All latrines included in the
                  analysis, however, will contain at least five scats per
                  sampling period.

                  Because sample variance is unknown, it is not possible to
                  specify the total number of samples necessary to adequately
                  describe food habits at this time.      Reduction in variation
                  of.the mean with increasing sample size (of latrines) will
                  be monitored for important food items to ensure that all
                  proportions are estimated within 0.05 of their true value
                  95% of the time (Kershaw 1964).

            Habitat Use

            B6-   Activity patterns of radio equipped river otters will be
                  used to test for changes in the availability of prey between
                  oiled and control study areas. A digital recorder linked to
                  a radio receiver will be operated to record activity of
                  otters.

            B7_   Data on home range and habitat selection of individuals will
                  be collected daily using radio-locations of telemetered
                  animals. Telemetry will be conducted from a small boat, and
                  the entire coastline of both study-areas (oiled and control)
                 'will be sampled each day.           Because river otter are
                  distributed immediately along coastal areas (Larsen 1983,
                  Johnson   1985),   telemetry    "fixes"   will   be made over
                  relatively short distances, and multiple "legs" can be-used
                  in triangulation.     Consequently, error polygons should be
                  small and biases from animal movements during triangulation
                  will be minimal. Locations determined via telemetry will be
                  confirmed visually whenever possible.



                                              255









             The time at which a telemetry transect starts will be
             randomized each day to help minimize any bias from duel
             activities of the mustelids on estimates of home range size
             and habitat selection.   Further, aerial telemetry will be
             conducted as needed to determine locations of individuals
             that cannot be located by boat. Telemetry transmitters will
             be equipped with a mortality signal that will allow the
             speedy recovery of dead animals.

       Methods for analyzing data are detailed below for each objective.

       Direct Effects

       Al-   A cause of death will be assigned each mink or river otter
             carcass based upon a necropsy report and lab analysis of
             tissue specimens.  Hydrocarbon levels will be presented for
             all usable samples.

       A2_   A one-tailed Z test for proportions (Snedecor and Cochran,
             1980) will be used to test this hypothesis.

       Population Change

       B1_   Analysis will follow methods described by Dennis et al., (In
             Press) for sampling a closed population with replacement.
             Population size and 95% confidence intervals for both
             control and oil affected areas will be estimated.     A one-
             tailed' Z statistic will be used to determine if the
             population density is lower in the oiled area versus the
             control area.     This test assumes that the population
             estimates are normally distributed and have equal variance
             (Seber 1982).

       B2_   Differences in rates of scat deposition between oiled and
             control study areas will be tested (a = 0.05) with a single
             factor covariance analysis model (Neter et al. 1983).     The
             response variable will be rate of scat deposition and the
             covariate will be the number of latrine sites.           Main
             effects will include oiling and months of study.     Since a
             one-tailed hypothesis is being tested with regard to the
             oiling main effect, the critical region for this section of
             the ANOVA table will be one-tailed.    If variances are not
             homogeneous, either a ranked procedure will be employed or
             the data will be transformed to obtain homogeneous variance
             or normality.

       B3    Estimation and analysis of survival distributions for radio
             marked individuals will follow standard procedures (Pollock
             et al. 1989). Model assumptions include a random sample of
             animals, that survival times are independent for different
             animals, and that censoring mechanisms are random.





                                       256












            Food Habits

            B4 and B5_ Statistical analysis will include only food
                 items that compose at least 10% of the diet. Comparisons of
                 food habits between oiled and control areas and among months
                 will be made with the Quade test including multiple
                 comparisons of food items (Conover 1980).

            Habitat Use

            B6-  It is hypothesized that if availability of forage fishes in
                 the subtidal zone were reduced due to oil, otters would
                 spend more time foraging to obtain a diet equivalent to that
                 in the control area. Because study areas were selected that
                 contained similarly high populations of otters, it is
                 presumed that both otters and their food were abundant prior
                 to the oil spill. The oil spill may have reduced both river
                 otters and their prey.         Consequently, the foraging
                 activities of otters could be expected to change with both
                 their population size and that of their prey.

                 Although this procedure will allow assessment of a reduction
                 in otters or a reduction in their prey, it will not detect a
                 simultaneous reduction in both.

                 Differences in activity of river otters (stratified by sex
                 and age class) between oiled and unoiled study areas will be
                 tested (a = 0.05) with a two-tailed Mann-Whitney test
                 (Conover 1980: 216).

            B7_  The procedures of Swihart and Slade (1985a,b) will be used
                 to correct for auto correlation among home range locations
                 and to determine the time interval to achieve independence
                 of observations.    An adequate number of relocations to
                 assess the seasonal home range of an individual will be
                 determined by obtaining an asymptotic relationship between
                 home range size with increasing number of relocations. once
                 the proper time interval and sample size have been
                 determined, the method of Dixon and Chapman (1980) will be
                 used to calculate 25%, 50%, 75% and 95% isoclines of home
                 range use.

                 Isoclines of home range use will be overlaid on detailed
                 maps of coastal habitats.     The 95% use isocline will be
                 employed to determine the habitats available for a
                 particular animal.   Proportional weighing by 25%, 50% and
                 75% isoclines within each habitat will determine use. Thus,
                 habitat use and availability will allow a determination of
                 habitat selection for each telemetered individual. Testing
                 for differences in habitat selection (rather       than use)
                 between oiled and control areas is essential       because a
                 difference in habitat use may occur as a           result of
                 differential availability of habitats independent  of effects
                 of oiling. A knowledge of habitat selection by river otters

                                           257









               is essential for extrapolating from our study areas to
               effects on habitat oiled in other areas.              Consequently,
               habitat selection will be inferred from a significant
               difference (P < 0.05) in use and availability matrices
               compared simultaneously with Hotelling's T         2  statistic; a
               posteriori comparisons of individual habitat types will be
               accomplished using Bonferroni multiple tests (Johnson and
               Wichern 1988:188).         similarly, comparisons of habitat
               selection in oiled     and control areas will be made with a
               multivariate analysis of variance (MANOVA) again using
               Bonferroni multiple contrasts.



                                       BIBLIOGRAPHY


               Adorjan, A.S. and G.B. Kolenosky.        1969.   A manual for the
                     identification of hairs of selected Ontario mammals.
                     Ontario Dept. Lands and Forests, Res. Rep. (Wildlife)
                     No. 901 64pp.

               Bowyer, R.T. and K.D. Curry.       1983.   Use of a roller press
                     to obtain cuticular impressions of guard hairs on
                     acetate strips. J. Mammal. 64:531-532.

               Bowyer, R.T., S.A. McKenna, and M.E. Shea.         1983.    Seasonal
                     changes in Coyote food habits as determined by fecal
                     analysis. Amer. Midland Nat. 109:266-273.

               Bunham, K. P. and W. S. Overton.        1978.   Estimation of the
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               Chao, A. 1989. Estimating population size for sparse data
                     in    capture-recapture      experiments.           Biometrics
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               Conover, W. J.    1980.    Practical nonparametric statistics.
                     John Wiley & Sons, New York, 493pp.

               Crabtree, R.L.,   F.G. Burton, T.R. Garland, D.A. Cataldo, and
                     W.H. Rickard. 1989. Slow-release radioisotope implants
                     as individual markers for carnivores. J. Wildl. Manage.
                     53: 949-955.

               Davis, D.E. and R.L. Winstead. 1980. Estimating the numbers
                     of wildlife populations.            Pp.   221-245    in    S.D.
                     Schemnitz, ed. Wildlife Management Techniques Manual.
                     Fourth Ed. The Wildl. Soc., Washington, DC.

               Day, M. G.       1966.    Identification of hair and feather
                     remains in the gut and feces of stoats and weasels. J.
                     Zool. (Lond.) 148:201-217.

               Dennis, B., R.L. Crabtree, and E.V. Eartan. In Press.


                                            258








                       Statistical methods for closed population estimation
                       using radioisotope tagging. J.Widl. Manage.

                  Dixon, K.R. and J.A. Chapman. 1980. Harmonic mean measure of
                       animal activity. Ecology 61:1040-1044.

                  Fleiss, J.L.      1973.    Statistical methods for rates and
                       proportions. John Wiley & Sons, New York, 223pp.

                  Gilbert, F.F. and E.G. Nancekivell.       1982.   Food habits of
                       mink (Mustela vision) and otter (Lutra canadensis) in
                       northeastern Alberta. Can. J. Zool. 60:1282-1288.


                  Hall, R.E. and K.R. Kelson.          1959.     Mammals of North
                       America. Ronald Press, New York, 1983pp.

                  Johnson, R.A. and D.W. Wichern. 1988. Applied multivariate
                       statistical analysis.        Prentice Hall, New Jersey,
                       606pp.

                  Kershaw, K.K.     1964.    Quantitative and dynamic ecology.
                       Edward Arnold, London, 1983pp.

                  Knaus, R.M., N. Kinler, and R.G. Linscombe. 1983. Estimating
                       river otter populations: the feasibility          of 65 Zn to
                       label feces. Wildl Soc. Bull. 11:375-377.
                  Kruuk 15 H., M. Gorman, and T. Parrish.       1980.    The use of
                        zn for estimating populations of carnivores.            Oikos
                       34:206-208.


                  Larsen, D.N. 1983. Habitats, movements, and foods of river
                       otters in coastal southeastern Alaska.         Unpubl. M. S.
                       Thesis, Univ. of Alaska Fairbanks, 149pp.

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                       to the dorsal guard hairs of California mammals. Amer.
                       Midland Nat. 48:480-512.

                  Melquist, W.E. and M.G. Hornocker.          1979.    Methods and
                       techniques for studying and censusing river otter
                       populations.    Tech Report 78, Forest, Wildl. and Range
                       Exper. Station, University of Idaho, Moscow, Idaho,
                       17pp.

                  Morrow, J.E. -' 1979.   Preliminary keys to otoliths of some
                       adult fishes of the Gulf of Alaska, Bering Sea, and
                       Beaufort Sea. NOAH Tech. Report NMFS Circ. No. 420

                  Murie, O.J.      1954.     A field guide to animal tracks.
                       Houghton Mifflin, Boston, 374pp.

                  Neter, J., W. Wasserman, and M.H. Kutner. 1983.            Applied
                       linear statistical methods.            Richard    D.    Irwin,
                       Homewood, Illinois, 1127pp.

                                              259







               Pollock, K.H., S.R. Winterstein and M.J. Conroy.               1989.
                     Estimation and analysis of survival distributions for
                     radio-tagged animals. Biometrics 45:99-109.

               Seber, G.A.F. 1982. The estimation of animal abundance and
                     related parameters. Macmillan, New York.

               Shirley, M.G., R.G. Linscombe, N.W. Kinler, R.M. Knaus,
                      and V.L Wright. 1988. Population estimates of river
                      otters in a Louisiana coastal marshland. J. Wild.
                      Manage. 52: 512-515.

               Snedecor, G. W.1 and W. G. Cochran.           1980.     Statistical
                     methods, 7th ed.      Iowa State University Press, Ames
                     Iowa 507pp.

               Swihart, R.K. and N.A. Slade. 1985a. Testing for
                      independence of observations in animal movements.
                      Ecology 66:1176-1184.

               Swihart, R.K. and N.A. Slade. 1985b. Influence of sampling
                     interval on estimates of home range size.          J. Wildl.
                     Manage. 49:1019-1025.

               White, G. C., D. R. Anderson, K. P. Burnham, and D. L. Otis.
                     1982.     Capture-recapture and removal methods for
                     sampling closed populations.        Los Alamos Natl. Lab.
                     Publ. LA-8787-NERP. 235pp.

               Woolington, J.D. 1984. Habitat use and movements of river
                     otters at Kelp Bay, Baranof Island, Alaska.            Unpubl.
                     M.S. Thesis, Univ. of Alaska Fairbanks, 47pp.

               BUDGET: ADF&G


               Personnel                      $     125.4
               Travel & per them                     20.0
               Contract                             166.7
               Commodities                           35.5

               Total                          $     347.6















                                            260








            I




           TERRESTRIAL MAMMAL STUDY NUMBER 4

           Study Title:     Assessment of the EVOS on Brown Bear Populations
                            on the Alaska Peninsula


           Lead Agency:     ADF&G

           Cooperating Agencies: DOI, NPS, FWS

                                        INTRODUCTION

           Relatively high densities of brown bears (Ursus arctos) occur
           along the 120-mile section of shoreline on the southern edge of
           the Alaska Peninsula that was impacted by crude oil from the
           EVOS.    There has been no objective estimate of the number of
           bears in the affected areas, but it is suspected that densities
           along the oil-contaminated Katmai coast are higher than those
           reported from other coastal brown bear populations: 1 bear/1.1
           mi near Terror Lake on northern Kodiak Island (Barnes- et al.
           1988) and I bear/2.0 Mi2 near Black Lake on the southern Alaska
           Peninsula (Miller and Sellers 1989).             These bears are an
           important economic and aesthetic resource.             On the Alaska
           Peninsula, Alaska residents and guided non-residents harvest
           about 250 bears annually, spending an estimated $2.2 million on
           those hunts (ADF&G files). Thousands of visitors from around the
           world come to Katmai National Park and the McNeil River State
           Game Sanctuary to observe and photograph bears.

           Brown bears are omnivorous, opportunistic feeders near the top of
           the food chain.     They may ingest oil directly by eating mousse
           and tar balls washed ashore, by eating oiled plants and clams, by
           scavenging oiled carcasses of animals killed offshore and
           deposited on beaches, or by grooming oiled fur.         Bears may also
           consume animals that have been physiologically contaminated by
           sublethal doses of oil. Effects of oil ingestion on individuals
           could range from quick death from acute toxic effects to long-
           term suppression of reproduction.       Experimental work with oiled
           polar bears in Canada (Oritsland, et al. 1981) indicated that two
           of three animals died from organ failure after grooming. Effects
           of oil contamination on bear populations could range from sharp,
           immediate declines to subtle long-term reductions as chronic
           effects from hydrocarbons stored in fat are expressed.

           To continue the determination of impact of EVOS on individual
           brown bears and the coastal Alaska peninsula brown bear
           population, a study area along the coast of Katmai National Park
           was selected.      This project will capture and radio-collar an
           additional sample of bears in oiled areas and will compare the
           natural mortality rate of this sample with that of coastal
           populations on Kodiak Island and near Black Lake further south on
           the Alaska Peninsula which were not exposed to large amounts of
           crude oil.      Dead bears found incidentally and radio-collared

                                             261









       bears that die will be necropsied, tissue samples taken, and the
       cause of death determined.      Extent of oil ingestion and the
       physiological effects will also be examined.

                                   OBJECTIVES

       A.   Test the hypothesis that radio-collared brown bears in an
            oil-contaminated area of the Alaska Peninsula (Katmai coast)
            ingested hydrocarbons (as evidenced by the level of
            hydrocarbons in fecal samples) at higher concentrations than
            radio-collared bears in an area on the Peninsula that was
            not contaminated (Black Lake).

       B.   Test the hypothesis that natural mortality rates of female
            brown bears near oiled areas of the Katmai coast occurred at
            a higher rate than females in other coastal brown bear
            populations inhabiting non-oiled areas during a period of
            three years after EVOS.

       C.   Test the hypothesis that some of the mortality of
            brown bears near the Katmai coast can be attributed to the
            physiological effects of ingesting hydrocarbons.

       D.   Estimate the adult brown bear population density of the
            study area (approximately 150 square miles) through a
            cooperative project with the NPS using a modified capture-
            recapture technique (Miller et al. 1987) with the goal of
            obtainihg a coefficient of variation of 0.10.

                                     METHODS

       Bears will be captured in the spring of 1990 by using a f ixed-
       wing spotter aircraft to locate bears and direct a helicopter
       with an immobilizing team to the site.         Each bear will be
       measured (skull length and width), weighed, tattooed (lips and
       groin), and fitted with ear tags and a rad i o -transmitter with
       mortality sensor.

       Blood and fecal samples will be collected from bears captured
       along the Katmai coast and near Black Lake during the spring.
       Whole blood will be collected in heparinized and non-heparinized
       collecting tubes.   Packed cell volume and percent hemoglobin in
       the blood will be determined in accordance with standard
       operating procedures and serum will be frozen and sent into an
       approved laboratory for analysis.

       During 1990, radio-collared bears will be relocated by a fixed-
       winged aircraft at scheduled two-three day intervals until over
       75% of the radio-collared bears are in winter dens.     One flight
       per month will be scheduled during the denning period.       Radio-
       tracking flights should continue for two years.       During 1991,
       flights will be made at two week intervals while bears are active

                                       262









            and monthly during denning.     A sample of at least 30 radio-
            collared bears will be followed into dens each year.         It is
            anticipated that at least 40 bears must have functioning radio-
            collars in the spring to achieve a sample of 30 in the fall. To
            maintain this sample size, collaring operations will be necessary
            during the spring of 1990 and possibly in 1991.

            Mortality data will be collected during radio-tracking flights-.
            When a dead bear is observed in the study area, gross necropsies
            will be performed in the field.    Data on sex, age, and probable
            cause and time of death will be recorded.      Tissue samples from
            recent mortalities will be collected for histopathological and
            hydrocarbon analysis.      Annual survival, distributions, and
            mortality rates will be calculated using modified Kaplan-Meier
            techniques (Pollock et al. 1989). Results will be compared with
            mortality rates from the Black Lake and Terror Lake (Kodiak)
            study areas.

            The density estimate (Miller et al. 1987) will be conducted in
            the spring of 1990.      Prior to the recapture portion of the
            procedure, a representative sample of 50 radio-collared bears
            will be required to serve as marks for the estimator.        Collars
            will be distributed proportionally to the estimated proportion of
            bears in various reproductive categories (e.g. lone adult males,
            lone adult females, subadult males, subadult females, females
            with cubs -of -the-year, females with yearling or older offspring)
            in the population. only independent observations of individuals
            will be used in the estimator.

            Data obtained from the density estimate and mortality rate
            calculations will be used to estimate the total number of bears
            that were killed by the effects of EVOS by comparison between
            years and between the oiled area and the control area. A
            subsequent population estimate, using the same methods in the
            same oil-conta-minated area, will be derived in the spring of
            1992.   It has been reported that capture-recapture techniques,
            such as the proposed density estimate procedure, tend to
            underestimate the known size of big game populations (deer) by
            10-20% in most instances; but, the estimators can be used to
            detect population trends by comparing estimates over time from
            the same area (Becker 1989).      Due to suspected heterogeneity
            among bear classes and lower sightability of bears, compared to
            deer, we suspect that the true number of bears in the population
            will be underestimated by an unknown amount somewhat greater than
            the 10-20% reported in the literature.

            Throughout all phases of this study we will attempt to identify
            potential alternative methods and strategies for restoration of
            lost use, populations, or habitat if injury is identified.       The
            final report will include a listing of suggested ways to address
            long-term restoration projects.


                                            263









         A two sample, one-sided T-statistic (Snedecor & Cochran, 1980)
         will be used to test the oil ingestion hypothesis (objective A).
         This statistic assumes the means are normally distributed.            if
         necessary, transformations will be used to ensure that the
         normality assumption is met.

         The natural mortality rate hypothesis (Objective B) will be
         tested using a log-rank test to compare the two Kaplan-Meier
         survival functions (Pollack et al. 1989). This statistic assumes
         that differences in survival functions are the result of a
         constant shift parameter (Cox & Oakes 1988).          This assumption
         will be examined by cumulative hazard plots of the two
         distributions and possibly involve analysis with time dependent
         covariates (Cox & Oakes 1988).            It is assumed that bear
         populations near Black Lake and Terror Lake are more likely to be
         shot by hunters, so all hunter-killed radio collared bears will
         be censored (as outlined in Pollock et al. (1989) for animals
         that emigrated from a study area or were otherwise lost).

         Tissues will be collected and analyzed as outlined by the EVOS
         Histopathological Technical Group and the Hydrocarbon Technical
         Group to test the hydrocarbon mortality hypothesis (Objective C).
         All statistical analyses will be performed at an alpha level of
         0.05.  A sample size will be used that is adequate to detect at
         least one bear affected by oil contamination with a given
         percentage   of   certainty,    for varying proportions        of the
         population contaminated by hydrocarbons.           These sample size
         calculation's are based on a binomial distribution (Mendenhall,
         Schaffer and Wackerly 1981) and assume that the sample size is
         very small compared to the population total. For the purposes of
         this study it will be assumed that at least 10% of the bear
         population was affected by EVOS; therefore, a total sample size
         of at least 29 bears will have to be followed by radio-telemetry
         to be 95% certain of following at least one bear affected by
         hydrocarbons.

         The Lincoln-Peterson estimator (Overton 1971) will be used to
         estimate daily adult population levels (Objective D) .        The mean
         of the estimates and its standard error will be used as the point
         estimate   and standard deviation.         The assumptions of this
         estimator  are (White et al. 1982):

              1)    all radio-collars are retained;
              2)    all animals are correctly classified as marked or
                    unmarked;
              3)    the recaptures (sightings) of adult bears are
                    independent;
              4)    the population is geographically and demographically
                    closed;
              5)    all bears have equal capture probabilities that are
                    constant over time.



                                           264









            The geographic closure assumption will be met by determining the
            number of radio-collared bears in the study area on a daily
            basis, and assuming that the proportion of marked bears in the
            area is representative of the unmarked bears. Assumption #5 can
            be relaxed to: average probability of capturing a marked animal
            equals the average probability of capturing an unmarked animal
            (Overton 1971).    If capture   heterogeneity exists, which it
            probably does with brown bears, then mark and recapture estimates
            tend to be biased low, because the animals that are easier to
            catch are over represented in the marked sample.       Because of
            this, calculated confidence intervals will. not retain their
            statistical validity, and as a result, the precision goal was
            stated in terms of the coefficient of variation.


                                      BIBLIOGRAPHY

            Barnes, V.G. Jr., R.B. Smith, and L.J. Van Daele. 1988.
                  Density estimates and estimated population of brown
                  bears on Kodiak and adjacent islands, 1987.
                  Unpublished report to the Kodiak Brown Bear Research
                  and Habitat Maintenance Trust. Anchorage, AK. '34pp.

            Becker, E.F. 1989. Mark recapture estimates versus known
                  populations. Memo to ADF&G bear researchers, dated
                  Aug. 11, 1989. 13pp.

            Cox, D. R. and D. Oakes. 1988. Analysis of statistical data.
                 Chapman and Hall. London. 201 pp.

            Mendenhall, W., R.L. Schaffer, and D.D. Wackerly. 1981.
                  Mathematical Statistics with Applications. Duxbury
                  Press. Boston, Mass. 686 pp.

            Miller, S.D., E.F. Becker, and W.B. Ballard. 1987. Black
                  and brown bear density estimates using modified
                  capture-recapture techniques in Alaska. Int. Conf.
                  Bear Res. and Manage. 7:23-35.

            Miller, S.D. and R.A. Sellers. 1989. Brown bear density on
                  the Alaska Peninsula at Black Lake, Alaska.
                  Unpublished preliminary report for the National Pa  rk
                  Service, U.S. Fish and Wildlife Service, and the
                  Alaska Department of Fish and Game. Anchorage, AK.
                  36 pp.

            Oritsland, N.A., F.R. Engelhardt, F.A. Juck, R.J. Hurst, and
                  P.D. Watts. 1981. Effect of Crude Oil on Polar Bears.
                  Can. Dept. Indian Affairs & North Devel. Publ. No.
                  QS-8283-020-EE-Al.

            Overton, W. S. 1971. Estimating the number of animals in
                  wildlife populations IN wildlife management techniques.

                                           265









                R. H. Giles ed. The Wildl. Soc. Washington, D. C.
                Pp. 403-456.

          Pollock, K.H., S.R. Winterstein, C.M. Bunck, and P.D.
                Curtis. 1989. Survival analysis in telemetry studies:
                The staggered entry design. J. Wildl. Manage.
                53(l):7-15.


          Snedecor, G.W. and W.G. Cochran. 1980. Statistical
                Methods; Seventh Edition. Iowa St. Univ. Press.
                Ames, Iowa. 507 pp.

          White, G. C., D. R. Anderson, A. P. Burnham, and D. L. Otis.
                1982. Capture-Recapture and Removal Methods for Sampling
                Closed Populations. Los Alamos Natl. Lab. Publ. LA-8787-
                NERP. 235 pp.

