[Federal Register Volume 66, Number 189 (Friday, September 28, 2001)]
[Proposed Rules]
[Pages 49794-49816]
From the Federal Register Online via the Government Publishing Office [www.gpo.gov]
[FR Doc No: 01-24374]



[[Page 49793]]

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Part VI





Environmental Protection Agency





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40 CFR Part 136



Guidelines Establishing Test Procedures for the Analysis of Pollutants; 
Whole Effluent Toxicity Test Methods; Proposed Rule

  Federal Register / Vol. 66, No. 189 / Friday, September 28, 2001 / 
Proposed Rules  

[[Page 49794]]


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ENVIRONMENTAL PROTECTION AGENCY

40 CFR Part 136

[FRL-7069-7]


Guidelines Establishing Test Procedures for the Analysis of 
Pollutants; Whole Effluent Toxicity Test Methods

AGENCY: Environmental Protection Agency (EPA).

ACTION: Proposed rule.

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SUMMARY: Today, EPA proposes to ratify its approval of several analytic 
test procedures measuring ``whole effluent toxicity,'' which the Agency 
standardized in an earlier rulemaking. Today's proposal also would 
modify some of those test procedures. EPA is proposing today's notice 
to satisfy obligations in a settlement agreement designed to resolve 
litigation over that earlier rulemaking. The proposed changes are 
intended to improve the performance of whole effluent toxicity (WET) 
tests, and thus increase confidence in the reliability of the results 
obtained using the test procedures.

DATES: Comments on this proposal must be postmarked, delivered by hand, 
or electronically mailed on or before November 27, 2001. Comments 
provided electronically will be considered timely if they are submitted 
electronically by 11:59 p.m. Eastern Standard Time (EST) on November 
27, 2001.

ADDRESSES: Send written or electronic comments on the proposed rule to 
``Whole Effluent Toxicity (WET) Test Method Changes'' Comment Clerk 
(WETEU-IX); Water Docket (4101); Environmental Protection Agency; Ariel 
Rios Building; 1200 Pennsylvania Avenue, NW; Washington, DC--P 20460. 
EPA requests that commenters submit copies of any references cited in 
comments. Commenters also are requested to submit an original and three 
copies of their written comments and enclosures. Commenters that want 
receipt of their comments acknowledged should include a self-addressed, 
stamped envelope. All written comments must be postmarked or delivered 
by hand. No facsimiles (faxes) will be accepted. Hand deliveries should 
be delivered to EPA's Water Docket at 401 M Street, SW, Room EB57, 
Washington, D.C. 20460.
    Comments may be submitted electronically to: [email protected]. 
Electronic comments must be submitted as a Word Perfect 5/6/7/8 file or 
an ASCII file, avoiding the use of special characters and any form of 
encryption. Comments and data also will be accepted on disks in 
WordPerfect 5/6/7/8 or ASCII file format. Electronic comments on this 
proposed rule may be filed online at any Federal Depository Library. 
All electronic comments must be identified by docket number (WET-IX). 
Electronic comments will be transferred into a paper version for the 
official record. EPA will attempt to clarify electronic comments if 
there is an apparent error in transmission.
    The record for this rulemaking has been established under docket 
number WET-IX. A copy of the supporting documents cited in this 
proposal is available for review at EPA's Water Docket, East Tower 
Basement (Room EB 57), 401 M Street, SW, Washington, DC 20460. For 
access to docket materials, call (202) 260-3027 on Monday through 
Friday, excluding Federal holidays, between 9:00 a.m. and 3:30 p.m. EST 
to schedule an appointment.
    This Federal Register document has been placed on the Internet for 
public review and downloading at the following location: http://www.epa.gov/fedrgstr/. The final report of EPA's WET Interlaboratory 
Variability Study, Volumes 1 and 2 (USEPA, 2001a; USEPA, 2001b) and the 
document titled, Proposed Changes to Whole Effluent Toxicity Method 
Manuals (USEPA, 2001d), which is referenced in today's rule and 
provides details of proposed changes, also are available on the 
Internet at http://www.epa.gov/waterscience/WET.

FOR FURTHER INFORMATION CONTACT: For regulatory information regarding 
this proposal, contact Marion Kelly, Engineering and Analysis Division 
(4303), Office of Science and Technology, Office of Water, U.S. 
Environmental Protection Agency, 1200 Pennsylvania Avenue, NW, 
Washington, DC 20460 (e-mail: [email protected]) or call (202) 260-
7117. For technical information regarding method changes proposed in 
today's rule, contact Teresa J. Norberg-King, National Health and 
Environmental Effects Research Laboratory, Mid-Continent Ecology 
Division, Office of Research and Development, U.S. Environmental 
Protection Agency, 6201 Congdon Boulevard, Duluth, MN 55804 (e-mail: 
[email protected]) or call (218) 529-5163.

SUPPLEMENTARY INFORMATION:

Potentially Regulated Entities

    EPA Regions, as well as State, Territories and Tribes authorized to 
implement the National Pollutant Discharge Elimination System (NPDES) 
program, issue permits that comply with the technology-based and water 
quality-based requirements of the Clean Water Act. In doing so, the 
NPDES permitting authority, including authorized States, Territories, 
and Tribes, make a number of discretionary choices associated with 
permit writing, including the selection of pollutants to be measured 
and, in many cases, limited in permits. If EPA has ``approved'' (i.e., 
promulgated through rulemaking) standardized testing procedures for a 
given pollutant, the NPDES permitting authority must specify one of the 
approved test procedures or an approved alternate test procedure for 
the measurements required under the permit. In addition, when a States, 
Territory, or authorized Tribe provides certification of Federal 
licenses under CWA section 401, measurements required by such 
certifications must be made using the approved testing procedures. 
Categories and entities that may be regulated include:

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                                             Examples of potentially
                Category                   affected/regulated entities
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States, Territorial, and Indian Tribal   States, Territories, and Tribes
 Governments.                             authorized to administer the
                                          NPDES permitting program;
                                          States, Territories, and
                                          Tribes that certify Federal
                                          licenses.
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    This table is not intended to be exhaustive, but rather provides a 
guide for readers regarding entities likely to be regulated by this 
action. This table lists the types of entities that EPA is now aware 
could potentially be regulated by this action. Other types of entities 
not listed in the table also could be regulated. If you have questions 
regarding the applicability of this action to a particular entity, 
consult the persons listed in the preceding FOR FURTHER INFORMATION 
CONTACT section.

[[Page 49795]]

Outline of Preamble

I. Statutory Authority
II. Regulatory Background
III. Explanation of Today's Action
    A. Introduction
    B. Proposed Method Changes
    1. Updates
    a. Incorporation of Previous Addenda and Errata
    b. Update of Method Precision Data
    2. Minor Corrections and Clarifications
    3. Specific Stakeholder Concerns
    a. Blocking by Known Parentage
    b. pH Drift
    c. Concentration-Response Relationships
    d. Nominal Error Rates
    e. Confidence Intervals
    f. Dilution Series
    g. Dilution Waters
    h. Pathogen Interference
    C. Ratification or Withdrawal of Methods
    1. WET Variability Study
    2. Ceriodaphnia dubia Acute Test, Ceriodaphnia dubia Survival 
and Reproduction Test, Fathead Minnow Acute Test, Fathead Minnow 
Larval Survival and Growth Test, Sheepshead Minnow Acute Test, 
Sheepshead Minnow Larval Survival and Growth Test, and Inland 
Silverside Acute Test
    3. Inland Silverside Larval Survival and Growth Test
    4. Champia parvula Reproduction Test
    5. Mysidopsis bahia Survival, Growth, and Fecundity Test
    6. Selenastrum capricornutum Growth Test
    7. Holmesimysis costata Acute Test
IV. Regulatory Requirements
    A. Executive Order 12866--Regulatory Planning and Review
    B. Unfunded Mandates Reform Act
    C. Regulatory Flexibility Act (RFA), as Amended by the Small 
Business Regulatory Enforcement Fairness Act of 1996 (SBREFA), 5 
U.S.C. 601 et seq.
    D. Paperwork Reduction Act
    E. National Technology Transfer and Advancement Act
    F. Executive Order 13045--Protection of Children From 
Environmental Health Risks and Safety Risks
    G. Executive Order 13175--Consultation and Coordination With 
Indian Tribal Governments
    H. Executive Order 13132--Federalism
    I. Executive Order 13211--Energy Effects
    J. Plain Language Directive
V. Request for Comments and Available Data
    A. pH Drift
    B. Percent Minimum Significant Difference
    C. Other Method Modifications
VI. References

I. Statutory Authority

    Today's proposal is pursuant to the authority of sections 101(a), 
301, 304(h), 402, and 501(a) of the Clean Water Act (CWA), 33 U.S.C. 
1251(a), 1311, 1314(h), 1342, 1361(a) (the ``Act''). Section 101(a) of 
the Act sets forth the ``goal of restoring and maintaining the 
chemical, physical, and biological integrity of the Nation's waters'' 
and prohibits ``the discharge of toxic pollutants in toxic amounts.'' 
Section 301 of the Act prohibits the discharge of any pollutant into 
navigable waters unless the discharge complies with a National 
Pollutant Discharge Elimination System (NPDES) permit, issued under 
section 402 of the Act. Section 304(h) of the Act requires the 
Administrator of the EPA to ``promulgate guidelines establishing test 
procedures for the analysis of pollutants that shall include the 
factors which must be provided in any certification pursuant to section 
401 of this Act or permit applications pursuant to section 402 of this 
Act.'' Section 501(a) of the Act authorizes the Administrator to 
``prescribe such regulations as are necessary to carry out his function 
under this Act.''

II. Regulatory Background

    Standardized analytical procedures for monitoring and reporting 
required in NPDES permits (40 CFR part 122, Secs. 122.21, 122.41, 
122.44, and 123.25), and in the implementation of the pretreatment 
standards issued under section 307 of the Act (40 CFR part 403, 
Secs. 403.10 and 402.12) appear at 40 CFR part 136. There may be 
discharges that require limitations for certain parameters using test 
procedures not yet approved under 40 CFR part 136. Under 40 CFR 
122.41(j)(4) and 122.44(i)(1)(iv) permit writers may include, through 
permit proceedings, parameters requiring the use of test procedures 
that are not approved part 136 methods. EPA also may include such 
parameters in accordance with the provisions prescribed at 40 CFR 
401.13, ``Test Procedures for Measurements.'' Permits may include, for 
example, effluent limitations for WET using standardized testing 
procedures other than those published at 40 CFR part 136 that are 
approved for nationwide use. In such cases, use of the particular test 
species and test protocols would remain subject to challenge on a case-
by-case basis in permit proceedings (except, for example, if an 
authorized State conducted rulemaking to standardize a particular 
testing procedure applicable within the State).
    In 1995, EPA amended the ``Guidelines Establishing Test Procedures 
for the Analysis of Pollutants,'' 40 CFR part 136, to add a series of 
standardized whole effluent toxicity (WET) test methods to the list of 
Agency approved methods for CWA data gathering and compliance 
monitoring programs (60 FR 53529; October 16, 1995) (WET final rule). 
The WET final rule amended 40 CFR 136.3 (Tables IA and II) by adding 
acute toxicity methods and short-term methods for estimating chronic 
toxicity. These methods measure the toxicity of effluents and receiving 
waters to freshwater, marine, and estuarine organisms. Acute methods 
(USEPA, 1993b) generally use death of the test organisms during 24 to 
96 hour exposure durations as the measured effect of an effluent or 
receiving water. The short-term methods for estimating chronic toxicity 
(USEPA, 1994a; USEPA, 1994b) use longer durations of exposure (up to 
nine days) to ascertain the adverse effects of an effluent or receiving 
water on survival, growth, and/or reproduction of the organisms. For 
this rulemaking notice, the short-term methods for estimating chronic 
toxicity will be referred to as chronic methods for ease of notation.
    Standardized test procedures for conducting the approved acute and 
chronic WET tests are provided in the following three method manuals, 
which were incorporated by reference in the WET final rule: Methods for 
Measuring the Acute Toxicity of Effluents and Receiving Water to 
Freshwater and Marine Organisms, Fourth Edition, August 1993, EPA/600/
4-90/027F (acute method manual); Short-Term Methods for Estimating the 
Chronic Toxicity of Effluents and Receiving Water to Freshwater 
Organisms, Third Edition, July 1994, EPA/600/4-91/002 (freshwater 
chronic method manual); and Short-Term Methods for Estimating the 
Chronic Toxicity of Effluents and Receiving Water to Marine and 
Estuarine Organisms, Second Edition, July 1994, EPA/600/4-91/003 
(marine chronic method manual).
    After promulgation of the WET methods, a variety of parties filed 
suit challenging the EPA rulemaking (Edison Electric Institute v. EPA, 
No. 96-1062 (D.C. Cir.); Western Coalition of Arid States v. EPA, No. 
96-1124; Lone Star Steel Co. v. EPA, No. 96-1157 (D.C. Cir.)). To 
resolve that litigation, EPA entered into settlement agreements with 
the various parties. EPA proposes actions today to fulfill obligations 
under some of those settlement agreements.
    In February 1999, EPA published a technical corrections notice that 
incorporated into the WET final rule an errata document to correct 
minor errors and omissions, provide clarification, and establish 
consistency among the WET final rule and method manuals (64 FR 4975; 
February 2, 1999). Further background on the WET test methods and these 
technical documents are included in the Federal Register notices cited 
above (60 FR 53529 and 64 FR 4975).

[[Page 49796]]

III. Explanation of Today's Action

A. Introduction

    Today's proposal would make a number of revisions to the currently 
approved WET test methods. See section III.B. Also in today's action, 
EPA presents final results of an interlaboratory variability study of 
WET test methods and, based on these results, proposes to ratify 11 of 
the 12 methods evaluated in the study (see section III.C). Today's 
proposal requests public comment on the inclusion of additional 
technical changes to the approved WET test methods and on EPA's 
proposal to ratify 11 of 12 WET test methods.
    Although today's action fulfills portions of settlement agreements 
resolving litigation over the 1995 WET test method rulemaking, EPA 
acknowledges that some stakeholders still have significant concerns 
related to implementation of WET control strategies through NPDES 
permits. By today's proposal, EPA intends to focus only on analytic 
testing methodologies to measure WET, not on WET implementation 
generally.
    Since the 1995 WET final rule, EPA and authorized States have taken 
additional actions to improve and enhance implementation of WET control 
strategies. EPA, for example, has published additional guidance on the 
conduct of a toxicity identification evaluation (TIE) and a toxicity 
reduction evaluation (TRE), as well as guidance on the circumstances 
that trigger such evaluations (USEPA, 1999c; USEPA, 2001g).
    Other questions have arisen about the significance of EPA action to 
standardize WET testing procedures through rulemaking. For example, 
some stakeholders question whether, by promulgating WET test methods, 
EPA has published recommended water quality criteria (pursuant to CWA 
section 304(a)) for ``toxicity.'' To respond and clarify, EPA's 
promulgation of WET test procedures are not water quality criteria 
recommendations under section 304(a). When States develop and implement 
water quality standards, including narrative water quality criteria, 
States should translate those criteria into measurable expressions of 
toxicity. The test methods themselves are not per se translators of the 
narrative criterion: ``no toxics in toxic amounts.'' The test methods 
are merely the measurement tools according to which such criteria may 
be translated.
    Today's proposed revisions include changes to the three method 
manuals (USEPA, 1993b; USEPA, 1994a; USEPA, 1994b) incorporated by 
reference in the WET rule (60 FR 53529; October 16, 1995) and amend the 
``Guidelines Establishing Test Procedures for the Analysis of 
Pollutants'' (40 CFR part 136) to reference the updated editions of the 
method manuals. Modifications to the method manuals are intended to 
update the methods, provide additional minor corrections and 
clarifications, and address specific stakeholder concerns (see Section 
III.B). EPA proposes to update the methods (1) by incorporating 
previous method addenda and errata and (2) by revising method precision 
statements to reflect results from recent EPA studies (USEPA, 2000d; 
USEPA, 2001a). In addition to corrections identified in previous method 
addenda and errata, EPA proposes to correct other minor technical 
errors and omissions. EPA also seeks comment on an additional 
modification to WET test methods that would require the application of 
upper and lower bounds on the percent minimum significant difference 
(PMSD) calculated in WET tests (see section V.B).
    EPA also proposes method revisions in response to specific 
stakeholder concerns. Specifically, these revisions include: requiring 
``blocking'' by known parentage in the Ceriodaphnia dubia Survival and 
Reproduction Test; adding procedures to control pH drift that may occur 
during testing; incorporating review procedures for the evaluation of 
concentration-response relationships; clarifying allowable nominal 
error rate adjustments; clarifying limitations in the generation of 
confidence intervals; adding guidance on dilution series selection; 
clarifying dilution water acceptability; and adding procedures for 
determining and minimizing the impact of pathogens in the Fathead 
Minnow Survival and Growth Test. These are summarized below in section 
III.B and detailed in the document titled, Proposed Changes to Whole 
Effluent Toxicity Method Manuals (USEPA, 2001d). Proposal of these 
revisions partially fulfills the requirements of two settlement 
agreements between stakeholders and EPA (Edison Electric Institute, et 
al. v. EPA, No. 96-1062 & consolidated case (D.C. Cir.), Settlement 
Agreement, July 24, 1998; Lone Star Steel v. EPA, No. 96-1157 (D.C. 
Cir.), Settlement Agreement, March 4, 1998).
    EPA requests public comment on the proposed changes to the WET test 
methods and on the proposal to ratify the WET test methods (see section 
V). When EPA takes final action on today's proposal, the Agency intends 
to incorporate the modifications proposed today into the text of new 
editions of each of the WET method manuals.

