/by Thomas K. Sawyer ... [et al.]. Sawyer, Thomas K. Northeast Fisheries Center (U.S.) United States.Environmental Protection Agency. United States.Food and Drug Administration. Maryland.Energy & Coastal Zone Administration. United States.National Oceanic and Atmospheric Administration.Office of Ocean-Dumping Program. Oxford, Md.:Northeast Fisheries Center,[1979?] Acanthamoeba. Marine sediments. DISTRIBUTION OF ACANTHAMOEBA (PROTOZOA: ACANTHAMEOEBIDAE) IN MARINE AND FRESHWATER SEDIMENTS 1979 QL 368 A5 D57 1979 DISTRIBUTION OF-ACANTHAMOEBA (PROTOZOA: ACANTHPJ,'IOEBIDAF) IN MARINE AND FRESHWATER SEDIMENTS by Thomas K. Sawyer and E. J. Lewis (Guest Worker) National Marine Fisheries Service Northeast Fisheries Center Oxford, Maryland 21654 Donald Lear and Mauria 0 .1alley Environmental Protection Agency Annapolis, Maryland 21401 and Cpt. Willard N. Adams and Cdr. J. Gaines 'A Food and Drug Administrat ion Davisville, Rhode Island 02854 Ll -1 'A Informal Report: Prepared for Energy and Coastal Zone Administration, Department of Natural Resources, Annapolis, Maryland, and Office of Ocean-Dumping Program, National Oceanic and'Atmospheric Administration, Rockville, Maryland. 2roperty of CSC Libr-.y Preliminary Draft Not To Be Cited Ik Introduction 'T Small free-living amoebae of the genus Acanthamoeba are well-known inhabitants of soil and water. The first described species,. A. castellanii, was more or less discovered accidentally when it was observed on an agar plate streaked with a soil-sample that was being studied. for bacteria and fungi. Very large numbers of the amoebae may begrown in the laboratory on agar plates and in sterile liquid ,culture medium. Although various species of Acanthamoeba have been known to science for over .50 years, intensive research on them began in the 1950's. Jahnes et al (1957) first observed Acanthamoeba in cultures of monkey kidney cells, and Culbertson et al (1959) found that amoebae from such cultures could kill laboratory animals when inoculated intranasally. In the mid 1960's it was discovered that several species 6f Acanthamoeba were responsible for disease or death in humans and domestic or wild animals. Griffin (1978) has published an extensive review on the role of free-living amoebae in human and animal disease. The genus Acanthamoeba has attracted worldwide attention and cases of disease have been reported from many countries including the United States, Russia, Korea, England, India, etc. Sawyer (1970) discovered that A. castellanii and A. polyphaga would grow experimentally on full-strenoth oceanic seawater and produce cysts which bad lost their diagnostic characters. Later, Sawyer (1971) described thelfirst marine species, A.. griffini in honor of Dr. Joe Griffin, Armed Forces Institute of Pathology, who made the original 2 isolation from a sediment sample collected in Long Island Sound. 'The observation that marine waters were.a new environmental source for Acanthamoeba then led. to discovery that a pathogenic species., A. culbertsoni, was present in sewage sediments of the Atlantic Ocean near New York State (Sawyer et 1977). Singh & Das (1972) previously had shown that several species of Acanthamoeba were present in municipal sewage wastes in India. Davis et al (1978) cultured seawater samples from -rep the open ocean and -orted that Acanthamoeba was one of three genera which were observed most commonly in their study. The discovery of both pathogenic and non-pathogenic species of Acanthamoeba in the marine@ environment suggested that a major study should be made to determine whether or not there is an association between the presence of sewage wastes in the sea and the presence of potentially pathogenic Acanthamoeba. Our report summarizes the results of a comprehensive study on the distribution of Acanthamoeba in sediments from inshore, coastal, and offshore environments. Our choice of study areas withdiffering characteristics included the St., Martins River-isle of Wight Bay complex which represents a shallow fresh- to seawater gradient; the New York Bight apex which represents a nearshore marine habitat which receives both municipal and barged sewage, and the 44 Philadelphia-Camden sewage disposal site which represents an offshore impacted enviTonment with sewage-free adjoining waters. The present -report provides the first extensive account of the distribution of Acanthamoeba species in oceanic bottom-sediments and demonstrates that such amoebae are potential -residents of the ocean when bacteria, including the. coliform group, are available as a source of food. Methods Sediment samples in or'distant from the Philadelph .ia-Camden acid or sew-age dump-sites were collected during 5 separate Environmental Protection Agency cruises (Table 1). A total of M sediment sub- samples were taken aseptically from bottom grabs made with a Smith- .McIntyre sampler. Subsamples were transferred to !