          BUDGET: ADF&G


          Salaries                   $     44.6
          Travel                            4.3
          Services                         47.0
          Commodities                       9.8
          Equipment                        20.0

          Total                      $    125.7





























                                            266











           TERRESTRIAL MAMMAL STUDY NUMBER 6

           Study Title:    Influence of Oil Hydrocarbons on Reproduction of
                           Mink (Mustela vision)

           Lead Agency:    ADF&G



                                       INTRODUCTION

           The mink is a carnivorous mammal inhabiting the margins of streams,
           lakes, marshes, and marine islands throughout most of North
           America. It is at the top of the food chain and thus exposed to a
           wide variety of environmental contaminants.      Certainly the most
           dramatic ef f ect of a toxicant or pollutant is outright death of the
           animal. An equally devastating effect on the animal population,
           however, is for apparently healthy animals to fail to reproduce or
           to   produce weakened offspring. Studies with ranched mink have
           documented that these animals are sensitive to many chemical and
           biological compounds (Sundqvist et al.1989). Some of those known
           to interfere with reproduction include heavy metals, halogenated
           hydrocarbon pesticides and other aromatic, halogenated hydrocarbons
           (Ringer, 1981; Sundqvist et al.1989). In the mid-1960s a decline
           in reproductive performance in ranched mink (Hartsough, 1965) was
           eventually traced to high polychlorinated biphenyl (PCB) content of
           Great Lakes f ish -used in commercial mink diets (Aulerich et
           al.1971; Aulerich et al.1973). Ranched mink fed 2-5 ppm PCBs, 1-
           2.5 ppm polybrominated biphenyls, or 5-25 ppm hexachlorobenzene
           suffered complete reproductive failure, significantly reduced
           litter size and/or excessive kit mortality (Aulerich et al.1985;
           Aulerich, and Ringer, 1979; Aulerich, and Ringer, 1977; Bleavins et
           al..1984; Bleavins et al.1980; Hornshaw et al.1983; Ringer, 1981;
           Ringer et al.1972; Rush et al.1983; Wren et al.1987). Particularly
           dangerous are compounds like PCBs that accumulate in the
           subcutaneous fat (Hornshaw et al.1983). Therefore, it is possible
           that hydrocarbons in crude oil ingested by mink and other
           carnivores may interfere with reproduction.

           Crude oil released into the environment is immediately subjected to
           a variety of weathering processes (Payne and McNabb, 1984). Within
           a few weeks of an oil spill the majority of the more toxic, lower
           molecular weight compounds are eliminated, primarily through
           evaporation (Payne and McNabb, 1984). However, heavier distillate
           products not subject to significant evaporative loss persist in the
           environment and are more likely to enter the food chain in
           signif icant quantities.    It is the ef f ect of ingestion of this
           weathered oil that will be studied.

           Mink feed on fish., small mammals, frogs, aquatic insects, and
           occasionally birds. These prey species live in areas impacted by
           the EVOS.    Therefore, it is highly probable that mink will be
           exposed to oil hydrocarbons.         But direct cause and effect

                                            267









         relationships such as the influence of oil hydrocarbons on
         reproduction are difficult to demonstrate in a field study. The
         complexity of a natural setting imposes too many uncontrolled
         variables. Even if field studies measure hydrocarbons from wild
         animals or show a change in the population dynamics of one or more
         species, there will always be the unanswered question: Was it the
         oil or some other unmeasured factor that influenced reproductive
         performance?   A controlled experiment in the laboratory will be
         conducted to define the effects of short-term and long-term
         ingestion of non-lethal amounts of weathered Prudhoe Bay crude oil
         (WPBC) using ranched mink as a model species. mink will be used
         for three reasons:      1) mink are known to be sensitive to
         hydrocarbon pollutants, 2) mink inhabit the PWS area and are thus
         at risk for exposure to oil hydrocarbons in feed, and 3) the mink
         is a well-established model for laboratory research and thus there
         is excellent documentation of their reproductive physiology (Enders
         1952; Sundqvist, et al., 1989).     Ranched mink are not domestic
         animals, and their physiological response to oil ingestion in the
         laboratory setting can be predicted to be no different from that of
         their wild counterparts.

                                      OBJECTIVES


         A. Short-Term Ingestion of Weathered Prudhoe Bay Crude Oil

         Test the hypothesis that short-term (seven day) , low-level (100
         ppm) ingestion of WPBC oil during pre-estrus, diapause, gestation,
         or lactation does not produce a significant (P < 0.05) difference
         in the reproduction of female mink. Reproductive variables will be
         the number of kits per litter, kit survival, kit growth and
         maturation, and histology of adult and kit reproductive tracts.

         B. Long-Term Ingestion of Weathered Prudhoe Bay Crude Oil

         Test the hypothesis that continual, low-level (100 ppm) ingestion
         of WPBC oil starting during pre-estrus and continuing through to
         the weaning of kits does not produce a significant (P < 0.05)
         difference in reproduction of female mink. Reproductive variables
         will be the number of kits per litter, kit survival, kit growth and
         maturation, and histology of adult and kit reproductive tracts.

                                       METHODS

         Crude oil from the Exxon Valdez will be weathered by placing it in
         a flat bottomed, solvent-rinsed vessel to a depth no greater than
         2 cm.  The oil will be gently agitated in a fume hood for seven
         days at room temperature.

         All mink will be fed commercial mink ration consisting of ground
         fish, chicken and beef by-products. WPBC oil will be mixed into the
         feed at a rate of 100 ppm. In order to measure such small amounts
         accurately, we will first dilute the WPBC in salmon oil. . The

                                          268









           resulting oiled diet will contain 100 ppm WPBC and 10 ml salmon oil
           per kg commercial mink ration. The level of 100 ppm WPBC oil was
           chosen because it did not alter food palatability and the oiled
           food was readily consumed by the mink. In addition, this level of
           contamination does not cause the mink to suffer or exhibit any
           clinical symptoms.

           For the short-term study, mink will be fed either 0 WPBC (control
           group) or 100 ppm WPBC for seven days at a time specif ic to the
           reproductive    cycle   (pre-estrus,    diapause,   pregnancy,     and
           lactation).   A total of 70 female mink will be used.        Mink (20
           each) will be randomly assigned to the control and pre-estrus
           groups prior to the breeding season. Successfully mated mink from
           the remaining 20 animals will be randomly assigned to the diapause,
           pregnancy, and lactation groups following breeding.       The female
           mink will be bred to untreated males. Males will be checked for
           fertility by palpation of testes, demonstration of copulatory
           behavior, and evidence of motile sperm in vaginal smears following
           mating.   Females will be allowed to rear their young to weaning
           age. After weaning, adults and selected kits will be euthanized
           and tissue and blood samples will be collected for histopathology,
           hydrocarbon analyses and liver cytochrome P450 analyses. Estrous
           behavior will be analyzed by comparing the number of females
           successfully bred and the time of breeding of the 20 control
           animals to the 20 pre-estrus animals.       For these tests all 20
           animals in each group will be included. For all other tests, only
           those animals successfully bred will be included.       The response
           variables to be analyzed on mated females in each group include
           number of kits born, number of live kits born, birth weight of
           kits, survival rate of kits, and growth rate of kits.

           For the long-term study twenty female mink will be fed 100 ppm WPBC
           oil in their diets beginning in February, 1990 and continuing until
           kits are weaned in June, 1990. The control group (n=20) from the
           short-term study will also serve as a control for this study. All
           mink in this long-term exposure study will be bred to untreated
           males and will be allowed to rear their young to weaning age.
           Adults and selected kits will be euthanized at weaning, and tissue
           and blood samples will be collected for histopathology, hydrocarbon
           analyses and liver cytochrome P450 analyses.            The response
           variables to be examined in this study are identical to those in
           the short-term exposure study:     mating activity, number of kits
           born, number of live kits born,    birth weight of kits, survival,
           growth and maturation of kits.

           The University of Alaska Fairbanks, (UAF), who will conduct this
           research, has on file with the Office for Protection Against
           Research Risks, National Institutes of Health, an "Assurance of
           Compliance with Public Health Service Policy on Humane Care and Use
           of Laboratory Animals".     The University's Animal Facilities are
           licensed by the United States Department of Agriculture and are


                                            269










        subject to twice yearly, unannounced inspections by USDA
        veterinarians to ensure compliance with the Animal Welfare Act.

        UAF has a full-time, staff veterinarian who supervises the
        veterinary care program as outlined in the Regulations of the
        Animal Welfare Act.      The University also has an Institutional
        Animal Care and Use Committee mandated by Public Health Service
        Policy and the Animal Welfare Act. This project was reviewed and
        approved, without modification, by the committee.

        Because of the nature of the response variables being examined, a
        number of different statistical tests will be used. comparisons
        between two groups involving binomial variables, such as number of
        females bred, will be tested by chi-square. Analysis of variables
        involving kits as an experimental unit will be tested by analysis
        of variance.    When there are significant differences, treatment
        groups will be compared to the control group by Dunnet's test.
        Because kit birth weight and kit growth rate are expected to vary
        with litter size unrelated to specific treatment effects, analysis
        of covariance with litter size as the covariate will be done.
        Statistical significance of differences between variables involving
        the long-term exposure group and the control animals will be
        determined by T-test or by chi-square as appropriate.

                                     BIBLIOGRAPHY

        Aulerich, R.J., Bursian, S.J., Breslian, W.J., Olson, B.A., and
              Ringer,   R.K.    (1985).   Toxicological    manifestations      of
              2 , 4 , 5 , 2     4 1 , 5    - 2 , 3 , 6 , 2 1 , 3 1 , 6 1 a n d
              3,4,5,31,41,51-hexachlorobiphenyl and aroclor 1254 in mink.
              J.Toxicol.Environ.Health 15, 63-79.

        Aulerich, R.J. and Ringer, R.K. (1977). Current status of PCB
              toxicity to mink, and effect on their reproduction. Archs
              environmental Contamination Toxic. 6, 279-292.

        Aulerich, R.J. and Ringer, R.K. (1979). Toxic effects of dietary
              polybrominated    biphenyls on mink. Archs environmental
              Contamination Toxic. 8, 487-489.

        Aulerich, R.J., Ringer, R.K., and Iwamoto, S. (1973). Reproductive
              failure and mortality in mink fed on Great Lakes fish.
              J.Reprod.Fert. Suppl.19, 365-376.

        Aulerich, R.J., Ringer, R.K., Seagran, H.L., and Youatt, W.G.
              (1971). Effects of feeding coho salmon and other Great Lake
              fish on mink reproduction. Can.J.Zool._ 49, 611-616.

        Bleavins, M.R., Aulerich, R.J., and Ringer, R.K.                  (1980).
              Polychlorinated biphenyls (Aroclors 1016 and 1242): effects on
              survival and reproduction in ferrets. Archs environmental
              Contamination Toxic. 9, 627-635.

                                          270









             Bleavins, M. R. , Aulerich, R. J. , and Ringer, R. K. (1984) . Ef f ects of
                   chronic dietary hexachlorobenzene exposure on the reproductive
                   performance and survivability of mink and European ferrets.
                   Archs environmental Contamination Toxic. 13, 357-365.

             Enders, R.K. (1952). Reproduction in the mink. Proc.Am. Phil. Soc.
                   96, 691-755.

             Hartsough, G.R. (1965). Great Lakes fish now suspect as mink food.
                   Am.Fur Breeder 38, 25-27.

             Hornshaw, T.C. Aulerich, R.J., and Johnson, H.E. (1983). Feeding
                   Great Lakes fish to mink:    effect on mink and accumulation and
                   elimination of PCB's by mink. J. Toxicol. Environ. Health 11,
                   933-946.

             Payne, J.R. and McNabb, G.D.JR. (1984). Weathering of petroleum in
                   the marine environment. MTS Journal 18(3), 1-20.

             Ringer, R.K. (1981). The effects of environmental contaminants on
                   reproduction in the mink (Mustela vison). In:" Environmental
                   factors in mammal reproduction" (Gilmore, D., and Cook, B.,
                   Eds.) pp. 232-237. University Park Press, Baltimore.

             Ringer, R.K., Aulerich, R.J., and Zabik, M. (1972). Effect of
                   dietary polychlorinated biphenyls on growth and reproduction
                   in the mink. Am.Chem.Soc.Nat.Meeting 12, 149-152.

             Rush, G.F., Smith, J.H., Maita, K., et al (1983). Perinatal
                   hexachlorobenzene toxicity in the mink. Environ. Res. 31,
                   116-124.


             Sundqvist, C., Amador, A.G., and Bartke, A. (1989). Reproduction
                   and fertility in the mink (Mustela vison). J.Reprod.Fertil.
                   85, 413-441.

             Wren, C.D., Hunter, D.B., Leatherland, J.F., and Stokes, P.M.
                   (1987). The effects of polychlorinated biphenyls and
                   methylmercury, singly and in combination on mink. II:
                   Reproduction and kit development. Archs environmental
                   Contamination Toxic. 16, 449-454.



             BUDGET: ADF&G


             Salaries                     $     23.7
             Travel                               0.0
             Contracts                          62.3
             Supplies                           48.0
             Equipment                            0.0

             Total                              134.0


                                                  271













                              BIRD INJURY ASSESSMENT

        The EVOS resulted in the death of a large number of migratory
        birds, especially seabirds, waterfowl, and bald eagles.      In the
        months following the spill it became apparent that the vast
        populations of numerous bird species that inhabit or utilize the
        spill zone remained at risk to direct mortality as well as
        sublethal, long-term damages.

        Fourteen studies were developed and deployed during 1989 to
        document damages to migratory birds.    It was recognized early in
        the process that it was not possible to study all the bird species
        potentially affected by the oil spill nor the full scope of effects
        to any species. Therefore, efforts were concentrated on studying
        key species or groups of species where injury was most evident and
        valid damage assessment could be determined in a reasonably cost-
        effective manner.


        Seven of these studies will be continued in 1990.     Some studies
        were not continued because it was concluded that all data pertinent
        to assessing damages likely to be gathered had indeed been
        gathered. Some studies, such as Bird Studies B6, B8 and B9 were
        either integrated into the remaining studies or are being conducted
        independent of the NRDA process.

        Continuing studies have been expanded and/or modified in response
        to comments from reviewers and the public.       The beached bird
        survey, for example, was carefully designed to provide essential
        data on bird drift and sinking rates that will increase the
        accuracy of total bird mortality estimates.     Both the eagle and
        peregrine falcon studies will provide information on loss to
        breeding populations, as well as reproductive -success. The seabird
        colony and seabird and waterfowl surveys will provide a means to
        compare pre and post spill populations.     Finally, the sea duck
        study will provide important information on sublethal effects of
        the spill on harlequin ducks.















                                        272













             BIRD STUDY NUMBER 1

             Study Title:   An Assessment of Damage to Seabirds in PWS and the
                            western GOA Resulting from the EVOS.

             Lead Agency:   FWS

                                        INTRODUCTION

             This study will assess mortality of marine birds following the EVOS
             by adapting existing bird damage assessment models to estimate
             total seabird mortality. The proposed field studies will examine
             the general characteristics of the decomposition and disappearance
             of bird carcasses under environmental conditions typical of PWS and
             the GOA to determine the rate at which carcasses are lost and the
             factors affecting that rate. It is not possible to simulate the
             exact environmental conditions prevailing during the spill, in
             particular the wind and current conditions and the presence of
             large floating mats of oil.



                                         OBJECTIVES



             A.   Synthesize available information on the beachcast-bird
                  recovery effort.

             B.   Determine the number of birds that died as a consequence of
                  oiling.

             C.   Determine the rate of carcass loss at sea and the time-course
                  of sinking, decomposition and scavenging.

             D.   Develop an estimate of total seabird mortality.



                                           METHODS

             The modeling approach is based on risk assessment models developed
             for the Pribilof Islands, Kodiak Island, and the Southern
             California Bight, and on damage assessment models developed for the
             T/V Puerto Rican and T/V Apex Houston oil spills (Ford et al. 1982,
             Weins et al. 1982, Ford 1984, Dobbin et al. 1986, Ford et al. 1987,
             Page et al. 1990).

             The carcasses of oiled seabirds may be assumed to have encountered
             one of three fates:


                  (1) beached but not recovered,

                  (2) lost at sea without making landfall,

                                             273










               (3) beached and recovered.

         The general approach will be to utilize data for birds which were
         beached and were recovered to estimate the total number of birds
         which were beached, and, with the aid of oil spill trajectory
         information, to work backwards from the beaches where birds were
         recovered in order to estimate the total number of birds which were
         directed toward the beach but were lost at sea before being
         beached.

         Unless a beach is thoroughly searched at frequent intervals, some
         of the beached birds will not be recovered.          During the Apex
         Houston oil spill in California, Page and Carter (1986) found that
         only 60% of the carcasses persisted on the beach face from one day
         to the next.     Beached birds may not be recovered due to the
         scavenging, burial, or reflotation of carcasses.          A model to
         estimate the actual number of carcasses deposited on a beach based
         on the observed number of carcasses on the beach is presented at
         Page et al. 1986. Data sufficient for this type of modeling are:

               (1)  The arrival rate of carcasses on a given beach as a
                    function of time.

               (2)  The frequency with which the beach was searched.

               (3)  The likelihood that a carcass will not be detectable as
                    a function of the length of time spent on the beach and
                    the age of the carcass when it is first cast.

         some of the data types listed above will need to be derived from
         information collected during the spill, and others will be
         estimated as a part of the proposed field studies. The arrival of
         bird carcasses on a given beach typically peaks rapidly, and tapers
         of f over a period of up to a week.     A typical arrival curve can
         usually be constructed from more frequently searched beaches and
         applied to less frequently searched beaches.             Data on the
         disappearance rates of beached carcasses and the distance moved by
         a refloated carcass would be collected as a part of the proposed
         study.

         In addition to carcasses that were not recovered on searched
         beaches, many carcasses were probably deposited on beaches which
         were not searched at all.     This effect will be accounted for by
         identifying a number of beached bird recovery areas based on
         coastal location and geomorphology. Within a recovery area, it is
         assumed that the average lineal density of carcasses for a given
         beach type in the unsampled area was the same as that in the
         sampled area within each sector. Using a Geographical Information
         System, the lineal extent of searched and unsearched beaches within
         each sector will be determined from a digital coastline.

         Even after correcting the estimate of beached birds for birds that

                                           274









           were beached but were not recovered, a large fraction of the
           mortality will remain unmeasured.     Carcasses that are drifting
           landward may not beach because they sink or are scavenged along the
           way. The process of carcass loss both at sea and on the beach f ace
           is poorly understood, but is critical to the estimate of total
           mortality since this appears to have been the fate of a large
           proportion of the birds killed in other spills. The component of
           the at-sea loss from sinking, scavenging, or other causes will be
           computed by taking the number of birds (corrected for recovery on
           the beach), estimating the time spent at sea, and correcting for
           the loss in transit.    Time spent at sea will be estimated using
           trajectory data.    These sources will be used to determine the
           likely path followed by a drifting seabird carcass to reach a given
           beach. The distribution of time spent at sea for carcasses found
           on that beach will be estimated by integrating the likely path of
           the oil reaching that beach with a density surface describing the
           at-sea distribution of birds through which the trajectory passed.
           The at-sea distribution will be estimated using historical data.
           Distributional data of this type for the GOA and PWS is known to
           have a high degree of variability both because of sample size and
           inter-annual variation.       The large scale aspects of bird
           distributions, however, such as the relationship of concentrations
           of birds relative to the shelf break or to colony sites can be
           expected to be conservative and can be adequately described by
           existing data. As part of the sensitivity analysis, the effect of
           alternative at-sea distributions of birds on model results will be
           tested. ongoing studies suggest that the loss of carcasses at sea
           increases with time.     The process of entering the near shore
           environment or beaching probably accelerates the disappearance
           rate, especially for older carcasses.

           Some carcasses will also be carried out to sea by winds and
           currents. The size of this component of the at-sea loss will be
           estimated using trajectory and bird distribution data.

           The effect of changing the value of a single input parameter on
           model results will be examined for all single-valued parameters for
           which the exact value is arguable. This will be done by running
           the model with a series of different values for a given parameter
           and plotting the estimated number of birds killed as a function of
           the input value for that parameter.       Examples of the kind of
           parameters which could be analyzed in this way include the
           disappearance rate of carcasses on the beach face, the proportion
           of birds at risk which actually died, etc.

           The effect of simultaneously using best-case and worst-case values
           for all appropriate parameters will be examined in order to
           generate maximum and minimum estimates of total mortality.       The
           effect of varying some of the inputs which are not single valued,
           such as the distribution of birds at sea based on historical data,
           will also be examined.          This would be done by manually
           constructing alternative distributions which are consistent with

                                           275










        the biology of various species, but which would lead to either
        increased or decreased values of total mortality.

        An alternative is to use a Monte Carlo type approach based on the
        use of probability density functions for arguable parameters (Ford
        et al. 1982).

        Detailed information describing recovery efforts will be used to
        correct the model input values for variation in level of effort.
        To the extent necessary, a general description of the recovery
        effort will be pieced together from records accompanying the frozen
        specimens. These records indicate where the particular birds were
        recovered and when.

        Logbooks will be reviewed to determine the distribution of search
        effort along the coastline impacted by EVOS, although in some cases
        it may be necessary to estimate search effort from other sources
        including interviews and returns of beachcast birds.

        Dead birds collected following the spill are presently bagged and
        stored in freezers in Anchorage.    Seabirds die f or a variety of
        reasons and may have been oiled secondarily.              A sample
        (approximately 10%) of recovered birds will be examined to
        quantify the proportion of unoiled birds that may have been
        recovered in different areas and times after the spill, and to
        describe the degree of oiling and state of decomposition for a
        number of birds from several locations. Some necropsy work may be
        performed on unoiled or lightly-oiled birds to determine whether
        oil had been ingested.

        Field studies will be conducted to determine an estimate of loss
        rate of oiled birds at sea. The loss of birds increases over time,
        and, ultimately, would reach 100% for birds not beached. The loss
        rate of birds oiled at sea can be estimated by radio-tracking of
        free-drifting carcasses.     The study will be conducted in two
        phases, the first focusing on PWS and the second on the Alaska
        Peninsula where much of the impact on seabird communities occurred.

        Free-drifting carcasses will be radio-tracked to obtain data on the
        rate of loss of oiled carcasses following release. The proportion
        of the sample group that is deposited on land will be determined.
        Carcasses will be released from known locations along different
        stretches of coastline.     over the duration of the study, the
        position of a carcass will be monitored until it has become
        beachcast or the signal is lost.    Since the failure rate of the
        radio-tags is very low, loss of signal indicates that a carcass has
        become submerged (taking the radio with it) or has drifted out of
        range. In order to distinguish between these two possibilities, a
        set of "decoys" will be released as a control.          In theory,
        carcasses and decoys will drift in the same manner but one group
        will begin to sink (the carcasses) and the other will not.        By
        comparison of data from the two groups, the loss rate of carcasses

                                        276









           over time can be calculated.      To the limits of data, we will
           correlate rate of loss with species, degree of decomposition, and
           degree of oiling.

           Some beached carcasses will be inspected by the boat crew for
           locations that can be safely visited. Beached carcasses and decoys
           will be photographed and scored for visibility and other factors
           that would affect their collection during beach clean-up effort and
           will be left in place and monitored from the air to determine if
           they are refloated.

           Each carcass and decoy will be equipped with a radio-tag that
           floats upright. Each radio unit weighing approximately 15 grams,
           has an effective range of about 03 nmi at 2000 feet above sea level
           (ASL) flight altitude, with a battery life of 50 days.            The
           transmitters are placed in devices that are completely self-
           righting and have neutral buoyancy. Radio-tags are activated by
           removal of an external magnet. Each radio-tag has a characteristic
           frequency and pulse rate.

           A scanner-receiver in an aircraft equipped with paired antenna
           mounted on the wings will be used to find the signal from each
           transmitter. The operator can select for a particular frequency or
           the instrument will automatically scan for all available
           frequencies.

           Determination of the precise location of a transmitter at sea is
           not necessary to achieve the objectives of the -study since the
           principal interest is in obtaining a frequent inventory of
           carcasses and decoys present in the area. If a carcass or decoy
           appears to be present on a beach, effort will be made to determine
           its precise position and assess the likelihood of its recovery.
           The approach used will be to f ly parallel to the beach over the
           surf zone.   If the transmitter is offshore, the signal will be
           received by only the outboard antenna. If the transmitter is on
           the beach or in the surf, the signal will be received by only the
           inboard antenna.   Flight altitude of the tracking aircraft      over
           open water will typically be 3,000 to 5,000 ft. ASL, but will
           decrease to 500 ft ASL or less near the beach. On subsequent     days
           following the release, a search area will be identified taking   into
           consideration the last known location of transmitters and the
           results of a trajectory model. The model to be used is the       NOAA
           Oil Spill Simulation Model (OSSM), using the most appropriate
           current field and winds data.

           Because of the great number of radio-tags deployed, a special
           computer data-logging system will be used for output from the
           receiver-data processor equipment.

           Calculation of the coordinates of the transmitter will be
           accomplished using a mathematical function describing the decrease
           in signal amplitude with distance.       This relationship must be

                                            277











       described for each kind of transmitter.


                                  BIBLIOGRAPHY

       Burger, A.E. 1989. Effects of the Nestucca oil spill (January
            1989) on seabirds along the southwest cost of Vancouver
            Island.

       Report to Environment Canada. Contract No. KA601-8-4184. 26 pp
            and Appendices.

       Carter, H.R., G. W. Page, and R.G. Ford. 1987. The importance of
            rehabilitation center date in determining the impacts of the
            1986 oil spill on marine birds in central California.
            Wildlife Journal 10:9-14.

       Dobbin, J.A., H.E. Robertson, R.G. Ford,  K.T. Briggs, and E.H.
            Clark 11. 1986. Resource'damage assessment of the T/V Puerto
            Rican oil spill incident. Unpubl. report, James Dobbin Assoc.
            Inc.1 Alexandria, Virginia.