B. Proposed Method Changes

    Today, EPA proposes to revise each of the WET method manuals 
(USEPA, 1993b; USEPA, 1994a; USEPA, 1994b). Proposed method changes 
include: (1) updates to the methods, (2) minor corrections and 
clarifications, and (3) modifications to address specific stakeholder 
concerns. These method changes are described in Sections 1 through 3 
below and are detailed in the document titled, Proposed Changes to 
Whole Effluent Toxicity Method Manuals (USEPA, 2001d), which is 
included in the docket supporting today's rule and is available online 
at http://www.epa.gov/waterscience/WET.
1. Updates

a. Incorporation of Previous Addenda and Errata

    Subsequent to promulgating the WET final rule in 1995, EPA issued 
several documents to correct and amend that rule and its supporting 
documentation. Specifically, in February 1999, EPA published a final 
rule that incorporated into the WET rule an errata document (USEPA, 
1999a) to correct minor errors and omissions in the WET method manuals 
(64 FR 4975; February 2, 1999). In addition, a 1996 addenda document 
(USEPA, 1996a) revised the 1993 acute method manual (USEPA, 1993b). 
Today, EPA proposes to incorporate the changes noted in the errata and 
the addenda documents into the text of the appropriate method manuals 
by issuing revised editions of each of the three method manuals. EPA 
plans to issue the revised editions when it takes final action on this 
proposal. The incorporation of the errata and addenda into the method 
manual text would not further alter the methods. This action would 
simply assist users of the method manuals by incorporating all previous 
corrections into updated editions.

b. Update of Method Precision Data

    Since publishing the WET method manuals, EPA has conducted two 
large-scale studies of WET test method precision. During 1999 and 2000, 
EPA conducted an interlaboratory variability study (the WET Variability 
Study) of 12 of the 17 WET test methods promulgated at 40 CFR part 136. 
This study generated data from more than 700 blind samples tested in 55 
laboratories. EPA published interlaboratory precision results from the 
WET Variability Study in 2000 (USEPA, 2000b; USEPA, 2000c) and 
submitted the study results for expert

[[Page 49797]]

peer review in 2001 (USEPA, 2001c). Following expert peer review, EPA 
published a final study report (USEPA, 2001a; USEPA, 2001b).
    In addition to the WET Variability Study, EPA conducted a study of 
intralaboratory WET test precision based on routine laboratory 
reference toxicant test data. EPA compiled a database of more than 
1,800 reference toxicant tests conducted for 23 different methods 
between 1988 and 1999 in 75 laboratories. EPA used this database to 
quantify estimates of precision for each of the WET methods. EPA 
published this precision data and additional guidance on reducing 
method variability in a guidance document titled, Understanding and 
Accounting for Method Variability in Whole Effluent Toxicity 
Applications Under the National Pollutant Discharge Elimination System 
Program (USEPA, 2000d) (the Variability Guidance Document).
    In today's action, EPA proposes to modify the WET method manuals by 
updating statements and inserting tables regarding the multi-laboratory 
(interlaboratory) and single-laboratory (intralaboratory) precision of 
the methods using data from the WET Variability Study and the 
Variability Guidance Document. Results from these two studies represent 
the most current and complete data available on intralaboratory and 
interlaboratory precision of WET test methods. The proposed changes 
would modify the chronic method manuals (USEPA, 1994a; USEPA, 1994b) by 
revising subsections on precision and accuracy for several test 
methods. The proposed changes also would modify Section 4 (Quality 
Assurance) of each of the method manuals (USEPA, 1993b; USEPA, 1994a; 
USEPA, 1994b) to update statements on test method variability and 
precision. The specifics of the proposed method manual changes related 
to updating precision statements are detailed in the document titled, 
Proposed Changes to Whole Effluent Toxicity Method Manuals (USEPA, 
2001d).
2. Minor Corrections and Clarifications
    In addition to the incorporation of changes identified in the 1999 
errata (USEPA, 1999a) and the acute manual addenda (USEPA, 1996a), EPA 
proposes to correct additional minor errors and omissions in the WET 
method manuals. All of the minor corrections and clarifications 
identified to date are detailed in the document titled, Proposed 
Changes to Whole Effluent Toxicity Method Manuals (USEPA, 2001d). This 
list may not be exhaustive, and EPA proposes to correct additional 
minor errors and omissions that become apparent during the correcting 
or revising of sections of the WET method manuals.
3. Specific Stakeholder Concerns
    Today, EPA also proposes to modify the WET method manuals to 
address specific stakeholder concerns. The proposed modifications are 
summarized in Sections a through h below and are detailed in the 
document titled, Proposed Changes to Whole Effluent Toxicity Method 
Manuals (USEPA, 2001d), which is included in the docket supporting 
today's rule and is available online at http://www.epa.gov/waterscience/WET. Proposal of these revisions partially fulfills the 
requirements of two settlement agreements between stakeholders and EPA 
(Edison Electric Institute, et al. v. EPA, Settlement Agreement, July 
24, 1998; Lone Star Steel v. EPA, Settlement Agreement, March 4, 1998).

a. Blocking by Known Parentage

    EPA proposes to amend the Ceriodaphnia dubia Survival and 
Reproduction Test (section 13 of USEPA, 1994a) to require that test 
organisms be allocated using ``blocking by known parentage.'' Blocking 
by known parentage is a block randomization technique for allocating 
test organisms among test chambers such that offspring from a single 
female are distributed evenly among the test treatments (one per 
treatment). In this arrangement, a block consists of the set of six 
test chambers (one for each test treatment) containing organisms 
derived from a single female parent.
    Currently, the promulgated method describes a blocking by known 
parentage procedure for use in test setup, but the method does not 
require the use of this procedure. Today's proposal would require the 
use of blocking by known parentage by using compulsory terms such as 
``must'' and ``shall.'' The procedure described for test setup in the 
current promulgated method would be retained as an example of how 
blocking by known parentage may be accomplished.
    In association with a blocking by known parentage requirement, 
today's proposal also would add guidance on the treatment of males that 
may occur in tests. The proposed changes would require exclusion of an 
entire block from reproduction analysis (i.e., calculation of the no 
observed effect concentration for reproduction and the 25% inhibition 
concentration for reproduction) when 50% or more of the surviving 
organisms in that block are identified as males. If less than 50% of 
surviving organisms in a block are identified as males, only those 
males would be excluded from the reproduction analysis. The proposed 
changes also would stipulate that a test is invalid if fewer than eight 
replicates remain in the control after excluding individual males and 
necessary blocks (i.e., those having 50% or more of surviving organisms 
identified as males). The specifics of all proposed method manual 
changes related to blocking by known parentage are detailed in the 
document titled, Proposed Changes to Whole Effluent Toxicity Method 
Manuals (USEPA, 2001d).
    Blocking by known parentage provides at least two benefits to the 
performance of the Ceriodaphnia dubia Survival and Reproduction Test 
(USEPA, 2001e). First, this technique of test organism allocation 
ensures that any ``brood effect'' is evenly distributed among the test 
treatments. Brood effects include differences in organism fecundity or 
sensitivity that may be attributed to the health or genetics of the 
parent organism. Blocking by known parentage minimizes any potential 
bias that may be caused by one test treatment receiving an inordinate 
number of underperforming (or overperforming) young from the same 
parent organism. In an analysis of 389 tests from EPA's reference 
toxicant test database (USEPA, 2000d) and 102 tests from EPA's WET 
Variability Study (USEPA, 2001a), 9% and 25% of tests, respectively, 
showed statistically significant (alpha = 0.05) block effects on the 
reproduction endpoint (USEPA, 2001e). This means that, for these tests, 
the number of offspring produced by test organisms was significantly 
affected by the parental source of those test organisms. The blocking 
by known parentage technique distributes this effect evenly across the 
test treatments to ensure that observed differences in reproduction 
between treatments are due to the effect of the treatment and not the 
parental source of test organisms.
    A second benefit of blocking by known parentage would be that it 
provides a means of minimizing the impact of male production on test 
performance. In healthy cultures, Ceriodaphnia dubia generally 
reproduce parthenogenetically to produce cloned females for use in 
testing. Under conditions of environmental stress, however, cladocerans 
(such as Ceriodaphnia dubia and Daphnia magna) are known to produce 
males (Pennak, 1989), which can negatively affect the performance of 
toxicity tests designed to measure reproductive

[[Page 49798]]

effects (Haynes et al., 1989). When using blocking by known parentage, 
males produced by a given brood female are contained within a single 
block of the test rather than randomly scattered throughout the test. 
If a large number of males are produced from a given brood female, the 
associated block may be removed from the analysis of reproduction, 
thereby minimizing the effect of those males on the test. Blocking by 
known parentage also allows the source of males to be identified, so 
that potential problems with culture health can be more easily 
isolated.

b. pH Drift

    During the conduct of static or static-renewal WET tests, the pH in 
test containers may fluctuate or drift from the initial pH value. This 
pH drift may be upward or downward depending upon test conditions and 
sample characteristics. For instance, the addition of food substances 
such as algae may cause a decrease in pH, while the loss of carbon 
dioxide (CO2) from supersaturated effluent samples may cause 
an increase in pH. A change in pH during testing means that an effluent 
sample might be tested for toxicity at a different pH than the effluent 
sample pH at the point of discharge. Under certain circumstances, this 
pH drift could influence sample toxicity and be considered a test 
interference. For this reason, EPA is proposing to provide guidance in 
the chronic method manuals (USEPA, 1994a; USEPA, 1994b) on how to 
identify if pH drift is a test interference and how to control test pH 
if artifactual toxicity due to pH drift is confirmed.
    For most tests, the range of pH drift is small, is well within the 
organisms' tolerance range, and does not interfere with the analysis of 
whole effluent toxicity. In EPA's WET Variability Study (USEPA, 2001a), 
daily pH drift in blank samples averaged only +0.1 units (with a range 
of -0.3 to +0.8 among 35 tests) in the Ceriodaphnia dubia Survival and 
Reproduction Test and -0.1 units (with a range of -1.4 to +0.7 among 25 
tests) in the Fathead Minnow Larval Survival and Growth Test. For 
effluent samples (municipal wastewater spiked with KCl) analyzed in 
EPA's WET Variability Study, pH drift in the 100% sample increased 
slightly for the Ceriodaphnia dubia Survival and Reproduction Test, 
averaging +0.3 units (with a range of -0.2 to +1.1 among 28 tests). For 
the Fathead Minnow Larval Survival and Growth Test, daily pH drift in 
effluent samples averaged -0.1 units (with a range of -0.6 to +0.4 
among 28 tests), the same degree of drift observed in blank samples. 
Ninety percent of Ceriodaphnia dubia Survival and Reproduction Tests 
(126 tests) experienced absolute pH drift (up or down) of less than 0.7 
units, and 90% of Fathead Minnow Larval Survival and Growth Tests (105 
tests) experienced absolute pH drift of less than 0.5 units.
    While pH drift was relatively mild for most samples analyzed in the 
WET Variability Study (USEPA, 2001a), other effluent samples may 
routinely exhibit a greater degree of pH drift. For example, municipal 
wastewater from Publicly-Owned Treatment Works (POTW) is typically 
discharged at a pH of 7.2-7.4, but the pH may equilibrate after contact 
with air and stabilize at 8.0-8.5 (USEPA, 1992). In a 1998 survey of 
433 POTWs, 39% of respondents indicated that upward drift of effluent 
sample pH had been observed during acute or chronic WET testing 
(DeGraeve et al., 1998). Upward pH drift in POTW effluent is generally 
caused by dissipation of CO2 from the sample. Biological 
treatment often produces an effluent that is supersaturated with 
CO2. As dissolved CO2 in the supersaturated 
sample equilibrates with the atmospheric CO2 concentration, 
CO2 is lost from the sample. Because dissolved 
CO2 acts as a weak acid, pH increases as CO2 is 
lost. In cases where pH drift is due to the effluent characteristics, 
the degree of drift will be greatest in the 100% effluent concentration 
and will decrease with decreasing test concentrations.
    EPA does not consider pH drift alone to be an interference in WET 
testing if pH is within the organism's tolerance range (typically pH 6 
to 9). Belanger and Cherry (1990) showed that Ceriodaphnia dubia 
survival and reproduction did not differ significantly in receiving 
water tests conducted at pH values ranging from 6 to 9. The degree of 
pH drift typically observed in effluent samples should generally only 
interfere with test results if the sample contains a compound with 
toxicity that is pH dependent and at a concentration that is near the 
toxicity threshold. Compounds with pH-dependent toxicity are those with 
chemical characteristics that allow sufficient differences in 
dissociation, solubility, or speciation to occur within a 
physiologically tolerable pH range of 6 to 9 (Schubauer-Berigan et al., 
1993). Examples of such compounds include ammonia, metals, hydrogen 
sulfide, cyanide, and ionizable organics. Ammonia, for instance, is 
very common in effluent samples, and its toxicity changes sharply 
within the typical effluent pH range of 7 to 8.5. As pH increases and 
the temperature is held relatively constant, the percent of total 
ammonia in the un-ionized form increases (USEPA, 1994a; Emerson et al., 
1975). Because the un-ionized form of ammonia (NH3) is significantly 
more toxic than the ionized form (NH4+), toxicity 
increases as pH increases. For metals, toxicity may increase or 
decrease with increasing pH. Lead and copper were found to be more 
acutely toxic at pH 6.5 than at pH 8.0 or 8.5, while nickel and zinc 
were more toxic at pH 8.5 than at pH 6.5 (USEPA, 1992). pH-dependent 
toxicity is likely to be affected by temperature, dissolved oxygen, 
CO2 concentrations, and total dissolved solids (USEPA, 
1992). When pH-dependent compounds are present at concentrations near 
the threshold for toxicity, pH drift during WET testing may produce 
artifactual toxicity, or toxicity that would not have been observed if 
the initial test pH had been maintained.
    In addition to the issue of pH drift affecting toxicity in the 
presence of pH-dependent compounds, stakeholders have raised concerns 
about daily pH drift and sample renewal cycles producing toxicity even 
in the absence of pH-dependent compounds. The circumstance of concern 
would be in static-renewal tests, where the pH may change between the 
time test organisms are placed into the test solutions and the time at 
which the test solution is renewed. At renewal, the pH of test 
solutions may be quickly returned to the initial sample pH. For chronic 
tests that require daily renewal, a daily cycle of pH drift and renewal 
may be established. Stakeholders expressed concern that, if the 
difference in pH between the test solution and the renewal solution is 
great, these adjustments in pH at renewal may cause shock to the test 
organisms. Because the control treatment does not always experience the 
same pH drift as effluent treatments, any shock resulting from daily 
renewal would be experienced only in effluent treatments and 
artifactual toxicity could result. In a 1998 settlement agreement with 
these stakeholders (Edison Electric Institute, et al. v. EPA, 
Settlement Agreement, July 24, 1998), EPA agreed to propose changes to 
the WET methods that would provide methodological solutions for 
controlling pH drift.
    Currently, the WET method manuals (USEPA, 1993b; USEPA, 1994a; 
USEPA, 1994b) provide guidance for effluent samples that arrive (i.e., 
at the testing laboratory prior to testing) with a pH outside of the 
6.0 to 9.0 range. This range represents the general organism tolerance 
range, so pH values outside of this range may produce toxic effects due 
to pH alone. For samples that arrive

[[Page 49799]]

with a pH outside of this range, the current method manuals require 
adjustment of the sample to pH 7 for freshwater testing or pH 8 for 
marine testing. The method manuals also suggest brief aeration of 
samples prior to use if dissolved oxygen levels are not at or near 
saturation. Aeration provides the benefit of bringing other dissolved 
gases (e.g., CO2) into equilibrium with the atmosphere and 
stabilizing pH, but use of aeration should be minimized to reduce the 
loss of volatile chemicals.
    In 1996, EPA issued additional guidance on ammonia and pH control 
in chronic testing (USEPA, 1996b). This guidance recognized that the 
analyst has flexibility to control artifactual toxicity caused by pH 
drift in chronic tests provided that the analyst verifies that the 
source of toxicity is, in fact, artifactual. To verify that the 
toxicity is artifactual, EPA recommended parallel testing using one 
test with an adjusted pH and one test without an adjusted pH. If 
toxicity is removed or reduced when pH is adjusted, the source of 
toxicity could be artifactual and pH could be controlled in the testing 
of the effluent. This guidance acknowledged that pH could be controlled 
during testing with procedures that do not significantly alter the 
nature of the sample.
    Today, EPA proposes to modify the chronic method manuals (USEPA, 
1994a; USEPA, 1994b) to incorporate procedures for controlling pH drift 
in static-renewal tests when sample toxicity is confirmed to be 
artifactual and caused by pH drift. EPA proposes adding guidance that 
is consistent with the 1996 USEPA guidance on pH and ammonia control in 
chronic testing (USEPA, 1996b), and extending this guidance to include 
situations where artifactual toxicity is caused by pH drift in the 
absence of ammonia.
    The proposed method changes would require that, prior to the use of 
pH control techniques, the analyst must confirm that observed toxicity 
is artifactual and caused by pH drift. Evidence of artifactual toxicity 
would be demonstrated by conducting parallel tests: one with controlled 
pH and one with uncontrolled pH. Several such parallel tests conducted 
on a given effluent may be required by the regulatory authority to 
verify that the toxicity observed in that effluent is artifactual and 
caused by pH drift (as opposed to variability in effluent samples). 
Following this determination, the regulatory authority may allow pH 
control in subsequent chronic toxicity testing of the effluent. The 
proposed method changes would specify the use of acid/base addition 
and/or a CO2-controlled atmosphere technique for adjusting 
and controlling pH in chronic tests.
    The CO2-controlled atmosphere technique that is proposed 
for pH control in chronic tests is conducted using enclosed test 
chambers with CO2 injected into the headspace above the test 
solution (USEPA, 1991a; USEPA, 1992; USEPA, 1996c; Mount and Mount, 
1992). An enriched-CO2 environment increases the dissolution 
of CO2 into the sample, which acts as a weak acid to prevent 
pH increases. This technique uses the natural carbonate buffering 
system to control pH and requires minimal alteration of the sample. 
This technique is one method recommended for adjusting pH in toxicity 
identification evaluations (TIEs) (USEPA, 1991a; USEPA, 1992; USEPA, 
1996c).
    In acute testing, the proposed method changes would recommend the 
use of static-renewal testing or flow-through testing when artifactual 
toxicity due to pH drift is suspected. The use of static-renewal 
testing may reduce the degree of pH drift (compared to static non-
renewal tests), and flow-through testing should eliminate pH drift that 
could occur due to static testing conditions. In flow-through testing, 
new sample is continually added to the test chambers, so drift from the 
initial sample pH should not occur. Flow-through testing also 
eliminates any potential for organism shock from pH drift and renewal 
cycles, because test renewal is continuous. Because flow-through 
testing provides an available option for reducing pH drift in acute 
tests without modifying the sample, EPA does not propose additional 
techniques (such as acid/base addition and/or CO2-controlled 
atmosphere techniques that are proposed for chronic test methods) for 
pH control in acute test methods.
    The specifics of all proposed method manual changes related to pH 
drift are detailed in the document titled, Proposed Changes to Whole 
Effluent Toxicity Method Manuals (USEPA, 2001d). The proposed changes 
related to pH drift will affect all methods in the freshwater chronic 
method manual (USEPA, 1994a), except for the Selenastrum capricornutum 
Growth Test; and all methods in the marine chronic method manual 
(USEPA, 1994b), except for the Arbacia punctulata Fertilization Test 
and the Champia parvula Reproduction Test. The Selenastrum, Arbacia, 
and Champia tests do not require test solution renewal, so daily pH 
fluctuations should not be a concern. Proposed changes to the acute 
method manual (USEPA, 1993b) would simply recommend the use of static-
renewal testing or flow-through testing when artifactual toxicity due 
to pH drift is suspected. EPA invites comments on how pH drift would 
and should be addressed in WET testing (see Section V.A).