§terile 60-mm plastic dishes and stored on ice until processed in the laboratory. A total of 36 subsamples were made in the New York Bight ---apex during 3 cruises with personnel from the Food and Drug Administration (Table 2). The subsamples were taken aseptically from bottom grabs made with a Ponar grab.sampler and stored on ice until processed. A total.of 77 sub- amples were coll t. Martins River-Isle s ected from the S of Wight complex -rips. (TAble 3). The samples were collected with a. during 4 sampli.ngt Ponar grab sampler, or with an.improvis .ed device made up of a short length of pipe attached to a wooden handle. The device was designed for use.in shallow-water stations which did not require the use.of a boat., Dishes containing sediment samples were,weiRbed before.and aft.er transferring small amounts of sediment to culture dishes in order,to, determine the weight of sediment used in each culture. Culture 1,7 4 procedures and media were the same as described previously by Sawyer St al (1977). In some cases each sediment was placed on agar media made with distilled water and on media made with low salinity river water (4 - 8 o/oo) to estimate whether or not the composition of culture media influenced the number of cultures that were positive for amoebae. Occasionally 2 or 3 grab samples were taken from the same station to estimate how frequently amoebae could be isolated from a single location. All cultures were examined several times a week to check for the growth of amoebae and cultures which were negative after 2 weeks were discarded. Identifications based on cyst characteristics were made on original cultures when only one species was present. Sub- cultures were made when more than one species was present to obtain pure cultures and facilitate identifications. Species of Acanthamoeba that were identified as known pathogens were grown at 400 C to confirm their ability to grow.at temperatures approximating or exceeding mammalian body tempe ratures. Selected strains were donated to the American Type Culture Collection, Rockville, Maryland, or to the Communicab le Disease Center, USPHS, Atlanta, Georgia, for animal pathogenicity tests. Amoebae which did not have the identifying characters for known species were studied further to determine if they were new to science. -5- Results Twenty-two of 177 stations sampled (12 percent) in the offshore Philadelphia-Camden dump-site yielded cultures of Acanthamoeba (Table 1), including two pathogenic species A. culbertsoni and A. hatchetti. It was of interest to note that only 2 of 119 control stations yielded amoebae while 17 of 42 stations in or near the sewage spoil area were positive. Most of the stations in the sewage area which yielded Acanthamoqeba also were positive for either fecal or non-fecal coliform. bacteria (Table 4). Positive cultures from stations identified as in or near the acid-waste site (3 of 16 stations) were of interest since coliform bacteria, if present, were below detectable limits with the methods employed. The location of all stations sampled in the vicinity of the sewage dump-site is shown in Fig 1. Analyses of the data showed that 40 percent of the sewage stations yielded Acanthamoeba in contrast to less than 2 percent for the non-sewage stations and 19 percent for the acid-waste site. Twenty-four of 36 stations sampled (67 percent) in the New York Bight apex (Tables 2 and 5) yielded cultures of Acanthamoeba, including the two pathogenic species. Bacteriological studies showed that 33 of the stations were positive for non-fecal coliforms and 23 of them were positive for fecal-coliforms; station locations are shown in Fig. 2. In this sampling area 29 of the 36 stations were located in the sewage 6 disposal site or in the closed area and 