       Fleming, T., D. Varoujean, and S. Speich. 1990. Performance and
            reliability of radio-tags for Marbled Murrelets. Abstract.
            Proceedings of Meetings of the Pacific Seabird Group, February
            1990, Victoria, B.C., Canada.

       Ford, R.G. 1984. Southern California Marine Mammal and Seabird
            Risk Ahalysis.     Final Report prepared for the Minerals
            Management Service, U.S. Department of the Interior, by
            Ecological Consulting. Contract #14-12-0001-30224. 240 pp.

       Ford, R.G., J.A. Wiens, D. Heinemann, and G.L. Hunt, Jr. 1982.
            Modeling the sensitivity of colonially breeding marine birds
            to oil perturbation. J. Appl. Ecol. 19:1-31/

       Ford, R.G., G.W. Page, and H.R. Carter.    1987. Estimating
            mortality of seabirds from oil spills. Pp. 848-51. In Proc.
            1987 Oil Spill Conference, American Petroleum Institute,
            Washington, D.C.

       Ford, R.G. and J.L. Casey. 1989. Preliminary Draft Report:
            Seabird Mortality Resulting from the Nestucca oil Spill
            Incident, Winter 1988-89. Prepared for Washington Department
            of Wildlife. 37 pp.

       Gould, P.D., D.J. Forsell, and C.J. Lensink. 1982. Pelagic
            distribution and abundance of seabirds in the Gulf of Alaska
            and the eastern Bering Sea. Fish and Wildlife Service, U.S.
            Dept. of the Interior. FWS/OBS-82-48.

       Page, G.W. and H.R. Carter (eds). 1986. Impacts of the 1986 San
            Joaquin Valley crude oil spill on marine birds in central

                                       278










                 California.   Unpubl. report, Point Reyes Bird Observatory,
                 Stinson Beach, California.

           Page, G.W., H.R. Carter, and R.G. Ford. 1990. Numbers of seabirds
                 killed or debilitated in the 1986 Apex Houston oil spill in
                 central California. Studies in Avian Biology. In press.

           Wiens, J.A., R. G. Ford, D. Heinemann, and C. Fieber. 1982. A
                 statistical analysis of the distribution of seabirds in the
                 vicinity of Kodiak Island, Alaska.           In Environmental
                 Assessment of the Alaska Continental Shelf. Final Reports.

           Budget:    FWS

           Personnel                   $46.0
           Travel                       21.5
           Contractual                 521.1
           Commodities                   9.4
           Equipment                     0.0

           Total:                     $598.0


































                                            279












       BIRD STUDY NUMBER 2

       Study Title:    Surveys to Determine Distribution and Abundance of
                       Migratory Birds in PWS and the Northern GOA

       Lead Agency:    FWS

                                   INTRODUCTION

       This study will continue to examine whether the EVOS caused a
       decline in the distribution and abundance of water birds in the
       waters and shorelines affected by the spill. Potential injuries to
       waterbirds from exposure to the EVOS include direct mortality,
       changes in behavior, and decreased productivity. Surveys by small
       boats and airplanes will collect information on the seasonal
       distribution and abundance of waterbirds in the spill zone for
       several seasons following the spill. These post-spill data will be
       compared to data collected using similar methods in prior years to
       determine whether the oil spill affected waterbird distribution and
       abundance.

       This proposal describes the boat survey and aerial survey work that
       will be accomplished in the second year of this study. The study
       will continue portions of Bird Studies Number 6 (Marbled Murrelets)
       and Number 9 (Pigeon Guillemot), and will also record observations
       of marine mammals in the study area which could prove valuable to
       other ongoing studies.

                                    OBJECTIVES
       A.    Aerial Surveys

             1.   To determine seasonal distribution and estimate relative
                  abundance of waterfowl and waterbirds in PWS and KP.

             2.   To compare relative abundance and distribution of
                  waterfowl and other waterbirds in oiled vs. non-oiled
                  areas between historical (1971) and recent (1989 and
                  1990) surveys. To monitor changes in the distribution and
                  abundance of waterfowl and other waterbirds between oiled
                  and non-oiled areas in PWS and KP.

             3.   To estimate the long- and short-term recovery rates of
                  waterfowl and waterbird populations impacted by the oil
                  spill.

       B.    Boat Surveys

             1.   To determine distribution and estimate abundance (with
                  95% confidence limits) of waterbirds in PWS and the
                  northern GOA.

             2.   To test the null hypothesis that estimates of waterbird

                                        280









                       relative abundances, using new and comparable historic
                       data, are not significantly lower (a = 0.05) in oiled
                       than non-oiled areas in PWS and the northern GOA.

                  3.   To estimate the long- and short-term trends of
                       populations that were determined in previous objectives
                       to be reduced by the oil spill.

                  4.   To test the null hypothesis that the total number of
                       Pigeon Guillemots attending colonies at Naked Island,
                       PWS, following the EVOS is Dot significantly lower than
                       the total number attending in prior years.

                  5.   To test the null hypothesis that the abundance index of
                       Marbled Murrelets on five transects on the western side
                       of Naked Island, PWS, following the EVOS is not
                       significantly lower than the abundance index on each
                       transect in prior years.

                                              METHODS


            A.    Aerial Survey Sampling Methods

                  Three surveys will be conducted during the 1990-1991 oil spill
                  year: the spring survey during May; the fall survey during
                  September; and the winter survey during February. A fourth
                  survey (summer survey during July) may be undertaken. Survey
                  dates are selected to count the spring and fall populations at
                  or near the peak of their migration and to count winter (and
                  possibly summer) populations when they are most stable. Three
                  single-engine fixed-wing aircraft, will be used for each
                  survey.

                  The aircraft will be flown at approximately 150 feet above
                  water level and 200 -meters offshore, following the shoreline
                  as closely as possible given the aircraft's capabilities, and
                  maintaining an airspeed of 95 - 100 mph. All birds and marine
                  mammals will be observed within 200 meters from each side of
                  the aircraft.   Date, survey beginning and stop time, wind
                  speed and direction, air temperature, cloud cover, ceilings
                  and visibility will be recorded for each survey date.

                  Survey weather minimums will be restricted to 1,500 feet or
                  greater ceilings, 10 miles horizontal visibility, surface
                  winds of 15 knots or less, and seas no larger than wavelets.

                  The entire coastline of PWS and southern KP, including
                  Kachemak Bay, will be surveyed during each of the three
                  seasonal surveys.

                  The visibility bias associated with counting birds from low
                  flying aircraft will result in an underestimate of population

                                             281









              size and bird densities, but this bias will be the same in
              both oiled and non-oiled areas and remain relatively constant
              between surveys. Therefore, the bias will have no effect on
              comparisons between areas and years.

              Estimates    of  waterfowl    and   waterbird   abundance     and
              distribution will be done using direct comparisons appropriate
              for complete shoreline survey indexes.        If a significant
              change in a species group or individual species (such as sea
              ducks or goldeneye) is detected, then subsets of data
              stratified by habitat will be analyzed to assess oil effects
              in comparable oiled and non-oiled habitat for each survey.

              Short- and long-term recovery rates, if there is a significant
              oil effect, will be calculated using a repeated measures
              ANOVA.     Trends may also be compared using regression
              techniques.

         B.   Boat Survey Sampling Methods

              Surveys will be conducted from small boats manned by an
              operator and two observers. observers will record all birds
              and marine mammals within 100 meters on each side of the boat
              within survey transects.         The survey window extends
              approximately 40-50 m ahead of and 100 m above the moving
              boat. Date and time of survey, and environmental variables
              including wind velocity and direction, air and water
              temperature, weather, observation conditions, sea state, tide,
              presence or absence of oil, and human activity will be
              recorded for each transect.


              a.     PWS

              A stratified random sampling design, including shoreline,
              coastal/pelagic and pelagic strata, will be used to meet
              Objectives 1-3. Approximately 29% of the shoreline and 25% of
              coastal/pelagic and pelagic plots will be surveyed once in
              March 1990 and three times (one survey each in June, July and
              August) during the summer of 1990. Surveys will be conducted
              jointly with Marine Mammal Study Number 6.        All birds and
              marine mammals within transect boundaries will be recorded.

              The shoreline stratum includes all water within 200 m of any
              shoreline, and will be surveyed by traveling 100 m offshore,
              parallel to the coast, at 5-10 knots. The shoreline stratum
              is divided into transects consistent with those used by D.
              Irons during 1984-1985 surveys.

              Coastal/pelagic and pelagic strata consist of plots of water
              delineated by 5-minute intervals (latitude and longitude) on
              NOAA charts and exclude any water within 200 m of the coast.
              Coastal/pelagic     and   pelagic   plots    differ    in    that

                                          282









                coastal/pelagic plots include more than approximately 1 nm
                (nautical mile) of shoreline, whereas pelagic plots contain
                less than 1 nm of shoreline.    For plots that are 5 minutes
                wide (east to west), two north-south transects extending 100
                m on each side of the boat and located 1 minute inside the
                east and west boundaries of the plot will be steered by a
                combination of compass heading and LORAN-C coordinates. For
                plots that are less than 5 minutes wide due to intersection
                with land, either one or two north-south transect lines will
                be surveyed, depending on plot size.

                b.   Kodiak Island

                Shoreline and pelagic transects will be surveyed off the west
                and north coasts of Kodiak Island to meet Objectives 1-3. The
                shoreline was divided into transects based on habitat type
                following the criteria of Irons et al. (1988) and then a
                simple random sample consisting of 25% of the transects was
                chosen. These shoreline transects, and all pelagic transects
                surveyed by Forsell and Gould (1981) will be surveyed in July
                1990 and February 1991.

                C.   Naked Island

                To meet Objective 4, the number of Pigeon Guillemots in the
                Naked Island group will be determined by circumnavigating each
                island in a small boat between 50 and 100 m from shore and
                counting all Guillemots on land and water. Surveys will be
                done during peak attendance, (between 0500 and 1000 hours),
                during the first week in June, when breeding birds are in
                attendance, and during the last week in July, when
                non-breeding birds are in attendance as well.

                Ancillary observations on Pigeon Guillemot reproduction will
                be made.   objectives of such observations include nesting
                success, fledgling weight, the rate at which chicks are fed,
                and identification of fish species fed to chicks as food.

                The number of Marbled Murrelets (and other birds and marine
                mammals) on five inshore transects on the western side of
                Naked Island will be counted from a small boat (Zodiac) to
                meet Objective 5. The transect routes will be those used by
                Bird Study 9 in 1989, and in three years previous to the oil
                spill to allow comparison with all previously collected data.

                Estimates of waterbird abundance and variances (Objective 1)
                will be made using ratio estimators and statistics appropriate
                for stratified random sampling. This technique will be used
                if the number of birds is positively correlated with transect
                length. If the- correlation between bird numbers and transect
                length is poor, simple means and variances will be calculated.


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                Differences in waterbird abundance between oiled and non-oiled
                areas (Objective 2) will be tested by examining the change in
                abundance on each transect between 1984 and 1989. One-tailed
                t-tests or the Mann-Whitney test will be used. If areas are
                stratified further based on habitat type then ANOVA will be
                used. ANOVA will be used to make comparisons between pre- and
                post-oil spill data with respect to oiled and non-oiled areas.

                Short- and long-term recovery rates (Objective 3), if there is
                a significant oil effect, will be analyzed using a repeated
                measures ANOVA. Trends may also be compared using regression
                techniques.

                The outlier t-test will be used to test whether the number of
                guillemots counted at Naked Island in 1990 is significantly
                lower than the mean of mean counts in prior years (objective
                4).    Counts will be logarithmically transformed prior to
                testing.

                The outlier t-test will be used to test whether the abundance
                indices of Marbled Murrelets in 1990 are significantly lower
                than the mean of mean indices for prior years (Objective 5).


                                           BIBLIOGRAPHY



          Alaska    Department     of   Environmental    Conservation.         1989.
                Unpublished preliminary digital maps of oil-impacted shore
                based on aerial and boat surveys during early ADEC response
                activities. (ARC/INFO data file].

          Bowden, D.C. 1973. Review and evaluation of May waterfowl
                breeding ground survey.       Unpubl. Manu. Fish and Wildlife
                Service. Anchorage, Alaska. 74 pages.

          Butler, W. 1989. Prince William Sound and Kenai Peninsula
                Migratory Bird Aerial Survey Project. Unpublished data. U.S.
                Fish and Wildlife Servicef Anchorage, Alaska.

          Cochran, W.G. 1977. Sampling Techniques. John Wiley and Sons,
                Inc. New York, New York. 428 pages.

          Conant, B., J.G. King, J.L. Trapp, and J.I. Hodges.                  1988.
                Estimating populations of wintering waterfowl in southeast
                Alaska.    U.S. Fish and Wildlife servicef Juneau, Alaska.
                Unpublished Report. 16 pages.

          Dwyer, T.J., P. Isleib, D.A. Davenport, and J.L. Haddock. 1975.
                Marine Bird Populations in Prince William Sound Alaska. U.S.
                Fish and Wildlife Service, Anchorage,, Alaska.          Unpublished
                Report, 21 pages.

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          Forsell, D.J. and P.J. Gould. 1981. Distribution and abundance
                of marine birds and mammals wintering in the Kodiak area of
                Alaska. U.S. Fish and Wildlife Service, Office of Biological
                Services, Washington, D.C. FWSIOBS-81113. 81 pages.

          Haddock, J.L., C. Evans, L.W. Sowl, M.E. Isleib, P. Havens,
                J. Reynolds, and D. Johnson. Unpublished 1971 Prince William
                Sound aerial survey data. U.S. Fish and Wildlife Service,
                Anchorage, Alaska.

          Hogan, M.E. and J. Murk. 1982. Seasonal distribution of marine
                birds in Prince William Sound, based on aerial surveys, 1971.
                U.S. Fish and Wildlife Service, Anchorage, Alaska. Unpublished
                Report.

          Irons, D.B., D.R. Nysewander, andJ.L. Trapp. 1988. Princewilliam
                Sound sea otter distribution. U.S. Fish and Wildlife Service,
                Anchorage, Alaska. Unpublished Report, 31 pages.

          Irons, D.B., D.R. Nyeswander, and J.L. Trapp. ms. Prince William
                Sound waterbird distributions in relation to habitat type.
                U.S. Fish and Wildlife Service, Anchorage, Alaska. 24 pages.

          Klosiewski, S. and L. Hotchkiss. 1990. Assessment of injury to
                waterbirds from the Exxon Valdez oil spill:          Surveys to
                determine distribution and abundance of migratory birds in
                Prince William Sound and the Northern Gulf of Alaska. Bird
                Study Number 2.    Preliminary Status Report.     U.S. Fish and
                Wildlife Service, Anchorage, Alaska.

          Nishimoto, M. and B. Rice. 1987. A re-survey of seabirds and
                marine mammals along the south coast of the Kenai Peninsula,
                Alaska during the summer of 1986.      U.S. Fish and Wildlife
                Service, Alaska Maritime National Wildlife Refuge, Homer,
                Alaska. Unpublished Report, 79 pages.



          BUDGET

          Category        Aerial Survey          Boat Survey        Total
                          Budget                                    Budget

          Personnel            $59.0                $207.0          $266.0
          Travel                5.0                  14.0              19.0
          Contractual           47.0                 64.0              111.0
          commodities           3.0                  60.0              63.0
          Equipment             0.0                  12.0              12.0

          Total               $114.0                $357.0          $471.0



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       BIRD STUDY NUMBER 3

       Study Title:    Population Surveys of Seabird Nesting Colonies in
                       PWS, the Outside Coast of the KP, Barren Islands,
                       and Other Nearby Colonies.

       Lead Agency:    FWS



                                   INTRODUCTION



       The 1989 oil spill in PWS and its drift to the west along the
       Alaskan coast prompted resurvey in 1989 of seabird colonies in PWS,
       the Chiswell Islands, the Barren Islands, sites along the Alaska
       Peninsula, some sites near Kodiak Island, and the Semidi Islands.
       Most of these colonies had been censused at least two and up to six
       different years out of the last 17 years, providing some base line
       for determining changes that may have occurred on the colonies
       surveyed.   Murres and kittiwakes on one colony site, Middleton
       Island, have been censused 11 of the last 17 years.

       Diving seabirds are known to be easily impacted by oil spills (King
       and Sanger, 1979). In addition, these species are long-lived with
       low reproductive rates, thus making any mortality of adults a
       critical factor in these species' ability to recover from loss.
       There are at least 320 seabird colonies that occur within the area
       affected by the oil spill. They contained about 1,121,500 breeding
       seabirds of which approximately 319,000 were murres (USFWS, Catalog
       of Alaskan Seabird Colonies --Computer Archives 1986).       Some of
       these colonies are among those most visited by tourists in    Alaska.
       Cliff-nesting seabirds, like murres, are an important part   of this
       human use/tourism.

       This year, the study will concentrate more on murre populations and
       less on other species than it did last year. This will facilitate
       increased replicate counts and improve statistical evaluation. The
       breeding season censuses will occur at the Chiswell Islands, Barren
       Islands, specific sites along the Alaska Peninsula and the Semidi
       Islands. Egg laying is the accepted time to census murres since
       their colony attendance is too variable at other times (Hatch and
       Hatch, 1988; Byrd, 1989; Nysewander, 1989).

                                    OBJECTIVES


       Determine whether the numbers of selected species of breeding
       colonial seabirds within the oiled area have decreased compared to
       numbers previously censused at these sites.       Non-oiled nesting
       colonies will be surveyed as a control.




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                                           METHODOLOGY

              Assessment of injury to murre populations is being conducted in
              four general areas:     1) Chiswell Islands, 2) Barren Islands, 3)
              Alaska Peninsula sites, and 4) Semidi Islands, a non-oiled control
              area.     Middleton Island is not part of this particular
              investigation, but an ongoing study at that location will provide
              numbers of murres present on those colonies for comparison with
              affected areas. Changes in numbers of breeding adult seabirds will
              be documented, with primary emphasis placed on murres.

              Total counts are not feasible at large colonies like the Semidi and
              Barren Islands; hence previously established plots will be
              utilized. Two strategies will be used: 1) counts of adult murres
              on plots from land-based observation points; and 2) counts from
              boat-based observation vantage points where land-based observations
              are not possible.     These plots may also serve as a correction
              factor for total counts or estimates that may be needed for
              comparison with past estimates.

              Specifically, the two strategies used in 1989 will be implemented
              again in 1990 (Nysewander, 1989), as follows:

              1)   A combination of total subcolony or island counts and counts
                   of sample plots counted from boats will occur at colony sites
                   like the Barren Islands and Chiswell Islands because the
                   colonies are much larger, in very exposed waters, have a poor
                   history of censusing, and require counts from boats. Previous
                   sample plots were established on the basis of accessibility
                   and visibility.

              2)   Land-based plots will be continued at the Semidi Islands
                   because these colonies are too large for total counts, and
                   land plots are feasible and have been used for over 10 years.
                   Sample plots were previously selected on the basis of
                   accessibility.     The Alaska Peninsula murre colonies will
                   require a combination of both applications since some portions
                   of the colonies are visible from land, but most aspects of the
                   colony will require boat counts.

              Colonies will be recensused using standard FWS methodology for
              either land-based or boat-based counts of seabirds (Byrd 1989;
              Hatch and Hatch 1988 and 1989; Irons et al. 1987; Nishimoto and
              Rice 1987) .    Efforts will be made to complete at least three
              replicate counts of colonies or plots after eggs are laid. Counts
              will be conducted on three separate days between the hours of 1000
              and 1600.   Plot counts and photographs of plots will be used to
              establish correction factors of total subcolony or island counts,
              comparisons with past counts of plots, and for evaluation of future
              recovery or change.

              At least three observers will make the counts by binoculars from

                                                287










       the boat. Each observer counts each section of the cliff at least
       two times and all counts are compared to determine if sections of
       the plot were missed (differences in counts by observers cannot be
       greater than 5%) and need more replicate counts.

       Standard procedures and assumptions used by the FWS for colony
       counts in the Alaska Maritime National Wildlife Refuge are
       described by Garton 1988 and Byrd 1989. Several key assumptions
       are: 1) Plots, by necessity, are not random and selection is based
       on accessibility; hence this study makes the assumption that counts
       within plots are representative of the way the counts varied on the
       entire colony; 2) Counts of plots or entire colonies from boats
       are very difficult for large colonies and replications of counts by
       several observers on the same day and different days illustrate the
       need to refine the accuracy and the variation recorded. This means
       that even counts of entire colonies are also considered to be
       indices, but this study assumes that changes in these indices
       represent the changes occurring in the colony; 3) Counts of things
       are very unlikely to be normally distributed and are more likely to
       be skewed and clumped.    This type of data requires either very
       large sample sizes, the use,of a non-parametric test, or the data
       needs to be transformed logarithmically and then tested by the
       appropriate parametric test. This transformation normalizes the
       data and is required for valid application of statistical tests on
       small sample sizes (Fowler and Cohen 1986, D. Robson pers. comm.).

       Standard FWS procedures mentioned prefer to compare trends between
       years using numerous replicate counts where all plots are censused
       each count day and these counts are replicated on successive days.
       The average of daily counts on the Semidi Islands will be used to
       calculate a confidence interval for the estimate as was done on the
       Semidi Islands data in the past (Hatch and Hatch 1988; Hatch and
       Hatch 1989).     At other sites where there were fewer replicate
       counts, past procedures which averaged available counts, will be
       followed.   The important question is whether the "after oiling"
       response is outside the anticipated annual variation in colony
       numbers that would be expected from past historical data without
       oiling effects.

                                   BIBLIOGRAPHY


       Byrd, G.V. 1989. Seabirds in the Pribilof Islands, Alaska:
            trends and monitoring methods. M.S. thesis, Univ. of Idaho,
            Moscow, Idaho. 96pp.

       Garton, E.O. 1988. A statistical evaluation of seabird monitoring
            programs at three sites on the Alaska Maritime National
            Wildlife Refuge. Univ. of Idaho, Moscow, Idaho. Unpubl. Rept.
            from contract with the refuge. 15pp.




                                       288









             Fowler, J. and L. Cohen. - 1986.      Statistics for Ornithologists.
                   British Trust for Ornithology, BTO Guide No. 22, Tring,
                   Hertfordshire. 175pp.

             Hatch, S.A. and M.A. Hatch. 1989. Attendance patterns of common
                   and thick-billed murres at breeding sites: implications for
                   monitoring. J. Wildl. Mgmt. 53(2):483-493.

             Hatch, S.A. and M.A. Hatch. 1988. Colony attendance and
                   population monitoring of black-legged kittiwakes on the Semidi
                   Islands, Alaska. Condor 90:613-620.

             Irons, D.B., D.R. Nysewander, and J.L. Trapp. 1987. Changes in
                   colony size and reproductive success of black-legged kit-
                   tiwakes in Prince William Sound, Alaska, 1972-1986.         U. S.
                   Dept. Interior, Fish and Wildl. Serv., Anchorage, Alaska,
                   Unpubl. Rept. 37pp.

             King, J.G. and G.A. Sanger. 1979. Oil vulnerability index for
                   marine oriented birds. Pp. 227-239 in J.C. Bartonek and D.N.
                   Nettleship eds. Conservation of marine birds of northern North
                   America.    U. S. Fish and Wildl. Serv., Washington, D.C.
                   319pp.

             Nishimoto, M. and B. Rice. 1987. A re-survey of seabirds and
                   marine mammals along the south coast of the Kenai Peninsula,
                   Alaska during the summer of 1986. U. 'S. Fish and Wild. Serv.,
                   Alaska Maritime National Wildl. Refuge, Homer, Alaska.
                   Unpubl. Rept. 79pp.

             Nysewander, D. 1989. Population surveys of seabird nesting colonies
                   in Prince William Sound, the outside coast of the Kenai
                   Peninsula, Barren Islands, and other nearby colonies.
                   U.S. Fish and Wildlife Serv., Alaska Maritime National
                   Wildlife Refuge, Homer, Alaska. Unpubl. Rept. 52pp.

             U. S. Fish and Wildlife Service. 1986. Catalog of Alaskan seabird
                   colonies--computer archives.     U.S. Fish and Wildl. Serv.,
                   Migratory Bird Management, Anchorage, Alaska 99503.


             BUDGET:    FWS


             Personnel                $87.5
             Travel                    12.3
             Contractual              111.2
             Commodities               24.1
             Equipment                 16.0

             Total                   $251.1



                                               289












         BIRD STUDY NUMBER 4

         Study Title:   Assessing the Effects of the EVOS on Bald Eagles

         Lead Agency:   FWS

                                    INTRODUCTION

         The area af f ected by the EVOS provides year-round habitat f or
         approximately 5000 adult bald eagles and seasonal habitat for an
         additional estimated 2500 immatures.    An unknown number of bald
         eagles from breeding areas in south-central Alaska probably also
         winter in the PWS.

         Bald eagles are closely associated with intertidal habitats that
         have been heavily impacted by the EVOS. Nearly all nests in the
         spill area occur within 100 meters of the beach and eagles commonly
         forage in intertidal habitats on fish and marine invertebrates.
         Eagles that breed elsewhere, but spend winters in the spill area,
         will also use the affected intertidal habitats for foraging.