c. Concentration-Response Relationships

    The concentration-response relationship established between the 
concentration of a toxicant and the magnitude of the response is a 
fundamental principle of toxicology. This principle assumes that there 
is a causal relationship between the dose of a toxicant (or 
concentration for toxicants in solution) and a measured response. A 
response may be any measurable biochemical or biological parameter that 
is correlated with exposure to the toxicant. The classical 
concentration-response relationship is depicted as a sigmoidal-shaped 
curve with detrimental responses increasing as the concentration of the 
toxicant increases. Not all concentration-response relationships, 
however, are represented by the classical sigmoidal-shaped curve. A 
corollary of the concentration-response concept is that every toxicant 
should exhibit a concentration-response relationship, given that the 
appropriate response is measured and given that the concentration range 
evaluated is appropriate. Use of this concept can be helpful in 
determining whether an effluent sample causes toxicity and in 
identifying anomalous test results.
    In July 2000, EPA published guidance on evaluating concentration-
response relationships to assist in determining the validity of WET 
test results (USEPA, 2000a). This document explained the concentration-
response concept and provided review steps for 10 different 
concentration-response patterns that may be encountered in WET test 
data. Based on the results of the review, the guidance anticipates one 
of three determinations: (1) that calculated effect concentrations are 
reliable and should be reported; (2) that calculated effect 
concentrations are anomalous and should be explained; or (3) that the 
test was inconclusive and should be repeated with a newly collected 
sample.
    In today's action, EPA proposes to require the review of 
concentration-response relationships generated for all multi-
concentration WET tests reported under the NPDES program. EPA proposes 
to modify section 10 of the two chronic method manuals (USEPA, 1994a; 
USEPA, 1994b) and section 12 of the acute method manual (USEPA, 1993b) 
to incorporate this required test review procedure. The modified 
sections would explain the

[[Page 49800]]

concentration-response concept, require the review of concentration-
response relationships, and reference EPA guidance (USEPA, 2000a) 
describing various forms of concentration-response relationships and 
review procedures. Use of the concentration-response review procedures 
(USEPA, 2000a) would ensure that a valid concentration-response 
relationship is demonstrated prior to the determination of toxicity. 
EPA intends to maintain the review procedures described in the guidance 
document (USEPA, 2000a) as ``guidance'' because these procedures may be 
revised as new information on the review of concentration-response 
relationships (including additional forms of concentration-response 
relationships) becomes available.
    To demonstrate the effectiveness of the proposed concentration-
response review steps, EPA used the guidance on concentration-response 
relationships (USEPA, 2000a) in the review and reporting of results 
from EPA's WET Variability Study (USEPA, 2001a). In this study, 635 
valid tests (i.e., those that met test acceptability criteria) were 
reviewed according to the proposed concentration-response evaluation 
procedures. Based on these review procedures, the calculated effect 
concentrations in 14 tests were determined to be anomalous, and the 
effect concentrations calculated in 9 tests were determined to be 
inconclusive. Eight of the 23 test results that were considered 
anomalous or inconclusive had erroneously indicated toxicity in blank 
samples. These results would have been reported as false positives if 
the concentration-response review procedures had not been used. This 
study indicates that the proposed concentration-response review 
procedures are effective in reducing the incidence of false positives 
in WET testing. The use of these review procedures reduced the rate of 
reported false positives in the WET Variability Study from 11.1% to 
3.7% for the Ceriodaphnia dubia Survival and Reproduction Test; from 
12.5% to 4.35% for the Fathead Minnow Larval Survival and Growth Test; 
from 14.3% to 0% for the Mysidopsis bahia Survival, Growth, and 
Fecundity Test; and from 14.3% to 0% for the Inland Silverside Larval 
Survival and Growth Test.
    In addition to requiring the review of concentration-response 
relationships, EPA proposes to modify section 12 of the acute method 
manual (USEPA, 1993b) and section 10 of the two chronic method manuals 
(USEPA, 1994a; USEPA, 1994b) to consolidate other important test review 
components that are described elsewhere in the method manuals. These 
revised sections, titled ``Report Preparation and Test Review,'' would 
describe the review of sample collection and handling conditions, test 
acceptability criteria, test conditions, statistical methods, 
concentration-response relationships, reference toxicant testing, and 
test variability. The specifics of the proposed method manual changes 
related to concentration-response relationship evaluation and other 
test review components are detailed in the document titled, Proposed 
Changes to Whole Effluent Toxicity Method Manuals (USEPA, 2001d).
    The quality of WET Variability Study data (USEPA, 2001a; USEPA, 
2001b) used to make decisions for this rulemaking is of primary 
importance to the Agency and to stakeholders. These data and the test 
review and acceptance criteria used in the WET Variability Study are 
detailed in a final study report contained in the record for this 
rulemaking (USEPA, 2001a). Some stakeholders believe that EPA 
improperly applied different standards in accepting or rejecting data 
generated in the WET Variability Study and departed from the stated 
objectives of the study design. EPA is proposing test review procedures 
consistent with the test reviews that EPA conducted on data developed 
in the WET Variability Study (though EPA notes that the objectives of 
the study differ from those associated with compliance monitoring). EPA 
proposes modifications to standardize the minimum elements of WET test 
review. While some of these test review components provide specific 
criteria for the acceptance or rejection of test results (e.g., the 
method test acceptability criteria), others (e.g., review of test 
conditions, reference toxicant testing, and concentration-response 
relationships) must be reviewed within the context of the test 
objective. Also, State and/or regional regulatory authorities may 
require additional test review components and criteria to further 
standardize the reporting and review of WET test data. EPA requests 
comment on the acceptance, interpretation, and use of the WET 
Variability Study data and on the proposed section of the method 
manuals titled, ``Report Preparation and Test Review''.

d. Nominal Error Rates

    WET test results (i.e., effect concentrations) may be determined by 
point estimation or hypothesis testing techniques (USEPA, 1994a; USEPA, 
1994b). Hypothesis testing techniques compare responses in the control 
treatment with responses in other treatments to test the ``null 
hypothesis'' that there is no statistically significant difference 
between the treatments (i.e., that the effluent is not toxic). To 
determine when a difference between treatments is large enough to be 
statistically significant, the statistician or analyst must select a 
nominal error rate. The nominal error rate, or alpha level, is an 
intended upper bound on the probability of incorrectly concluding that 
the treatments are different when, in fact, they are not (a Type I 
statistical error). The larger the alpha level, the greater the 
probability of incorrectly rejecting the null hypothesis (i.e., 
determining that the effluent is toxic when, in fact, it is not). For 
all WET tests, EPA recommends using an alpha level of 0.05, which 
corresponds to a 5% probability of making a Type I error.
    In response to stakeholder concerns that an alpha level of 0.05 
does not adequately protect against Type I errors (Moore et al., 2000; 
Edison Electric Institute, et al. v. EPA, Settlement Agreement, July 
24, 1998), EPA published guidance on nominal error rate selection 
(USEPA, 2000a). This guidance clarifies that the alpha level may be 
reduced to 0.01 in specific circumstances. These circumstances include 
instances when sublethal endpoints from Ceriodaphnia dubia or fathead 
minnow tests are reported under NPDES permit requirements, or when WET 
permit limits (based on any WET method) are derived without allowing 
for receiving water dilution. Even under these circumstances, however, 
the alpha level may be reduced only in tests that meet a fixed 
criterion for test sensitivity because reductions in the alpha level 
also reduce statistical power. Specifically, the percent minimum 
significant difference (PMSD) calculated for the test using an alpha 
level of 0.01 should be less than or equal to criteria set forth in the 
guidance document (USEPA, 2000a). The document also provides guidance 
on determining the need for additional test replication to meet PMSD 
criteria and guidance on the decision process for reducing the nominal 
error rate in hypothesis testing.
    In today's action, EPA proposes to modify the chronic WET method 
manuals (USEPA, 1994a; USEPA, 1994b) to clarify the circumstances under 
which the recommended alpha level may be reduced. The proposed change 
would modify subsection 9.4.6 (Recommended Alpha Levels) of the two 
chronic method manuals (USEPA, 1994a; USEPA, 1994b). This subsection 
would maintain the current recommendation that an alpha level of 0.05 
be used for hypothesis testing. In

[[Page 49801]]

addition, the subsection would identify the specific circumstances 
where the alpha level used for hypothesis testing could appropriately 
be reduced from 0.05 to 0.01. The subsection would describe these 
circumstances and reference the published guidance (USEPA, 2000a) for 
information on determining adequate test sensitivity and determining 
the appropriateness of reductions in the alpha level. The specifics of 
the proposed method manual changes related to nominal error rates are 
detailed in the document titled, Proposed Changes to Whole Effluent 
Toxicity Method Manuals (USEPA, 2001d).

e. Confidence Intervals

    Point estimation techniques described in the WET method manuals are 
used to generate effect concentrations and associated 95% confidence 
intervals (USEPA, 1993b; USEPA, 1994a; USEPA, 1994b). Software used to 
conduct these statistical procedures occasionally do not provide the 
associated confidence intervals. This situation may arise when test 
data do not conform with specific assumptions required by the 
statistical methods, when point estimates are outside of the test 
concentration range, and when specific limitations imposed by the 
software are encountered. In July 2000, EPA published guidance on the 
specific circumstances under which confidence intervals are not 
generated or are not suitable (USEPA, 2000a).
    In today's action, EPA proposes to modify the WET method manuals to 
clarify the circumstances under which confidence intervals are not 
generated by point estimation techniques and to reference the published 
guidance on this issue (USEPA, 2000a). The proposed change would modify 
subsection 9.3.2 (Point Estimation Techniques) of the two chronic 
method manuals (USEPA, 1994a; USEPA, 1994b) and subsection 11.2 
(Determination of the LC50 from Definitive, Multi-Effluent-
Concentration Acute Toxicity Tests) of the acute method manual (USEPA, 
1993b). The specifics of the proposed method manual changes related to 
confidence intervals are detailed in the document titled, Proposed 
Changes to Whole Effluent Toxicity Method Manuals (USEPA, 2001d).

f. Dilution Series

    In multi-concentration (definitive) WET tests, organism effects are 
measured in a range of effluent concentrations. The dilution series 
selected for the test defines the concentrations of effluent tested. 
The WET methods recommend preparing test concentrations using a 
dilution factor of greater than or equal to 0.5 and provide an example 
dilution series of 100%, 50%, 25%, 12.5%, and 6.25% effluent. While 
this particular dilution series is commonly used in WET testing, test 
concentrations for each test should be selected independently based on 
the objective of the study, the expected range of toxicity, the 
receiving water concentration (or instream waste concentration), and 
any available historical testing information on the effluent. The 
dilution series should be selected to optimize the precision of 
calculated effect concentrations and assist in establishing 
concentration-response relationships. In July 2000, EPA published 
guidance on selecting appropriate dilution series for WET testing 
(USEPA, 2000a).
    In today's action, EPA proposes to modify the WET method manuals to 
reference the published guidance on selecting dilution series (USEPA, 
2000a) and to clarify that dilution series should be selected 
independently for each test based on the objective of the study, the 
expected range of toxicity, the receiving water concentration (or 
instream waste concentration), and any available historical testing 
information on the effluent. The proposed change would modify 
subsection 8.10 (Multi-concentration [Definitive] Effluent Toxicity 
Tests) of the two chronic method manuals (USEPA, 1994a; USEPA, 1994b) 
and subsection 9.3 (Multi-concentration [Definitive] Effluent Toxicity 
Tests) of the acute method manual (USEPA, 1993b). The specifics of the 
proposed method manual changes related to dilution series selection are 
detailed in the document titled, Proposed Changes to Whole Effluent 
Toxicity Method Manuals (USEPA, 2001d).

g. Dilution Waters

    Test concentrations in definitive WET tests are prepared by 
diluting the effluent sample with an appropriate dilution water. The 
WET methods allow the use of natural receiving waters or synthetically 
prepared waters for dilution. Because the choice of dilution water can 
affect WET test results (Cooney et al., 1992; Belanger et al., 1989; 
DeLisle and Roberts, 1988), selecting an appropriate dilution water is 
important. To assist in this process, EPA published guidance on 
dilution water selection (USEPA, 2000a) that clarifies what EPA 
considers to be an acceptable dilution water. An acceptable dilution 
water is one that is appropriate for the objectives of the test; 
supports adequate performance of the test organisms with respect to 
survival, growth, reproduction, or other responses that may be measured 
in the test (i.e., consistently meets test acceptability criteria for 
control responses); is consistent in quality; and does not contain 
contaminants that could produce toxicity. The guidance also provides 
recommendations on how to select an appropriate dilution water based on 
the objectives of the test, the condition and quality of ambient 
receiving water, in-stream dilution potential, and recommendations or 
requirements from local regulatory authorities. Lastly, the guidance 
explains the use of dual controls when dilution water differs from 
organism culture water.
    In today's action, EPA proposes to modify the WET method manuals by 
clarifying the definition of acceptable dilution waters and referencing 
the published guidance (USEPA, 2000a) for more information on selecting 
appropriate dilution waters. The proposed change would modify 
subsection 7.1 (Types of Dilution Water) of each of the method manuals 
(USEPA, 1993b; USEPA, 1994a; USEPA, 1994b). The specifics of the 
proposed method manual changes related to dilution waters are detailed 
in the document titled, Proposed Changes to Whole Effluent Toxicity 
Method Manuals (USEPA, 2001d).

h. Pathogen Interference

    WET testing is designed to measure the aggregate toxicity of an 
aqueous test sample. The presence of pathogens and/or parasites in the 
test sample, however, may confound this measurement of toxicity by 
causing sporadic mortality among test organisms. Today, EPA proposes to 
modify the Fathead Minnow (Pimephales promelas) Larval Survival and 
Growth Test to provide guidance on the adverse effects of pathogens 
and/or parasites on test performance (i.e., pathogen and/or parasite 
test interference). EPA proposes procedures to control pathogen and/or 
parasite effects without compromising the capacity of the test to 
measure the toxicity of the test sample. The proposed method 
modifications are summarized below and detailed in the document titled, 
Proposed Changes to Whole Effluent Toxicity Method Manuals (USEPA, 
2001d).
    Pathogens that interfere with the test may come from the receiving 
water used for test dilutions, from the effluent, or from the receiving 
water that is used as intake water. Most receiving waters contain all 
the common fish pathogens, but these fish pathogens do not cause a 
problem in the stream. At times, however, the test conditions during

[[Page 49802]]

