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At the present time the Bureau of Commercial Fisheries is conducting a research program to determine the effects of pesticides on commercial fishery resources. I should like to discuss certain phases of this research and their relation to field applications of pesticidal chemicals. The value of the estuarine environment to commercial fishery resources can not be over emphasized. Our most important commercial species of finfish and shellfish spend all or part of their life span in estuarine waters. Sessile or slightly motile forms as oysters and clams are necessarily restricted to these inshore waters. Motile species such as crabs, shrimp, menhaden and mullet spend much of their early life in lower salinity waters near sources of natural land drainage. Heavy spraying of certain chemicals to control larvae in or near estuarine waters can be very destructive to these marine forms both through direct kills and destruction of their food supply. However, the amount of insecticides used in such coastal operations is far outweighed by that used on forest and farmlands. It seems inevitable that some of these chemicals or their metabolites will reach estuarine waters. One of the major objectives of our research program has been the determination of acute toxicity of the commonly used pesticides to representative marine species. These bioassays are conducted under controlled laboratory conditions. The development of uniform testing methods has been an important part of this research. Since several phyla of marine organisms are used as bioassay animals, different criteria of toxicity must be used. Each chemical is tested on oysters, shrimp, phytoplankton and at least one species of marine fish.

Lowe, Jack I. 1965. Some Effects of Endrin on Estuarine Fishes. In: Proc. 19th Annu. Conf. Southeast. Assoc. Game Fish Comm., Oct. 10-13, 1965, Tulsa, OK. James W. Webb, Editor. Southeastern Asociation of Game and Fish Commissioners, Columbia, SC. Pp. 271-276. (ERL,GB 063).

Laboratory experiments were conducted to determine the acute and chronic effects of endrin to estuarine fishes. Short-term bioassays in flowing seawater determined 24-hour LC50's for spot (Leiostomus xanthurus), mullet (Mugil cephalus), menhaden (Brevoortia patronus), longnose killifish (Fundulus similis), and sheepshead minnows (Cyprinodon variegatus). A population of spot was exposed continuously for eight months to a sublethal concentration (0.05 ppb.). No pathology was found in the spot after seven months of exposure, but, a three-week exposure to a near-lethal concentration (approximately 0.075 ppb.) produced pathology characterized by systemic lesions involving the brain and spinal cord, liver, kidneys and stomach. Residue analyses (gas chromatography) of spot exposed to 0.05 ppb. endrin for five months revealed an accumulation of 78 ppb. (micrograms/kilogram). No endrin could be detected in these fish after a 13-day holding period in uncontaminated water. Chronic exposure of spot to endrin did not affect their tolerance to sudden changes of salinity. Endrin-exposed fish were also able to endure extended periods of starvation. Sublethal concentrations of endrin do not appear to affect the general physical condition of spot. The threshold of toxicity is extremely critical and the importance of time must not be underestimated in determination of a sublethal concentration of endrin for fish.

Butler, Philip A. 1965. Pesticides. In: Sixteenth Annual Report (1964-1965) of the Gulf States Marine Fisheries Commission to the Congress of the United States and to the Governors and Legislators of Alabama, Florida, Louisiana, Mississippi, Texas. Gulf States Marine Fisheries Commission, New Orleans, LA. Pp. 35-36. (ERL,GB 066).

Research projects are continuing to refine older techniques and develop new methods for evaluating the effects of synthetic organic pesticides on marine biota and detecting the presence of pesticide pollution in the environment. Substantial progress is being made in methods for detecting the organophosphate compounds which are both highly toxic and relatively transitory in the estuary. The screening program is being broadened to include more test on microscopic plants that serve as food for oyster larvae, as well as tests on the larvae themselves. Modernization of an existing structure has added 1600 feet of efficient laboratory facilities for this microbiological work. Additional fiberglass tanks have been installed outdoors to increase our holding facilities for the shrimp and fish used in bioassay work. During the year, laboratory staff members discussed pesticide problems and research progress at six meetings in the Gulf states. Twelve research reports were published or approved for publication.

West, Walter L. and Philip A. Butler. 1968. Mechanical Testing and Bioassay of Adhesive/Sealants for Use in an Aquatic Environment. Drum and Croaker (Wash.). 68(1):9-10. (ERL,GB 089).

In 1965, specific tests were begun to determine the material best suited and commercially available as an adhesive/sealant for large and small scale aquarium use. In most cases, the materials were not initially intended for aquarium or underwater uses. The testings were designed for two purposes: 1. Determine the toxicity of the materials to aquatic organisms. 2. Determine the suitability of the materials to seal hair-like cracks on the water side of concrete tanks, ease the removal of algae, and determine if these materials could be used to seal the periphery of viewing glass. The testing under (1) above was done under contract at the Steinhart Aquarium, San Francisco and at the Gulf Breeze, Florida, laboratory of the U.S. Bureau of Commercial Fisheries. The testing under (2) above was by the NFCA staff and the National Fish Hatchery, Pisgah Forest, North Carolina.

Duke, Thomas W. 1970. Estuarine Pesticide Research--Bureau of Commercial Fisheries. In: Proc. Gulf Caribb. Fish. Inst., 22nd Annual Session, Nov., 1969, Miami Beach, FL. James B. Higman, Editor. University of Miami, Gulf and Caribbean Fisheries Institute, Miami, FL. Pp. 146-153. (ERL,GB 100).