7 stations were not in close proximity to either area; the bacteriological studies showed 6 of the 7 stations harbored coliforms. All stations sampled were located in or near the area of the Bight apex that has been closed.to shellfishing and none of them could be considered as control stations. ..Twelve of 19 stations sampled (63 percent) in the St. Martins-Isle of Wight Bay complex, yielded cultures of Acanthamoeba including the two pathogenic species and one new species (Lewis and Sawyer, in press). The -river-bay system (Fig. 3) had vnry few characteristics in common., with@the ocean-dumping stations other than being recognized as a polluted environment which has been closed to the harvesting of shellfish.' All of the stations ranged from only 1 to 9,feet in depth, from 0 to 30 o/oo salinity; sediments ranged from foul, black muds to clean sand. The 6stations iocated in the Isle of Wight Bay.were sampled In from 1 to 3 -replicates to yield a total of 2S.subsamples and only 2. of them were positive for Acanthamoeba. In contrast to the bay stations, the 13 river stations were sampled to yield 52 subsamples of which 30 (60 percent) were positive for the amoebae..- Comparative analyses of data from-the 3 different Ollecting sites supported one singularly important hypothesis, i.e., the presence of -.Pathogenic or non-pathogenic species of Acanthamoeba depends,-in part, 6n the availability of suitable bacteria ds food organisms. The requirement for such food sources is met, in part, by the introduction of sewage-associated bacterial species. The recovery of amoebas from 7 sediments that are not impacted by sewage additions appeared to depend on the presence of bacteria which occur naturally as a consequence of. ..natural biogenic decay of organic materials,.a phenomenon that is enhanced.artificially by the introduction and decay of sewage-Telated m aterials. Discussion The design of.our studies had a twofold purpose,.(I) to demonstrate that the ocean disposal of barged sewage wastes alters the natural microbial flora of the seabottowin such a way as to enhance the, recovery of pathogenic and non-pathogenic amoebae from impacted collection sites, and (2) to show that the.distribution of such amoebae in the marine environment depends on the presence of.an adequate supply of bacterial food organisms whether or not such bacteria.are.associated with sewage input. The recovery of pathogenic and non-pathoge-nic species of Acanthamoeba ,.from the marine environment-, and the relationship of their distribution -,to the ocean disposal of-sewage sludge., was observed more precisely in the Philadelphia-Camden-study area than in the New York sewage dump-site or the St. Martins River-Isleof Wight Bay complex. The distribution of fecal and non-fecal coliform bacteria in sediments from the offshore study area showed precisely that such bacteria were introduced in the. sewage sludge--and not by potential run-off from municipal di-schargeg. In contrast, the New York Bight apex study area showed distribution 8 patterns indicative of bacterial input from both sewag& sludge and municipal discharges. Thus, many stations in the Philadelphia-Camden area could be considered suitable as control sites while none of the stations in the Bightapex were suitable as controls. Both dump-sites were similar with respect to salinity in the sense that they were not subject to dilution,by freshwater run -off. The.St. Martins River@lsle. of Wight complex provided parameters for analyses that were not present in the ocean.stations: (1) it was not impacted by sewage sludge deposited .,.by barges, (2) water depth ranged from only I to 9 feet in depth, (3) certain stations were impacted by residential or commercial pollution,', (4) stations ranged from salt marshes to polluted muds, and (5) historical records showed that fecal coliforms and very low levels of dissolved oxygen were found in the low salinity to freshwater uppermost stations. 'Cumulative data derived from the Philadelphia-Camden and New York Bight study areas showed that less than 2 percent of the non-sewage impacted samples (2/119) yielded*cultures of Acanthamoeba. In marked contrast, 54 percent of the sewage impacted stations (40/74) were. positive for the amoebae. The results are remarkable since the amount of sediment inoculated onto agar plates seldom exceeded 0.5 g in weight. When the vastness of the seabottom. and the wide distribution in station Jocation-.is taken into account, it is apparent that our approach is extremely cons'ervative-. Regardless of salinity, water dept.h, coliform bacteria, etc., 58 of 232 stations (25 percent) were positive.for one or more species of Acanthamoeba. 