WET tests (e.g., 24 hour durations between sample renewals, beakers 
used for seven days without change, or uneaten brine shrimp) may 
promote bacterial growth. Some opportunistic bacteria take advantage of 
these conditions and flourish or ``bloom.'' The bacteria that bloom may 
be harmless or they may be fish pathogens. Blooms may even differ 
between replicates. In some cases, the presence of uncontrolled 
pathogen and/or parasite effects in the WET test may suggest the 
selection of a different test species.
    Stakeholders have identified particular concerns with the adverse 
effect of pathogens on the performance of the Fathead Minnow Larval 
Survival and Growth Test. A typical indication that pathogen 
interference has occurred in a WET test is when test organisms exhibit 
``sporadic mortality.'' This sporadic mortality phenomenon is 
characterized by an unexpected concentration-response relationship 
(i.e., effects that do not increase with increasing effluent 
concentration) and fathead minnow survival that varies greatly among 
replicates and among effluent dilutions. The observed sporadic 
mortality among replicates tends to occur in receiving water controls 
and in lower effluent concentrations (or occasionally in the full-
strength effluent samples) on day three or day four of the Fathead 
Minnow Larval Survival and Growth Test. EPA does not have evidence of 
such sporadic mortality occurring in concurrently conducted chronic 
tests using the cladoceran, Ceriodaphnia dubia, or concurrent acute 
tests with the fathead minnow, C. dubia, or other acute test species.
    When sporadic mortality is observed, often a fungal growth occurs 
directly on the fish, especially in the gill area. This growth 
interferes with measuring toxicity in the WET test. Biological test 
interference due to this type of fungal growth may occur during the 
toxicity test when effluents and water samples tested are derived from 
the receiving water (i.e., their source is a receiving water intake) or 
when the receiving water is used as the diluent. The fungal growth has 
been attributed to Saprolegnia sp. (Downey et al., 2000) which may be a 
secondary infection following infection from a known fish pathogen. 
Microbiological evaluations on receiving waters, the fish, and their 
food indicated the ubiquitous nature of pathogenic organisms (e.g., 
Flexibacter spp., Aeromonas hydrophila). Eradicating these types of 
organisms from the test through the decontamination of the fish and 
their food has not been practical (Geis et al., 2000a).
    Data from the WET test must be reviewed carefully to ascertain if 
pathogens are suspected. The key indicators that pathogen interference 
has occurred are the presence of an unexpected concentration-response 
relationship (i.e., effects that do not increase with increasing 
effluent concentration), and organism survival that varies greatly 
among replicates and among effluent dilutions. The analyst should 
evaluate the test data to determine a cause for any unexpected 
concentration-response pattern and subsequently to determine the 
validity of calculated results (USEPA, 2000a). Normal, reversed, or 
bimodal concentration-response relationships are not considered 
indicators of test interference by pathogenic bacteria (USEPA, 2000a). 
The analyst also should evaluate the responses at each test 
concentration for unusually high mortality and/or for unevenness of 
mortalities among replicates. If the within-treatment coefficient of 
variation (CVs) for survival in an effluent treatment is greater than 
40% and relatively low for control replicates in standard synthetic 
water, pathogen interference should be considered. Following data 
evaluations, additional testing would be required to ascertain that 
sporadic mortality observed in the WET test is due to interference by 
pathogenic bacteria. Parallel tests should be conducted using 
reconstituted water and receiving water as diluents with the effluent.
    Before modifying any test procedures that will allow the analyst to 
account for pathogen interference, all available options within the 
flexibility of the method should be exhausted. Samples should be 
filtered through a 2-4 mm mesh opening (as described in Subsection 
8.8.2 of the freshwater chronic method manual (USEPA, 1994a)) to remove 
indigenous organisms. Tests should be conducted using separate 
glassware, pipettes, and siphons for each concentration to minimize 
cross contaminating replicates of all treatments. The analyst also must 
keep laboratory equipment clean and dry when not in use. Use of 
reconstituted laboratory waters instead of receiving waters may 
eliminate the interference, and the use of reconstituted water would be 
preferable to invalid tests. However, for those instances when 
receiving water is required as the diluent or when the effluent and the 
subsequent dilutions exhibit the interference, EPA recommends modifying 
the test design to prevent the spread of the pathogen among the test 
chambers during the test.
    Once pathogenic test interference has been confirmed by additional 
testing, the proposed modifications to the Fathead Minnow Larval 
Survival and Growth Test would recommend use of an altered test design 
to minimize the effects of the pathogenic interference. The use of 
fewer fish per test chamber and new test chambers daily has been the 
most effective technique for controlling the effects of pathogenic 
bacteria in the Fathead Minnow Larval Survival and Growth Test. Use of 
small plastic 30-ml cups containing two fish per cup showed the 
greatest improvement to the test method, removing the pathogenic effect 
91% of the time (Geis et al., 2000a). For instance, use of 20 ml of 
test solution in a 1 ounce plastic cup and two fish per beaker 
significantly reduced the sporadic mortality not attributed to the 
effluent toxicity. The total number of fish tested is not reduced 
(i.e., 40 per treatment), and the fish are combined at the end of the 
test into the typical number of replicates so that data analysis 
following the test method manuals is unchanged.
    When parallel testing has confirmed pathogen interference and the 
modifications to the test design for the number of fish per chamber 
does not reduce the pathogen interference, the regulatory authority may 
allow modifications of the effluent samples to remove or inactivate the 
pathogens. The analyst should apply TIE filtration steps (USEPA, 1991a; 
USEPA, 1992) in combination with various sterilization techniques 
listed below to ascertain and control adverse influences on tests 
caused by pathogens in the intake or receiving waters used for 
dilution. For some samples, one or more techniques such as irradiation 
with ultraviolet light, pasteurization, filtration (0.2  m 
pore size), and addition of antibiotics has been shown to improve 
survival and reduce variability among replicates effectively (SETAC, 
1999). EPA cautions that some treatment methods that might control 
pathogens in the test, (e.g., ultraviolet light treatment or the 
addition of antibiotics (Downey et al., 2000)) may also improperly 
reduce or increase the toxicity of the sample. Filtration also may 
remove some toxicity in the sample as shown in toxicity identification 
evaluations (USEPA, 1991a; 1992; 1993a). The use of ultrafiltration on 
an effluent sample containing particulate matter to which process-
induced metals have adsorbed may improperly remove a significant source 
of process-related toxicity. Also, chlorination and dechlorination may 
be

[[Page 49803]]

a treatment option where pathogenic bacteria are suspected as the sole 
source of toxicity in the ambient intake waters. However, when the 
analyst prepares samples using techniques of chlorination and/or 
dechlorination, potential exists for oxidation and reduction of other 
compounds (USEPA, 1991a; 1992). All toxicity tests conducted on 
modified samples (e.g., sterilized) must include an additional blank 
preparation (control) consisting of similarly treated reconsituted 
laboratory water (USEPA, 1991a; 1992).
    Procedures to control the adverse influences of pathogens must not 
be used to reduce process-related sources of toxicity. With effluents 
and ambient waters, the pathogen(s) may mask the presence of a chemical 
that is, by itself, toxic. It is also possible that the pathogen 
infection is induced by some predisposing factor in the receiving water 
and would not occur without that factor. The need to evaluate both 
intake water and effluent samples to determine the cause of the 
pathogen or the source of pathogens is essential before applying any 
pathogen/parasite control technology and cannot be overemphasized. The 
analyst must evaluate whether the intake water is contributing the 
interference observed in the toxicity test of the final effluent.
    The method modifications proposed today provide techniques to 
assess and control the effects of pathogens in the Fathead Minnow 
Larval Survival and Growth Test. Today's proposal does not address, 
however, the determination as to the conditions under which this 
control is appropriate for purposes of NPDES permit compliance. By 
today's proposal, EPA does not concede that the discharge of toxic 
biological agents to waters of the US is appropriate or authorized but 
merely that pathogens in test samples may confound measurement of whole 
effluent toxicity.

C. Ratification or Withdrawal of Methods

    In a 1998 settlement agreement with Edison Electric Institute et 
al. (Edison Electric Institute, et al. v. EPA, No. 96-1062 & 
consolidated case (D.C. Cir.), Settlement Agreement, July 24, 1998), 
EPA agreed to conduct an interlaboratory variability study of 12 of the 
17 approved WET test methods (the WET Variability Study). The 12 
methods evaluated in the study (Table 1) represent a combination of 
acute and chronic test methods; freshwater and marine test methods; and 
invertebrate, fish, and algal species. EPA conducted the WET 
Variability Study in 1999 through 2000, and published preliminary 
results from the study in October 2000 (USEPA, 2000b; USEPA, 2000c). In 
2001, EPA submitted the preliminary results of the study for expert 
peer review (USEPA, 2001c). The peer review comments and EPA's response 
to those comments are included in the record established for this 
rulemaking (see Addresses section of this rule). Based on peer review 
comments, EPA revised the preliminary study report to produce a final 
study report. In conjunction with today's action, EPA is publishing a 
final study report (USEPA, 2001a; USEPA, 2001b) that presents the final 
results of EPA's WET Variability Study. These results are discussed in 
section III.C.1 below.
    The settlement agreement (Edison Electric Institute, et al. v. EPA, 
Settlement Agreement, July 24, 1998) also required that EPA propose to 
ratify or withdraw each of the 12 WET test methods evaluated in the WET 
Variability Study. Based on the results of the WET Variability Study, 
consideration of peer review comments, and an overall evaluation of the 
WET program, EPA proposes to ratify 11 of the methods evaluated in the 
WET Variability Study. EPA proposes to ratify nine of these methods, in 
an amended form, as described in Section III.B of this rule. EPA 
proposes to ratify two other methods (the Selenastrum capricornutum 
Growth Test and the Mysidopsis bahia Survival, Growth and Fecundity 
Test) with additional modifications (i.e., in addition those described 
in Section III.B of this rule) to improve the performance of the 
methods. EPA proposes to withdraw and propose a new Holmesimysis 
costata Acute Test method. The Holmesimysis costata Acute Test method 
was promulgated and tested in the WET Variability Study using acute 
test procedures designed for the Mysidopsis bahia Acute Test (except at 
a temperature of 12 deg.C, instead of 20 deg.C or 25 deg.C; and a 
salinity of 32-34, instead of 5-30). Results of the 
WET Variability Study revealed that acute test procedures designed for 
Mysidopsis bahia were insufficient for successful test conduct using 
Holmesimysis costata. For this reason, EPA proposes to withdraw 
Holmesimysis costata as an acceptable species for use in the Mysidopsis 
bahia Acute Test method and to propose it as an acute toxicity test 
method designed specifically for Holmesimysis costata. Sections 2-7 
below discuss the proposed ratification and/or withdrawal of each 
method evaluated in the WET Variability Study.

  Table 1.--Whole effluent toxicity test methods included in EPA's WET
                            Variability Study
------------------------------------------------------------------------
                                                             Test method
           Test method              Common test method name     No. a
------------------------------------------------------------------------
Cladoceran, Ceriodaphnia dubia,    Ceriodaphnia-- dubia      ...........
 Acute Test.                        Acute Test.
Cladoceran, Ceriodaphnia dubia,    Ceriodaphnia dubia             1002.0
 Survival and Reproduction Test.    Survival and
                                    Reproduction Test.
Fathead Minnow, Pimephales         Fathead Minnow Acute      ...........
 promelas, Acute Test.              Test.
Fathead Minnow, Pimephales         Fathead Minnow Larval          1000.0
 promelas, Larval Survival and      Survival and Growth
 Growth Test.                       Test.
Green Alga, Selenastrum            Selenastrum                    1003.0
 capricornutum, Growth Test.        capricornutum Growth
                                    Test.
Mysid, Mysidopsis bahia,           Mysidopsis bahia               1007.0
 Survival, Growth, and Fecundity    Survival, Growth, and
 Test.                              Fecundity Test.
Sheepshead Minnow, Cyprinodon      Sheepshead Minnow Acute   ...........
 variegatus, Acute Test.            Test.
Sheepshead Minnow, Cyprinodon      Sheepshead Minnow Larval       1004.0
 variegatus, Larval Survival and    Survival and Growth
 Growth Test.                       Test.
Inland Silverside, Menidia         Inland Silverside Acute   ...........
 beryllina, Acute Test.             Test.
Inland Silverside, Menidia         Inland Silverside Larval       1006.0
 beryllina, Larval Survival and     Survival and Growth
 Growth Test.                       Test.
Red Macroalga, Champia parvula,    Champia parvula                1009.0
 Reproduction Test b.               Reproduction Test.
Mysid, Holmesimysis costata,       Holmesimysis costata      ...........
 Acute Test b c.                    Acute Test.
------------------------------------------------------------------------
a Test method numbers were not designated for acute test methods in
  USEPA, 1993b.
b Due to insufficient laboratory support, interlaboratory data were not
  obtained for this method.
c The EPA-approved acute test with Holmesimysis costata was performed
  using the test conditions for the Mysidopsis bahia Acute Test method
  (except at a temperature of 12 deg.C, instead of 20 deg.C or 25 deg.C;
  and a salinity of 32-34, instead of 5-30).


[[Page 49804]]

    In ratifying WET test methods, EPA reaffirms the conclusion 
expressed in the 1995 WET final rule (60 FR 53529; October 16, 1995), 
that these methods are applicable for use in NPDES permits. In the 1995 
WET final rule, this conclusion was based on the well-established use 
of the methods, the existence of extensive guidance on quality 
assurance and routine quality control activities, and validation data 
from a number of studies conducted by EPA, State programs, and 
universities. Since promulgation of the methods, this basis for 
approval has been strengthened by more widespread use of the methods, 
additional guidance on quality assurance and quality control issues 
(USEPA, 2000a; USEPA, 2000d), and the WET Variability Study to confirm 
method performance data from original validation studies (USEPA, 2001a; 
USEPA, 2001b).
1. WET Variability Study
    EPA designed the WET Variability Study to characterize 
interlaboratory variability, the rate of successful test completion, 
and the rate of ``false positive'' incidence (i.e., the measurement of 
toxicity in non-toxic blank samples) for the 12 test methods listed in 
Table 1. For two of these methods (the Champia parvula Reproduction 
Test and the Holmesimysis costata Acute Test), EPA was unable to obtain 
interlaboratory data due to laboratory unavailability (i.e., EPA was 
unable to contract with a minimum of six laboratories qualified and 
willing to conduct these test methods within the time frame of the 
study). Intralaboratory data were obtained for the Champia parvula 
Reproduction Test, but no valid intralaboratory or interlaboratory data 
were obtained for the Holmesimysis costata acute test. For each of the 
remaining 10 methods, 7 to 35 laboratories participated in multi-
laboratory testing of 3 or 4 ``blind'' test samples. Laboratories 
received some combination of the following test sample types: reagent 
water (or ``blank''); reference toxicant; municipal or industrial 
effluent; and receiving water. Participant laboratories were required 
to analyze each blind test sample according to the promulgated WET test 
method manuals and specific instructions in participant laboratory 
standard operating procedures developed for the study (appendix B, 
USEPA, 2001b). In total, the study generated interlaboratory precision 
data from testing more than 700 blind samples among 55 participant 
laboratories. EPA had not previously conducted a study of this 
magnitude with these objectives in this time frame.
    The results of the WET Variability Study (Table 2) supported the 
conclusions of the 1995 WET final rule and confirmed the acceptability 
of the WET test methods for use in NPDES permits, except as noted below 
in sections 2 through 7. The analysis of successful test completion 
rates revealed that most WET test methods could be consistently and 
reliably performed by qualified testing laboratories. For the purposes 
of the study, EPA defined successful test completion rates to be the 
percentage of initiated and properly terminated tests that met the test 
acceptability criteria as specified in the WET method manuals. 
Successful test completion rates were above 90% for 8 of the 10 methods 
evaluated during interlaboratory testing. Only the Ceriodaphnia dubia 
Survival and Reproduction Test method (see section 2 below) and the 
Selenastrum capricornutum Growth Test method (see section 5 below) 
produced successful test completion rates less than 90%.
    The analysis of false positive rates revealed that the WET test 
methodologies, including applicable guidance on reviewing WET test 
results (USEPA, 2000a), effectively control the incidence of falsely 
identifying toxicity in non-toxic ``blank'' samples. False positive 
rates were defined as the percentage of valid tests conducted on blank 
samples that indicated toxicity by producing LC50 (median lethal 
concentration), NOEC (no observed effect concentration), or IC25 (25% 
inhibition concentration) values of less than 100% sample. False 
positive results were reported for three test methods, and the rates of 
false positives were below the theoretical false positive rate of 5% 
(based on the recommended 0.05 alpha level for hypothesis testing) for 
all but the Selenastrum capricornutum Growth Test conducted without 
EDTA.
    The analysis of interlaboratory precision data revealed that the 
WET test methods are sufficiently precise for use in NPDES permits. 
Interlaboratory coefficients of variation (CVs) calculated in the WET 
Variability Study ranged from 10.5% to 58.5% (Table 2). This observed 
range of interlaboratory variability is consistent with the range of 
variability reported for chemical methods approved at 40 CFR part 136 
(USEPA, 1991b). For chemical methods measuring metals at the low end of 
the detection range, interlaboratory CVs range from 18% to 129%, with a 
median CV of 45%. Interlaboratory CVs for chemical methods for organic 
analyses range from greater than 12% to 91%, and interlaboratory CVs 
for nonmetal inorganic analyses range from 4.6% to 70%.

   Table 2.--Summary of Test Results From EPA's WET Variability Study
------------------------------------------------------------------------
                                Successful
                                   test        False     Interlaboratory
         Test method            completion    positive    precision  (%
                                rate  (%)    ratea (%)        CV) b
------------------------------------------------------------------------
Ceriodaphnia dubia Acute Test         95.2         0.00           29.0
Ceriodaphnia dubia Survival           82.0         3.70           35.0
 and Reproduction Test.......
Fathead Minnow Acute Test....          100         0.00           20.0
Fathead Minnow Larval                 98.0         4.35           20.9
 Survival and Growth Test....
Selenastrum capricornutum             63.6         0.00           34.3
 Growth Test (with EDTA) c...
Selenastrum capricornutum             65.9         33.3           58.5
 Growth Test (without EDTA) c
Mysidopsis bahia Survival,          d 97.7         0.00           41.3
 Growth, and Fecundity Test..
Sheepshead Minnow Acute Test.          100         0.00           26.0
Sheepshead Minnow Larval               100         0.00           10.5
 Survival and Growth Test....
Inland Silverside Acute Test.         94.4         0.00           38.5
Inland Silverside Larval               100         0.00           43.8
 Survival and Growth Test....
Champia parvula Reproduction            ND           ND           f ND
 Test e......................
Holmesimysis costata Acutee..           ND           ND            ND
------------------------------------------------------------------------
a False positive rates reported for each method represent the higher of
  false positive rates observed for hypothesis testing or point estimate
  endpoints.