         Contamination of these intertidal habitats may result in serious
         impacts to bald eagles. Effects may include direct mortality of
         adults and immatures from ingestion of oil -contaminated food or
         from preening of oil from feathers.    Eagles that become heavily
         oiled or entrapped in oil may die. Mortality of embryos can occur
         when eggs are contaminated with oil carried to the nests on the
         plumage of the adults. Decreases in the abundance of prey such as
         herring, eulachon, salmon, or marine invertebrates may increase the
         vulnerability of eagles to starvation or to disease induced by
         weakened physical condition.      Significant losses of breeding
         adults, eggs, nestlings and non-breeding eagles are expected.

         This study is designed to document the magnitude and duration of
         these impacts and determine whether these impacts are a result of
         oil contamination. Estimates for the number of eagles occupying
         the spill area after the spill will be compared with historical
         data to identify changes in the population.          Occupancy and
         reproduction surveys will be conducted to determine productivity
         and to document differences in production between oil-affected and
         non-oiled areas.   Nestling and adult bald eagles from oiled and
         non-oiled areas will be radio-tagged and monitored to estimate
         survival ratest distribution and exposure to oiled areas, and
         determine causes of mortality.

         Because eagles mature slowly and are long-lived, impacts to the
         population may not be readily apparent. It may require an extended
         period of study to substantiate the long-term impact of oil
         contamination on bald eagles.




                                         290











                                         OBJECTIVES

           A.   Estimate numbers of resident bald eagles such that the
                estimate is within 10% of the actual size 95% of the time-,
                determine whether changes in population size have occurred in
                the oil-affected areas since 1982 and test whether the change
                in number of eagles in oil-affected areas is different than
                changes in non-oiled areas.

           B.   Test the hypothesis that productivity of bald eagles is the
                same in oiled and non-oiled areas (a = 0.05).

           C.   Test the hypothesis that survival rates are the same for bald
                eagles in oiled and non-oiled areas (a = 0.05).

           D.   Determine toxic and sublethal effects of oiling on eagles and
                eggs.


                                          METHODS



           Population surveys (objective A).      Surveys of randomly selected
           plots roughly 50 square miles in size will be conducted from
           Malaspina Glacier to Cape Elizabeth in early May, following
           methodology discussed in Hodges et al. (1984). All shorelines in
           each selected plot will be flown at an altitude of about 200 feet
           and an airspeed of 90-100 knots using fixed-wing aircraft. Eagles
           will be classified as either white-headed or immature. "White-
           headed" eagles will include sexually mature adults and near-adults
           that have predominately white heads. This survey will not directly
           estimate the number of immatures, therefore we will assume that our
           ability to detect all age classes is equal for birds in flight, and
           a ratio of adults to immatures observed flying will be used to
           estimate the number of immatures.

           A parametric two-sample t-test (Steel and Torrie 1960) which does
           not require equal variances will be used to test the above
           hypotheses.    Analysis of variance will be used for multiple
           comparisons.  Assumptions necessary for valid application of the t-
           test will be  checked (e.g., test for normality). If assumptions
           are violated, we will use either an appropriate transformation or
           an equivalent non-parametric test.

           Productivity  surveys   (Obiective B) .    Two surveys to determine
           productivity will be conducted in the oil spill area (PWS, KP,
           Kodiak/Afognak Islands and the Alaska Peninsula) and in the Copper
           River Basin, an area used by eagles that may winter in the oil
           spill area. The first aerial survey will be flown during mid-May
           to estimate the number of adults that attempt to breed. The second
           survey will be flown in mid-July to estimate the number of
           successful nests and the number of young produced. Surveys will be
           conducted from helicopter at an altitude of 80-200 feet at 40-60

                                            291









         kts. airspeed to determine nest status.          Data collected will
         include number of nests surveyed, number of nests occupied, number
         of nests that successfully produce young, and number of young
         produced (Postupalsky 1974).

         Nests that fail will be climbed to collect dead eggs or nestlings
         and to identify the cause of failure.

         The hypothesis that there is no difference in the observed
         production among treatment groups compared with what would be
         expected if nests were assigned randomly to each of the treatment
         groups will be tested using a non-parametric permutation test.

         Beaches within 1/2 mile of bald eagle nest sites are representative
         of bald eagle home range in coastal Alaska (USFWS, unpubl. data).
         The length of shoreline within the "home range" will be measured
         and the lengths of segments classified as heavily, moderately,
         lightly, or unoiled will be totalled for each "home range". These
         values will be used in a multiple regression to identify
         relationships among the degree of oiling, nest occupancy, and
         productivity parameters.      Data on productivity from the Copper
         River Basin will be compared with data from coastal areas.
         Productivity data from southeastern Alaska will also be used for
         comparative purposes.

         Survival Studies (Objective C):       To estimate survival rates, 60
         eagles (15 adults and 15 nestlings each from oiled and non-oiled
         areas) will be tagged with radio transmitters (Pollock et al.
         1989).    Approximately 15 adult bald eagles will also be radio-
         tagged in the Copper River Basin to demonstrate whether        ' eagles
         breeding inland winter in areas affected by the oil spill. Weekly
         aerial flights will be made to relocate the transmitters using
         standard telemetry techniques (Gilmer et al.    1981) and to document
         eagle numbers, distribution and mortality within the study,area.
         Dead eagles will be retrieved and necropsied to determine the cause
         of death.

         A Z-test (Bart and Robson 1982) will be used to test for
         significant differences in survival rates between eagles marked in
         oiled areas and eagles marked in unoiled areas.           This Z-test
         requires the use of a transformation of the survival rate and
         standard error to normalize its distribution and allow use of a'Z
         statistic to test for differences in survival rates.           Accurate
         relocations    of   individual   radio-marked    eagles   will    allow
         appropriate classification of eagles into treatment groups based on
         the proportional amount of time they were located in- oiled or
         unoiled areas.

         Toxic and Sublethal Effects of Oiling (Oblective DL: All eagles
         found dead will be collected and necropsied to determine the cause
         of death, to note the extent of oiling and to look for ingested oil


                                           292









           or other signs of oil contamination.      Tissue samples from the
           collected specimens will be analyzed for contaminants.

           Unhatched eggs collected from failed nests will be examined for oil
           contamination of eggshells, egg contents, and the presence and
           development of embryos.

           Blood samples from free-flying birds will be collected by properly
           trained personnel and analyzed to determine concentrations of
           hydrocarbons   and   other   contaminants   associated   with    oil
           contamination. Approximately equal numbers of bald eagles will be
           sampled from oiled and non-oiled areas. Blood samples will also be
           analyzed for standard blood chemistry profiles, which will help
           identify sublethal impacts.

           Significant differences in levels of contaminants and blood
           characteristics between bald eagles from oiled and non-oiled areas
           will be tested using a 2-sample t-test (a = 0.05).      Assumptions
           necessary for valid application of the t-test will be checked
           (e.g., test for normality) . If assumptions are violated, either an
           appropriate transformation or an equivalent non-parametric test
           will be used.



                                      BIBLIOGRAPHY

           Bart, J. and D. S. Robson. 1982. Estimating survivorship when the
                subjects are visited periodically. Ecology 63:1078-1090.

           Bortolotti, G. R. 1984. Physical development of nestling bald
                eagles with emphasis on the timing of growth events. Wilson
                Bull. 96:524-542.

           Carpenter, in press.

           Gilmer, D. S., L. M. Cowardin, R. L. Duvall, L. M. Mechlin, C. W.
                Shaiffer, and V. B. Kuechle. 1981. Procedures for the use of
                aircraft in wildlife biotelemetry studies. U. S. Fish and
                Wildlife Service Resource Publication 140. 19 pp.

           Hodges, J. I., J. G. King, and R. Davies. 1984. Bald eagle breeding
                population survey of coastal British Columbia. J. Wildl.
                Manage. 48:993-998.

           Kaplan, E. L. and P. Meier. 1958. Non-parametric estimation from
                incomplete observations. J. Am. Stat. Assoc. 53:457-481.

           Pollock, K. H., S. R. Winterstein, C. M. Bunck, and P. D. Curtis.
                1989. survival analysis in telemetry studies: the staggered
                entry design. J. Wildl. Manage. 53:7-15.



                                           293









       Postupalsky, S. 1974. Raptor reproductive success: some problems
             with terminology. Raptor Research Report 2:21-31.

       Steel, R.G.D. and J.H. Torrie. 1960. Principals and procedures in
             statistics. McGraw-Hill, New York. 481 pp.


       Budget:    FWS

       Personnel             $120.0
       Travel                  17.0
       Contractual            503.0
       Commodities             25.0
       Equipment               10.0

       Total                 $675.0






































                                         294












             BIRD STUDY NUMBER 5


             Study Title:    Impact Assessment of the EVOS On Peale's Peregrine
                             Falcons


             Lead Agency:    FWS

             Cooperating Agency:      ADF&G




                                          INTRODUCTION



             Peale's falcons (Falco peregrinus pealei) occur along the
             southern coast of Alaska from the Aleutian Islands through
             southeastern Alaska.     The goal of this project is to determine
             whether the EVOS has had, or will have, a measurable impact on
             Peale's peregrine falcons in PWS and coastal KP.

             Peale's falcon populations in Alaska have been estimated at
             between 500-600 pairs (Schempf 1989, Ambrose pers. comm).             An
             estimated 40-60 pairs inhabit PWS and coastal KP (Janik & Schempf
             1985) and another 20-30 pairs occur in the Kodiak Archipelago,
             upper AP, and CI area, for a total of 60-90 pairs in coastal
             habitat affected by EVOS.

             Alcids, small gulls, and petrels are prime peregrine prey species
             that became oiled as a result of EVOS and may be taken by fal-
             cons.    Oil transferred to peregrine falcons could affect in-
             dividuals and the population through:       1)   coating of feathers
             and the resulting loss of insulation and flight capabilities; 2)
             reduced reproduction due to ingestion of hydrocarbons and trace-
             metals that affect the breeding physiology of adults;                 3)
             reduced reproduction due to transfer of oil from feathers of
             incubating adults to eggs;     4)   mortality of individuals due      to
             toxicity of oil; and      5)   reduced reproduction due to reduced
             prey availability when prey populations are impacted.

             This project will continue to provide information on the number
             of nest sites occupied by Peale's falcons and their productivity.
             These data, in combination with historical data for this area,
             will provide a basis to evaluate whether changes have occurred in
             the distribution, abundance, and/or productivity of falcons.
             Examination of secondary wing feathers taken from young, along
             with prey remains and eggs collected from occupied aerie, will
             provide evidence of whether crude oil was ingested or absorbed by
             falcons.   Analysis of wing feathers and prey remains collected
             several months after the oil spill will provide information on
             the bioaccumulation of trace-metals from crude oil, in marine and
             terrestrial food chains.



                                               295












                                    OBJECTIVES

       A.   Test   the   hypothesis    that   nest   site   occupancy    and
            productivity are lower in the project area as a result of
            EVOS than in other populations.

       B.   Test the hypothesis that the quantities of vanadium and
            nickel in peregrine feathers are the same for birds nesting
            in oiled and non-oiled areas.

       C.   Count and identify prey remains collected at aeries.

       D.   Test   the   hypothesis    that   the   level    of    pesticide
            contamination of egg clutches in the project area is less
            than contamination levels reported in scientific literature
            as causing reproductive failures in peregrine falcons.


                                      METHODS

       The project area will include the mainland shore and islands of
       PWS from Cape Hinchinbrook along the southern coast of the KP
       through Kachemak Bay.

       Two surveys of the project area will be conducted.         Guidelines
       have   been   formulated    to   standardize   survey     techniques,
       terminology,. and data collection.        The initial survey, to
       determine presence or absence of peregrines at coastal bluffs and
       to collect fresh egg samples for contaminant analysis, will take
       place in mid-April.     A boat and helicopter will be used for
       transportation.    If a helicopter is used at sites with large
       concentrations of cliff nesting seabirds, it will land far enough
       away from bluffs to minimize disturbance.           Observers will
       approach on foot to survey potential nesting habitat.

       The second survey via helicopter, in June, will cover the same
       area, but will focus on the sites determined to be occupied by
       peregrine falcons during the initial survey.         Nests will be
       located by observers on the ground and then visited to collect
       feather samples and to band nestlings with standard aluminum
       bands. Secondary feather samples from young will be collected at
       aeries during the June nest survey.     Prey remains and addled or
       broken eggs will be collected at nest sites.       During both sur-
       veys, investigators will document oil on falcons and look for
       bands on adults to learn where they were banded.

       Twenty-five prey remains will be examined for hydrocarbon con-
       tamination.   Samples collected for hydrocarbon analysis will be
       handled carefully.    Chain-of -custody will be maintained for all
       samples, and they will be stored in a secure facility at ADF&G in
       Anchorage until they can be sent to an approved laboratory for
       analysis.

                                       296









              Feathers grown by nestling peregrine falcons should contain
              trace-elements in an array of concentrations unique to the local
              ecosystem (Parrish et al. 1983).        High levels of nickel and
              vanadium have been associated with North Slope crude oil and
              these trace-metals are bioaccumulated in marine and terrestrial
              food chains (Minerals Management Service 1988). Predators at the
              top of food chains, such as the peregrine falcon, may encounter
              toxic levels of trace-metals because these elements are con-
              centrated with each step up the food chain. Toxic quantities of
              trace-metals have been implicated in population decline- of
              peregrines and other raptors (Newton- 1979).      Elevated levels of
              nickel in the diet will produce physiological effects similar to
              lead or mercury poisoning such as central nervous system disor-
              ders and reduced reproductive success (Williams, pers. comm.)
              Traces of these metals can be measured efficiently in birds
              feathers by instrumental neutron-activation analysis (INAA)
              (Wainerdi and Dubeau 1963).     Feather samples from peregrines not
              influenced by the oil spill from other regions of the state will
              serve as controls.

              Approximately 30 feather samples will be collected for trace-
              metal analysis.    The distal 1 cm of the fifth secondary remige
              will be collected from adult and nestling peregrines for INAA as
              described by Parrish et al. (1983).        Feather samples will be
              labeled and preserved.     Chain-of -custody will be maintained for
              all samples and they      will be stored in a secure facility at
              ADF&G in Anchorage until they can be sent to an approved laborat-
              ory for INAA.

              The decline of peregrine populations in North America during the
              1950's through the early 1970's was linked to organochlorine
              pesticides (Hickey 1969).     Substantial levels of biocides have
              been found in Peale's falcons in coastal British Columbia and it
              has been suggested that the depressed reproductive success on
              Langara Island was largely due to the effects of pollutants
              (Nelson   &  Myres    1976).      Since   trace-metals    may    affect
              reproduction in peregrines, similar to organochlorine pesticides,
              a pesticide monitoring program would help identify which factors
              are involved.    Thus collection of fresh eggs is necessary for
              pesticide analysis.

              Historically, about 35 aeries are thought to be occupied each
              year in the project area. The collection of 10 eggs will provide
              an adequate sample without significantly impacting productivity.

              Chain-of-custody will be maintained for all samples and they will
              be stored in a secure facility until they can be sent to an
              approved laboratory for chemical analysis as described by
              Cromartie et al. (1975) and Kaiser et al. (1980).

              Data analysis to achieve objective A involves a comparison of
              site occupancy and productivity in the project area with other

                                               297









          peregrine populations. In order to control for yearly variation
          in brood size, the mean brood size of peregrines in Norton Sound
          for 1989 and 1990, will be compared to the historical data with
          an analysis of variance (ANOVA) (Snedecor and Cochran 1980) using
          the appropriate linear contrasts to test the hypothesis that the
          mean brood sizes are equal between the historical data and Norton
          Sound.   Assuming the Norton Sound data do not differ from the
          historical data, a two sample T-test will be used to test the
          null hypothesis that mean brood size in PWS in 1990 is greater
          than or equal to mean brood size in Norton Sound.                  The
          alternative hypothesis is that mean brood size in 1990 in PWS is
          less than mean brood size in Norton Sound.        A similar analysis
          will be done for the 1990 productivity data.

          ANOVA and a two sample T-test have the following assumptions:

          1) The samples are random and independent;
          2) The distribution of the different means is normal; and
          3) The variances of the samples are equal.

          A Q-Q plot (Hoaglin et al. 1985) of the raw data will determine
          whether the data is approximately normal, in which case the
          Central Limit Theorum will insure that assumption two is met. If
          assumption two is not met, a non-parametric test will be employed
          (Conover 1980).     Bartlett's statistic will be used to test
          assumption three and a transformation employed, if necessary, to
          meet this assumption.

          Two separate Fisher's exact test (Ostle and Mensing 1982) will be
          used to determine whether PWS had lower nest occupancy rates than
          Norton Sound for 1989 and 1990.

          Data analysis to achieve objective B involves a two sample T-test
          (Snedecor and Cochran 1980) to determine whether trace-metal
          concentrations are lower in the project area than outside the
          project area.    The null hypothesis is that nickel and vanadium
          concentrations  in peregrine feathers from the project area in
          1990 is less than or equal to nickel and vanadium concentrations
          in peregrine feathers from elsewhere in Alaska in 1990.             The
          alternative hypothesis is that nickel and vanadium concentrations
          in peregrine feathers from the project area in 1990 were greater
          than nickel and vanadium concentrations in peregrine feathers
          from elsewhere in Alaska in 1990.

          For objective C, if hydrocarbon prey remains are observed, an
          estimate of the proportion of contaminated prey remains will be
          estimated at a 95% confidence interval. The confidence intervals
          require that the proportion be normally distributed.                 if
          necessary, transformations will be used to meet this assumption
          (Snedecor and Cochran 1980).

          The null hypothesis contained in Objective D states that levels

                                           298









          of pesticide contamination of peregrine eggs collected in the
          project area in 1990 are greater than or equal to the levels of
          pesticide contamination of peregrine eggs reported in literature
          as causing reproductive failures (Peakall et al. 1975).       The
          alternative hypothesis states that levels of pesticide contamina-
          tion of peregrine eggs collected in the project area in 1990 are
          less than the reported levels of pesticide contamination of
          peregrine eggs associated with reproductive failures.      A one-
          tailed, one sample T-test (Snedecor and Cochran 1980) will be
          used to test this hypothesis.   This test assumes the sample was
          randomly collected and the mean has a normal distribution.      if
          necessary, either a transformation will be used to meet the
          normality assumption or the Wilcoxon Signed Ranks test (Conover
          1980) will be employed to test this hypothesis.



                                     BIBLIOGRAPHY

          Conover, W. J. 1980. Practical nonparametric statistics, 2nd
               ed. John Wiley & Sons, New York, N. Y. 493 pp.

          Cromartie, E., W. L. Reichel, L. N. Locke, A. A. Belisle, T. E.
               Kaiser, T. G. Lamont, B. M. Mulhern, R. M. Prouty, and D. M.
               Swineford. 1975. Residues of organochlorine pesticides and
               polychlorinated biphenyls and autopsy data for Bald Eagles,
               1971-72. Pestic. Monit. J. 9: 11-14.

          Hickey, J. J. (ed) 1969. Peregrine Falcon Populations: their
               biology and decline.    Univ. of Wisconsin Press, Madison.
               596 pp.

          Hoaglin, D. C., F. Mosteller, and J. W. Tukey. 1985. Exploring
               data tables, trends, and shapes.     John Wiley & Sons, New
               York, N. Y. 527 pp.

          Janik, C. A. and P. F. Schempf. 1985. Peale's peregrine falcon
               (Falco. peregrinus pealei) studies in Alaska, June 12-24,
               1985.   Raptor Management Studies, U.S. Fish and Wildlife
               Service, Juneau, Alaska. 12 pp.

          Kaiser, T. E., W. L. Reichel, L. N. Locke, E. Cromartie, A. J.
               Krynitsky, T. G. Lamont, B. M. Mulhern, R. M. Prouty, C. J.
               Stafford, and D. M. Swineford. 1980. Organochlrine pesti-
               cide, PCB, and PBB residues and necropsy data for Bald
               Eagles from 29 states - 1975-77.      Pestic. Monit. J. 13:
               145-49.

          Minerals Management Service. 1988. Draft environmental impact
               statement, Outer Continental Shelf Mining Program, Norton
               Sound Lease Sale.   Minerals Management Service, Anchorage,
               Alaska.



                                         299









         Nelson, R. W. and M. T. Myres. 1976. Declines in populations of
               peregrine falcons and their seabird prey at Langara Island,
               British Columbia. Condor 78: 281-293.

         Newton, I. N. 1979. Population ecology of raptors, Buteo Books,
               Vermillion, South Dakota. 399 pp.

         Ostle, B. and R.W. Mensing. 1982. Statistics in research 3rd ed.
         The Iowa State Univ. Press, Ames IA. 596pp.

         Parrish, J. R., D. T. Rogers, Jr., and F. P. Ward. 1983.
               Identification of natal locales of peregrine falcons (Falco
               peregrinus) by trace-element analysis of feathers. Auk 100:
               560-567.

         Peakall, D. B., T. J. Cade, C. M. White, and J. R. Haugh. 1975.
               Organochlorine residues in Alaskan peregrines.            Pestic.
               Monit. J. 8: 255-260.

         Schempf, P. F. 1989. Raptors in Alaska. Pages 144-54 in
               proceedings of the western raptor management symposium and
               workshop.   National Wildlife Federation, Washington, D. C.
               320 pp.

         Snedecor, G. W. and W. G. Cochran. 1980. Statistical methods,
               7th ed. Iowa State University Press, Ames, Iowa. 507 pp.

         Wainerdi, R. E. and N. P. DuBeau. 1963. Nuclear activation
               analysis. Science 139: 1027-1033.


         Personal Communications

         Ambrose, R. E. U.S. Fish and Wildlife Service, Endangered
               Species Branch, Fairbanks, Alaska.

         Williams, D. Quantum Medicine, Eagle River, Alaska.



         BUDGET:    FWS


         Personnel                            $ 32.2
         Travel & per them                        3.5
         services                                69.5
         Commodities                              2.5
         Equipment                                0.0

         TOTAL                                 $107.7






                                           300












           BIRD STUDY NUMBER 11

           Study Title:    Injury Assessment of Hydrocarbon Uptake by Sea
                           Ducks in PWS



           Lead Agency:    FWS

           Cooperating Agency: ADF&G



                                      INTRODUCTION

           This study will focus on the effects of petroleum hydrocarbon
           ingestion by Harlequin Ducks (Histronicus histronicus), Barrow's
           Goldeneyes (Bucephala islandica), Common Goldeneyes (Bucephala
           .clangula), Black Scoters (Oidemia nigra), and Surf Scoters
           (Melanitta perspicillata) in PWS as a result of the EVOS. PWS is a
           major wintering area for these sea duck species (Isleib and Kessel,
           1973).    It is also an important migration area for sea ducks in
           spring and fall and a breeding site for resident Harlequin Ducks
           during the summer (Hogan, 1980).     Harlequin Ducks in particular,
           because of their resident status and intertidal foraging habits,
           are considered substantially at risk to effects of the EVOS (King
           and Sanger, 1979).        Goldeneyes and Surf Scoters, although
           migratory, are also - at , risk because of their intertidal and
           subtidal foraging habits.

           The five sea duck species included in this plan are heavily
           dependent on intertidal and subtidal marine invertebrates (Vermeer
           and Bourne, 1982).      Surf Scoters and goldeneyes utilize blue
           mussels (Mytilus) and, like Harlequins, consume a wide variety of
           clams, snails, and limpets (Koehle, Rothe and Dirksen, 1982;
           Dzinbal and Jarvis, 1982).     Bivalves, particularly blue mussels,
           and small clams (Macoma) are well-known for their ability to
           concentrate pollutants at high levels (Shaw et al., 1976).          The
           EVOS may cause severe damage to marine invertebrates that support
           sea ducks throughout the year (Stekoll, Clement, and Shaw, 1980)
           and bioaccumulation in the food chain may result in uptake of
           petroleum hydrocarbons by sea ducks over a long period (Dzinbal and
           Jarvis, 1982; Sanger and Jones, 1982).     This study will determine
           levels of petroleum hydrocarbon ingestion by sea ducks, and will
           predict resultant physiological and life-history effects (Hall and
           Coon, 1988). A predictive model may be constructed for sea duck
           reproductive losses, for instance, based upon physiological ef f ects
           of petroleum contamination resulting from the EVOS. Pre-oil spill
           baseline data is available on petroleum contaminant levels of
           Harlequin Ducks in PWS (Irons, FWS, pers. comm.).





                                            301












                                   OBJECTIVES

       A.   Continue to develop a data base describing f ood habits of the
            five species of sea ducks in PWS.

       B.   Obtain data from other NRDA studies of petroleum hydrocarbon
            levels in marine invertebrates, particularly blue mussels,
            from the PWS area; Relate this data to the levels of petroleum
            hydrocarbons f ound by chemical analysis of invertebrates in
            gut samples from sea ducks collected in oil spill and control
            areas; and Test the hypothesis (at alpha = 0. 05) that the
            incidence of petroleum hydrocarbons in gut samples from
            collected sea ducks is higher in the oil spill areas than in
            the control areas investigated in 1989-90 (Oil Year 1).