9 Characteristics of the Philadelphia-Camden Dumping Grounds. The. sewage disposal site situated 40 miles offshore from Maryland, Delaware, and New Jersey has been utiliz6d only since 1972. 'In contrast to the New York Bight apex 9tation which has been.used for over 40 years, this. site rarely yields bottom sediments which uniformly are made up of black silty sludge. Thus, there does not yet appear to be an extensive sludge l'blanket" overlaying the bottom sediments. Furthermore, fecal and non- fecal coliform sediment MPN's vary from less than 2 to over 300 bacteriaP s ggesting that such sewage contamination is erratic as a result of the u "youth". of the dump-tite. In this study area high'bacterial counts during one cruise mayhave reflected recent dumping activity, and,low counts obtained during other cruises may have reflected differences in the dispersion patterns of settled sewage. In spite of differences in sediment characteristics and the numbers of countable coliform bacteria, a ve.ry.high rate of recovery for Acanthamoeba species was recorded for the area. In the immediate vicinity of the dump-site bottom, bacterio- gical studies.and studies on the presence of amoebae indicate that sludge may have spread to the'northeast and southwest of the dump-site. In. contrast, other stations situated some distance from the sewage'site Consistently were negative for coliforms and rarely yielded cultures of the amoebae. Charact6kistics of the New York Bight Apex Dumping Grounds. The sewage disposal site situated approximately 12 miles from.the shores of Long Island Sound, New York has been used for sludge dumping for about 10 .40 years. Bottom sediments taken from the site have no resemblance to naturally occurring seabottom and have been described as having the appearance of "black mayonnaise.11 Fecal co.liform sediment MPN's ranging to several thousand are not unusual. Measurable amounts of the human fecal"steral "cop-rostanol" also are present throughout the area that is Influenced by barged sewage sludge. Shoreward in all directions from the dump-site varying numbers of coliform bacteria aie present in sediments as a result of land-based sewage discharges. Acanthamoeba routinely was isolated from the dump-site and from stations situated between the site and the adjacent shoreline. In marked contrast, similar amoebae rarely were.obtained from sediments shoreward from the Philadelphia-Camden disposal site. Characteristics of the St. Martins River-Isle of Wight Bay Lnple The river-bay complex which.runs inland -from Ocean City, Maryland was selected for study because it represented a system that is used extensively for recreational, residential, industrial, and agricultural purposes. The most inland stations that were sampled were in or near the town of Bishopvillewhere the river is considered to be highly polluted and subject to concern by state health personnel. Stations downriver from Bishopville as the river empties into the Isle of Wight Bay werebordered by farms, residential communities, salt marshes, a. dead-end canal at Ocean.Pines, etc. Additionally, stations along the selected traA--kect represented a full range of salinity from oceanic-to brackish or freshwater and sediments ranged from,foul, black mud to clean mud or sand. The study area, although not impacted by sewage@ sludge dumping practices, was chosen to provide an inland study area With known pollutants, including fecal coliform bacteria which, theoretically, couldbe associated with the presence of Acanthamoeba.. Thus, the river-bay system was studied to determine whether point sources of pollution could be identified as sources for amoebae which might be -flushed downriver and seed the coastal marine seabott.om. General Conclusions. 