[[Page 49805]]

 
b Coefficients of variation (CVs) reported for each method represent the
  CV of LC50 values for acute test methods and IC25 values for chronic
  test methods. CVs reported are based on total interlaboratory
  variability (including within-laboratory and between-laboratory
  components of variability) and averaged across sample types.
c The Selenastrum capricornutum Growth Test method was conducted with
  and without ethylenediaminetetraacetic acid (EDTA) as a component of
  the nutrients added to test and control treatments. Due to improved
  test performance with the addition of EDTA, EPA is proposing to
  recommend the addition of EDTA in the Selenastrum capricornutum Growth
  Test.
d Successful test completion for the optional fecundity endpoint was
  50%.
e ND = not determined. Due to insufficient laboratory support,
  interlaboratory data were not obtained for the Champia parvula
  Reproduction Test method and the Holmesimysis costata Acute Test
  method.
f While interlaboratory test data were not obtained for the Champia
  parvula Reproduction Test method, intralaboratory data was obtained
  from the referee laboratory. Intralaboratory CVs were 27.6%, 49.7%,
  and 50.0% for reference toxicant, receiving water, and effluent sample
  types, respectively.

2. Ceriodaphnia dubia Acute Test, Ceriodaphnia dubia Survival and 
Reproduction Test, Fathead Minnow Acute Test, Fathead Minnow Larval 
Survival and Growth Test, Sheepshead Minnow Acute Test, Sheepshead 
Minnow Larval Survival and Growth Test, and Inland Silverside Acute 
Test
    Today, EPA proposes to ratify its previous rulemaking standardizing 
the following WET test methods: Ceriodaphnia dubia Acute Test, 
Ceriodaphnia dubia Survival and Reproduction Test, Fathead Minnow Acute 
Test, Fathead Minnow Larval Survival and Growth Test, Sheepshead Minnow 
Acute Test, Sheepshead Minnow Larval Survival and Growth Test, and the 
Inland Silverside Acute Test. At the time of method promulgation, 
interlaboratory precision data were available for each of these test 
methods. Based on these precision data, EPA concluded that toxicity 
tests are no more variable than chemical analytical methods in 40 CFR 
part 136, and that toxicity tests provide reliable indicators of whole 
effluent toxicity. At that time, EPA also anticipated that laboratory 
performance would improve with use of the methods over time. Results 
from the WET Variability Study not only confirmed the level of 
precision previously cited for these methods, but indicated that the 
methods currently exhibit even lower variability than estimated at the 
time of method promulgation (60 FR 53529; October 16, 1995). Such data 
also confirm EPA's assumptions regarding the likely improvement in 
laboratory performance over time. The average of interlaboratory CVs 
reported (in the WET method manuals and/or the Technical Support 
Document for Water Quality-based Toxics Control (USEPA, 1991b)) for 
each method at the time of promulgation ranged from 34% to 44.2% (Table 
3). Interlaboratory CVs reported for these methods in the WET 
Variability Study ranged from 10.5% to 38.5%. For each method, 
interlaboratory variability measured in the WET Variability Study was 
lower than that cited at the time of promulgation (Table 3). 
Interlaboratory CVs measured in the WET Variability Study were 4% to 
34% lower than average values cited in the method manuals for the same 
methods. On average, interlaboratory variability measured in the WET 
Variability Study was 15% lower than originally reported at the time of 
method promulgation. These results strongly confirm EPA's conclusions 
that these methods provide sufficient precision for use in NPDES 
permits.

   Table 3.--Comparison of Interlaboratory Method Precision at the Time of Method Promulgation and Measured in
                                           EPA's WET Variability Study
----------------------------------------------------------------------------------------------------------------
                                              Interlaboratory
                                                 precision         Updated
                                              estimates (%CV)  interlaboratory
                   Method                        at time of       precision            Improved precision?
                                                   method      estimates (%CV)
                                                promulgation         \b\
----------------------------------------------------\a\---------------------------------------------------------
Ceriodaphnia dubia Acute Test...............           44.2             29.0    Yes
Ceriodaphnia dubia Survival and Reproduction             42             35.0    Yes
 Test.
Fathead Minnow Acute Test...................             35             20.0    Yes
Fathead Minnow Larval Survival and Growth                34             20.9    Yes
 Test.
Selenastrum capricornutum Growth Test.......         \c\ NR         \d\ 34.3    NA \e\
Mysidopsis bahia Survival, Growth, and               \c\ NR             41.3    NA \e\
 Fecundity Test.
Sheepshead Minnow Acute Test................             42             26.0    Yes
Sheepshead Minnow Larval Survival and Growth           44.2             10.5    Yes
 Test.
Inland Silverside Acute Test................           42.2             38.5    Yes
Inland Silverside Larval Survival and Growth         \c\ NR             43.8    NA \e\
 Test.
Champia parvula Reproduction Test...........         \c\ NR           \c\ NR    NA \f\
Holmesimysis costata Acute Test.............         \c\ NR           \c\ NR    NA \f\
----------------------------------------------------------------------------------------------------------------
\a\ Precision estimates represent an average of all interlaboratory CVs reported for a given method in the WET
  method manuals (USEPA, 1993b; USEPA, 1994a; USEPA, 1994b) and/or the Technical Support Document for Water
  Quality-based Toxics Control (USEPA, 1991b). The number of significant figures displayed differs because these
  data are obtained from various sources, which reported results to either two or three significant figures.
\b\ Precision estimates were obtained from EPA's WET Variability Study conducted in 1999-2000 (USEPA, 2001a).
\c\ NR = None reported.
\d\ Precision estimates for the Selenastrum capricornutum Growth Test method are based on conduct of the test
  with Ethylenediaminetetraacetic acid (EDTA) as a component of the nutrients added to test and control
  treatments.
\e\ NA = not applicable. Improved precision could not be determined because estimates of interlaboratory
  precision were not reported at the time of method promulgation.
\f\ NA = not applicable. Improved precision could not be determined because estimates of interlaboratory
  precision were not reported at the time of method promulgation or determined in the WET Variability Study.


[[Page 49806]]

    Other test performance characteristics measured in the WET 
Variability Study also confirmed EPA's conclusions that these methods 
are applicable for use in NDPES permits. False positive rates for these 
methods were below the theoretical false positive rate of 5% (based on 
the recommended 0.05 alpha level for hypothesis testing), indicating 
that the methods do not routinely indicate toxicity in non-toxic 
samples. Successful test completion rates for these methods were also 
at acceptable levels (82.0% to 100%), with 6 of these 7 methods 
exhibiting successful test completion rates above 90%. While the 82.0% 
successful test completion rate for the Ceriodaphnia dubia Survival and 
Reproduction Test method was lower than for most other methods 
evaluated in the WET Variability Study, this rate is consistent with 
successful test completion rate information available for this method 
at the time of promulgation. The 82.0% successful test completion rate 
observed in the WET Variability Study is consistent with the 80% rate 
reported for this method in a 1989 interlaboratory study (USEPA, 1991b) 
and represents tremendous improvement from a 1987 interlaboratory study 
that reported a successful test completion rate of 56% (DeGraeve et 
al., 1992).
    The overall successful test completion rate observed for the 
Ceriodaphnia dubia Survival and Reproduction Test method in the WET 
Variability Study was also suppressed by poor performance in a subset 
of laboratories. Only 10 of the 34 participant laboratories performed 
invalid tests, but 8 of these laboratories performed invalid tests on 
50% or more of the samples tested. The low rate of successful test 
completion in these 8 laboratories may have been influenced by the 
study's strict testing schedule, which required each test to be 
conducted on a given day and all tests to be conducted within a 15-day 
time period. When invalid tests conducted in a given laboratory were 
due to marginal or poor health of the test organism cultures, then it 
was logical that the laboratory would fail a high percentage of tests 
during this study because culture health was unlikely to fully recover 
within 15 days. EPA believes that successful test completion rates for 
this method improve when testing laboratories are allowed flexibility 
in the timing of sample collection and can avoid initiating tests 
during periods of marginal to poor culture health.
3. Inland Silverside Larval Survival and Growth Test
    EPA proposes to ratify the Inland Silverside Larval Survival and 
Growth Test method. Similarly to the methods listed in section 2 above, 
the Inland Silverside Larval Survival and Growth Test method exhibited 
acceptable successful test completion rates and false positive rates 
(Table 2). No false positives were observed for the method in the WET 
Variability Study, and the successful test completion rate was 100%. 
Unlike the methods listed in section 2 above, however, EPA cannot 
compare interlaboratory precision data cited at the time of method 
promulgation and data reported from the WET Variability Study because 
EPA did not rely on interlaboratory precision data for this method at 
the time of promulgation (Table 3). Instead, EPA relied on 
intralaboratory data for the method. The Agency's previous experience 
with method variability evaluations supported EPA's assumption that, 
though WET tests typically have lower CVs (higher precision) in 
intralaboratory studies than in interlaboratory studies, acceptable 
ranges of precision demonstrated in intralaboratory studies tend to 
subsequently be confirmed by interlaboratory studies.
    In the WET Variability Study, an interlaboratory CV of 43.8% was 
reported for the Inland Silverside Larval Survival and Growth Test 
method. While interlaboratory variability for this method is higher 
than for other methods reported in the study, it is within the range of 
interlaboratory CVs (34% to 44.2%) cited for other WET methods at the 
time of promulgation (Table 3). It is also within the range of 
interlaboratory CVs reported for chemical methods approved at 40 CFR 
part 136 (USEPA, 1991b). Therefore, EPA reaffirms the conclusions that 
this method is no more variable than chemical analytical methods 
approved at 40 CFR part 136 and that this method is applicable for use 
in NPDES permits (60 FR 53529; October 16, 1995).
4. Champia parvula Reproduction Test
    In the WET Variability Study, insufficient participant laboratory 
support was available to conduct interlaboratory testing of the Champia 
parvula Reproduction Test method within the time frame of the study. In 
addition to the referee laboratory, only one laboratory submitted the 
necessary quality control information to prequalify for participation 
in the interlaboratory study of this method. Due to insufficient 
laboratory support and failure to meet the study's data quality 
objective of a minimum of six laboratories, EPA canceled 
interlaboratory testing of the Champia parvula Reproduction Test 
method. Though interlaboratory testing was canceled, the referee 
laboratory conducted single-laboratory testing of the Champia parvula 
Reproduction Test method. In the 1995 WET rule, EPA addressed the issue 
of limited laboratory availability for conduct of the Champia parvula 
Reproduction Test method. EPA predicted that as the requirements for 
use of this organism in the NPDES permit program increased, the 
resulting increase in market demand would result in an increase in the 
number of laboratories capable of performing this test. However, the 
number of permits requiring the Champia parvula Reproduction Test 
method has remained low (DeGraeve et al., 1998), so few laboratories 
have invested in developing Champia parvula cultures or standard 
operating procedures for conduct of the method.
    EPA believes that the limited use of the Champia parvula 
Reproduction Test method does not reduce the value of the test method. 
The Champia parvula Reproduction Test represents the only approved test 
method for a marine plant species. Maintaining an approved test method 
for this functional group (marine/plant/chronic test) is important for 
proper implementation of the WET program. The Technical Support 
Document for Water Quality-Based Toxics Control (USEPA, 1991b) 
recommends the use of at least three marine species representing three 
different phyla (e.g., a fish, an invertebrate, and a plant) for 
testing the toxicity of effluents discharged to estuarine and marine 
environments.
    The limited use of the Champia parvula Reproduction Test method 
also does not affect the performance of the test method in laboratories 
that are qualified to conduct the test. While the WET Variability Study 
did not provide interlaboratory precision data for the Champia parvula 
Reproduction Test method, referee laboratory data confirmed the 
estimates of intralaboratory precision cited at the time of method 
promulgation (USEPA, 1994b). Intralaboratory CVs cited in the method 
manual for Champia parvula Reproduction Tests conducted using copper 
sulfate and sodium dodecyl sulfate averaged 63%. In preliminary testing 
for the WET Variability Study, the referee laboratory achieved an 
intralaboratory CV of 27.6% for 3 reference toxicant tests using copper 
sulfate, and an intralaboratory CV of 49.7% for 4 tests of spiked 
receiving water. Only one pair of replicate

[[Page 49807]]

effluent samples was tested using the Champia parvula Reproduction Test 
method. Tests of these duplicate effluent samples yielded a CV of 
50.0%. All other testing of the effluent sample type was conducted on 
samples from different sampling dates, so additional precision 
measurements were not obtained for this sample type. In addition to 
intralaboratory test data from the WET Variability Study, EPA's 
Variability Guidance Document (USEPA, 2000d) reported an 
intralaboratory CV of 59% for the Champia parvula Reproduction Test 
based on 23 reference toxicant tests conducted in 2 laboratories. 
Intralaboratory data from both the WET Variability Study and the 
Variability Guidance Document support the intralaboratory precision 
data previously cited in the method manual (USEPA, 1994b) for the 
Champia parvula Reproduction Test method. Based on the confirmation of 
intralaboratory precision data cited at the time of method 
promulgation, EPA proposes to ratify the Champia parvula Reproduction 
Test method.
5. Mysidopsis bahia Survival, Growth, and Fecundity Test
    The Mysidopsis bahia Survival, Growth, and Fecundity Test uses 
three test endpoints to evaluate toxicity: survival, growth, and 
fecundity (or reproduction). The survival and growth endpoints are 
required endpoints and specific test acceptability criteria for these 
endpoints must be met (80% survival and mean weight of 0.20 mg in the 
control treatment) to produce a valid test. The fecundity endpoint is 
optional and may be used if the test acceptability criterion for 
fecundity (egg production by 50% or more of control females) is met. 
Failure to meet the test acceptability criterion for fecundity does not 
invalidate a test but means that the fecundity endpoint may not be used 
in calculating test results. In the WET Variability Study, 97.7% of 
tests met the required test acceptability criteria for survival and 
growth, but only 50% of tests met the test acceptability criterion for 
fecundity. While failure to generate fecundity data does not invalidate 
a test, it may affect the sensitivity of the measurement. Researchers 
have shown that the fecundity endpoint is often the most sensitive 
endpoint and that the test most effectively estimates the chronic 
toxicity of effluents when all three endpoints are used (Lussier et 
al., 1999).
    EPA proposes to ratify the Mysidopsis bahia Survival, Growth, and 
Fecundity Test method with an additional modification to improve the 
performance of the method. EPA proposes to add guidance to improve the 
success of obtaining fecundity data. The specifics of the proposed 
method manual changes to implement this modification are detailed in 
the document titled, Proposed Changes to Whole Effluent Toxicity Method 
Manuals (USEPA, 2001d). The additional guidance would recommend 
optimizing temperature, feeding, and organism densities during the 
seven-day pre-test holding period and during the testing period. These 
factors are critical to the success of the fecundity endpoint, because 
they control the rate of mysid development and maturation. While these 
factors are typically controlled during the testing period, equal 
attention should be paid to these factors during the pre-test holding 
period to ensure maximum mysid development. Lussier et al. (1999) found 
that by increasing holding temperature and test temperature from 
26 deg.C  1 deg.C to 26 deg.C-27 deg.C and maintaining 
holding densities at 10 organisms/L, the percentage of tests 
meeting the test acceptability criteria for fecundity increased from 
60% to 97%.
    With the exception of the low successful test completion rate for 
the fecundity endpoint, other test method performance measures 
evaluated in the WET Variability Study for the Mysidopsis bahia 
Survival, Growth, and Fecundity Test were acceptable. No false 
positives were observed for the method, the successful test completion 
rate was 97.7% for the survival and growth endpoints, and 
interlaboratory variability (%CV) was 41.3% for the growth IC25 
endpoint (Table 2). No interlaboratory precision data were reported for 
the Mysidopsis bahia Survival, Growth, and Fecundity Test method at the 
time of method promulgation; therefore interlaboratory precision data 
from the WET Variability Study could not be compared to previously 
cited values for this method (Table 3). While interlaboratory 
variability for this method is higher than for most other methods 
reported in the study, it is within the range of interlaboratory CVs 
(34% to 44.2%) cited for other WET methods at the time of promulgation 
(Table 3). It is also within the range of interlaboratory CVs reported 
for chemical methods approved at 40 CFR part 136 (USEPA, 1991b). 
Therefore, EPA reaffirms the conclusions that this method is applicable 
for use in NPDES permits (60 FR 53529; October 16, 1995).
6. Selenastrum capricornutum Growth Test
    In the WET Variability Study, the Selenastrum capricornutum Growth 
Test method was conducted with and without the addition of 
ethylenediaminetetraacetic acid (EDTA). In the approved Selenastrum 
capricornutum Growth Test method, EDTA is an optional component of the 
nutrient mixture that is added to test and control treatments. While 
algal growth is enhanced by the addition of EDTA, the method recommends 
excluding EDTA from the nutrient mixture when testing samples that may 
contain metals. EDTA is a chelating agent that effectively binds 
metals, thereby potentially reducing the toxic effect of metals present 
in the analyzed sample. Because the presence of metals in WET samples 
is often unknown at the time of testing, laboratories often conduct the 
Selenastrum capricornutum Growth Test method without the addition of 
EDTA.
    Results from the WET Variability Study revealed that Selenastrum 
capricornutum Growth Test method performance was substantially better 
when EDTA was added to the nutrient mixture than when it was excluded. 
No false positives were observed when EDTA was used, but 2 of the 6 
blank samples (33.3%) analyzed without EDTA produced false positive 
results (USEPA, 2001a). Interlaboratory variability of the Selenastrum 
capricornutum Growth Test method was also much lower with EDTA (34.3%) 
than without EDTA (58.5%). When conducted with EDTA, the Selenastrum 
capricornutum Growth Test method exhibited interlaboratory precision 
similar to other chronic methods evaluated in the WET Variability 
Study. No interlaboratory precision data were reported for the 
Selenastrum capricornutum Growth Test method at the time of method 
promulgation, so interlaboratory precision data from the WET 
Variability Study could not be compared to previously cited values for 
this method. When compared to interlaboratory precision cited for other 
WET test methods at the time of promulgation, the Selenastrum 
capricornutum Growth Test method (conducted with EDTA) was well within 
the range (Table 3).
    The successful test completion rate of the Selenastrum 
capricornutum Growth Test method was low for tests conducted with and 
without EDTA (63.6% and 65.9%, respectively), however, the low 
successful test completion rates were in part due to laboratory 
inexperience in using both the with and without-EDTA techniques. Two 
laboratories that cultured organisms without EDTA and generally 
conducted tests without EDTA showed poor successful test completion 
rates