       C.   Estimate by chemical analysis the petroleum hydrocarbon levels
            in collected sea duck tissues and body fluids within 10% of
            the actual value 95% of the time.

       D.   Test the hypothesis (at alpha = 0.05) that the incidence of
            petroleum hydrocarbons in tissues of collected sea ducks is
            significantly higher in 1989-91 in the oil spill areas than in
            the control areas.

       E.   Estimate the ingested petroleum hydrocarbon effects on
            morbidity, mortality, and reproductive potential of sea ducks.
            This information may be related to other studies to identify
            changes' in abundance and distribution within the affected
            areas.


                                     METHODS

       This study will compare levels of petroleum hydrocarbons          in
       tissues of five species of ducks collected in four study areas.
       The areas exposed to petroleum are western PWS and southwestern
       Kodiak Island. The unexposed control sites are southeastern PWS and
       southeastern Alaska, north of Juneau. Only PWS and the Juneau
       control site are to be investigated in Oil Year two (March 1990-
       February 1991). Tissues will be collected for evidence of either
       histopathology or chemical contamination. Additional seaducks will
       be collected in "clean" areas within western PWS. This is to
       provide a secondary control for ducks collected at known sites for
       heavy oiling of seaduck intertidal forage species.

       Seaduck collection in oiled areas of PWS will be integrated with
       the collection sites of blue mussels in oiled areas in order to
       demonstrate that seaducks feed in contaminated areas on
       contaminated prey. Ten such sites are located in western PWS. At
       each site where petroleum exposure status is documented for
       intertidal organisms, approximately ten Harlequin Ducks are to be
       collected in the summer of 1990. These ducks will be sampled for
       petroleum contamination of food items in the proventriculus, as

                                       302









             well as histopathology.

             Data on petroleum hydrocarbon levels in marine invertebrates
             (especially blue mussels) and the degree. of oiling at selected
             sites will be acquired from the Fish/Shellfish Studies 1 and 13,
             the Coastal Habitat Study, the Air/Water Studies, and Technical
             Services Study Number 3.

             ANOVA (Snedecor and Cochran, 1980) will be used to test the
             hypothesis that incidence of petroleum hydrocarbons in gut samples
             from collected sea ducks is higher in-the oil spill areas than in
             the control areas.

             Cumulative logit loglinear models (William and Grizzle, 1972;
             Agresti, 1984) on a per species basis will be used to model the
             incidence of petroleum hydrocarbons using the area in which
             collected as the explanatory variable.       Hypotheses concerning
             differences by area in incidence of petroleum hydrocarbons will be
             tested with a conditional likelihood ratio statistic for nested
             models (Agresti, 1984). A Bonferroni (Snedecor and Cochran, 1980)
             Z-statistic (Agresti, 1984) will be used to determine the nature of
             the differences among areas if the main effect is significant.

             Physiological effects will be classified as none, slight, moderate
             or severe. Loglinear models (Agresti, 1984) will be used to model
             the distribution of physiological classification by area by
             species.   A conditional likelihood ratio statistic for nested
             models will be used to test the hypothesis that physiological
             classification is independent of area. If area and physiological
             classifications are dependent, a Bonferroni (Snedecor and Cockran,
             1980) Z-statistic (Agresti, 1984) will be used to determine
             differences among areas while controlling for physiological effect.

             If possible, an exact test for contingency tables (Agresti, et al,
             1990) with ordered responses will be used to determine whether
             ducks in the oiled area were in significantly poorer physiological
             condition than ducks in the control area. If it is not feasible to
             perform the exact test because of unavailability of appropriate
             methodology, a cumulative logit analysis (Agresti, 1984) will be
             used to test this hypothesis.

             Tissues will be collected for either chemical analysis (presence,
             absence, or degree of petroleum residue) or histopathology.
             Results will be compared to unexposed specimens from "clean"
             (unexposed control) areas. Choice of materials and tissues,
             handling, and discussion of results will follow published,
             guidelines for interpreting residues of petroleum hydrocarbons in
             wildlife tissues (Hall and Coon, 1988).





                                             303













                                    BIBLIOGRAPHY

       Agresti, A. 1984. Analysis of ordinal categorical data.
             John Wiley & Sons, New York. 287 pp.

       Agresti, A., C.R. Mehta, and N.R. Patel. 1990. Exact
             inference for contingency   tables with ordered categories. J.
             Amer. Statist. Assoc. 85:   in press.

       Dzinbal, K.A. and R.L. Jarvis.    1982. Coastal feeding ecology
             of Harlequin Ducks in PWS,  Alaska, during summer. pp. 6 - 10
             in Marine birds: their feeding ecology and commercial
             Fisheries relationships.    Nettleship, D.A., G.A. Sanger, and
             P.F. Springer, eds.      Proc. Pacific Seabird Group Symp.,
             Seattle, WA., 6 - 8 Jan. 1982. Can. Wildl. Serv. Spec. Publ.

       Sanger, G.A. and R.D. Jones, Jr. 1982. Winter feeding
             ecology and trophic relationships of Oldsquaws and White-
             winged Scoters  on Kachemak Bay, Alaska. pp. 20 - 28    in Marine
             birds: their feeding ecology and commercial fisheries
             relationships.    Nettleship, D.A,      G.A. Sanger,    and P.F.
             Springer, eds.   Proc. Pacific Seabird Group Symp., Seattle,
             WA., 6 - 8 Jan  1982. Can. Wildl. Serv. Spec. Publ.

       Hall, R.J. and N.C.   Coon. 1988. Interpreting residues of
             petroleum hydrocarbons in wildlife tissues.       U.S.  Fish and
             Wildl. Serv., Biol. Rep. 88(15). 8 pp.

       Hogan, M.E. 1980. Seasonal habitat use of Port Valdez,
             Alaska by marine birds. Unpublished administrative report.
             U.S. Fish and Wildl. Serv., Anchorage, Ak. 25 pp.

       Isleib, M.E. and B. Kessel. 1973. Birds of the North Gulf
             Coast - Prince William Sound Region, Alaska. Biol. Pap. Univ.
             Alaska 14.

       King, J.G. and G.A. Sanger. 1979. Oil vulnerability index
             for marine oriented birds. pp. 227-239 in J.C. Bartonek and
             D.N. Nettleship (eds.). Conservation of marine birds in
             northern North America. U.S. Fish and Wildl. Serv., Wildl.
             Res. Rep. 11. Washington, D.C.

       Koehl, P.S., T.C. Rothe, and D.V. Derksen.      1982. Winter
             food habits of Barrow's Goldeneyes in    southeast Alaska. pp.
             1 - 5 in marine birds: their feeding     ecology and commercial
             fisheries relationships. Nettleship,    D. N., G.A. Sanger, and
             P.F. Springer, eds.      Proc. Pacific Seabird Group Symp.,
             Seattle, WA., 6-8 Jan. 1982. Can. Wildl. Serv. Spec. Publ.

       Sanger, G.A. and R.D. Jones, Jr. 1982. Winter feeding
             ecology and trophic relationships of Oldsquaws and White-

                                         304









                   winged Scoters on Kachemak Bay, Alaska. pp. 20-28 in Marine
                   birds: their feeding ecology and commercial T17sheries
                   relationships.     Nettleship, D.N., G.A. Sanger, and P.F.
                   Springer, eds. Proc. Pacific Seabird Group Symp., Seattle,
                   WA., 6-8 Jan. 1982. Can. Wildl. Serv. Spec. Publ.

             Shaw, D.G., A.J. Paul, L.M. Cheek, and H.M. Feder. 1976.
                   Macoma balthica: an indicator of oil pollution. Mar. Poll.
                   Bull. 7 (2): 29-31.

             Snedecor, G.W. and W. G. Cochran. 1980. Statistical
                   methods. Iowa State University Press. Ames, Iowa. 507 pp.

             Stekoll, M.S., L.E. Clement, and D.G. Shaw. 1980. Sublethal
                   effects of chronic oil exposure on the intertidal clam Macoma
                   balthica. Mar. Biol. 57: 51-60.

             Vermeer, K. and N. Bourne. 1982. The White-winged Scoter diet in
                   British Columbia: resource partitioning with other scoters.
                   pp. 30 - 38 in Marine birds: their feeding ecology and
                   commercial fisheries relationships.       Nettleship, D.A.,G.A.
                   Sanger, and P.F. Springer, eds. Proc. Pacific Seabird Group
                   Symp., Seattle, WA., 6-8 Jan. 1982. Can. Wildl. Serv. Spec.
                   Publ.

             Williams, O.D. and J.E. Grizzle. 1972. Analysis of contingency
                   tables having ordered response categories. J. Amer. Statist.
                   Assoc. Vol. 67: 55-63.






             BUDGET:    FWS


             Salaries              $75.0
             Travel                 25.0
             Contracts              35.0
             Supplies                5.0
             Equipment              10.0

             TOTAL                $150.0













                                               305












         BIRD STUDY NUMBER 13

         Study Title:    Preliminary Survey of Passerine Birds in PWS to
                         Assess Impact of the EVOS.

         Lead Agency:    FWS

         Cooperating Agency: ADF&G

                                      INTRODUCTION


         Several year-round resident passerine species are heavily dependent
         upon shoreline and intertidal areas in PWS, and may have become
         oiled and suffered injury.      These species include the gray jay,
         (Perisoreus canadensis) , Steller's jay (Cyanocitta Steller) , black-
         billed   magpie    (Pica   Pica),   common raven      (Corvus corax),
         northwestern crow (Corvus caurinus), and others. Other migratory
         passerines that use intertidal and shoreline areas also may be
         similarly affected by oil contamination. These passerines include
         swallows (Hirundini dae) , thrushes (Muscicap idea) , several species
         of    blackbirds    and   sparrows    (Emberizidae),    water     pipits
         (Motacillidae). These birds occur in the hundreds of thousands.
         In addition to direct lethal effects of oiled plumage, birds may be
         subject to sublethal effects of oil contamination, which could
         affect overall health and reproductive potential.              Passerine
         species have major intrinsic and recreational (viewing) value.

         This study is a reconnaissance survey only.         It is designed to
         provide preliminary information that will assist in determining
         whether additional, more rigorous, studies of passerines are
         needed. It was originally proposed in the 1989 damage assessment
         plan to collect a wider scope of information. However, that study
         was not implemented because of logistical constraints that arose
         during the 1989 field season.



                                       OBJECTIVES


         A.    observe, record, and report the presence or absence of
               passerine species in oiled and non-oiled study sites in PWS.

         B.    Compare count data for 1990 between oiled and non-oiled sites.


         C.    Compare count data for 1990 with historical data.


                                         METHODS

         oiled and non-oiled shores will be selected for survey, with
         selection cognizant of available data and logistical support.
         observations of passerines will be recorded along oiled and non-

                                           306









           oiled beaches on Perry Island in PWS.


           Counts and observations will be made from stationary locations and
           transects that were established and surveyed in previous years.
           This will allow comparison between pre-oiled years, 1982-86, and
           post-oiled years 1989-90.



           BUDGET:    FWS


           Personnel                     $ 3.0
           Travel                          0.5
           Contracts                       5.2
           Supplies                        0.8
           Equipment                       0.5

           Total                          $10.0






































                                            307












         ASSESSMENT OF DAMAGE TO HISTORIC PROPERTIES AND ARCHEOLOGICAL
         RESOURCES


         Lead Agency: USFS

         Cooperating Agencies: DNR, FWS, NPS

                                     INTRODUCTION

         Holocene richness and diversity of resources resulted in the
         development of the largest prehistoric populations in Alaska along
         the Pacific mainland and island coasts.       Kodiak Island had the
         largest, most dense prehistoric population of Eskimo peoples in the
         world.   similar ecological abundance suggests PWS and mainland
         coasts also supported major human populations. The region of oiled
         beaches includes large areas where few archeological surveys have
         been done. To determine damage, specific information is needed on
         the location, number, and character of historic sites within the
         EVOS area. This information is obtainable through intensive on-
         the-ground sample surveys and direct testing.

                                      OBJECTIVES


         This study includes activities designed to identify and quantify
         injury to cultural resources from a scientific standpoint and to
         develop the foundation for a meaningful program to restore and
         rehabilitate archeological resources.      To determine the injury
         caused by the spill the study will focus on the following:

         A.    Impacts on soil chemistry (pH, Calcium, Phosphate)

         B.    Impacts on soil structure and inclusions (stratigraphy;
               charcoal)

         C.    Impacts on artifacts including petroglyphs, bone, wood,
               ceramic, fiber and shell

         D.    Impacts on vegetative cover of sites, including new or
               increased erosion on the sites

         E.    Occurrence of theft or vandalism on sites, including new or
               increased incidences


                                        METHODS


         1.    Activities will be performed in a manner consistent with the
               Secretary of the Interior's Standards and Guidelines for
               Archeology and Historic Preservation (48 Fed. Reg. 44716-
               44740, September 29, 1983).

         2.    Through a literature search and in-field surveys, an estimate
               of the number, type, character, and the significance of

                                          308









                historic properties in the area affected by the oil spill will
                be determined.

           3.   Develop topologies based on site type, time period, and
                location.

           4.   Using the topologies developed, a representative sample of
                historic properties types and locations to be investigated for
                impacts, will be selected. The sample will include sites in
                non-oiled areas to serve as control sites.

           5.   Conduct archeological investigations at the selected sites and
                locations.

           6.   Oil spill response workers and government employees will be
                interviewed concerning impacts to historic properties and
                archeological resources.

           7.   A laboratory analysis of the effects of the oil on the
                physical characteristics of the soil column will be performed.
                Attention will be given to its component parts to determine
                changes in preservation, soil compaction, stratification, and
                obscuration of the stratigraphy, as well as leaching and the
                chemical breakdown of organic materials.

           8.   Radiocarbon age determinations and soil sample analyses for
                pH, calcium, and phosphate will be performed.

           9.   Pre- and post-spill vandalism and erosion data will be
                compiled and evaluated to establish rates and effects of
                vandalism and erosion.


                                       DISCUSSION

           To assess the potential injury to historic properties along the
           coast, three physical zones can be established: submerged (below
           the lowest low tide), intertidal (between the lowest low and the
           highest high tides), and shore margin uplands (above the highest
           high tide).   The greatest potential for damage exists through
           direct deposition of oil in the intertidal zone.           Secondary
           transport into adjacent submerged areas and uplands may also injure
           historic properties. Upland historic properties are also subject
           to contamination from transportation of oil by wind, storm tide
           inundation, migration of contaminants in ground water, oiled bird
           and animal movement from the feeding/travel corridor of the
           intertidal zone, and their death and decomposition on archeological
           deposits. Theft of artifacts and vandalism to historic properties
           and archeological resources is a potential danger in the intertidal
           and upland zones. The intertidal zone contains historic properties
           of great variety, numbers, and susceptibility to oil damage.
           Shipwrecks, eroded/scattered artifacts, inundated stratified
           archeological deposits, prehistoric rock art, prehistoric fish

                                           309









       weirs, and remnants of structures or objects deliberately placed in
       the intertidal zone are among the site types known to exist. The
       shore margin uplands may contain all the previously mentioned site
       types, plus burials, above-ground structures, and recognizable
       resource collection locations such as culturally modified trees.

       In the two higher elevation zones, a major potential injury
       resulting from oil contamination is interference with traditional
       archeological dating techniques.      Radiocarbon dating depends on
       comparison of the ratio of radioactive carbon 14 to carbon 12 in
       the sample being analyzed.      Because petroleum contains abundant
       radioactively- inert carbon from organisms dead for millions of
       years, and the use of radiocarbon dating for dates up to 35,000
       years ago, contamination by even a small amount of ancient carbon
       is expected to result in age determinations that are significantly
       older than the archeological event being dated.            This would
       seriously compromise radiocarbon dating as a technique for dating
       human activities and paleoenvironmental events and conditions. The
       potential for affecting age determination may be significant even
       in areas where only a sheen exists and may be investigated in
       assessing injury.     In  cases of oil contamination in stratified
       archeological deposits,   masking of the visibility and alteration of
       the chemical components of the microstratigraphy may also affect
       archaeologists' ability to trace strata.

       Both direct and indirect injuries to historic properties may have
       occurred from response and treatment activities, as well as from
       increased activities in the resource areas.        Further, increased
       access of personnel to remote areas may have increased the
       knowledge of site locations and potentially may accelerate
       vandalism, theft of heritage resources, and damage to the
       scientific value of the sites.


       BUDGET: USFS


       Personnel                             $    382.0
       Travel                                        12.0
       Contracts                                  300.0
       Equipment  & Supplies                      238.0

       TOTAL                                 $    932.0


       BUDGET:


       Personnel                             $    123.0
       Travel                                         4.0
       Contracts                                     96.0
       Equipment  & Supplies                         77.0

       TOTAL                                 $    300.00

       TOTAL BOTH AGENCIES:                  $  1,232.00

                                         310












                                      TECHNICAL SERVICES




             The hydrocarbon analysis, histopathology, and mapping projects
             described in this section are designed to provide high quality
             technical services to studies described in other portions of the
             NRDA plan.       Hydrocarbon analytical services includes the
             generation, archival, and retrieval of all chemical analytical
             data.   Histopathology involves continuing the quality assurance
             program begun in 1989 and facilitating the analysis work of
             approved contractors employed by individual 'studies.          Mapping
             includes implementing and managing a geographic information system
             to record and process data collected by NRDA studies.

             Appropriate information on exposure of the resource to hydrocarbon
             residues from the spill is required to determine and quantify
             injury.   It can be demonstrated by detailed information on the
             distribution and evolving chemical composition of the spilled oil
             through time, in concert with analyses of petroleum hydrocarbons or
             their metabolites in the tissues of organisms.

             Samples of water, sediments and tissues for chemical analysis are
             being collected by individual studies throughout the entire region
             impacted by the EVOS.    Selected samples are being analyzed by a
             team of participating laboratories in accord with a centralized
             QA/QC program (Appendix A) which will help ensure that all data are
             of known, defensible, and verifiable quality and comparability.

             Information on the incidence of oil-induced histopathological
             conditions is required by many of the studies described under
             Fish/Shellfish, Birds, Marine Mammals, and Terrestrial Mammals.
             This information is being gathered under strict quality assurance
             guidelines (Appendix B) by a group of contracted histopathologists
             to ensure compatibility of results and evaluations throughout the
             NRDA program.

             The mapping project will continue to develop the damage assessment
             geographic information system. Good progress has been made on the
             collection and verification of the primary data layers.
             Additionally, large scale production and transmittal of map
             products has begun.











                                              311













       TECHNICAL SERVICES STUDY NUMBER I

       Study Title:     Hydrocarbon    Analytical    Support    Services     and
                        Analysis of Distribution and Weathering of Spilled
                        Oil

       Lead Agency: USFWS, NOAA

                                     INTRODUCTION

       In order to document the exposure of resources in the PWS and GOA
       ecosystems to spilled oil, NRDA projects are collecting samples
       from a wide variety of environmental matrices to be analyzed for
       petroleum hydrocarbons. The data resulting from the analysis of
       these samples will not only be used to demonstrate an impact on
       that particular resource and support the individual project, but
       also to produce an integrated synthesis of the distribution of the
       oil in space and time, i.e., provide information on subsurface
       transport, residence time, and mass balance. Both of these uses
       require that the analytical data be accurate and precise, as well
       as of demonstrable quality. Analysis of the distribution of oil
       requires that the data be comparable from project to project
       through the entire NRDA process. The large number of samples and
       the length of time involved in the NRDA process require the use of
       more than one laboratory to provide analytical data. Rather than
       make each project responsible for analyzing their samples, TS 1 is
       responsiblefor analysis of all samples collected for hydrocarbon
       chemistry. This requires the generation, archival, and retrieval
       of all chemical analytical data.

       To date, TS 1 has:

             Developed and implemented an analytical chemistry QA/QC plan.

             Developed and implemented a sample inventory and tracking
             system.    This system forms part of the overall database
             management system for TS 1.      Sample collection and quality
             data and analytical and associated QA/QC data are other parts
             of this system.

             Contracted with the National Institute of Standards and
             Technology (NIST) to develop and supply calibration standards
             and control materials.

             Initiated the measurement of petroleum hydrocarbons and their
             metabolites in water, sediment, tissues and bile.              Some
             samples have already been analyzed and results comminuted to
             PIS.    The remaining samples in the inventory have been
             assigned an analytical priority and will be released for
             analysis in that order.


                                          312









                   Conducted a performance audit with participating analytical
                   laboratories.

                   Initiated a synthesis of the distribution and composition of
                   spilled oil with TS 3.

                                           OBJECTIVES


             A.    Measure petroleum hydrocarbons, hydrozarbon metabolites and
             other appropriate chemical/biochemical measures of hydrocarbon
             exposure in water, sediment, and biota. collected through the NRDA.

             B.    Prepare a QA/QC plan that establishes detailed procedures and
             protocols' for sample collection, sample identification, chain of
             custody, and shipping.

             C.    Oversee and develop a centralized QA/QC program to assist the
             analytical laboratories in providing quality data and demonstrate
             the accuracy, precision, and comparability of all data developed by
             the program.

             D.    Provide technical on-site system audits of field and
             laboratory data collection activities.

             E .   Develop and provide appropriate instrument calibration
             standards and natural matrix control materials.

             F.    Develop an integrated synthesis of the distribution and
             chemical compositionof spilled oil, as it weathers through-time,
             to provide a detailed basis for final exposure assessment.

                                   METHODS AND DATA ANALYSIS

             Objectives'A-E.;     @This information is provided i:n 7"Analytical
             Chemistry QA/QC11 (Appendix A).

             Objective F.     Data will be integrated and displayed by means of
             TS 3 mapping capability.

                                    SCHEDULES AND PLANNING

             Additional analytical support both in terms of number of
             laboratories, and types of analyses, e.g. UV/P@are being actively
             sought. At least one and perhaps two more facilities will be added
             to add analytical capability.

             TS I will conduct a series of training sessions on the oil spill
             collection and handling of samples before the projects begin year
             two field work.

             A proposal to transfer bar-coding sample-tracking technology will
             be considered.


                                               313








          Discussions on a sample holding time study have been initiated.


          BUDGET: NOAA


          Salaries                   $     58.7
          Travel                            3.5
          Contractual                     832.5
          Equipment                        15.0
          Supplies                          4.5

          Total                      $    914.2


          BUDGET: FWS


          Salaries                   $     62.0
          Travel                            5.2
          Contractual                     999.0
          Equipment                         7.0
          Supplies                         16.0

          Total                      $ 1,089.2































                                            314












           TECHNICAL SERVICES STUDY NUMBER 2

           Study Title:   Histopathology:    Examination of Abnormalities in
                          Tissues from Bird, Mammals, Finfish, and Shellfish
                          Exposed to the Spilled Oil

           Lead Agency:   ADF&G



                                       INTRODUCTION

           Histopathology is an important tool used in determining mechanisms
           of death and sublethal effects caused by infectious agents and
           toxic substances.   A number of histopathological conditions are
           known to result from exposure to oil. Evidence of these conditions
           will be documented in tissue samples taken by individual NRDA
           studies as one means of demonstrating spill-related injury.
           Histopathology technical services will support that effort by
           continuing the quality assurance program begun in 1989 and by
           facilitating analyses by approved contractors.


                                        OBJECTIVE

           Measure the incidence of histopathological conditions and external
           lesions in selected species of birds, mammals, finfish, and
           shellfish collected by NRDA studies.


                                          METHODS

           Standard histological methods for collection, preservation,
           processing, and interpretation, as specified in the quality
           assurance program (Appendix B) , will be continued. Assistance will
           be provided to NRDA studies in selecting and contracting with labs
           or individuals to complete analyses.


           BUDGET:   ADFG

           salaries                       $ 20.0
           Travel                            5.0
           Contracts                         70.0
           supplies                          5.0
           Equipment                      -  0.0

           Total                           $100.0






                                           315












         TECHNICAL SERVICES STUDY NUMBER 3

         Study Title:   Implement and Manage a Geographic Information System
                         (GIS) to Record and Process NRDA Data

         Lead Agency:    DNR and FWS

         Cooperating Agency: USFS and DEC

                                     INTRODUCTION

         The purpose of Technical Services Number 3 (TS3) remains unchanged:
         the group is charged with implementing and managing the geographic
         information system (GIS) to record and process data collected in
         NRDA studies.    Good progress has been made on the collection and
         verification of the primary data layers.      Additionally, TS3 has
         begun large scale production and transmittal of NRDA map products.


                                      OBJECTIVES

         1.   Produce and disseminate requisite maps and analytical products
              for participants in the natural resource damage assessment
              process.

         2.   Create and maintain, throughout the damage assessment process,
              a data base or data pertinent to the overall damage assessment
              process, in a way that is accessible to all of the
              participating agencies.

                                        METHODS

         Methods are essentially the same as described in the 1989 study
         plan. In addition to the data layers described in the 1989 study
         plan, data layers have been or will be prepared for study site
         locations, sampling locations, beach segment locations and multi-
         thematic atlases of pre-spill data from various sources.
         Additional data layers will be added as needed by investigators and
         the Trustee Council to enable geographic-based compilation of study
         results and other pertinent data.