'The first report of the presence of potentially patho genicA. culbertsoni.from the marine environment (Sawyer eVal, 1977) suggested that there might be a cause-and-effect relationship between the ocean disposal of sewage wastes and the introduction of sewage- associated pathogenic protozoans into otherwise clean bottom sediments. Such a -relationship must be studied-further because barged sewage sludge -has not been examined for the presence of the amoebae or their resistant cyst stages.. Our extensive survey data clearly demonstrate,, however, that there is a high correlation between the presence of fecal and no.n- fecal coliforms in bottom sediments and the successful isolation of Acanthamoeba from the marine and freshwater environments. The Philadelpbia-Camden sewage disposal site may be considered as a geographi- cal source for Acanthamoeba since it is approximately 40 miles offshore and 40 percent (17/42) sewage-associated sediment samples yielded the amoebae. In contrast, less than 2 percent (2/119) of the stations 12 sampled from the periphery Of the dump-site to'less than I mile from shore yielded cultures of amoebae. It is evident from.studies in the Philadelphia-Camden area that sewage-i.nduced bacterial loading of the seabottom, has measurable effects on the distribution of Acanthamoeba in the sea. Conclusions based on the findings in the Philadelphia-Camden dump-site have been confirmed by further studies made in the New York Bightapex near New Jersey and New York. Thirty-six stations that were sampled either in the dump-site or in adjacent waters that have been -closed to shellfishing yielded a 67 percent recovery of Acanthamoeba (24/3,6 stations). -The association of Acanthamoeba.with coliform bacteria was confirmed by the--'fact that in the New York stations 92 percent (33/36) of.the stations harbored non-fecal colif6rms, and 64 percent (23/36) harbored fecal coliforms. Bacteriological studies showed that coliforms were widely distributed in bottom sediments.that were taken shoreward from the dump-site. Results of the study in the St. Martins River-Isle of Ifight complex, although obtained in an,entirely different ecosystem.from that of.the sewage dump-sites, supported our hypothesis that bacterial loading influenced the frequency with which Acanthamoeba could be recovered from bottom sediments.. Culture studi es showed that the presence of the amoebas was independent of water depth,.salinity,.and sediment character- .istics.. The studies showed, however, that sediments wliicfi yielded the amoeba most frequently were those that were in close proximity to sources of humanand industrial activity. 13 The final, and most important, conclusion to be reached from our studies concerns the fact that the presence of Acanthamoeba in the coastal or oceanic environment does not depend exclusively on the introduction of bacteria-laden sewage sludge. Davis et al (1978) isolated amoebae from waters of the open ocean and found that species of Acanthamoeba were among the most frequently isolated organisms of the identifiable protozoa encountered in their.study. Superficially, the data suggest that species of Acanthamoeba appear.to be among the most common inhabitants of the marine environment. Rather curiously, however, among the 76 species of marine amoebae included in a recent.key to the'.marinb amoeba:e_(Bovee and--.Sawyer, 1979), and among the 13 species isolated by Davis et al (1978), only members of the genus Acanthamoeba are known to form resistant cysts. Thus, it is possible that.a culture which yields Acanthamoeba may have originated from a single cyst and prevalence or frequency data would not serve as an indication of the abundance or population density of Acanthamoeba. There remains a very important requirement that known amounts of bottom sediment or seawater be analyzed tg.determine how many of the amoebae are present per unit.of weight of sediment or volume of seawater.. There appears to" be very.little doubt that population densities will be determined,*in part, by the extent of bacterial loading in the test sample. Summary 1. Pathogenic.or non-pathogenic species of Acanthamoeba may be isolated from bottom sediments, the open ocean, and the nearshore environment. 14 2. The opportunity or chance of obtaining positive cultures depends on the presence of resistant cyst stages in the@sample, or on the presence of suitable bacteria that provide a source of food.. .3. The disposal of barge-delivered sewage wastes in the marine environment introduces fecal coliform bacteria into marine ecosystems which, otherwise, would harbor only naturally-occurring marine species. .4. It is not yet known whether Acanthamoeba species are present in barge-delivered sewage wastes, or whether bacteria present in the waste material encourage the growth of protozoan species to levels which exceed naturally-occurring ambient levels. 