[[Page 49808]]

(failing eight of eight tests) when EDTA was used. These laboratories 
failed all eight tests conducted with EDTA and passed all but one test 
(seven) without EDTA. When these two laboratories were removed from the 
analysis, the successful test completion rate for tests conducted with 
EDTA increased to 77.8%.
    Based on WET Variability Study results, EPA proposes to ratify the 
Selenastrum capricornutum Growth Test method with a modification to 
recommend the addition of EDTA to the nutrient mixture added to control 
and test treatments. The specifics of the proposed method manual 
changes to implement this modification are detailed in the document 
titled, Proposed Changes to Whole Effluent Toxicity Method Manuals 
(USEPA, 2001d). This method modification will improve overall test 
method performance by reducing false positives and increasing 
interlaboratory precision. EPA also believes that recommending the use 
of EDTA will improve successful test completion rates for the method as 
laboratories consistently culture and test with EDTA. In addition to 
improving test method performance, the method modification to recommend 
the use of EDTA is consistent with other established Selenastrum 
capricornutum toxicity testing protocols. Both ASTM (1992) and 
Environment Canada (1992) methods for toxicity testing using 
Selenastrum capricornutum recommend the use of EDTA.
    EPA recognizes that the proposed modification to the Selenastrum 
capricornutum Growth Test method may cause the method to underestimate 
the toxicity of metals. EPA believes, however, that this modification 
is necessary to ensure adequate performance of the Selenastrum 
capricornutum Growth Test method. EPA also believes that under 
appropriate implementation of the WET program, this modification will 
not significantly reduce environmental protection. The Technical 
Support Document for Water Quality-based Toxics Control (USEPA, 1991b) 
recommends that permitting decisions be based on testing using a 
minimum of three species representing three different phyla (e.g., a 
fish, invertebrate, and plant). This recommendation is based on the 
recognition that species differ in their sensitivity to toxicants. By 
using a battery of species to test the toxicity of an effluent, 
permitting decisions can be made to protect the most sensitive species 
tested. Using this approach, the addition of EDTA in the Selenastrum 
capricornutum Growth Test method would affect environmental protection 
only when Selenastrum capricornutum is determined to be the most 
sensitive species and when the effluent contains metals whose toxicity 
is reduced by the addition of EDTA. This situation should be 
infrequent, and result in only minor decreases in test sensitivity. 
Geis et al. (2000b) showed that Ceriodaphnia dubia was more sensitive 
than Selenastrum capricornutum to three of five metals tested (copper, 
nickel, and cadmium), and Selenastrum capricornutum was only slightly 
more sensitive than Ceriodaphnia dubia to zinc and lead.
7. Holmesimysis costata Acute Test
    Holmesimysis costata is a Pacific coast mysid species that was 
elevated from the supplemental species list in the previous acute 
method manual and added to the list of approved acute toxicity test 
species at the time of the WET final rule (60 FR 53529; October 16, 
1995). This species was added in response to comments that the 
recommended test species in the acute method manual did not include any 
invertebrate species indigenous to Pacific coastal waters. One 
commenter also submitted data showing that Holmesimysis costata was at 
least as sensitive to toxicants as the recommended acute toxicity test 
species. Based on these comments, the acute method manual was modified 
to add a footnote listing Holmesimysis costata as an acceptable species 
for use with the Mysidopsis bahia Acute Test procedures. The footnote 
to the table of test conditions for the Mysidopsis bahia Acute Test 
states that ``Holmesimysis costata can be used with the test conditions 
in this table, except at a temperature of 12 deg.C, instead of 20 deg.C 
or 25 deg.C, and a salinity of 32-34, instead of 
5-30, where it is the required test organism in 
discharge permits.'' Because the acute method manual was incorporated 
by reference in the final rule, the incorporation of this footnote 
established Holmesimysis costata as an approved acute toxicity test 
species. The WET final rule (60 FR 53529; October 16, 1995) clarified 
this by stating that ``EPA accepts the use of * * * Holmesimysis 
costata in place of Mysidopsis bahia, with the same test conditions 
(except at a temperature of 12 deg.C, instead of 20 deg.C or 25 deg.C, 
and a salinity of 32-34, instead of 5-
30).''
    EPA decided to evaluate the Holmesimysis costata Acute Test method 
in the WET Variability Study according to the protocol as the method 
was promulgated, i.e., using the test conditions for Mysidopsis bahia 
(except at a temperature of 12 deg.C, instead of 20 deg.C or 25 deg.C, 
and a salinity of 32 to 34, instead of 5 
to 30). Sufficient participant laboratory support, however, 
was not available to conduct interlaboratory testing of the 
Holmesimysis costata Acute Test method within the time frame of the 
study. In addition to the referee laboratory, only two laboratories 
submitted the necessary quality control information to prequalify for 
participation in the interlaboratory study of this method. This method 
is required only in NPDES permits issued in California, so few 
laboratories currently conduct this test routinely. Due to insufficient 
laboratory support and failure to meet the study's data quality 
objective of a minimum of six laboratories, EPA canceled 
interlaboratory testing of the Holmesimysis costata Acute Test method. 
Though interlaboratory testing was canceled, the referee laboratory did 
attempt to conduct single-laboratory testing of the Holmesimysis 
costata Acute Test.
    During the WET Variability Study, the referee laboratory initiated 
five Holmesimysis costata acute tests. The referee laboratory did not 
initiate additional tests due to difficulties in obtaining test 
organisms. Juvenile Holmesimysis costata used for testing are generally 
obtained from field-collected gravid females. The referee laboratory 
was unable to collect sufficient numbers of gravid females during most 
of the time frame for the WET Variability Study (September 1999 through 
April 2000). Of the five tests that were initiated, none successfully 
met test acceptability criteria and required test conditions. Three 
tests failed to meet test acceptability criteria for control survival, 
and two tests failed to meet requirements for the age of test organisms 
(all within 24 hours). These test failures demonstrated the inadequacy 
of Mysidopsis bahia Acute Test procedures for use in conducting acute 
tests with Holmesimysis costata. EPA has since concluded that modified 
test procedures are needed for successful conduct of the Holmesimysis 
costata Acute Test. These modifications include more detailed organism 
collection and holding procedures, specific dilution water 
requirements, revised temperature requirements, and less restrictive 
test organism age requirements.
    Today, EPA proposes to withdraw Holmesimysis costata as an 
acceptable species for use in the Mysidopsis bahia Acute Test method 
and proposes a separate Holmesimysis costata Acute Test method. This 
proposal would add

[[Page 49809]]

to the acute method manual a table of test conditions specific to 
Holmesimysis costata and information in Appendix A.3 on the morphology, 
taxonomy, collection, holding, culturing, feeding, and testing of 
Holmesimysis costata. The specifics of the proposed Holmesimysis 
costata Acute Test method and the method manual changes necessary to 
implement the addition of this method are detailed in the document 
titled, Proposed Changes to Whole Effluent Toxicity Method Manuals 
(USEPA, 2001d).
    The proposed Holmesimysis costata Acute Test method is based on 
method development data from the California Water Resources Control 
Board's Marine Bioassay Project (State Water Resources Control Board, 
1990) and from peer-reviewed literature (Martin et al., 1989; Hunt et 
al., 1997). These data show that given the appropriate test procedures 
and test conditions, the Holmesimysis costata Acute Test can produce 
reliable and sensitive toxicity results with adequate precision. 
Single-laboratory testing of zinc with the Holmesimysis costata Acute 
Test method yielded intralaboratory precision (CVs) of 19% and 23% in 
48-h and 96-h acute tests, respectively. Multi-laboratory testing of 
zinc with the Holmesimysis costata Acute Test method yielded 
interlaboratory precision (CVs) of 24% and 1% in 2 separate trials.
    In addition to the proposed Holmesimysis costata Acute Test method, 
EPA requests comment on the applicability of similar methods published 
by voluntary consensus standard bodies. A mysid toxicity test method 
with specific test procedures for Holmesimysis costata is published in 
Standard Methods for the Examination of Water and Wastewater (APHA et 
al., 1998), and a West Coast mysid toxicity test method is published by 
the American Society for Testing and Materials (ASTM, 1993). EPA does 
not believe that these methods from voluntary consensus standard bodies 
provide the detailed requirements necessary for routine use in 
compliance monitoring, so EPA is proposing a new Holmesimysis costata 
Acute Test method for inclusion in EPA's acute method manual (USEPA, 
1993b). EPA invites comment, however, on whether to approve the other 
organizations' testing procedures, including comment on their use for 
compliance monitoring.

IV. Regulatory Requirements

A. Executive Order 12866--Regulatory Planning and Review

    Under Executive Order 12866 (58 FR 51735 (October 4, 1993)), the 
Agency must determine whether a regulatory action is ``significant'' 
and therefore subject to Office of Management and Budget (OMB) review 
and the requirements of the Executive Order. The Executive Order 
defines ``significant regulatory action'' as one that is likely to 
result in a rule that may: (1) Have an annual effect on the economy of 
$100 million or more or adversely affect in a material way the economy, 
a sector of the economy, productivity, competition, jobs, the 
environment, public health or safety, or State, local, or tribal 
governments or communities; (2) create a serious inconsistency or 
otherwise interfere with an action taken or planned by another agency; 
(3) materially alter the budgetary impact of entitlements, grants, user 
fees, or loan programs or the rights and obligations of recipients 
thereof; or (4) raise novel legal or policy issues arising out of legal 
mandates, the President's priorities, or the principles set forth in 
the Executive Order.''
    Pursuant to the terms of Executive Order 12866, it has been 
determined that this rule is not a ``significant regulatory action.'' 
Therefore, this action is not subject to OMB review.

B. Unfunded Mandates Reform Act

    Title II of the Unfunded Mandates Reform Act of 1995 (UMRA), Public 
Law 104-4, establishes requirements for Federal agencies to assess the 
effects of their regulatory actions on State, local, and tribal 
governments and the private sector. Under section 202 of the UMRA, EPA 
generally must prepare a written statement, including a cost-benefit 
analysis, for proposed and final rules with ``Federal mandates'' that 
may result in expenditures to State, local, and tribal governments, in 
the aggregate, or to the private sector, of $100 million or more in any 
one year. Before promulgating an EPA rule for which a written statement 
is needed, section 205 of the UMRA generally requires EPA to identify 
and consider a reasonable number of regulatory alternatives and adopt 
the least costly, most cost-effective or least burdensome alternative 
that achieves the objectives of the rule. The provisions of section 205 
do not apply when they are inconsistent with applicable law. Moreover, 
section 205 allows EPA to adopt an alternative other than the least 
costly, most cost-effective or least burdensome alternative if the 
Administrator publishes with the final rule an explanation of why that 
alternative was not adopted. Before EPA establishes any regulatory 
requirements that may significantly or uniquely affect small 
governments, including tribal governments, it must have developed, 
under section 203 of the UMRA, a small government agency plan. The plan 
must provide for the notification of potentially affected small 
governments, enabling officials of affected small governments to have 
meaningful and timely input in the development of EPA regulatory 
proposals with significant Federal intergovernmental mandates, and 
informing, educating, and advising small governments on compliance with 
the regulatory requirements.
    EPA has determined that today's proposed rule does not contain a 
Federal mandate that may result in expenditures of $100 million or more 
for State, local, and tribal governments, in the aggregate, or the 
private sector in any one year. Today's rule proposes revisions to WET 
test methods that are currently approved for use in NPDES permits and 
certification of Federal licenses under the CWA. The revisions are 
minor and the cost to implement them is minimal. Thus, today's rule is 
not subject to the requirements of sections 202 and 205 of the UMRA.
    EPA has determined that this rule contains no regulatory 
requirements that might significantly or uniquely affect small 
governments. It would not significantly affect them because any 
incremental costs incurred are minimal, and it would not uniquely 
affect them because it would affect entities of all sizes required to 
test for whole effluent toxicity by a regulatory authority the same. 
Further, whole effluent toxicity monitoring by small entities is 
generally expected to be less frequent than such monitoring by larger 
entities. Therefore, today's rule is not subject to the requirements of 
section 203 of UMRA.

C. Regulatory Flexibility Act (RFA), as Amended by the Small Business 
Regulatory Enforcement Fairness Act of 1996 (SBREFA), 5 U.S.C. 601 et 
seq.

    The RFA generally requires an agency to prepare a regulatory 
flexibility analysis of any rule subject to notice and comment 
rulemaking requirements under the Administrative Procedure Act or any 
other stature unless the agency certifies that the rule will not have a 
significant economic impact on a substantial number of small entities. 
Small entities include small businesses, small organizations, and small 
governmental jurisdictions.
    For purposes of assessing the impacts of today's rule on small 
entities, small entity is defined as: (1) a small business as defined 
by the U.S. Small Business Administration definitions at 13 CFR 
121.201; (2) a small governmental jurisdiction that is a government of 
a city, county, town, school district or special district with a 
population of less

[[Page 49810]]

than 50,000; and (3) a small organization that is any not-for-profit 
enterprise which is independently owned and operated and is not 
dominant in its field.
    After considering the economic impacts of today's proposed rule on 
small entities, I certify that this action will not have a significant 
economic impact on a substantial number of small entities. Today's rule 
proposes revisions to WET test methods that are currently approved for 
use in NPDES permits and certification of Federal licenses under the 
CWA. The revisions are minor and the cost to implement them is minimal. 
The proposed revisions are intended to improve the performance of WET 
tests, and thus increase confidence in the reliability of the results 
obtained using the test methods. EPA estimates that any incremental 
costs associated with the proposed revisions would be alleviated by a 
potential reduction in retesting that may result from improved test 
performance and increased confidence in the reliability of testing 
results. We continue to be interested in the potential impacts of the 
proposed rule on small entities and welcome comments on issues related 
to such impacts.

D. Paperwork Reduction Act

    This action does not impose an information collection burden under 
the provisions of the Paperwork Reduction Act, 44 U.S.C. 3501 et seq. 
It does not contain any information, collection, reporting, or record 
keeping requirements.
    Burden means the total time, effort, or financial resources 
expended by persons to generate, maintain, retain, or disclose or 
provide information to or for a Federal agency. This includes the time 
needed to review instructions; develop, acquire, install, and utilize 
technology and systems for the purposes of collecting, validating, and 
verifying information, processing and maintaining information, and 
disclosing and providing information; adjust the existing ways to 
comply with any previously applicable instructions and requirements; 
train personnel to be able to respond to a collection of information; 
search data sources; complete and review the collection of information; 
and transmit or otherwise disclose the information.
    An Agency may not conduct or sponsor, and a person is not required 
to respond to a collection of information unless it displays a 
currently valid OMB control number. The OMB control numbers for EPA's 
regulations are listed in 40 CFR part 9 and 48 CFR chapter 15.

E. National Technology Transfer and Advancement Act

    Section 12(d) of the National Technology Transfer and Advancement 
Act of 1995 (NTTAA), Public Law 104-113, section 12(d) (15 U.S.C. 272 
note) directs EPA to use voluntary consensus standards in its 
regulatory activities unless to do so would be inconsistent with 
applicable law or otherwise impractical. Voluntary consensus standards 
are technical standards (e.g., materials specifications, test methods, 
sampling procedures, and business practices) that are developed or 
adopted by voluntary consensus standards bodies. The NTTAA directs EPA 
to provide Congress, through OMB, explanations when the Agency decides 
not to use available and applicable voluntary consensus standards.
    Today's action would revise existing EPA WET test methods and add a 
new Holmesimysis costata Acute Test method. For the methods that EPA is 
proposing to revise, the Agency did not conduct a search to identify 
potentially applicable voluntary consensus standards, because the 
revisions EPA proposes today would merely incorporate more specificity 
and detail into already approved EPA test methods. EPA invites comment, 
however, on the extent to which voluntary consensus standard 
organizations' methods would be consistent with the EPA methods for 
which revisions are proposed today. For the new Holmesimysis costata 
Acute Test method, the Agency reviewed applicable voluntary consensus 
standards and identified two mysid methods (ASTM, 1993; APHA et al., 
1998) that provide specific test procedures for use with Holmesimysis 
costata. While EPA requests comment on the applicability of these 
voluntary consensus standards, the Agency does not believe that these 
methods would provide the additional detailed requirements EPA proposes 
today. For this reason, EPA proposes a new EPA Holmesimysis costata 
Acute Test method. EPA welcomes comments on this aspect of the proposed 
rulemaking and, specifically, invites the public to identify 
potentially-applicable voluntary consensus standards and to explain why 
such standards should be used in this regulation.