         Quality control will continue to be emphasized, with review of
         information in data layers against qualified data sources and full
         documentation of source data and review procedures. A data backup
         system which includes redundant backup and off-site storage has
         been implemented and will be maintained.







                                          316













           BUDGET: DNR


           Salaries                   $    332.4
           Travel                           11.2
           Contracts                        69.6
           Supplies                         67.0
           Equipment                       112.0

           Total                      $    592.2


           BUDGET: FWS

           All Activities             $    200.0

           Total                      $    792.2








































                                            317



























 I
 I




















                                        PART II


                                       ECONOMICS












                                ECONOMIC STUDIES

       The studies in this section are federal studies designed to assess
       the economic value of injury to natural resources associated with
       the EVOS. State studies designed to assess the economic value of
       injury to natural resources resulting from the EVOS are not
       discussed in this document.

       The federal studies cover seven major areas: (1) commercial
       fishing, (2) public land values, (3) recreation, (4) subsistence,
       (5) intrinsic values, (6) research programs and (7) archaeological
       resources.







































































                                      318












             ECONOMICS STUDY NUMBER I

             Study Title: Commercial Fisheries Losses Caused by the EVOS



                                        INTRODUCTION

             This study combines the studies previously designated as Economics
             Study Number 1 (Estimated Price Effects on Commercial Fisheries),
             Economics Study Number 2 (Fishing Industry Costs) and Economics
             Study Number 3 (Bioeconomic Models f.or Damage Assessment) with
             primary emphasis on former Economics Study Number 1.

             The EVOS may have resulted in substantially reduced seafood
             production at, among others, Cordova, Seward, Kodiak, Kenai, and
             Homer, which are some of the most important commercial fishing
             ports in the United States.     Both short term impacts, through
             closure of certain fisheriesf and long term effects, such as
             reductions in population that will not become apparent for several
             years as well as through continued exposure to contaminants, may
             occur. These impacts may affect both the supply of and demand for
             seafood.

             For example, changes in quality (both real and perceived) may have
             occurred, which could adversely affect seafood markets.      In the
             case of several important commercial salmon fisheries, the spill
             resulted in harvests being confined to "terminal" areas, thus
             restricting traditional fishing patterns and timing of the harvest.

             Terminal area harvests occur in close proximity to the salmon's
             spawning grounds.   The result can be a significant reduction in
             quality, as compared to salmon harvested in more typical
             circumstancef i.e., more distant from, but en route to, spawning
             sites. The reduction in quality may affect the salmon's overall
             marketability and/or its appropriateness and acceptability for
             specific product forms. In either case, seafood consumers at every
             market level incur losses.

             Salmon is only one commercial species group which may have been
             adversely affected.    others may include Pacific halibut, Pacific
             herring, sablefish, shellfish and groundfish.

                                         OBJECTIVES

             Measure the economic loss to seafood consumers caused by the EVOS.

                                           METHODS


             The species most affected by the spill must first be determined.
             Conceptual   models    of   consumer    preferences   and     market
             characteristics for certain seafood products must be created.
             Furthermore, a methodology to assess statistically changes in the

                                             319










       level and quality of harvest must also be established. Next, the
       data appropriate for the models must be collected, assembled and
       assessed. The models will then be used to estimate the demand for
       various seaf ood products, the price changes associated with the
       spill, and the effects of seafood quality and quantity changes on
       consumers.


       BUDGET

       Salaries                   $ 103.0
       Travel                         15.0
       Contracts                      95.0
       supplies                        7.0
       Equipment                       9.0

       Total                      $ 229.0









































                                        320












             ECONOMICS STUDY NUMBER 4

             Study Title: Effects of the EVOS on the Value of Public Land




                                        INTRODUCTION

             The EVOS affected subtidal, intertidal, tidal and uplands areas on
             the shores of PWS and the GOA. This study will assess the lost
             market value of publicly held lands attributable to the oil spill.
             It will estimate market demand for leases and sales of land in the
             impacted areas, and project changes in total value of public lands.

                                         OBJECTIVES

             Determine the change in market values of public lands.

                                           METHODS

             Land appraisals are a common method of assessing the market value
             of land.   Appraisers usually estimate the market value of land
             parcels from the selling price of similar parcels. Because no two
             parcels are identical, adjustments are required to achieve
             comparability.   For the purposes of appraisal, market value is
             generally defined as the amount in cash, or in terms reasonably
             equivalent to cash, for which, in all probability, the property
             would be sold by a knowledgeable owner who is willing but not
             obligated to sell to a knowledgeable purchaser, who desires the
             property but is not obligated to buy.     Using this definition of
             market valuel the effect of the oil spill on land values will be
             estimated as the difference between the pre- and post- spill
             selling prices.

             This study will proceed in several stages.     First, a conceptual
             model will be developed to determine the total public value of a
             land parcel and to show how land appraisals fit within that model.
             Next an attempt will be made to identify instances where land
             valu@ studies can provide estimates of value changes that are not
             captured by other economic studies.       Third, the adequacy of
             appraisals as a method of assessing changes in land values due to
             the spill will be evaluated.

             If appraisals are warranted, they will be conducted by obtaining
             data on ownership patterns in areas affected by the oil spill,
             gathering data on previous oil spills and their effect on land
             values, estimating the effect of the oil spill on the value of
             property through use of paired-scale data, and inspecting areas
             affected by the oil spill.




                                             321












         BUDGET


         Salaries             $ 96.0
         Travel                  20.0
         Contracts                50.0
         supplies                  5.0
         Equipment                 9.0

         Total                 $180.0
















































                                          322











           ECONOMICS STUDY NUMBER 5

           Study Title: Economic Damages to Recreation


                                       INTRODUCTION

           The EVOS has impacted natural resources that support a wide range
           of recreational activities including fishing, hunting, boating,
           hiking, camping, and sightseeing.        Because of their unique
           attributes, these resources attract recreationists from throughout
           the United States and other countries to PWS and the GOA coast.

           The oil spill may result in economic damage to those resources,
           recreational   services   in   two principal     ways:      1)    some
           recreationists who otherwise would have gone to the area will
           choose a substitute activity and/or area, thereby potentially
           suffering a loss in personal satisfaction and possibly incurring
           increased costs, and 2) recreationists who visit the area may
           suffer reduced satisfaction because of the oil spill's adverse
           impacts on recreational services that the natural resources
           otherwise would have provided. These types of losses may have been
           experienced by sea kayakers, users of charterboat services,
           recreational fishers, cruise ship patrons and general tourists.

           While relatively few in number, sea kayakers may have been
           significantly affected by the oil spill. Kayaking trips are taken
           from Valdez, Kodiak, Homer, Whittier and Seward to the western
           portion of PWS and the bays along the Kenai peninsula and Kodiak
           Island. A typical trip involves charter boat transportation to a
           site some distance from port. Most trips last more than one day
           and  thus   include    both   kayaking   and   wilderness    camping.
           Southcentral Alaska includes some of the premier kayaking areas in
           the world.

           The potential effect of the oil spill on kayakers could take
           several forms:

             -  beaches used for wilderness camping are oiled and unusable;
             -  wilderness scenery is despoiled and sense of pristine
                environment is lost;
             -  wildlife viewing opportunities are reduced;
             -  areas un-oiled suffer from increased congestion;
             -  clean-up activities make boats for transport expensive or
                impossible to charter; and
             -  clean-up activities spoil the wilderness nature of the
                experience.

           All of these potential effects may have applied during the 1989
           season; some may remain for several years.

           Recreational activities that use the services of charterboats and


                                            323









          other private boats for hire are typically less intense than sea
          kayaking, but they are f ar more numerous.     Vessels for hire and
          charterboats range from the standard six passenger charterboat
          called a 11sixpack" to large tour boats carrying over a hundred
          passengers. All types of vessels for hire have been impacted by
          cleanup activity. For brevity in this proposal, this entire group
          is referred to as "charterboats." Charterboat related recreational
          activities include salmon and halibut fishing, sightseeing and
          viewing marine wildlife and ferrying for wilderness camping in the
          PWS, KP and Kodiak areas. Charterboats go out of Valdez, Whittier,
          Homer, Kodiak, Seward and the smaller villages in southcentral
          Alaska.

          Because access to the general area is not easy, there are
          potentially substantial impacts which can be measured through a
          careful study of the charter fleet. The purpose of such a study
          would be to determine the reduction in the use of the PWS
          environment through the charter fleet as a consequence of the oil
          spill.

          The level of participation in recreational fishing among the
          residents of Alaska is far greater than among the residents of any
          other state in the United States.      Marine recreational fishing
          originates in all major towns on the PWS as well as Cook Inlet,
          Kodiak Island and the KP and the Alaska Peninsula. Fishing trips
          are taken in several ways -- from shore, from private boats. and
          from charter vessels.    Because access by car from Anchorage is
          relatively easy, shore fishing and private boat fishing 'on the
          Kenai is quite popular. All kinds of fishing draw large numbers of
          tourists to Alaska.

          The previous study of@charterboats willaddress only part of the
          potential recreational fishing effects. ' It is possible that the
          oil spill had detrimental effects on shore and private boat
          recreational fishing, as well. For example,

          a)   fishing trips in the potentially oiled areas may have declined
               due to fear of contaminated fish and waters;
          b)   anglers may not have been able to find accommodations in areas
               where they wanted to fish because of cleanup related
               activities.,
          c)   -the value of particular fishing trips out of the potentially
               oiled zones   may have declined because sites became more
               congested.

          Each season, a number of cruise ships pass through PWS on their way
          from Seattle or 'Juneau' to'Whittier where, they discharge their
          passengers for the train trip to Anchorage.' The likelihood that.
          these individuals were directly affected by the oil spill is small,
          but many have canceled their trips, because of fear that the oil
          spill would spoil the experience.


                                           324









           The general tourist activity sub-component of the proposal dif f ers
           from the others in that it is not directed toward one specific
           recreational activity.      Here the goal is to determine, from
           aggregate level data, the extent to which general tourist activity
           in the area of the spill may have been dislocated because of clean-
           up activities. There will have been losses to recreationists if
           these activities were diverted away from areas thought to be
           contaminated by the spill or affected by the congestion and lost
           services associated with clean up. Some of the marine related part
           of this damage will be captured in the investigation of the
           charterboats and kayaking. However, those people who do not plan
           to use boats but rather state parks or other facilities will not
           have been covered.


                                        OBJECTIVES


           Develop estimates of economic injuries to recreationists.

                                          METHODS

           The study will look at the types of consumptive and nonconsumptive
           recreational activities.

           Sea kayaking:    This study contains several stages.      First, the
           relevant sea kayaking population will be identified.        Second, a
           survey instrument which will contribute to both recreational demand
           and simple contingent valuation analysis will be created. Third,
           the survey instrument will be pretested. Fourth, the survey will
           be administered. Fifth, the survey results will be analyzed.

           Charterboat activities: Data for this study will also be collected
           through a survey instrument. After development of a theoretical
           framework for damage measurement, the sample frame will be defined.
           A survey instrument will be designed to determine the periodic
           recreational and cleanup activities undertaken by each charter
           vessel, the number of recreationists served, the extent of
           cancellations and the amount of time the vessel      was involved in
           clean up activity.     Vessel owners may also be interviewed in
           person. Finally, the   data will be analyzed.

           Recreational fishing: There is an existing model for recreational
           f ishing in the KP area.      This model will be investigated to
           determine whether it can be usefully applied to the ef f ects of this
           oil spill.

           Cruise ship tours:      Cruise ship firms will be contacted to
           determine whether demand f or cruise ship tours to PWS were af f ected
           by the oil spill. If there is evidence of substantial reductions
           in  demand,   methods of estimating the actual            losses to
           recreationists will be explored.

           General tourist activity:      Assuming that aggregate ef f ects on

                                            325









       tourism may be accurately estimated, this study will compare those
       aggregate effects with the results of the activity directed
       substudies to determine whether important categories of losses have
       been missed.


       BUDGET


       Salaries              $ 229.0
       Travel                    27.0
       Contracts                 20.0
       Supplies                   8.0
       Equipment                 10.0

       Total                 $ 294.0











































                                        326












             ECONOMICS STUDY NUMBER 6


             Study Title: Losses to Subsistence Households



                                          INTRODUCTION

             several communities on the shores of PWS, LCI, Kodiak Island, and
             the Alaska Peninsula, and in or near the EVOS area, are highly
             dependent upon noncommercial fishing, intertidal food gathering,
             marine mammal hunting, and land mammal hunting for subsistence
             uses.    Among the small subsistence communities are Tatitlek,
             Chenega Bay, English Bay, Port Graham, Ouzinkie, Port Lions, Larsen
             Bay, Karluk, Akhiok, Old Harbor, and Chignik Bay.                Larger
             subsistence communities include Cordova, Valdez, Seldovia, and
             Kodiak. Subsistence uses are defined as rural Alaska residents'
             customary and traditional uses of wild, renewable resources for
             direct personal or family consumption as food, shelter, fuel,
             clothing, tools, or transportation; for the making and selling of
             handicraft articles out of nonedible byproducts of fish and
             wildlife resources taken for personal or family consumption; for
             barter, or sharing for personal or family consumption; and for
             customary trade. Those uses are designated as the priority public
             consumptive use of wild resources.

             Following the oil spill, subsistence harvests were reduced in
             several communities because of health concerns. This could have
             important ramifications in the economy and social order of the
             communities.     Potentially important economic losses to the
             communities include (1) subsistence losses, (2) local inflation
             affecting harvests and food procurement, (3) damage to subsistence
             property and (4) loss of intrinsic value to subsistence users.

                                           OBJECTIVES

             A.   Conduct a literature review and compile base line information.

             B.   Document extent of oil contact and clean-up on or near
                  historic harvest sites.

             C.   Document changes in subsistence use through time (i.e.,
                  species selection; harvest timing, quantities, areas, methods,
                  and efficiency; and household participation rates in harvest,
                  use, sharing, barter, and exchange)-

             D.   Document local social and economic changes that affect
                  subsistence use, including wage/labor patterns, income levels,
                  inflation rates in the -villages for goods and services,
                  cleanup work, outside agency demands, and industry demands.

             E.   Assign monetary values to losses to subsistence households.



                                              327,












                                     METHODS


      Field observations and interviews will be used to collect
      information. Changes in subsistence use and socioeconomic patterns
      will be determined by conducting systematic household surveys and
      interviews, and comparing these data to historic information.
      Where applicable market prices and price imputation will be used to
      estimate damages. For marketed goods, the cost of replacing the
      goods injured by the spill will normally be the measure of economic
      damage. However, the adverse effects of the spill extended beyond
      marketed goods. To determine the economic damages to non market
      goods and services, survey methodologies, similar to those
      described in Study 7, will be employed.

      BUDGET


      Salaries             $  255.5
      Travel                    48.0
      Contracts               237.5
      Supplies                151.4
      Equipment               192.6

      Total                $  885.0
































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             ECONOMICS STUDY NUMBER 7


             Study Title: Loss of Intrinsic Values Due to the EVOS



                                          INTRODUCTION

             Intrinsic values include existence value, option value, and bequest
             value. These values are independent of the economic values arising
             from direct use of natural resources and cannot be measured by
             observing use of the area affected by the EVOS.       Resources with
             intrinsic values include fish, birds and mammals,     along with the
             wilderness character, ecological integrity and/or scenic quality of
             certain areas. These values are only imperfectly captured by the
             prices of goods traded in markets. Accordingly, non-market methods
             must be used to calculate intrinsic values. This study is designed
             to use the contingent valuation method to determine the loss in
             intrinsic values resulting from the oil spill.


                                           OBJECTIVES


             Determine the loss of intrinsic value of natural resources
             attributable to the EVOS.



                                            METHODS

             The contingent valuation method involves use of surveys to
             determine the values that people place on goods that are not traded
             in markets. This study will require development of a conceptual
             framework for contingent valuation survey design and analysis of
             survey results. Next, research will be conducted to determine the
             most accurate survey instrument for assessing intrinsic values.
             This research will involve consultation with economists and survey
             design experts. Substantial preliminary testing of survey formats
             will be conducted among small groups of people to verify the
             accuracy of the survey instrument.      A nationwide survey will be
             conducted using a professional survey research firm. Econometric
             analysis will be used to interpret the results of the survey.

             BUDGET


             Salaries              $  515.0
             Travel                   145.0
             Contracts                680.0
             Supplies                 295.0
             Equipment                375.0

             Total                  $2,010.0



                                              329












        ECONOMICS STUDY NUMBER 8

        Study Title: Economic Damage Assessment of Research Programs
                       Affected by the EVOS



                                     INTRODUCTION

        The oil spill affected research programs in the vicinity of the
        spill, resulting in damage to or loss of various research and
        resource monitoring studies.        Opportunities to study natural
        resource systems in the affected area may have been lost or
        diminished as a result of the spill.       Research studies underway
        before   the    Spill   and   conducted,   permitted,     cooperatively
        participated in, sponsored or funded by the federal government
        likely were impacted. One example is a study involving tagging of
        fish that was in progress in an affected area of PWS.
        Determination of the set of studies affected and the extent or
        degree of damage will require careful evaluation and study.

                                      OBJECTIVES

        Assess damage to and economic loss of research investigations, and
        account for the cost of resources expended in affected studies,
        focusing on research-based expenditures made or committed to before
        the oil spill.

                                        METHODS

        The first step in this study is to identify the universe of studies
        that were underway in the affected area at the time of the spill.
        The next stage requires a determination of which studies were
        negatively impacted by the spill. Some of those impacts may have
        been so significant that the entire study was discontinued. Other
        studies may have been able to continue, but only at an increased
        cost caused by the impacts of the spill. For example, sample sets
        may have been destroyed or the study may have been moved to another
        area.     once the universe of affected research programs is
        identified, this study will value the destroyed and damaged
        research studies by looking first to total project costs, extra
        sums expended and amounts spent on each study prior to being
        impacted by the spill.

        BUDGET


        Salaries              $ 23.5
        Travel                   10.0
        Contracts                 10.0
        Supplies                   7.5
        Equipment                  0.0

        Total                 $ 51.0

                                          330












           ECONOMICS STUDY NUMBER 9

           Study Title: Quantification of Damage to Archeological Resources

                                      INTRODUCTION

           Archeological sites along the many miles of oiled coastline and
           intertidal zones may have been physically damaged by oil. Upland
           sites may have been damaged by erosion caused by destruction of
           site vegetation or transportation of the oil inland.        Loss to
           archeological resources includes direct and indirect oiling.
           Determination of the number of cultural resources impacted by the
           oil spill as well as the type and extent of injury to the
           archeological  sites has been moved to a separate science study.
           The economics study is now limited to quantifying the loss to
           archeological resources.

                                       OBJECTIVES

           Assess the economic damages to archeological sites.

                                         METHODS

           The archeological science study will create a data base containing
           listings of the oil impacted areas and a model for the kinds of
           cultural resources impacted, the degree of the impact and the
           physical setting of the damaged resource. Both use and intrinsic
           values of archeological resources may have been impacted.

           Use Value


                I .  Effects on the scientific value of the archeological
                resource.    The magnitude of this damage depends on the
                uniqueness of the affected site, the original quality of
                information available at the site, the nature of the impacts,
                and the willingness of the scientific community to pay for the
                lost information.    If the site is unique and substitute
                sources of similar information do not exist, the value of the
                damage may be large.

                2.   Loss of value as tourist and educational attractions.
                unique or spectacular archeological sites have value as
                tourist attractions. All significant archeological sites have
                educational value as the focus of field trips and published
                descriptions. Archeological information and artifacts have
                value for museum interpretation and display.       Oil impacts
                could substantially reduce these values.

           Intrinsic Value

                1. Impacts on the religious, cultural or symbolic values for
                native groups.

                                           331









               2.   Loss of intrinsic value for the general, non-native
              population.

         BUDGET


         Salaries                         $ 25.0
         Travel                              15.0
         Contracts                            0.0
         Supplies                            10.0
         Equipment                            0.0

         Total                            $  50.0













































                                            332













































                                       PART III


                                RESTORATION PLANNING











                          RESTORATION PLANNING PROJECT


                                   INTRODUCTION

       The Trustees recognized from the beginning that restoration of the
       ecological health of areas affected by the oil spill is the
       fundamental purpose f or conducting the NRDA. Initially, studies to
       determine the injury to natural resources were emphasized, since
       that information is basic to a determination of damages, and
       finally, restoration of resources.

       Since late 1989, considerable effort has gone into specific
       restoration planning activities.      An interagency     Restoration
       Planning Work Group (RPWG) was f ormed to develop and coordinate
       what is envisioned to be a steadily growing level of activity
       throughout this year and next.      A variety of activities have
       already been initiated by the RPWG and several more are proposed
       to occur during 1990, as described in the following pages.         In
       addition, it is anticipated that restoration planning and project
       activities will be expanded further in 1991 and beyond.

                                    OBJECTIVES

       The overall goal of the Restoration Planning Project is to identify
       appropriate measures that can be taken to restore the ecological
       health of natural resources affected by the EVOS.         Among the
       objectives within this overall goal are:

       A.    Encourage and provide for public participation and review
             during the restoration planning process.

       B.    Identify and develop technically feasible restoration options
             for natural resources and services potentially affected by the
             oil spill.

       C.    Incorporate an "ecosystem approach" to restoration (i.e.,
             broadly focus on recovery of the ecosystems, as well as
             individual components).

       D.    Identify when active restoration measures may be warranted,
             and when it may be appropriate to rely on natural recovery.

       E.    Identify the costs associated with implementing feasible
             restoration measures, in support of the overall NRDA process.









                                       333












                                         DEFINITION

             Restoration is a broad term that can include direct restoration,
             replacement, or acquisition of resources or uses those resources
             provided that are    equivalent in terms of ecological or human
             services.

             Direct restoration refers to measures taken to restore an injured
             resource, and generally equates with on-site actions. An example
             would be to rehabilitate an oiled marsh ecosystem by supplementing
             natural plant and animal populations after removal of the oil.

             Replacement refers to the substitution of one resource for an
             injured resource of the same type.         An example is to use
             hatchery/aquaculture techniques to establish an entirely new
             fishery stock in place of one that has been severely damaged.
             Replacement activities may or may not be limited to the specific
             site or area where injury occurred.

             Acquisition of equivalent resources means to obtain or otherwise
             protect resources that are similar or related to the injured
             resources in terms of ecological value, functions, or uses.       An
             example is to obtain or protect undamaged wildlife habitats as
             alternatives to direct restoration of injured habitats. Equivalent
             resources could be acquired in locations removed from the immediate
             vicinity of the injured resource.

                            1990 RESTORATION PLANNING ACTIVITIES

             Several major activities have been initiated or -are proposed under
             the Restoration Planning Project in 1990. Each major activity area
             is described in this section.

             Public Participation

             In part as a response to public comments on the 1989 NRDA Plan,
             several avenues have been developed for public involvement in the
             restoration planning process.    TheiRPWG has conducted a public
             Restoration Symposium, and held public information and scoping
             meetings in several Alaskan communities directly affected by the
             oil spill. Additional public meetings may be held outside Alaska
             during 1990 as well. The RPWG has also begun to contact interest
             groups and other organizations that have expressed an interest in
             the restoration planning process, in order to gain a more direct
             and detailed understanding of their concerns and suggestions. An
             information flier and response form has been developed and
             distributed initially in Alaskan communities in order to encourage
             additional comments from residents of areas most directly affected
             by the spill.' Reports generated through the Restoration Planning
             Project will generally be distributed publicly.      The following
             paragraphs briefly describe the outcomes of the major public
             activities conducted to date.


                                             334









       Restoration Symposium

       A two-day public Restoration Symposium was held at the Egan Civic
       and Convention Center in Anchorage, Alaska on March 25 and 26,
       1990. The symposium was the first opportunity for environmental,
       industry, and other interest groups and members of the general
       public to present their views about the content of a restoration
       plan. Formal presentations were made by more than 30 individuals.
       A report documenting the presentations and comments given at the
       Restoration Symposium is scheduled to be publicly distributed in
       July 1990.

       Community Scoping Meetings

       An initial series of public information and scoping meetings was
       held beginning in April 1990. The RPWG travelled to eight Alaskan
       communities directly affected by the oil spill to provide an
       opportunity for residents to express their views about what a
       restoration plan should entail.     Evening meetings were held in
       Cordova, Valdez, Whittier, Seward, Kenai, Homer, Kodiak and
       Anchorage. The community scoping meetings resulted in a variety
       of restoration ideas being identified. Public comments received as
       a result of the community scoping meetings will be documented in
       the progress report scheduled for public distribution in July 1990.

       Technical Workshops on Restoration

       The RPWG conducted an initial three-day technical workshop on
       restoration in Anchorage in early April, 1990. The workshop
       provided a forum for the scientists most familiar with the effects
       of the oil spill, as well as other scientists with relevant
       knowledge, to focus their attention on potential restoration needs
       and opportunities. A second technical workshop is planned for the
       Fall 1990, and it is anticipated that more such opportunities will
       occur before the conclusion of the process. One purpose of the
       first technical workshop was to help identify and develop an
       initial set of potentially beneficial restoration techniques that
       could receive small-scale field testing during the Summer 1990. An
       array of potential feasibility study projects was identified, some
       of which are proposed to be initiated (see Restoration Feasibility
       Studies below). The results of the workshop will be documented in
       the progress report scheduled for public distribution in July 1990.