5. Most of the well-known species of Acanthamoeba are not limited in their distribution by factors such as salinity, water depth, and sediment characteristics. 6. Marine habitats which harbor fecal coliform bacteria have a 50 percent or betterchance of harboring Acanthamoeba than those which are not impacted by sewage-disposal practices. .7. Several species of Acanthamoeba which-thrive at mammalian body temperatures 'and are kn6wn.to cause disease or death in laboratory. animals and man wereisolated.from each.of the 3.areas employed in the present study- 8.. The Philadelphia-Camden sewage dump-siie situated 40 miles from shore harbors geographically isolated species.of Acainthamoeba which -rarely are fou'nd at 'shoreward stations which do not yield countable numbers of coliform bacteria. 9. Species of Acanthamoeba may prove to be of equal or greater value than coliforms for measuring the persistent effects of unnatural. loading of.the marine environment by bacteria. 10. Less than I 'g of sediment was used for isolating Acanthamoeba. Further studies with larger amounts of sediment.are likely to show that the amoebae are considerably more prevalent in the ocean than is. docuniented in our report. Acknowledgm The authors gratefully acknowledge L. Zeni, C. Ostrum, E. Hollis, and S. Bayley, Energy and Coastal Zone Administration,. Annapolis, Maryland for their interest and cooperation-during all of the research... conducted in the Philadelphia-Camden ocean disposal sites. All of the collections made in the St. Niartins River-Isle of Wight Bay complex WeTe made possible through the interest and cooperation of J. Casey and A. Wesche, Maryland Department of Natural Resources. We are especially grateful to the managers and directors of the State and Federal agencies involved in approving and supporting the requirements of our various research activities. LITERATURE CITED Bovee, E. C. and T. K. Sawyer. 1979. Marine flora and fauna of the Northeastern United States. Protozoa: Sarcodina: Amoebae. NOAA Tech. Rep. NMFS Circ. 419, 56 pp. Culbertson, C. G., J. W. Smith, H. K. Cohen, and J. R. Minner. 1959. Experimental infection of mice and monkeys by Acanthamoeba. Am. J. Pathol. 35: 185-197. Davis, P. G., D.. A. Caron,_*and J. KcN. Sieburth. 1978. Oceanic amoebae from the North Atlantic: culture, distribution, and taxonomy. Trans. Am. Microsc. Soc. 97: 73-88. Griffin, J. L. 1978. Pathogenic fri--e-living amoebae. In: J. P. Kreier, ed., Parasitic Protozoa, Vol. II. Intestinal Flagellates, Histomonads, Trichomonads, Amoebas, Opalinids, and Ciliates. Academic Press, New York. pp. 507-549. Jahnes, W. G., H. M. Fullmer, and C. P. Li. 1957. Free living amoebae, as contaminants in monkey kidney tissue culture. Proc. Soc. Exp. Biol. Med. 96: 484-488.@ @Lewis, E. J. and T. K. Sawyer. In press..@ Acanthamoeba tubiashi n.,sp., a new species of freshwater amoebida (Acanthamoebidae). Trans. Am. Microsc. Soc. Sawyer, T. K. 1970. The influence of seawater media on growth and encystment of Acanthamoeba polyphaga. Proc. Helminthol. Soc. Wash. 37: 182-188. Sawyer, T. K@ 1971. Acanthamoeba griffini, a new species of marine amoeba. J. Protozool. 18: 650-654. Sawyer, T. K., G. Visvesvara, and B. A. Harke. 1977. Pathogenic amoebas. from brackish and ocean sediments, with a description of Acanthamoeba hatchetti, n. sp. Science 196: 1324-1325. Singh, B. N., and S. R. Das. 1972. Occurrence of pathogenic Naegleria aerobia, Hartmannella culbertsoni .and H. rhysodes in sewage sludge samples of Lucknow. Curr. Sci. 41: 277-281. Table 1. Summary of Environmental,Protection Agency Cruises to@ Philadelphia-Camden Sewage Disposal, Acid-Waste and Control Stationsl Name of Control Acid-Waste Sewage Total No 2 Cruise Date Stations Stations Stations Positive Stations RIDGERUNNER Apr 1978 1/60 2/7 4/14 7/81 DIEKKO Aug 1978 1/5 1/4 2/9 HELMALEE Sep 1978 0/18 0/4 4/31 3 FDA Oct 1978 0/7 1/5 1/12 ENFIN Apr 1979 0/29 1/5 7/10 8/44 Totals .2/119 3/16 17/42 22/177 No. Stations Positive for Acanthamoeba/No. Stations