F. Executive Order 13045--Protection of Children From Environmental 
Health Risks and Safety Risks

    Executive Order 13045 (62 FR 19885, April 23, 1997) applies to any 
rule that: (1) Is determined to be ``economically significant'' as 
defined under Executive Order 12866, and (2) concerns an environmental 
health or safety risk that EPA has reason to believe may have a 
disproportionate effect on children. If the regulatory action meets 
both criteria, the Agency must evaluate the environmental health or 
safety effects of the planned rule on children, and explain why the 
planned regulation is preferable to other potentially effective and 
reasonably feasible alternatives considered by the Agency. This rule is 
not subject to the Executive Order because it is not economically 
significant as defined in Executive Order 12866, nor does it concern an 
environmental health or safety risk that EPA has reason to believe may 
have a disproportionate effect on children.

G. Executive Order 13175--Consultation and Coordination With Indian 
Tribal Governments

    Executive Order 13175, entitled ``Consultation and Coordination 
with Indian Tribal Governments'' (65 FR 67249; November 6, 2000), 
requires EPA to develop an accountable process to ensure ``meaningful 
and timely input by tribal officials in the development of regulatory 
policies that have tribal implications.'' ``Policies that have tribal 
implications'' is defined in the Executive Order to include regulations 
that have ``substantial direct effects on one or more Indian tribes, on 
the relationship between the Federal government and the Indian tribes, 
or on the distribution of power and responsibilities between the 
Federal government and Indian tribes.''
    This proposed rule does not have tribal implications. It will not 
have substantial direct effects on tribal governments, on the 
relationship between the Federal government and Indian tribes, or on 
the distribution of power and responsibilities between the Federal 
government and Indian tribes, as defined in Executive Order 13175. 
Today's proposed rule would revise WET test methods that are currently 
approved for use in NPDES permits and certification of Federal licenses 
under the CWA. The revisions are minor and the cost to implement them 
is minimal. Thus, Executive Order 13175 does not apply to this rule. In 
the spirit of Executive Order 13175, and consistent with EPA policy to 
promote communications between EPA and tribal governments, EPA 
specifically solicits comment on this proposed rule from tribal 
officials.

H. Executive Order 13132--Federalism

    Executive Order 13132, entitled ``Federalism'' (64 FR 43255; August 
10, 1999), requires EPA to develop an

[[Page 49811]]

accountable process to ensure ``meaningful and timely input by State 
and local officials in the development of regulatory policies that have 
federalism implications.'' ``Policies that have federalism 
implications'' is defined in the Executive Order to include regulations 
that have ``substantial direct effects on the States, on the 
relationship between the national government and the States, or on the 
distribution of power and responsibilities among the various levels of 
government.''
    This proposed rule does not have federalism implications. It will 
not have substantial direct effects on the States, on the relationship 
between the national government and the States, or on the distribution 
of power and responsibilities among the various levels of government, 
as specified in Executive Order 13132. Today's rule proposes revisions 
to WET test methods that are currently approved for use in NPDES 
permits and certification of Federal licenses under the CWA. The 
revisions are minor and the cost to implement them is minimal. Thus, 
Executive Order 13132 does not apply to this rule. In the sprit of 
Executive Order 13132, and consistent with EPA policy to promote 
communications between EPA and State and local governments, EPA 
specifically solicits comment on this proposed rule from State and 
local officials.

I. Executive Order 13211--Energy Effects

    This rule is not subject to Executive Order 13211, ``Actions 
Concerning Regulations That Significantly Affect Energy Supply, 
Distribution, or Use'' (66 FR 28355 (May 22, 2001)) because it is not a 
significant regulatory action under Executive Order 12866.

J. Plain Language Directive

    Executive Order 12866 requires each agency to write all rules in 
plain language. We invite your comments on how to make this proposed 
rule easier to understand. For example, have we organized the material 
to suit your needs? Are the requirements in the rule clearly stated? 
Does the rule contain technical language or jargon that isn't clear? 
Would a different format (grouping and order of sections, use of 
headings, paragraphing) make the rule easier to understand? Would more 
(but shorter) sections be better? Could we improve clarity by adding 
tables, lists, or diagrams? What else could we do to make the rule 
easier to understand?

V. Request for Comments and Available Data

    EPA requests public comments on this proposed rule. EPA invites 
comment on the technical merit, applicability, and implementation of 
the specific WET test method changes included in this proposal. EPA 
also invites comments on the ratification of the methods listed. EPA 
encourages commenters to provide copies of supporting data and/or 
references cited in comments.
    EPA recognizes that stakeholders continue to have concerns over a 
variety of issues related to implementation of whole effluent toxicity 
controls through NPDES permits. Today's notice, however, invites 
comments only on the conduct of WET test methods and not on the 
implementation of WET control strategies through NPDES permits. EPA is 
interested in comments on the extent to which some aspect(s) of the 
technical components of the method revisions proposed today may affect 
implementation of WET control strategies. For example, today's notice 
solicits comments related to the proposed application of percent 
minimum significant difference (PMSD) approaches to evaluate the 
precision of WET test results (see Section B below). Application of the 
PMSD approach is intended to control the within-test variability in WET 
methods. Nationwide, however, NPDES agencies have implemented other 
concepts, such as limits on CVs to control for within-test variability 
rather than the PMSD concepts about which EPA solicits comment today. 
It is not EPA's objective to create conflict with the current 
implementation of WET control strategies that do not presently apply 
the PMSD concepts, but instead to enhance ongoing implementation 
efforts by providing an additional review step for WET test results to 
promote WET test precision. To the extent that application of the PMSD 
concepts could result in conflicts with the current and ongoing WET 
implementation, EPA invites comments on how to ameliorate any such 
adverse effects on WET implementation.

A. pH Drift

    In particular, EPA requests comments and available data to support 
or refute test method changes related to pH drift (see Section 
III.B.3.b). EPA requests that commenters provide any data that show the 
value of proposed pH control measures in situations where ammonia or 
other pH-dependent toxicants are not present. EPA specifically requests 
chronic toxicity data from parallel controlled-pH and uncontrolled-pH 
tests on well-treated municipal or industrial effluents. Such data 
should include raw toxicity test data sheets, ammonia measurements on 
tested samples, and daily initial and final pH measurements on each 
test treatment. EPA also requests data from multiple tests conducted on 
a given effluent over time to demonstrate a trend of artifactual 
toxicity due to pH drift in that effluent. Data sets should include 
full strength effluent, as well as a range of effluent concentrations 
and a dilution water control. Electronic as well as hard copy formats 
of raw test data and statistical analysis are encouraged. Though EPA 
continues to search for and may yet develop data supporting the need 
for procedures to control pH drift in the absence of ammonia or other 
pH-dependent toxicants, if sufficient data are not available at the 
time of final action on today's proposal, EPA may not incorporate 
changes to the methods beyond the 1996 guidance in the final rule.

B. Percent Minimum Significant Difference

    The percent minimum significant difference (PMSD) is a measure of 
within-test variability and test sensitivity. The PMSD for a given WET 
test can be defined as the smallest percentage difference between the 
control and a treatment (an effluent dilution) that could be declared 
as statistically significant. As test variability increases, the 
ability of a test to detect small toxic effects diminishes and the test 
becomes a less sensitive measure of toxicity. Appendix C of the WET 
method manuals (USEPA, 1994a; USEPA, 1994b) describes the calculation 
of the minimum significant difference (MSD). The PMSD is simply the MSD 
expressed as a percentage of the control response (i.e., PMSD = MSD/
control mean * 100).
    In June 2000, EPA published guidance on WET test variability that 
recommended placing upper and lower bounds on the PMSD to control 
variability and ensure a specified range of test sensitivity (USEPA, 
2000d). This guidance derived lower and upper bounds as the 10th and 
90th percentiles, respectively, of PMSDs from a large number of 
reference toxicant tests. Based on this guidance, tests for which the 
PMSD exceeds an upper bound would be conducted again (with a newly 
collected sample), if the test leads to a decision that there is no 
significant toxicity at the concentration identified in the permit as a 
limit (``Instream Waste Concentration'' (IWC) or ``Receiving Water 
Concentration''). This

[[Page 49812]]

guidance also applies lower PMSD bounds for the purpose of determining 
the no observed effect concentration (NOEC). The purpose of the lower 
PMSD bound is to avoid declaring as ``significant'' toxic effects that 
are smaller than those that can generally and routinely be detected by 
the method as currently conducted by qualified laboratories. 
Application of a lower bound does not imply that EPA has knowledge 
that, or considers that, percent differences smaller than the lower 
bound represent non-toxic effects. The lower bound PMSD is used here 
not as a threshold for toxicity but as a measure of method precision.
    Today, EPA seeks comment on proposing to require the application of 
the upper and lower PMSD bounds for sublethal endpoints in the (1) 
Ceriodaphnia dubia Survival and Reproduction Test; (2) Fathead Minnow 
Larval Survival and Growth Test; (3) Mysidopsis bahia Survival, Growth, 
and Fecundity Test; and (4) Inland Silverside Larval Survival and 
Growth Test. The proposed requirement would apply to the determination 
of the NOEC and LOEC (lowest observed effect concentration) for 
sublethal endpoints in multi-concentration tests. In the proposed 
application, the upper and lower PMSD bounds would be used to determine 
when a treatment differs significantly from the control treatment. Any 
test treatment with a percentage difference from the control (i.e., 
[mean control response--mean treatment response]/ mean control response 
* 100) that is greater than the upper PMSD bound would be considered as 
significantly different. Any test treatment with a percentage 
difference from the control that is less than the lower PMSD bound 
would not be considered as significantly different. The specifics of 
method manual changes proposed to institute the required application of 
PMSD bounds are detailed in the document titled, Proposed Changes to 
Whole Effluent Toxicity Method Manuals (USEPA, 2001d). The PMSD 
procedures about which EPA invites comment today would not preclude 
application of the current recommended guidance (USEPA 2000d) on PMSD 
bounds because today's proposed procedures are less restrictive than 
the guidance recommendation. EPA will consider using additional sources 
of data for developing lower and upper bounds for PMSD, including, but 
not limited to, data from EPA's WET Variability Study (USEPA, 2001a).
    EPA considered the appropriateness of requiring PMSD bounds for the 
growth endpoints of the Sheepshead Minnow Larval Survival and Growth 
Test and the Selenastrum capricornutum Growth Test. At this time, EPA 
does not believe that requiring PMSD bounds for these test methods 
would be appropriate because: (a) These methods appear to achieve 
smaller PMSDs than the other chronic methods (USEPA 2000d), and (b) the 
PMSD bounds for these methods (USEPA 2000d) would be based upon fewer 
laboratories and tests (albeit a substantial number) than the PMSD 
bounds for the methods for which EPA invites comment today. EPA also 
considered the appropriateness of PMSD bounds for the survival 
endpoints of test methods for chronic toxicity, and test methods for 
acute toxicity. At this time, EPA does not believe that imposing PMSD 
bounds for the survival endpoints would be necessary because precision 
for survival endpoints appears to be, in most cases, better than 
precision for sublethal endpoints (USEPA 2000d). EPA seeks comment on 
the appropriateness of imposing PMSD bounds for four test methods and 
for sublethal endpoints.
    EPA considered other measures of test precision, including the 
standard deviations and coefficients of variation for treatments and 
control, MSD, and the mean square for error from the analysis of 
variance of treatment effects (USEPA 1994a, 1994b). EPA considers the 
PMSD to be the measure that would be most easily understood and that 
could be directly applied to determination of NOEC and LOEC values. The 
PMSD quantifies the smallest percentage difference between the control 
and a treatment (effluent dilution) that could be declared as 
statistically significant. It thus includes exactly that variability 
affecting determination of the NOEC and LOEC. The CV for the control or 
any one treatment, or for selected treatments, represents only a 
portion of the variability affecting the NOEC, LOEC, and point 
estimates. Some State or Regional WET programs have requirements on the 
CV for the control and the treatment representing the IWC 
concentration. Such requirements can provide finer control over the 
variability influencing a comparison, especially a direct comparison 
between the control and the IWC treatment. The PMSD upper bound 
provides control over the average variability and would be used here 
specifically for multi-concentration tests in which the NOEC or LOEC 
are determined by using the MSD. EPA seeks comment on (1) the need for 
increased within-test precision, (2) the merits and drawbacks of 
applying PMSD bounds as described above, and (3) additional or 
alternative applications of PMSD bounds to control test precision. 
Alternative applications of PMSD bounds could include quality control 
requirements for laboratories to track PMSD values over time (e.g., 
control charts for PMSD performance in reference toxicant and/or 
effluent tests); a requirement to demonstrate recent, ongoing precision 
(PMSD less than an upper bound) in multiple tests before starting an 
effluent test; and/or use of PMSD bounds as a component of test review. 
EPA also requests that commenters submit data (hard copy and electronic 
format) to support their comments or recommendations regarding the 
application of PMSDs.

C. Other Method Modifications

    In addition to the method modifications proposed today, EPA seeks 
comment and recommendations on other method modifications that would 
improve the performance of the WET test methods. Specifically, EPA 
requests comment and recommendations on (1) increasing the test 
acceptability criteria for mean control reproduction (number of young 
per surviving female) in the Ceriodaphnia dubia Survival and 
Reproduction Test; (2) increasing the test acceptability criteria for 
mean control weight (mean weight per original) in the Fathead Minnow 
Larval Survival and Growth Test; (3) increasing the number of replicate 
chambers per concentration from a minimum of three to a minimum of four 
in the Fathead Minnow Larval Survival and Growth Test Method, 
Sheepshead Minnow Larval Survival and Growth Test Method, the Inland 
Silverside Larval Survival and Growth Test Method, and the Sea Urchin 
Fertilization Test Method; and (4) increasing the minimum number of 
replicates in the Ceriodaphnia dubia Survival and Reproduction Test 
Method. Modifications to the minimum number of replicates would be made 
to improve the precision of the test methods. EPA intends to evaluate 
these and other options for improving WET test method performance using 
existing data (from the WET Variability Study and the Variability 
Guidance Document) and data submitted to EPA in response to this 
request. EPA requests comments and recommendations on any additional 
quality control measures that would increase test precision or the 
overall quality of data generated. Comments should be supported by data 
(hard copy and electronic format) and other technical information 
whenever possible. Comments that contain

[[Page 49813]]

suggestions that are not supported by submitted data will be 
considered, but will be given less weight than those supported by data. 
EPA also requests that commenters submit information on estimated 
increases in testing costs that may be associated with any recommended 
method modification.
    Lastly, EPA requests comment on the document titled, Study Report 
and Recommended Standard Operating Procedure (SOP) for Shipping Large 
Volume Samples at Less Than 4 deg.C (USEPA, 2001f), which is included 
in the record for this rulemaking (see Addresses section of this rule 
for more information on obtaining copies of referenced materials). This 
report presents data to support a recommended SOP for meeting sample 
temperature requirements (less than 4 deg.C) during shipping of WET 
samples.