       Literature Review

       The first phase of a comprehensive search of worldwide literature
       relevant to restoration of natural resources was initiated early in
       1990. "Phase 1,11 the initial search of key computerized literature
       data bases, identified several thousand potentially relevant
       references, which were narrowed to approximately one thousand of
       the most directly applicable citations. These references have been
       screened, and the most important ones have been flagged for

                                       335









            acquisition. These references will be reviewed in detail during
            the "Phase III' literature review, along with other references
            identified in an expanded search. Literature review activities are
            expected to continue throughout the restoration planning process.
            The results of "Phase I" will be summarized in the progress report
            scheduled for public distribution in July 1990. Updated results
            will be presented in subsequent reports.

            Feasibility Study Projects

            There are relatively few existing technologies for restoration of
            natural resources that can be immediately applied under Alaskan
            conditions with certainty of success. For this reason, feasibility
            study projects are among the most important aspects of restoration
            planning. A feasibility study project may be appropriate when a
            restoration idea has been developed that appears to be potentially
            beneficial, but for which there is substantial uncertainty of its
            success or benefit with local species or under the sub-arctic
            conditions of the spill area.

            The following pages present summaries for each of the initial
            feasibility study projects proposed for 1990. These projects were
            developed from ideas presented at the public 'symposium, the
            community scoping meetings, and the technical workshop. Factors
            considered in selecting 1990 studies included: the need to initiate
            the particular study as soon as possible, the ability to implement
            the project in a short time frame, reasonable likelihood of
            success, identified public concern, relationship to other NRDA
            studies, and budget priorities.

            Five restoration Feasibility Studies having a total budget of
            $326,400 are proposed for initial field testing in 1990. Two of
            these concern restoration of intertidal resources and communities!
            one addresses upland habitats used by wildlife affected by the
            spill, one involves stabilization and restoration in the supratidal
            zone, and one supports the potential acquisition of equivalent
            resources through review of land status, uses, and plans.

            Three restoration technical support projects with a budget of
            $236,500 are planned.    The first will institute a formal peer
            review process for restoration project results and planning. The
            second will compile shoreline status information from both response
            and NRDA sources to support selection of sites and habitats for
            future feasibility studies and restoration projects.     The third
            technical support project will fund development of detailed
            proposals   for   feasibility   studies  to   be   considered    for
            implementation in 1991.






                                            336













          BUDGET


          Restoration symposium                $   50.0
          Community scoping meetings               40.0
          Technical workshops                     200.0
          Literature collection/review             90.0
          Feasibility study projects              562.9
          Report preparation/publication          150.0
          Salaries                                600.0
          Travel                                   70.0

          TOTAL                                 $1,762.9



          Lead agencies:            EPA, ADF&G

          Cooperating Agencies:     DNR, DEC, DOA, DOI, DOC
































                                           337











          RESTORATION TECHNICAL SUPPORT PROJECT NUMBER 1

          Project Title: Peer Reviewer Process for Restoration Feasibility
                         Studies

          Lead Agency: RPWG

          Cooperating Agencies: DOJ, DOL

                                     INTRODUCTION

          The initial feasibility study projects to be conducted during the
          1990 field season were developed with the assistance of many of the
          scientists involved in the NRDA process, after considering comments
          received at the technical workshop and a series of public meetings
          held in Spring 1990 in Alaska. Due to the limited time available
          before projects need to be in the field, an additional more formal
          round of peer review is not possible.      This technical support
          project is designed to incorporate formal peer review in the
          design, implementation, and evaluation of 1991 and future
          feasibility studies. It will also provide for detailed review of
          1990 feasibility study results.

                                      OBJECTIVE

          Implement a peer reviewer process to assure the scientific quality
          of feasibility studies and restoration projects.

                                       METHODS

          Peer reviewers may include experts -already involved in the NRDA
          process, experts involved in the technical workshops on
          restoration, or other selected individuals. Peer reviewers would
          review and comment on feasibility study proposals (including
          overall design and detailed study plans) and results. The budget
          for 1990 is based on the services of 10 expert reviewers for five
          days each, plus expenses. It is anticipated that this technical
          support project will expand in 1991, as additional feasibility
          studies are initiated and as results from 1990 feasibility study
          projects become available.












                                          338











          BUDGET: DOJ, DOL

          Salaries:                  $   0.0
          Travel:                        0.0
          Contractual Services:          70.0
          supplies:                      5.0
          Equipment:                     0.0

          TOTAL:                        $75.0















































                                            339











          RESTORATION TECHNICAL SUPPORT PROJECT NUMBER 2

          Project Title: Assessment of Beach Segment Survey Data


          Lead Agency: DNR

          Cooperating Agencies: DEC, ADF&G, USFS, NPS, EPA

                                     INTRODUCTION

          There is a large volume of beach-survey information obtained
          through response activities (e.g., the fall and spring surveys) and
          NRDA studies (e.g., CH 1) . All of these data are being integrated
          into a standard NRDA data base. This information is being reviewed
          and summarized with respect to restoration planning needs and will
          complement and support Restoration Feasibility Study Number 5 (RF
          5). Together, this information will help identify potential sites
          at which (a) hands-on restoration projects may be carried out, and
          (b) equivalent resources may be acquired. Additionally, it should
          prove valuable in providing further information for analytical
          purposes in the development of the restoration planning matrix.

                                      OBJECTIVES

          A.   obtain and translate to maps, pertinent beach survey
               information that is important for feasibility studies and
               restoration projects.

          B.   Analyze possible trends in information for applicability to
               restoration feasibility studies.

          C.   Create a data base for future reference use in restoration
               projects.

          Relationships with Other Studies:

          This project relates directly to RF 5 and provides data of
          fundamental importance to the entire Restoration Planning Project.

                                        METHODS

          Research and map, using standard cartographic and G.I.S.
          techniques, all available information from the Fall 1989, Spring
          1990, and Fall 1990 walk-a-thon and shoreline assessment team
          surveys. Combined with RF 5, this will provide further support in
          the selection process for specific restoration sites and habitats.
          It may also prove advantageous for documenting natural recovery
          processes that may be occurring.      Care will be taken to not
          duplicate existing data bases and maps. The need is to integrate
          new information and summarize it in a form helpful to the
          Restoration Planning Project. This project will essentially add a

                                          340







        "restoration layer" to the existing NRDA data base.
        BUDGET: DNR

        Salaries                  $ 16.0
        Travel                        0.0
        Contractual Services          5.0
        supplies                      4.0
        Equipment                 -   0.0
        TOTAL                        25.0



































                                       341











             RESTORATION TECHNICAL SUPPORT PROJECT NUMBER 3

             Project Title: Development of Potential Feasibility Studies for
             1991


             Lead Agency: ADF&G and EPA

             Cooperating Agencies:   DNR, DEC, DOA, DOI, DOC

                                        INTRODUCTION

             A variety of potential restoration feasibility studies need to be
             undertaken before recommendations can be made in the Restoration
             Plan. Due to funding and timing constraints in 1990, it was
             possible to carry out only a limited number of such studies in
             the current season. There is much that can and needs to be done,
             however, to develop the substance of feasibility study proposals
             for possible implementation in 1991. A number of specific areas
             have been identified for development of study plans. These
             include (A) Monitoring "Natural" Recoveries, (B) Pink Salmon
             Stock Identification, (C) Herring Stock Identification/Spawning
             Site Inventory, (D) Artificial Reefs for Fish and Shellfish, (E)
             Alternative Recreation Sites and Facilities, (F) Historic Sites
             and Artifacts, and (G) Availability of Forage Fish. In addition,
             as new information becomes available through the NRDA process,
             public comments, and technical consultations, the RPWG expects to
             identify additional restoration ideas and areas of concern for
             which feasibility studies may be appropriate.

             Objectives:

             A.   To identify restoration ideas and areas of concern for which
                  feasibility studies may be necessary and appropriate.

             B.   To develop feasibility study plans and proposals which may
                  be considered for implementation in 1991 and beyond.

             Relationships with Other Studies:

             This project relates directly to Restoration Technical Services
             Project Number 1, implementation of a peer reviewer process, as
             well as the entire NRDA and Restoration Planning Project.

             Methods:


             Based on public comments, NRDA results, and consultations with
             technical experts, the RPWG anticipates that candidate restora-
             tion projects will be identified on an on-going basis. In order
             to fully evaluate some of these suggestions, it will be necessary
             to carry out feasibility studies. The RPWG then needs to convene
             ad hoc committees consisting of combinations of agency personnel,
             peer reviewers, and outside experts to more fully develop the

                                            342









      study plans and proposals. Support is needed to convene meet-
      ings, particularly involvin4 travel by outside experts. In some
      cases, site visits will be    eeded to examine particular problem
      areas related to the oil spill or successful restoration projects
      which have been implementedlelsewhere.

      BUDGET:


      salaries              $ 5.0
      Travel                   77.5
      Contractual Services     40.0
      Supplies                 11.0
      Equipment                 3.0

      TOTAL                   136.5




















































                                        343











             RESTORATION FEASIBILITY STUDY NUMBER I


             Study Title: Re-establishment of Fucus in Rocky Intertidal
                           Ecosystems

             Lead Agency:   EPA

             Cooperating Agency: USFS

                                       INTRODUCTION

             Qualitative evidence indicates that rockweed, the marine alga,
             Fucus, was damaged by both the spilled oil and the cleanup
             effort. Fucus is a critical structural component of the inter-
             tidal habitat in the oil-spill area, and it serves as an impor-
             tant spawning substrate for herring. Re-establishment of this
             species will increase the rate of recovery of other associated
             biotic communities.

             There may be a substantial delay in natural recovery of areas
             where populations were reduced over large areas (100-1000 m of
             shoreline), because dispersal of seeds is limited (< I m in most
             circumstances). Drift plants may increase this distance, but the
             importance of this mode is unknown.

             The reproductive and life history of Fucus is well known, and
             techniques for collection of seed are well established. In
             southern parts of the range plants are fertile year round, so the
             timing of the application of seeds may be relatively unimportant
             in the establishment of the plant. The specific life history
             cycle of the plant in PWS and the GOA is not known. It is
             expected, however, that the plants will be fertile for at least
             most of the spring and summer.

             Objectives:

             A.   Document the extent and magnitude of recruitment of Fucus in
                  areas subjected to alternative cleaning technologies.

             B.   Determine the feasibility of re-establishing Fucus in dam-
                  aged areas.

             C.   Develop and demonstrate potential large scale seeding tech-
                  niques to re-establish fucus.

             D.   Demonstrate the efficacy of seeding versus transplanting
                  Fucus.


             E.   Identify the costs of implementing a full-scale Fucus resto-
                  ration project.



                                            344








         Relationships with Other Studies:

         This study is fundamental to bringing an ecosystem approach to
         the restoration program. It relates directly to RF 2, re-estab-
         lishing critical intertidal fauna, and to various NRDA studies,
         particularly Coastal Habitat Study Number 1.

                                        Methods

         The study plan has two parts: (1) laboratory experiments that
         develop techniques for obtaining large quantities of embryos
         suitable for use in reseeding, and (2) field experiments to test
         the effectiveness of embryo reseeding and transplanting in
         habitats that experienced varying degrees of oiling and cleaning.

         Laboratory experiments will be conducted to determine embryo
         attachment strength over time. Since the seeds must remain in
         suspension, experiments will also be conducted to assure their
         viability in culture media for at least two weeks. Although
         techniques for obtaining Fucus embryos are simple and well known,
         these techniques will be modified and tested for the production
         and handling of the large numbers of embryos that would be
         necessary for a full-scale reseeding project.

         Field tests will then be conducted with various "seeding" proce-
         dures (e.g., dispersal of embryos, dispersal of embryos, and
         transplants of fertile adults). All three methods will be tested
         in one control and one habitat that was disturbed by oil and
         subsequently cleaned. Dispersal of embryos will then be tested
         in habitats with different combinations of oil and cleanup
         techniques (e.g., bioremediated, hot water wash). The experimen-
         tal design will use three replicates of each habitat type, three
         replicates of each procedure, and three replicates of controls to
         measure natural settlement. Variables to be measured include
         height of Fucus plants, numbers of plants, and percent
         vegetative cover. Maps prepared by the Damage Assessment
         Geoprocessing Group will be used to identify potential study
         sites. In the initial project, primary study sites will be in or
         near Herring Bay, PWS.

         BUDGET: EPA


         Salaries                  $ 2.0
         Travel                       11.0
         Contractual Services        135.0
         Supplies                      2.0
         Equipment                     0.0

         TOTAL                       150.0






                                           345











           RESTORATION FEASIBILITY STUDY NUMBER 2

           Study Title:   Re-establishment of Critical Fauna in Rocky
                          Intertidal Ecosystems

           Lead Agency: USFS

           Cooperating Agency: EPA

                                      INTRODUCTION

           Intertidal ecosystems on rocky shores, including both f auna and
           flora, were seriously affected by the oil spill and cleanup
           activities.   Initial results suggest that certain key faunal
           species, such as grazers and predators, that are likely to
           structure these intertidal communities, were moderately to heavily
           affected. Natural restoration processes in these communities will
           be limited by recolonization rates of these key species, which in
           some cases are known to be quite low. Re-establishment of Fucus
           alone may therefore not be sufficient to ensure a return to pre-
           spill conditions on ecologically meaningful time scales. Before a
           restoration plan is proposed, we should demonstrate the feasibility
           of enhancing the rate of recovery of the intertidal community by
           the re-establishment of key grazers and predators. If the natural
           recoveries of Fucus and intertidal fauna can be augmented by
           restoration projects, it will be of fundamental benefit to the
           marine ecosystem.

                                       OBJECTIVES

           A.   Compare rates of recovery of rocky intertidal communities with
                and without key faunal species and combinations of species.

           B.   Demonstrate the feasibility of restoring rocky intertidal
                communities by enhancing colonization by key faunal species.

           C.   Determine the costs of implementing a full-scale restoration
                project to re-establish key faunal species in rocky intertidal
                ecosystems.

           Relationships with Other Studies:

           This study will be carried out in conjunction with the Fucus study,
           R/F 1, and it is related to several other NRDA studies,
           particularly CH 1.

                                         METHODS

           Based on results of NRDA studies, limpets have been identified as
           important grazers that were harmed by the oil spill in rocky
           intertidal ecosystems.       Predators,     such as Nucella and
           Leptasterius, also could be important in structuring these

                                           346










         intertidal communities. Rates of recovery of intertidal areas with
         and without key species and combinations of species will be
         compared.    Grazer, predator, and grazer-predator exclusion and
         enhancement plots will be established in habitats that experienced
         differing degrees of oiling or were subjected to different cleanup
         techniques (e.g., bioremediated, hot-water high-pressure cleaned) .
         A key aspect of the study will be demonstrating the feasibility of
         enhancing colonization by key species.

         BUDGET: USFS


         Salaries                   $ 0.0
         Travel                        5.0
         Contractual Services         65.0
         Supplies                      2.0
         Equipment                     3.0

         TOTAL                        75.0

















































                                          347












          RESTORATION FEASIBILITY STUDY NUMBER 3

          Study Title:    Identification of Potential Sites for
                          Stabilization and Restoration with Beach Wildrye

          Lead Agency: DNR

          Cooperating Agencies: USFS

                                     INTRODUCTION

          The EVOS and associated cleanup efforts have affected supratidal
          beach ecosystems, of which a key component is the native grassl
          beach wildrye (Elymus mollis). The supratidal beach wildrye
          plant community is extremely important in the prevention of
          erosion in the coastal environment. Erosion can lead to the
          destabilization and degradation of cultural and recreational
          sites as well as of wildlife habitats (e.g., for ground-nesting
          birds). There are well established techniques for restoring rye
          grasses and other plants on coastal dune systems, including at
          some sites in Alaska. It is necessary, however, to first
          identify sites at which damage has occurred and restoration
          efforts appear to be feasible, and it is also necessary to
          establish the cost of a full-scale restoration project in the
          EVOS area.


                                      OBJECTIVES

          A.   Determine the distribution and areal extent of supratidal
               sites at which beach wildrye restoration efforts will be
               needed and feasible.

          B.   Identify potential sites for pilot projects to re-establish
               supratidal stands of beach wildrye.

          C.   Determine the costs of implementing a full-scale project to
               restore supratidal stands of beach wildrye.

          Relationships with Other Studies:

          This feasibility study addresses a key component in supratidal
          beach ecosystems. It relates directly to other feasibility
          studies and potential restoration projects in the areas of
          cultural, recreational, and avian resources.

                                       METHODS


          Beach segment survey data, aerial photographs, on-site
          inspections, and other sources of coastline status data will be
          used for a preliminary identification of sites where stands of
          beach wildrye have been injured and erosion is occurring or may
          occur as a result. Based on these preliminary results,

                                          348











       individual sites will be visited and evaluated for their
       potential as sites at which beach wildrye restoration techniques
       may be developed and tested. The on-ground activities will
       include documenting the size, type, and extent of damage and the
       depth of oil, if present, in the substrate. This study will
       enable development and evaluation of a proposal for a full-scale
       feasibility study of restoration methods in subsequent years.

       BUDGET: DNR


       Salaries                   $ 14.4
       Travel                        5.6
       Contractual Services          5.0
       Supplies                      3.1
       Equipment                     0.0

       TOTAL                      $ 28.1







































                                        349












            RESTORATION FEASIBILITY STUDY NUMBER 4

            Study Title:   Identification of Upland Habitats Used by Wildlife
                           Affected by the EVOS

            Lead Agency: FWS

            Cooperating Agency: ADF&G

                                       INTRODUCTION

            A variety of marine birds, waterfowl,'and other bird and mam-
            malian species were killed by the spill or injured by contami-
            nation of their prey and habitats. Many of these wildlife
            species are dependent on aquatic or intertidal habitats for such
            activities as feeding and resting, but they use upland habitats
            in forests, along streams, or above tree line to fulfill other
            life-history requirements (e.g., nesting, shelter). Through the
            public scoping process and technical workshop, many people have
            suggested that protection of upland wildlife habitats from
            further degradation may be an important way to help wildlife
            recover from the effects of EVOS. To explore this potential, it
            is necessary to learn more about the specific upland habitats
            upon which these species depend and how they use them. While
            such a feasibility study would be a large and complex under-
            taking, an initial study that primarily focuses on the marbled
            murrelet (Brachyrumphus marmoratus) and the harlequin duck
            (Histrionicus histrionicus) will be conducted in 1990. The
            results of this study will provide a basis for developing and
            evaluating a broader feasibility study proposal that will more
            fully explore the ecological relationship between marine-depen-
            dent wildlife and upland habitats.

                                        OBJECTIVES

            Objectives A-C specifically apply to both harlequin ducks and to
            marbled murrelets, the primary subjects of the 1990 study:

            A.   Develop and test methods for establishing the presence of
                 breeding birds.

            B.   Develop and test methods for locating nest sites.

            C.   Identify and characterize nest habitats and sites.

            D.   Define the parameters of and develop a proposal for a full-
                 scale upland habitat feasibility study for marine birds,
                 waterfowl, and other species.

            E.   Determine the costs of implementing a full-scale restoration
                 project concerning upland habitats used by marine-dependent
                 wildlife.


                                           350









        Relationships with Other Studies:

        This study relates directly to the results and field work of Bird
        Studies 2 and 11 and RF 5.


                                        METHODS

        Marbled murrelet: Naked Island in PWS will be the primary study
        site. The presence of breeding murrelets will be recorded by a
        stationary observer at dawn, at which times murrelets fly to
        inland nest sites. Murrelet altitude, behavioral, and other data
        will be recorded for each bird observed. Sites with high mur-
        relet activity will be identified and then searched for nests.
        The efficacy of the dawn detection technique will be evaluated.

        Harlequin duck: Streams in PWS will be selected for investigation
        based upon reported concentrations of ducks, survey data from
        NRDA projects, and interviews with knowledgeable field personnel.
        Once streams are identified as having a high potential for
        harlequin nests, there will be intensive ground searches for
        nests. As nests are located, the nest sites and habitats will be
        characterized by such parameters as distance from the stream and
        coast, topography, and vegetative cover.

        BUDGET: FWS, ADF&G

        Salaries                    $13.3
        Travel                        1.0
        Contractual Services          3.0
        Supplies                      2.5
        Equipment                     3.5

        Total                        23.3



























                                          351











             RESTORATION FEASIBILITY STUDY NUMBER 5

             Study Title:   Land Status, Uses, and Management Plans in Relation
                            to Natural Resources and Services


             Lead Agency: DNR

             Cooperating Agencies: USFS, NPS, ADF&G

                                        INTRODUCTION

             Through the restoration scoping process members of the public have
             suggested a wide variety of projects to acquire equivalent
             resources. Examples are the acquisition of timber or development
             rights, conservation easements, recreational and cultural sites,
             inholdings within state and federal protected areas, and buffer
             strips along streams and coasts.         In addition, scientists
             participating in the technical workshop found that in some cases
             habitat protection projects would be the best means of providing
             for the long-term restoration of injured wildlife resources. In
             order to begin to identify and evaluate potential restoration
             projects of this type, it is necessary to summarize existing
             information about the land status., uses, and management plans for
             both privately and publicly owned lands. This initial effort will
             focus on the oil-spill area and adjacent lands and will also serve
             to identify potential sites for other types of restoration
             projects.

                                         OBJECTIVES


             A.   Summarize and map the land status and ownership, land-use
                  designations, and existing and proposed uses of tidelands and
                  related uplands.

             B.   Summarize and map the extent and degree of oiling and coastal
                  morphology as necessary for restoration planning purposes.

             C.   Summarize and map natural resources and services, including
                  vegetation, fish and wildlife populations, habitats, and
                  sensitive areas, recreation, and commercial forestry.

             Relationships with Other Studies:

             These data are fundamental to the entire Restoration Planning
             Project and especially to those feasibility studies and potential
             restoration projects that concern the acquisition of equivalent
             resources.


                                           METHODS

             The DNR, through the NRDA Study TS 1, has compiled much of the
             necessary data on their computerized G.I.S. Additional resource

                                             352









         and land use information is available in state and federal
         management plans and resource inventories and from the Alaska
         Coastal Management Program. The RPWG and technical advisors will
         be consulted to define the specific area and information needs,
         which will then be obtained from the various existing data bases.
         After determining the most feasible means and best resolution to
         portray the information, it will be summarized, produced, and
         distributed, primarily in map form.