Sampled 2 No. Stations Positive for Acanthamoeba/No. Stations with Coliform Bacteria 10120 excludinj DEKKO for ;Which bacteriology was not.done. 3 FDA Cruise a cruise made outside of.the established sewage disposal site for purpose of determining horizontal spread of sewage as indicated by fecal and non-fecal coliform counts. Table 2. Summary of Cruises in New York Bight Apex Within or Near Area Closed to Shellfishing. No. Samples in No. Stations Total No@ 2 Trip.No. Date Closed Area Outside of Positive Stations Closed Area Jul 1978 S/8 5/8 2 Dec 1978 3/S 2/S 5/10 3 Jun 1979 13/16 1/2 14/18 Totals 21/29 3/7 24/36 No@ Stations Positive for Acanthamoeba/No- Stations Sampled. No. Stations Positive for Acanthamoeba/No. Stations with Acanthamoeba and Coliform Bacteria 22/24. Table 3. Summary Data from 4 Collecting Trips to the St. Martins River-Isle of Wight Bay Complex.1 Station Type of Positive or Negative No. Positive/ Number Station2 for Acanthamoeba No. Replicates 3 Pos. 1/3 2 Pos. 2/4 q -3 2 Pos. .4 2 Pos.. 4/4 2 Pos. 2/4 6 3 Pos. 1/5 7 3 Necr. O/S 8 3 Neg. 0/4 9 2 Pos. 3/S .10 Pos. 2/5 Neg. 0/1 12 1 Pos. 1/4. 13 Pos. 4/4 14 Neg. 0/4 is 2 Pos. 4/4 16 2 Neg. 0/4 17 2- Pos. 3/4 18 3 Neg. 0/4 .19 Neg. 0/4 Summary of 4 trips 19 stations with 77 subsamples (See below)._ 2 Type 1 - more than 3 miles from Bishopville, MD - 4/6 positive; 9/22 replicates Type 2 - within 3 miles of Bishopville, @M 6/7 positive; 21/30 replicates Type 3 - Isle of Wight Bay, MD 2/6 positive; 2/25 replicates. Table 4. Summary of Environmental Protection Agency Stations Positive for Acanthamoeba With or Without Countable Coliform Bacteria. Cruise Station No. Total Coliform. Fecal Coliform, Name Date and Typel Count Count RIDGERUNNER Apr 1978 NB-l (1) < 10 <. 10 11 (3) >230 >230 206 (3) 160 >230 407 (2) < 10 10 414 (2) < 10 <10 420 (3) 22 51 423 (3) 22 91 DEKKO Aug 78 206 (3) N.D. N.D. 33 (1) -N.D. N.D. HELMALEE Sep 78 IK3 (3) 120 91 423 (3) 22 10 E (31 < 10 <10 2 (3) < 21 .< 21 FDA @.Oct 78 97 (3) 2400 33 ENFIN Apr 1979 2 (3) 77 <37 20 (2) < 37 <37 203 (3). 37 37 210 13) 37 37 211 (3) < 37 <37 E-18 (3) < 37 <-37 E-12 (3) < 37 <37 206, (3) < 37 <37 (1) Control Stations, 2/119 positive for Acanthamoeba; no coliform' bactdj7ia (2) Acid-Ilaste Site, 3/16 positive for Acantha-moeba; no coliform bacteria (3) Sewage Dump-site, 17/42 positive for Acantbamoeba; 10/17 with coliform bacteria Table 5. Summary of New York Bight Apex Stations Positive for Acanthamoeba With or Without Countable ColifoTm Bacteria Station No. Total Coli- Fecal Coli- Date and Typel form Count form Count Jul 19782. 2 (DS) 170 <. 20 3 (DS) 78 45 4 (DS) 160,000 3,300 5 (DS) 4,600 700 6 (DS) 460 Dec 19783 2 (DS) 270 37 4 (DS) > 7,000 270 5 (DS) 630 37 6 (CA) 630 37 10 (CA) 630, 37 Jun -19794 53 (CA) 49 4.5 62 (CA) 4.5 < 2 103 (OCA) < 2 < 2 104 (CA) < 2 < 2 754 (CA) 13 < 2 d 756 (CA) 79 6.8 7S7 (CA) 13 < 2 761 (CA) 14 < 2 764 (CA) 7.9 2 765 (CA) 33 2 790 (CA) 2 < 2 .791 (CA) 4 2 792 (CA) 6.8 1385 (CA) 2 < 2 (DS) - Sewage disposal site (8/24) (CA) - Not a disposal site but closed to shellfishingr (15/24) (OCA) -,Outside of closed area (1/24) 2 Five of eight stations yielded amoebae;- 8/8 stations positive for coliform Vacteria. 3 Five of ten stations yielded amoebae; 10/10 stations positive for coliform bacteria. 4 'Fourteen of eighteen stations yielded amoebae; 15/18 stations positive for-coliform bacteria; 12/14 stations positive for amoebae also positive for coliform bacteria and 3 stations positive for coliforms but negative for amoebae. X ='-posil@IV-0 -for AcanthaToeb ........... -.o =-Negative for AcanthaToeb 7y _30 40, 15> 0, CA 117 0 % 177 Y2 V SOO SCSI 9 y_ of- So? 4w it* IK3 S. 6 05 @03 X X.-)AS !Zoe. x MY Z1. 3Y IF32 > S e Fig. 1. Representatitoe stitions sampled for' Acanthamoeba 9duTing 5 EPA-FDA cras:@s tq the Philadelphia-Cwtden dump-site. NEW y co A 0 Positi o- Negati 104, 103 CLOSED 62 Mo 792 TO G 765 b 7640 6 0 .763 053' 791 '761 1385 15V 756 NEW 7qo JERSEY 75 4 0 .753 Fig. 2. New York Bight apex stations FOOD AND sampled for,Acantham'oeba and coliform SHELLPIS 742 bacteria. NE CLOSUP JUNE 0 Sampl 10 M, MILES NZ TECHN Delaware Maryland Bisho Ille Assawoman Bay oil V Ix Positive for Acanthamoeba Negati.ve for Acanthamoeba. Isle of Wight Bay. ale Fia. 3. St. Martins River-Isle of Wight Bay complex. Ocean City 3 6668 14103 8804