VI. References

American Public Health Association (APHA), American Water Works 
Association (AWWA), and Water Environment Federation (WEF). 1998. 
Standard Methods for the Examination of Water and Wastewater, 20th ed. 
American Public Health Association, Washington, DC.
American Society for Testing and Materials. 1992. Standard guide for 
conducting static 96-h toxicity tests with microalgae. E 1218-90. In 
Annual Book of ASTM Standards, Vol. 11.04. American Society for Testing 
and Materials, Philadelphia, PA, pp 874-885.
American Society for Testing and Materials. 1993. Standard guide for 
conducting static and flow-through acute toxicity tests with mysids 
from the West Coast of the United States. E 1463-92. In Annual Book of 
ASTM Standards, Vol. 11.04. American Society for Testing and Materials, 
Philadelphia, PA, pp 1278-1299.
Belanger, S.E., J.L. Farris, and D.S. Cherry. 1989. Effects of diet, 
water hardness, and population source on acute and chronic copper 
toxicity to Ceriodaphnia dubia. Arch. Environ. Contam. Toxicol. 18: 
601-611.
Belanger, S.E. and D.S. Cherry. 1990. Interaction effects of pH 
acclimation, pH, and heavy metals on acute and chronic toxicity to 
Ceriodaphnia dubia (Cladocera). J. Crust. Biol. 10(2): 225-235.
Cooney, J.D., G.M. DeGraeve, E.L. Moore, B.J. Lenoble, T.L. Pollock, 
and G.J. Smith. 1992. Effects of environmental and experimental design 
factors on culturing and toxicity testing of Ceriodaphnia dubia. 
Environ. Toxicol. Chem. 11: 839-850.
DeGraeve, G.M., J.D. Cooney, B.H. Marsh, T.L. Pollock, and N.G. 
Reichenback. 1992. Variability in the performance of the 7-d 
Ceriodaphnia dubia survival and reproduction test: an intra- and 
interlaboratory study. Environ. Toxicol. Chem. 11: 851-866.
DeGraeve, G.M., G.J. Smith, W.H. Clement, D.O. McIntyre, and T. 
Forgette. 1998. WET Testing Program: Evaluation of Practices and 
Implementation. Project 94-HHE-1. Water Environment Research 
Foundation, Alexandria, VA.
DeLisle, P.F. and M.H. Roberts. 1988. The effect of salinity on cadmium 
toxicity to the estuarine mysid Mysidopsis bahia: role of chemical 
speciation. Aquat. Toxicol. 12(4): 357-370.
Downey, P.J., K. Fleming, R. Guinn, N. Chapman, P. Varner, and J.D. 
Cooney. 2000. Sporadic mortality in chronic toxicity tests using 
Pimephales promelas (Rafinesque): cases of characterization and 
control. Environ. Toxicol. Chem. 19(1): 248-255.
Edison Electric Institute et al. v. EPA, Settlement Agreement, July 24, 
1998. U.S. Court of Appeals, D.C. Circuit, No. 96-1062.
Emerson, K., R.C. Russo, R.E. Lund, and R.V. Thurston. 1975. Aqueous 
ammonia equilibrium calculations; effect of pH and temperature. J. Fish 
Res. Bd. Can. 32(12): 2380-2383.
Environment Canada. 1992. Biological test method: growth inhibition 
test using the freshwater alga Selenastrum capricornutum. Report EPS 1/
RM/25. Environment Canada, Ottawa, ON.
Geis, S., K. Fleming, A. Mager, and K. Schappe. 2000a. Investigation of 
the pathogenic effect in whole effluent toxicity (WET) chronic fathead 
minnow tests. SETAC Abstract Book, 21st Annual Meeting, 12-16 November, 
2000.
Geis, S.W., K.L. Fleming, E.T. Korthals, G. Searle, L. Reynolds, and 
D.A. Karner. 2000b. Modifications to the algal growth inhibition test 
for use as a regulatory assay. Environ. Toxicol. Chem. 19(1): 36-41.
Haynes, G.J., A.J. Stewart, and B.C. Harvey. 1989. Gender-dependent 
problems in toxicity tests with Ceriodaphnia dubia. Bull. Environ. 
Contam. Toxicol. 43(2): 271-279.
Hunt, J.W., B.S. Anderson, S.L. Turpen, A.R. Coulon, M. Martin, and F. 
Palmer. 1997. Precision and sensitivity of a seven-day growth and 
survival toxicity test using the west coast mysid crustacean, 
Holmesimysis costata. Environ. Toxicol. Chem. 16(4): 824-834.
Lone Star Steel v. EPA, Settlement Agreement, March 4, 1998. U.S. Court 
of Appeals, D.C. Circuit, No. 96-1157.
Lussier, S.M., A. Kuhn, and R. Comeleo. 1999. An evaluation of the 
seven-day toxicity test with Americamysis bahia (formerly Mysidopsis 
bahia). Environ. Toxicol. Chem. 18(12): 2888-2893.
Martin, M., J.W. Hunt, and B.S. Anderson. 1989. Experimental evaluation 
of the mysid Holmesimysis costata as a test organism for effluent 
toxicity testing. Environ. Toxicol. Chem. 8: 001-010.
Moore, T.F., S.P. Canton, and M. Grimes. 2000. Investigating the 
Incidence of Type I Errors for Chronic Whole Effluent Toxicity Testing 
Using Ceriodaphnia dubia. Environ. Toxicol. Chem. 19: 118-122.
Mount, D.R. and D.I. Mount. 1992. A simple method of pH control for 
static and static renewal aquatic toxicity tests. Environ. Toxicol. 
Chem. 11: 609-614.
Pennak, R.W. 1989. Fresh-water Invertebrates of the United States: 
Protozoa to Mollusca, 3rd ed. John Wiley & Sons, New York.
Schubauer-Berigan, M.K., J.R. Dierkes, P.D. Monson, and G.T. Ankley. 
1993. pH-dependent toxicity of Cd, Cu, Ni, Pb, and Zn to Ceriodaphnia 
dubia, Pimephales promelas, Hyalella azteca, and Lumbriculus 
variegatus. Environ. Toxicol. Chem. 12: 1261-1266.
Society of Environmental Toxicology and Chemistry (SETAC). 1999. 
Potential pathogenic interference in short-term chronic WET tests using 
fathead minnows. 7pp. http://www.setac.org/wetFAQs.html.
State Water Resources Control Board. 1990. Procedures Manual for 
Conducting Toxicity Tests Developed by the Marine Bioassay Project. 
Report 90-10WQ. California Environmental Protection Agency, Sacramento, 
CA.
U.S. Environmental Protection Agency. 1991a. Methods for Aquatic 
Toxicity Identification Evaluations: Phase I Toxicity Characterization 
Procedures, 2nd ed. EPA/600/6-91/003. U.S. Environmental Protection 
Agency, Office of Research and Development, Environmental Research 
Laboratory, Duluth, MN.
U.S. Environmental Protection Agency. 1991b. Technical Support

[[Page 49814]]

Document for Water Quality-Based Toxics Control. EPA/505/2-90/001. U.S. 
Environmental Protection Agency, Office of Water Enforcement and 
Permits, and Office of Water Regulations and Standards, Washington, 
D.C.
U.S. Environmental Protection Agency. 1992. Toxicity Identification 
Evaluation: Characterization of Chronically Toxic Effluents, Phase I. 
EPA/600/6-91/005F. U.S. Environmental Protection Agency, Office of 
Research and Development, Environmental Research Laboratory, Duluth, 
MN.
U.S. Environmental Protection Agency. 1993a. Methods for Aquatic 
Toxicity Identification Evaluations: Phase II Toxicity Identification 
Procedures for Acutely and Chronically Toxic Samples. EPA-600/R-92/080. 
U.S. Environmental Protection Agency, Office of Research and 
Development, Environmental Research Laboratory, Duluth, MN.
U.S. Environmental Protection Agency. 1993b. Methods for Measuring the 
Acute Toxicity of Effluents and Receiving Waters to Freshwater and 
Marine Organisms, 4th ed. EPA/600/4-90/027F. U.S. Environmental 
Protection Agency, Environmental Monitoring Systems Laboratory, 
Cincinnati, OH.
U.S. Environmental Protection Agency. 1994a. Short-term Methods for 
Estimating the Chronic Toxicity of Effluents and Receiving Waters to 
Freshwater Organisms, 3rd ed. EPA/600/4-91/002. U.S. Environmental 
Protection Agency, Environmental Monitoring Systems Laboratory, 
Cincinnati, OH.
U.S. Environmental Protection Agency. 1994b. Short-term Methods for 
Estimating the Chronic Toxicity of Effluents and Receiving Waters to 
Marine and Estuarine Organisms, 2nd ed. EPA/600/4-91/003. U.S. 
Environmental Protection Agency, Environmental Monitoring Systems 
Laboratory, Cincinnati, OH.
U.S. Environmental Protection Agency. 1995. Whole effluent toxicity: 
guidelines establishing test procedures for the analysis of pollutants, 
final rule. Fed. Reg. 60: 53529-53563.
U.S. Environmental Protection Agency. 1996a. Addenda for Acute Manual. 
In U.S. Environmental Protection Agency, Methods for Measuring the 
Acute Toxicity of Effluents and Receiving Waters to Freshwater and 
Marine Organisms, 4th ed. EPA/600/4-90/027F. U.S. Environmental 
Protection Agency, Environmental Monitoring Systems Laboratory, 
Cincinnati, OH.
U.S. Environmental Protection Agency. 1996b. Clarifications Regarding 
Flexibility in 40 CFR Part 136 Whole Effluent Toxicity (WET) Test 
Methods, April 10, 1996, memorandum from Tudor Davies, U.S. 
Environmental Protection Agency, Office of Science and Technology, 
Washington D.C.
U.S. Environmental Protection Agency. 1996c. Marine Toxicity 
Identification Evaluation (TIE): Phase I Guidance Document. EPA/600/R-
96/054. U.S. Environmental Protection Agency, National Health and 
Environmental Effects Research Laboratory, Narragansett, RI.
U.S. Environmental Protection Agency. 1999a. Errata for Effluent and 
Receiving Water Toxicity Test Manuals: Acute Toxicity of Effluents and 
Receiving Waters to Freshwater and Marine Organisms; Short-term Methods 
for Estimating the Chronic Toxicity of Effluents and Receiving Waters 
to Freshwater Organisms; and Short-term Methods for Estimating the 
Chronic Toxicity of Effluents and Receiving Waters to Marine and 
Estuarine Organisms. January 1999. EPA/600/R-98/182. U.S. Environmental 
Protection Agency, Office of Research and Development, Duluth, MN.
U.S. Environmental Protection Agency. 1999b. Whole effluent toxicity: 
guidelines establishing test procedures for the analysis of pollutants, 
whole effluent toxicity tests; final rule, technical correction. FR 64: 
4975-4991.
U.S. Environmental Protection Agency. 1999c. Toxicity Reduction 
Evaluation Guidance for Municipal Wastewater Treatment Plants. EPA/833/
B-99/002. U.S. Environmental Protection Agency, Office of Water, 
Washington, D.C.
U.S. Environmental Protection Agency. 2000a. Method Guidance and 
Recommendations for Whole Effluent Toxicity (WET) Testing (40 CFR Part 
136). EPA/821/B-00/004. U.S. Environmental Protection Agency, Office of 
Water, Washington, D.C.
U.S. Environmental Protection Agency. 2000b. Preliminary Report: 
Interlaboratory Variability Study of EPA Short-term Chronic and Acute 
Whole Effluent Toxicity Test Methods, Vol. 1. EPA/821/R-00/028A. U.S. 
Environmental Protection Agency, Office of Water, Washington, D.C.
U.S. Environmental Protection Agency. 2000c. Preliminary Report: 
Interlaboratory Variability Study of EPA Short-term Chronic and Acute 
Whole Effluent Toxicity Test Methods, Vol. 2: Appendix. EPA/821/R-00/
028B. U.S. Environmental Protection Agency, Office of Water, 
Washington, D.C.
U.S. Environmental Protection Agency. 2000d. Understanding and 
Accounting for Method Variability in Whole Effluent Toxicity 
Applications Under the National Pollutant Discharge Elimination System 
Program. EPA/833/R-00/003. U.S. Environmental Protection Agency, Office 
of Wastewater Management, Washington, D.C.
U.S. Environmental Protection Agency. 2001a. Final Report: 
Interlaboratory Variability Study of EPA Short-term Chronic and Acute 
Whole Effluent Toxicity Test Methods, Vol. 1. EPA/821/B-01/004. U.S. 
Environmental Protection Agency, Office of Water, Washington, D.C.
U.S. Environmental Protection Agency. 2001b. Final Report: 
Interlaboratory Variability Study of EPA Short-term Chronic and Acute 
Whole Effluent Toxicity Test Methods, Vol. 2: Appendix. EPA/821/B-01-
005. U.S. Environmental Protection Agency, Office of Water, Washington, 
D.C.
U.S. Environmental Protection Agency. 2001c. Summary Report: Peer 
Review of ``Preliminary Report: Interlaboratory Variability Study of 
EPA Short-term Chronic and Acute Whole Effluent Toxicity Test Methods'' 
(WET Study Report). U.S. Environmental Protection Agency, Office of 
Water, Washington, D.C.
U.S. Environmental Protection Agency. 2001d. Proposed Changes to Whole 
Effluent Toxicity Method Manuals. EPA/821/B-01/002. U.S. Environmental 
Protection Agency, Office of Water, Washington, D.C.
U.S. Environmental Protection Agency. 2001e. Report on the Analysis of 
Block Effects. U.S. Environmental Protection Agency, Office of Water, 
Washington, D.C.
U.S. Environmental Protection Agency. 2001f. Study Report and 
Recommended Standard Operating Procedure (SOP) for Shipping Large 
Volume Samples at Less Than 4 deg.C. U.S. Environmental Protection 
Agency, Office of Water, Washington, D.C.
U.S. Environmental Protection Agency. 2001g. Clarifications Regarding 
Toxicity Reduction and Identification Evaluations in the National 
Pollutant Discharge Elimination System Program. U.S. Environmental 
Protection Agency, Office of Water, Washington, D.C.

[[Page 49815]]

List of Subjects in 40 CFR Part 136

    Environmental protection, Reporting and recordkeeping requirements, 
Water pollution control.

    Dated: September 24, 2001.
Christine Todd Whitman,
Administrator.

    For the reasons set out in the preamble, title 40, chapter I of the 
Code of Federal Regulations, is proposed to be amended as follows:

PART 136--GUIDELINES ESTABLISHING TEST PROCEDURES FOR THE ANALYSIS 
OF POLLUTANTS

    1. The authority citation for Part 136 continues to read as 
follows:

    Authority: Secs. 301, 304(h), 307, and 501(a), Pub. L. 95-217, 
91 Stat. 1566, et seq. (33 U.S.C. 1251, et seq.) (The Federal Water 
Pollution Control Act Amendments of 1972 as amended by the Clean 
Water Act of 1977).

    2. Section 136.3 is amended:
    a. In Table IA paragraph (a) by revising entries 6 to 9.
    b. In paragraph (a) by revising footnotes 7-9 to Table IA.
    c. In paragraph (b) by revising references (34), (38), and (39). d. 
In paragraph (b) by removing and reserving reference (42).


Sec. 136.3  Identification of test procedures.

    (a) * * *

                                                     Table IA.--List of Approved Biological Methods
--------------------------------------------------------------------------------------------------------------------------------------------------------
                                                                             Standard
      Parameter and units            Method \1\              EPA           methods 18th,       ASTM            AOAC            USGS            Other
                                                                          19th, 20th Ed.
--------------------------------------------------------------------------------------------------------------------------------------------------------
 
                *                   *                   *                   *                   *                   *                  *
Aquatic Toxicity:
    6. Toxicity, acute, fresh    Daphnia,            Sec. 9 \7\
     water organisms, LC50,       Ceriodaphnia,
     percent effluent..           Fathead Minnow,
                                  Rainbow Trout,
                                  Brook Trout, or
                                  Bannerfin Shiner
                                  mortality.
    7. Toxicity, acute,          Mysidopsis bahia,   Sec. 9 \7\
     estuarine and marine         Holmesimysis
     organisms, LC50, percent     costata,
     effluent..                   Sheepshead
                                  Minnow, or
                                  Menidia spp.
                                  mortality.
    8. Toxicity, chronic, fresh  Fathead minnow      1000.0 \8\
     water organisms, NOEC or     larval survival
     IC25, percent effluent..     and growth.
                                 Fathead minnow      1001.0 \8\
                                  embryo-larval
                                  survival and
                                  teratogenicity.
                                 Ceriodaphnia        1002.0 \8\
                                  survival and
                                  reproduction.
                                 Selenastrum growth  1003.0 \8\
    9. Toxicity, chronic,        Sheepshead minnow   1004.0 \9\
     estuarine and marine         larval survival
     organisms, NOEC or IC25,     and growth.
     percent effluent..
                                 Sheepshead minnow   1005.0 \9\
                                  embryo-larval
                                  survival and
                                  teratogenicity.
                                 Menidia beryllina   1006.0 \9\
                                  larval survival
                                  and growth.
                                 Mysidopsis bahia    1007.0 \9\
                                  survival, growth,
                                  a fecundity.
                                 Arbacia punctulata  1008.0 \9\
                                  fertilization.
                                 Champia parvula     1009.0 \9\
                                  reproduction.
 
                *                   *                   *                   *                   *                   *                   *
--------------------------------------------------------------------------------------------------------------------------------------------------------
Notes to Table IA:

[[Page 49816]]

 
\1\ The method must be specified when results are reported.
\7\ USEPA. [Date: To be completed at final rule]. Methods for Measuring the Acute Toxicity of Effluents to Freshwater and Marine Organisms. Fifth
  Edition. U.S. Environmental Protection Agency, Environmental Monitoring Systems Laboratory, Cincinnati, Ohio. [EPA number: To be completed at final
  rule].
\8\ USEPA. [Date: To be completed at final rule]. Short-term Methods for Estimating the Chronic Toxicity of Effluents and Receiving Waters to Freshwater
  Organisms. Fourth Edition. U.S. Environmental Protection Agency, Environmental Monitoring Systems Laboratory, Cincinnati, Ohio. [EPA number: To be
  completed at final rule].
\9\ USEPA [Date: to be completed at final rule]. Short-term Methods for Estimating the Chronic Toxicity of Effluents and Receiving Waters to Marine and
  Estuarine Organisms. Third Edition. U.S. Environmental Protection Agency, Environmental Monitoring Systems Laboratory, Cincinnati, Ohio. [EPA number:
  To be completed at final rule]. These methods do not apply to marine waters of the Pacific Ocean.

* * * * *
    (b) * * *
    References, Sources, Costs, and Table Citations:
* * * * *
    (34) USEPA. [Date: To be completed at final rule]. Methods for 
Measuring the Acute Toxicity of Effluents and Receiving Water to 
Freshwater and Marine Organisms. Fifth Edition. [Date: To be completed 
at final rule]. U.S. Environmental Protection Agency, Environmental 
Monitoring Systems Laboratory, Cincinnati, Ohio [EPA number: To be 
completed at final rule]. Available from: National Technical 
Information Service, 5285 Port Royal Road, Springfield, Virginia 22161, 
Publ. No. [Publication number: To be completed at final rule]. Cost: 
$[Cost: To be completed at final rule]. Table IA, Note 7.
* * * * *
    (38) USEPA. [Date: To be completed at final rule]. Short-Term 
Methods for Estimating the Chronic Toxicity of Effluents and Receiving 
Water to Freshwater Organisms. Fourth Edition. [Date: To be completed 
at final rule]. U.S. Environmental Protection Agency, Environmental 
Monitoring Systems Laboratory, Cincinnati, Ohio. [EPA number: To be 
completed at final rule]. Available from: National Technical 
Information Service, 5285 Port Royal Road, Springfield, Virginia 22161, 
Publ. No. [Publication number: To be completed at final rule]. Cost: 
$[Cost: To be completed at final rule]. Table IA, Note 8.
    (39) USEPA. [Date: To be completed at final rule]. Short-Term 
Methods for Estimating the Chronic Toxicity of Effluents and Receiving 
Water to Marine and Estuarine Organisms. Third Edition. [Date: To be 
completed at final rule]. U.S. Environmental Protection Agency, 
Environmental Monitoring Systems Laboratory, Cincinnati, Ohio. [EPA 
number: To be completed at final rule]. Available from: National 
Technical Information Service, 5285 Port Royal Road, Springfield, 
Virginia 22161, Publ. No. [Publication number: To be completed at final 
rule]. Cost: $[Cost: To be completed at final rule]. Table IA, Note 9.
* * * * *
    (42) [Reserved]
* * * * *

[FR Doc. 01-24374 Filed 9-27-01; 8:45 am]
BILLING CODE 6560-50-P