         BUDGET: DNR


         Salaries                   $      34.0
         Travel                            1.0
         Contracts                         5.0
         supplies                          10.0
         Equipment                         0.0

         Total                      $      50.0






































                                           353












































                                        PART IV


                                         BUDGET









                      Budget Summary for the Exxon Valdez Oil Spill Damage Assessment - 1990
                                           Budgets are in 1000's of Dollars

   Budgets are costs for projects from 3-1-90 through 2-28-91

   Study
   Category             Number           Title                             Agency          Budget



   Coastal              CH1        Comprehensive Assessment               ADF&G            156.7
   Habitat                                                                USFS            9,113.0

   Air/Water            A/W2       Injury to Subtidal                     ADF&G            333.5
                                                                          NOAA             466.8

                        A/W3       Hydrocarbons in Water                  DEC                47.5
                                                                          NOAA             472.5

                        A/W6       Oil Fate and Toxicity                  NOAA             870.0

   Fisheries            FIS1       Salmon Spawning Area Injury            ADF&G            391.5

                        FIS2       Egg and Preemergent                    ADF&G            302.8
                                     Fry Sampling
                        F/S3.      Coded-Wire Tagging                     ADF&G           1,990.0

                        F/S4       Early Marine Salmon Injury             ADF&G            150.0
                                                                          NOAA             400.0

                        F/S5       Dolly Varden Injury                    ADF&G            290.0

                        F/S7a      Salmon Spawning Area Injury, LCI       ADF&G            117.6

                        F/S7b      Salmon Spawning Area Injury,           ADF&G            460.3
                                     Kodiak & Chignik

                                                          354









                    Budget Summary for the Exxon Valdez Oil Spill Damage Assessment Program
                                         Budgets are in 1000's of Dollars

 Budgets are costs for projects from 3-1-90 through 2-28-91

 Study
 Category              Number     Title                                 Agency          Budget



 Fisheries             F/S8a      Egg & Preemergent Fry
                                     Sampling, LCI                     ADF&G              71.0

                       F/S8b      Egg & Preemergent Fry
                                     Sampling, Kodiak & Chignik        ADF&G            149.3

                       F/S11      Herring Injury                       ADF&G            558.4

                       F/S13      Clam Injury                          ADF&G            229.2

                       F/S15      Spot Shrimp Injury                   ADF&G              65.0

                       F/S17      Rockfish Injury                      ADF&G            109.4

                       FIS18      Trawl Assessment in PWS              NOAA             186.1

                       F/S22      Crab Injury, Outside PWS             NOAA             110.0

                       F/S24      Trawl Assessment,
                                    Outside PWS                        NOAA             450.0


                       F27        Sockeye Salmon
                                    Overescapement                     ADF&G            392.0

                       F28        Run Reconstruction                   ADF&G            175.1




                                                         355








                     Budget Summary for the   'Exxon Valdez Oil Spill Damage Assessment Program
                                          Budgets are in 1000's of Dollars

  Budgets are costs for projects from 3-1-90 through 2-28-91


  Study
  Category             Number     Title                                 Agency            Budget



                       F30        Data Base Management                  ADF&G             120.0

  Marine               mmi        Humpback Whale                        NOAA               92.0
   Mammals
                       MM2        Killer Whale                          NOAA              255.8

                       MM4        Sea Lion                              NOAA              171.2

                       MM5        Harbor Seal                           NOAA              159.3

                       MM6a       sea otter Injury                      FWS              1,060.5

                       MM6b       Sea Otter Mortality
                                    comparisons                         FWS                11.0

                       MM6c       Sea Otter Drift Study                 FWS                33.5

                       MM7        Sea Otter Rehabilitation              FWS               147.0

  Terrestrial          TM1        Injury to Sitka Black-
    Mammals                         Tail Dear                           ADF&G             124.6

                       TM2        Injury to Black Bear                  ADF&G              10.0

                       TM3        Injury to River Otter
                                                                        ADF&G             347.6


                                                         356









                     Budget Summary for the Exxon Valdez,Oil Spill Damage Assessment Program
                                          Budgets are in 1000's of Dollars

   Budgets are costs for projects from 3-.1-90 through 2-28-91


   Study
   Category             Number           "Title                          Agency           Budget




                        TM4        Injury to  Brown Bear                 ADF&G            125.7

                        TM6        Reproduction of  Mink                 ADF&G            134.0

   Birds                B1         Beach Bird Survey                     FWS              598.0

                        B2         censuses & Seasonal
                                     Distribution                        FWS              471.0

                        B3         Seabird Colony Surveys                FWS              251.1

                        B4         Bald Eagles                           FWS              675.0

                        B5         Peale's Peregrine Falcons             FWS              107.7

                        B11        Sea,Ducks.                            FWS              150.0


                        B13        Passerines                            FWS                10.0

   Technical            TS1.       Hydrocarbon Analysis                  NOAA             914.2
     Services                                                            FWS             1,089.2

                        TS2        Histopathology                        ADF&G            100.0



                                                          357








                    Budget Summary for the Exxon Valdez Oil Spill Damage Assessment Program
                                         Budgets are in 1000's of Dollars

 Budgets are costs for projects from 3-31-90 through 2-28-91


 Study
 Category             Number           Title                           Agency           Budget




                      TS3        GIS                                   DNR              592.2
                                                                       FWS              200.0


                      ARCH1      Archeology                            USFS             932.0
                                                                       DNR              300.0

 Restoration          RP1        Restoration Planning'                 ALL             1,762.9
   Planning

 overhead                        State of Alaska                       ADF&G           1,745.0

                                 Dept. of Agriculture                  USFS            1,245.0

                                 Dept. of  Interior                    FWS              500.0

                                 Dept. of  Commerce                    NOAA             953.8.

                                 Environmental Protection
                                   Agency                              EPA                44.1

 Discontinued Studies
 Completion                      All Agencies                                           140.0






                                                        3.58








                     Budget Summary for the Exxon Valdez Oil Spill Damage Assessment Program
                                          Budgets are in 1000's of Dollars

  Budgets are costs for projects from 3-1-90 through 2-28-91


  Study
  Category                        Management Entity               Agency            Budget




  Economics             ECON1     commercial Fisheries Losses           ALL FED          229.0

                        ECON4     Public Land Value Effects             ALL FED          180.0

                        ECON5     Recreational U.ses Damage             ALL FED          294.0

                        ECON6     Subsistence Losses                    ALL FED          885.0

                        ECON7     Intrinsic Value Loss                  ALL FED         2,010.0

                        ECONS     Research Program Damage               ALL FED            51.0

                        ECON9     Archeological Resource Damage         ALL FED            50.0





  TOTALS                                                                              $37,330.2







                                                         359








                 Trustee Budget Summary for the Exxon Valdez Oil Spill Damage Assessment Program
                                         Budgets are in 1000's of Dollars

   Budgets are costs for projects from 3-1-90 through 2-28-91


   Trustee                                                        Budget



   State of Alaska                                          $      10,504.9
   Department of Agriculture                                       11,545.4
   Department of the Interior                                      5,559.4
   Department of Commerce                                          5,757.0
   Environmental Protection Agency                                    264.5
   All Federal                                                     3,699.0




   TOTALS                                                   $      37,330.2

















                                                        360






































                                      APPENDICES














                                   APPENDIX A


                       STATE/FEDERAL DAMAGE ASSESSMENT PLAN

                              ANALYTICAL CHEMISTRY


                   QUALITY ASSURANCE (QA)/QUALITY CONTROL (QC)



                                TABLE OF CONTENTS


        1.   QUALITY ASSURANCE FOR ANALYTICAL CHEMISTRY

             1.1  Study-Specific AQ Plans (QAP)
             1.2  Technical System Audits
             1.3  Standards and Quality Control Materials
             1.4  Analytical Performance Evaluations
             1.5  Data Reporting and Deliverables

        2.   MINIMUM REQUIREMENTS: SAMPLING AND SAMPLING EQUIPMENT

             2.1 Sampling Identification and Labeling
             2.2 Field Chain of Custody

        3.   MINIMUM REQUIREMENTS: ANALYSIS

        4.   MINIMUM REQUIREMENTS: REPORTING AND DATA DELIVERABLES










          Appendix A (continued)



          This document describes the Quality Assurance for the analytical
          chemistry portions of the Exxon Valdez Damage Assessment Process.
          it is to be used in conjunction with the Analytical Chemistry
          Quality Assurance Programs of the Trustee Agencies. It describes
          only those minimum requirements necessary to validate the data
          generated by analytical chemistry laboratories. Quality assurance
          requirements for other types of measurements are not addressed.
          For instructions in meeting the requirements described in @his
          document, please consult "Collection and Handling of Samples,"
          which was prepared by the Analytical Chemistry Group for use in
          training field personnel or the following Agency representatives:


          Carol-Ann Manen, National Oceanic and Atmospheric Administration,
          (907) 789-6014.

          Everett Robinson-Wilson, U.S. Fish and Wildlife Service,
          (907) 786-3493.

          Rolly Grabbe, Alaska Department of Environmental Conservation,
          (907) 364-2155.

          John Moore, U.S. Fish and Wildlife Service, (301) 497-0524.

























                                         2











       Appendix A (continued)



       1.   Quality Assurance for Analytical Chemistry

       Each Trustee agency through their individual standard documented
       QA programs and guidances shall ensure that all data generated by
       or for that agency and their contractors, in support of the Exxon
       Valdez Damage Assessment, are of known, defensible, and verifiable
       quality.

       These documented QA programs and guidances include but are not
       limited to:

            NOAA National Status and Trends Program, Mussel Watch Phase
                  4 Work/QA Project Plan
            Quality Assurance of Chemical Analyses Performed Under
                  Contract With the USFWS
            EPA SW-846, Chpt. 1, QA/QC Requirements
            EPA Guidelines and Specification for Preparing Quality
                  Assurance Project Plans, QAMS-005
            EPA Handbook for Sampling and Sample Preservation of Water and
                  Wastewater

       In addition, an interagency team of leading scientists from the
       Trustee agencies and the Environmental Protection Agency, hereafter
       referred to as the Analytical Chemistry Group (ACG), shall develop
       and oversee a centralized program which will demonstrate the
       quality and comparability of the chemical data obtained by the
       Trustee agencies.

       The major components of this centralized QA program will be:

       1.   Development of study-specific analytical chemistry QA plans.

       2.   Technical on-site system audits of field and laboratory data
            collection activities.

       3.   Development    and   provision   of   appropriate    instrument
            calibration standards and control materials.

       4.   Laboratory performance evaluations by means of intercomparison
            exercises.

       5.   Review of data deliverables and all supportive documentation
            to evaluate data quality.







                                        3











            Appendix A (continued)



            1.1 Study-Specific Quality Assurance Plans

            Prior to the initiation of each study, the study manager must
            prepare and submit a study-specific analytical chemistry QAP to the
            ACG for review and concurrence.     This plan shall specify each
            study's goals, sampling procedures, analytical procedures, and all
            quality control measures and acceptance criteria associated with
            those procedures.

            The QAP must be study-specific, however any documented QA guidance
            and/or appropriate Standard operating Procedures (SOP's) used by
            the Trustee agencies may form the basis of individual study QA
            plans.

            A Quality Assurance Plan must address the following:

                    Title Page - Includes the signatures of the individuals
                    responsible for the project and ACG concurrence.

                    Proiect Description and Sampling Objectives - Briefly
                    describes the what, where, and why of the project.

                    Data Needs - Describes what elements, compounds, classes
                    of compounds, and/or physical data are required.        Must
                    describe the desired detection limitst precision and
                    accuracy of the data for the study.

                    Sampling and Labelling Procedures - Describes sample
                    collection, including field QC and preservation. Estimates
                    the number and kind of samples to be collected. Minimum
                    requirements for sample collection are described in
                    Section 2.

                    Chain of CustodV - Describes Chain-of-Custody and
                    documentation procedures.      Minimum requirements are
                    described in Section 2.

                    Analytical Procedures - References or describes in detail
                    proposed method(s).

                    Internal Quality Control - Describes type and frequency of
                    internal quality control.       Minimum requirements are
                    described in Section 3.


                    Calibration Procedures and Frequency - Describes the
                    methods and frequency for calibrating field and laboratory
                    instruments. These must be specified in SOP's.



                                                4









      Appendix A (continued)



               Data Verification   Describes the data verification in SOP
               form and includes; (1) the methods used to identify and
               treat outliers, and (2) the data flow from generation of
               raw data through storage of verified results.

               Data Deliverables - Specifies reporting needs additional
               to the minimum requirements described in Section 4.

               Technical System and Performance Audits - Specifies'field
               or intra-laboratory audits planned by the responsible
               Agency.


      1.2 Technical System Audits

      On-site system audits may be performed without prior notification
      by the ACG after consultation with the responsible agency.


      1.3 Standards and Ouality Control Materials

      The National Institute of Standards and Technology (NIST) will
      develop and provide appropriate standards and quality control
      materials.



      1.4 Analytical Performance Evaluations

      Prior to the initiation of work, each analytical laboratory will
      be required to demonstrate its capability.           This will be
      accomplished by providing laboratory documentation on the
      performance of the proposed methods and through the analysis of an
      accuracy based material.    The results of this analysis must be
      within +/- 15% of the value of each analyte or measurement
      parameter.

      Any changes in analytical methodology from that proposed in the
      original QA plan shall be validated under agency procedures and
      documented to the ACG.

      A series of three intercomparison exercises, utilizing the blind
      analysis of gravimetrically prepared materials, extracts of
      environmental matrices (tissue, sediment and water) or the matrices
      themselves, will be conducted annually.     Participation in these
      exercised is mandatory.    Materials will be prepared by, and data





                                    5










             Appendix A (continued)



             returned to the NIST for statistical analysis.       The NIST will
             report to the chairperson of the ACG.     Unacceptable perf ormance
             will result in the discarding of the associated data.

             The ACG will review and provide written reports on the results of
             intercomparison studies to the Management Team.


             1.5 Data Reporting and Deliverables

             Data deliverables will be reviewed by the generating Agency to
             verify the quality and useability of the data. A QC report on each
             data set will be provided to the ACG for review.

             All data and associated documentation will be held in a secure
             place under chain-of -custody procedures until the Trustees indicate
             otherwise.



             2.  Minimum Requirements: Sampling and Sampling Equipment

             Sample collection activities must be described in SOP's.
             References to existing documents are acceptable.

             The method of collection should not alter the samples.

             Sample collection and storage devices shall not alter the sample.

             Samples shall be held in a secure place under appropriate
             conditions and under chain-of-custody until the Trustees indicate
             otherwise.



             2.1 Sampling Identification and Labelling

             An SOP will be in place for each study which describes procedures
             for the unique identification of each sample.      A sample tag or
             label will be attached to the sample container.       A waterproof
             (indelible) marker must be used on the tag or label. Included on
             the tag are the sample identification number, the location of the
             collection site, the date 'of collection and signature of the
             collector.


             The information above will also be recorded in a field notebook
             along with other pertinent information about the collection and
             signed by the collecting scientist.



                                                6







         Appendix A (continued)



         2.2 Field Chain-of-Custody

         The field sampler will be personally responsible for the care and
         custody of the samples collected until they are transferred to
         another responsible party.

         Samples will be accompanied by a chain-of-custody record or field
         sample data record.      When samples are transferred from one
         individual's custody to another's, the individuals relinquishing
         and receiving will sign, date and note the time on the record.

         Shipping containers will be custody-sealed for shipment. Whenever
         samples are split, a separate chain-of -custody record will be
         prepared for those samples and marked to indicate with whom the
         samples are being split.

         Samples shall be maintained in a manner that preserves their
         chemical integrity from collection through final analysis.

         Sample shipper will arrange for sample receipt.

         After analysis, any remaining sample and all sample tags, labels
         and containers shall be held under chain-of -custody procedure until
         the Trustees indicate otherwise.


         3.   Minimum Requirements: Analysis

         The applicable methodology must be referenced or described in
         detail in the SOP's for each measurement parameter.

         Method limits of detection must be calculated by matrix and
         analyte.

         Control of the analytical method in terms of accuracy and precision
         must be demonstrated.

         Calibration must be verified at the end of each analysis sequence.

         Samples must be quantified within the demonstrated linear working
         range for each analyte.

         Standard curves must be established with at least 3 points besides
         0.

         Field blanks, procedural blanks, reference materials, replicates
         and analyte recovery samples must be run at a minimum frequency of
         5 percent each per sample matrix batch.







          Appendix A (continued)




          A minimum list of the petroleum hydrocarbon compounds which are to
          be considered for identification and quantification in water,
          tissue and sediment include the volatiles, i.e., benzene, toluene,
          xylene and the polynuclear aromatic and aliphatic hydrocarbons
          listed below:


               Naphthalene                          n-dodecane
               2-Methylnaphthalene                  n-tridecane
               1-Methylnaphthalene                  n-tetradecane
               Biphenyl                             n-pentadecane
               2,6-Dimethylnaphthalene              n-hexadecane
               Acenaphthylene                       n-heptadecane
               Acenaphthene                         pristane
               2,3,5-Trimethylnaphthalene           n-octadecane
               Fluorene                             phytane
               Phenanthrene                         n-nonadecane
               Anthracene                           n-eicosane
               1-Methylphenanthrene
               Fluoranthene
               Pyrene
               Benz(a)anthracene
               Chrysene
               Benzo(b)fluoranthene
               Benzo(k)fluoranthene
               Benzo(a)pyrene                       Benzo(e)pyrene
               Indeno(1,2,3-c,d)pyrene              Perylene
               Dibenz(a,h)anthracene
               Benzo(g,h,i)perylene


          4.   Minimum Requirements: Reporting and Data Deliverables.

          Measurement resultst including negative results, as if three
          figures were significant must be reported.

          Results of quality control samples analyzed in conjunction with the
          study samples -must be reported.

          Documentation demonstrating analytical control of precision and
          accuracy on an analyte and matrix specific basis must be reported.









                                             8












                                      APPENDIX B


                    EXXON VALDEZ OIL SPILL DAMAGE ASSESSMENT PLAN
                              HISTOPATHOLOGY GUIDELINES

         Histopathology is an important tool used in determining mechanisms
         of death and sublethal effects caused by infectious agents and
         toxic substances.    A definitive diagnosis often does not result
         from histological examination, but can give strong support to other
         positive measurements.     Tissues deteriorate (autolyze) rapidly
         after an animal dies; therefore, to be of value, any samples taken
         for histological evaluation as part of the damage assessment of the
         Exxon Valdez oil spill must be collected, preserved, and processed
         under strict guidelines.

         Sample Collection and Preservation Protocols

         Standard protocols for necropsy and preservation of tissue samples
         for histopathology shall be used throughout the oil spill
         assessment studies.     Different protocols have been designed to
         accommodate the different groups of animals to be encountered in
         the assessment studies. Necropsy procedures have been established
         and provided to study managers under separate cover for a variety
         of different animal groups including finfish, bivalve mollusks,
         brachyuran and crab-like anomurans (i.e., king crabs) , shrimp,
         marine and terrestrial mammals, and migratory and nonmigratory
         waterfowl.

         Paired sampling of animals from oiled versus non-oiled sites will
         be done for comparative purposes.        Histopathological sampling
         should be done during any observed acute episodes of mortality or
         morbidity to determine the cause of death or abnormality. These
         types of samples are the most valuable in assessing acute toxicity
         affects and will be the most likely samples collected for birds and
         mammals due to their high visibility in the impacted areas.
         Because of the low visibility of fish and shellfish, many histology
         samples will consist of random collections in impacted and control
         areas with little prior obvious indication of morbidity or
         mortality.

         Any histological processing of samples collected from apparently
         normal shellfish will be performed after results of parallel
         hydrocarbon sampling are known; i.e., positive hydrocarbon results
         may merit further histopathology studies.        This would not be
         advisable for fish and other higher animals that possess an active
         mixed function oxidase (MFO) liver enzyme system which could
         metabolize hydrocarbons to other compounds providing negative
         hydrocarbon    resultsf    while   potentially    still     exhibiting
         toxicological lesions.    Analyses of enzyme function may show an
         activated MFO system      in exposed fish and higher animals.
         Consequently, histology   and hydrocarbon samples, as well as other
         appropriate samples, such as liver and bile, will be taken from the

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          same animal when possible for analyses of metabolites and enzyme
          function.   If certain fish and shellfish are too few or small,
          subsampling other animals from the same site at the same time will
          be necessary.

          Processing and Interpretation Protocols

          Histopathology assessment of birds and mammals will be done
          primarily on tissues from clinically affected animals using
          established criteria of cellular degenerative and necrotic changes
          recognized by a board certified veterinary pathologist.

          Histopathological analysis of finfish and shellfish tissues will
          include the criteria above as well as indices established in the
          Amoco Cadiz oil spill studies (Haensly, et al., 1982; Berthou, et
          al., 1987) to allow some quantification of potentially subtle
          degenerative changes in tissue histology of otherwise clinically
          normal animals. Briefly, these indices include mean concentration
          of mucus cells per mm.2 of gill lamellae (fish); mean concentration
          of mucus cells per mm of epidermis in 10 f ields (f ish) ; mean
          concentration of macrophage centers per mm of liver; mean
          concentration   of hepatocellular vacuolation due to             fatty
          degeneration (f ish) ; a mean and total tissue necrosis index
          (invertebrates) ; histological gonadal index (invertebrates) ; and
          differences in prevalences and intensities of incidental lesions
          caused by infectious agents (fish and invertebrates).

          Ouality Assurance in Field Collection of Samples and in
          Interpretation of Results

          Field Collection:

          Veterinary personnel trained in sample taking will be utilized for
          onsite necropsies of birds and mammals in order to ensure adequate
          quality control and standardized sample collection. The same high
          standards will be attainable in fish and invertebrates in that
          sample collection will be done by trained finfish and shellfish
          biologists.   A fish pathologist and technician are available to
          train field personnel and assist in necropsy and preservation of
          finfish and shellfish samples at collection sites.

          Finfish and shellfish samples can be coordinated through an ADF&G
          fish pathologist, Fisheries Rehabilitation, Enhancement and
          Development Division.

          Interpretation of Results:

          Quality control of all processed work will require independent
          blind reading of subsampled histology slides by two different
          laboratories.     Tissues with known lesions will be included
          periodically in groups of tissue samples for blind reading and
          determination of  competency in interpretation.

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       Chain of Custody Guidelines

       Due to the evidentiary nature of sample collecting investigations,
       the possession of samples will be traceable from the time the
       samples are collected until they are introduced as evidence in
       legal proceedings.   To maintain and document sample possession,
       chain of custody procedures will be followed.

       The field sampler will be personally responsible for the care and
       custody of the samples collected until they are transferred.     All
       samples will be accompanied by a chain of custody record and will
       be custody-sealed. This procedure includes use of a custody seal
       such that the only access to the package is breaking the seal.
       When samples are transferred from one individual's custody to
       another's, the individuals relinquishing and receiving will sign,
       date, and note the time on the record. This record documents the
       transfer of custody of samples from the sampler to another person
       and, ultimately, to a specified analytical laboratory.

       Shipping containers will also be custody-sealed for shipment. The
       seal shall be signed before the sample is shipped. The chain of
       custody record will be dated and signed to indicate transfer. The
       original record will accompany the shipment and a copy will be
       retained by the sample collector. Whenever samples are split, a
       separate chain of custody record will be prepared for those samples
       and marked to indicate with whom the samples are being split. If
       samples are being sent by common carrier, copies of all bills of
       lading or air bills must be retained as part of the permanent
       documentation.

       Subcontracting  for Histological Work

       Subcontracting    work    for   histopathology    processing     and
       interpretation  will be under the control of an interagency team
       referred to as  the Histology Technical Group which will determine
       if potential contractors are qualified to do the work.
       Qualifications  for mammal and avian samples will require a board
       certified veterinary pathologist. Finfish and shellfish work will
       require individuals with a demonstrated publication record in the
       field of histopathology.

       References

       Bell, T.A., and V.V. Lightner. 1988. A Handbook of Normal/Penaeid
            Shrimp Histology. The World Aquaculture Society, Baton Rouge,
            LA.

       Berthou, F., G. Balouet, G. Bodennec, and M. Marchand. 1987. The
            occurrence of hydrocarbons and histophatological abnormalities
            in oysters for seven years following the wreck of the Amoco
            Cadiz in Brittany (France). Mar. Environ. Res. 23:103-133.


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            CERCLA. 1988. Natural Resource Damage Assessments. 53 Federal
                 Regulation 5166 and 9769.

            Haensly, W.E., J.M. Neff, J.R. Sharp, A.C. Morris, M.F. Bedgood,
                 and P.D. Boem. 1982. Histopathology of Pleuronectes platessa
                 L. from Aber Wrac1h and Aber Benoit, Brittany, France: long-
                 term effects of the Amoco Cadiz crude oil spill. J. Fish Dis.
                 5:365-391.

            Sparks, A.K. 1985. Synopsis of Invertebrate Pathology Excluding
                 Insects. Elsevier Publ., New York.









































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                                    APPENDIX C


                           GLOSSARY OF TERMS, ACRONYMS








       ADF&G      Alaska Department of Fish and Game
       AFK        Armin F. Koernig Fish Hatchery
       AHs        Aromatic Hydrocarbons
       AHH        Aryl Hydrocarbon Hydroxylase
       ANOVA      Analysis of variance
       A/W        Air/Water
       AWL        Age, Weight, Length
       CERCLA     Comprehensive Environmental Response, Compensation and
                  Liability Act
       C/H        Coastal Habitat
       CI         Cook Inlet
       CIK        Cook Inlet/Kenai
       CTD        Conductivity/temperature/depth
       CWA        Clean Water Act
       CWT        Coded wire tag
       DEC        Alaska Department of Environmental Conservation
       DNR        Alaska Department of Natural Resources
       DOA        Department of Agriculture
       DOC        Department of Commerce
       DOI        Department of the Interior
       DOJ        Department of Justice
       DBMS       Database Management System
       EPA        Environmental Protection Agency
       EIS        Economic Study
       EVOS       Exxon Valdez Oil Spill
       FRED       Fisheries Rehabilitation, Enhancement and Development
                  Division, ADF&G
       FIS        Fish/Shellfish
       FWS        U.S. Fish and Wildlife Service
       GC-MS      Gas chromatography-mass spectrometry
       GOA        Gulf of Alaska
       KAP        Kodiak Archipelago/Alaska Peninsula
       KP         Kenai Peninsula
       LCI        Lower Cook Inlet
       Mro        Mix@d function oxiaase
       MLLW       Mean lower low water
       M/M        Marine Mammal
       NIOSH      National Institute of Occupational Safety and Health
       NMFS       National Marine Fisheries Service
       NOAA       National Oceanic and Atmospheric Administration
       NPH        Naphthalene
       NPS        National Park Service


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                                         APPENDIX C


                                GLOSSARY OF TERMS, ACRONYMS






            NRDA       Natural Resource Damage Assessment
            NSO        Nitrogen-sulphur-oxygen
            PED        Potential egg deposition
            PHN        Phenanthrene
            PI         Principal Investigator(s)
            PWS        Prince William Sound
            PWSAC      Prince William Sound Aquaculture
            QA/QC      Quality Assurance/Quality Control
            RPWG       Restoration Planning Work Group
            SCAT       Shoreline Cleanup Advisory Team
            SSAT       Spring Shoreline Assessment Team
            T/M        Terrestrial Mammals
            T/S        Technical Services
            USFS       United States Forest Service
            VFDA       Valdez Fisheries Development Association





























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