Pesticides that enter the estuarine environment can be accumulated by the fishery organisms that inhabit these fertile waters. Effects of pesticides on estuarine organisms and the fate of these chemicals in the estuarine environment are being studied at the Bureau of Commercial Fisheries (BCF) Pesticide Field Station, Gulf Breeze, Florida. Bioassay projects investigate the toxicity of new pesticides submitted by pesticide manufacturers and perform analyses requested by the Pesticide Review Board. Emphasis is placed on determining possible adverse effect of prolonged exposure (chronic studies) of fishery organisms to sublethal concentrations of selected pesticides. Ecological studies include the effects of a herbicide, Dichlobenil, on the ecology of an experimental pond. Experiments also are conducted to determine effects of sublethal levels of pesticides on the behavior of estuarine fish. For example, the capacity of fish to avoid water containing pesticides is tested in a special apparatus in the laboratory.

Nimmo, D.R., A.J. Wilson, Jr. and R.R. Blackman. 1970. Localization of DDT in the Body Organs of Pink and White Shrimp. Bull. Environ. Contam. Toxicol. 5(4):333-341. (ERL,GB 104).

Bioassays conducted at this laboratory have shown commercial penaeid shrimp to be one of the most sensitive crustaceans to organochloride pesticides. Nevertheless, little is known about residues in the organs of shrimp or the sites of pesticide localization. We therefore exposed shrimp to low concentrations of DDT (0.05 to 0.2 p.p.b., parts per billion) in the laboratory for extended periods to compare residues accumulated under controlled conditions with residues in natural populations.

Duke, Thomas W. 1970. Effects of Pesticides on Estuarine Organisms. Mar. Pollut. Bull. 1:126. (ERL,GB 115).

The Pesticide Field Station of the US Fish and Wildlife Service Bureau of Commercial Fisheries (BCF), an agency of the Department of the Interior, is located at Gulf Breeze, Florida, about 9 km south-east of Pensacola. BCF began a research and monitoring programme in 1958 at Gulf Breeze to study the effects of pesticides on estuarine animals in the laboratory, and to determine the concentrations of polychlorinated pesticides in these animals in many estuaries of the United States. Much information has been obtained on the relative toxicity of various pesticides to clams, oysters, shrimp, fish, and other estuarine animals. At present investigators are pursuing three lines of pesticide research: bioassay, ecological studies and physiological studies. Also, we participate in a nationwide monitoring programme.

Lewis, Michael A., David E. Weber and Roman S. Stanley. 1998. Comparative Animal and Plant Toxicities of 10 Treated Effluents Discharged to Near-Coastal Areas of the Gulf of Mexico. Water Environ. Res. 70(6):1108-1117. (ERL,GB 1016).

The chemical quality and acute and chronic toxicities of 10 effluents discharged to near-coastal areas in Northwest Florida were determined using standard and nonstandard toxicity tests. The primary objectives of the study were to evaluate and compare the toxicities of different types of effluents and to assess the ability of a variety of toxicity tests to differentiate effluent-specific effects. Focus was placed on animal-plant sensitivity comparisons because phytotoxicity is rarely determined for effluents discharged to coastal estuaries. The standard toxicity test organisms included two algae, two invertebrates and two fish. In addition, effluent toxicity was evaluated using three rapid bioassays and an early-seedling-growth toxicity test. Most concentrations of potentially toxic inorganic and organic contaminants in the effluents were low, a finding that contrasted with the sometimes elevated biochemical oxygen demand and nutrient concentrations. With the exception of their effects on algae, the toxicities of most effluents were considered moderate. Chronic effects on Ceriodaphnia dubia and Mysidopsis bahia were observed consistently for five effluents; the lowest first-effect levels were typically 35 or 71% effluent. Inhibitory effects on fish and macrophyte seedling growth were infrequent, as were those determined using the three rapid bioassays. Nine of the ten effluents were either phytotoxic or phytostimulatory to algae; first effects were noted in some cases at effluent concentrations less than 6%. Overall, the results indicate the importance of using invertebrates, but more so algae, as test species in the hazard-evaluation process for effluents discharged to Gulf of Mexico near-coastal areas, which are frequently nutrient-affected. Furthermore, additional development of tests using rooted aquatic plants is needed because the effects of effluents in this coastal area on plant-dominated coastal habitats (wetlands) and on plant species at risk (sea grasses) are not well understood.

Nimmo, D.R. and Robbin R. Blackman. 1972. Effects of DDT on Cations in the Hepatopancreas of Penaeid Shrimp. Trans. Am. Fish. Soc. 101(3):547-549. (ERL,GB 117).

We have observed two distinct symptoms of DDT poisoning in penaeid shrimp. In acute bioassays (concentrations of 0.15 parts per billion (ppb) or more), shrimp showed the nervous impairments--tremors, hyperexcitability, and finally paralysis--which are characteristic of arthropods. In chronic tests, when less DDT was used, shrimp became lethargic, refused food, and finally died, but at no time were nervous disorders noted. Analyses of shrimp in all tests showed that shrimp accumulated more DDT in the hepatopancreas than in other organs. We present data which demonstrate that when living shrimp were exposed to DDT, concentrations of some cations in the hepatopacreas became depressed.

Genthner, F.J., C.A. Chancy, J.A. Couch, S.S. Foss, D.P. Middaugh, S.E. George, M.A. Warren and J.A. Bantle. 1998. Toxicity and Pathogenicity Testing of the Insect Pest Control Fungus, Metarhizium anisopliae. Arch. Environ. Contam. Toxicol. 35(2):317-324. (ERL,GB 1027).

Renewed interest in the use of Metarhizium anisopliae and its toxins for instect control prompted the following safety assessment. A neutral extract (methylene chloride, pH 7.2), derived from M.anisopliae cultures, was evaluated for toxicity and mutagenicity using aquatic animal bioassays and by the Ames test. The average LC50 of the neutral extract obtained in static, acute 96-h tests conducted with < or =24-h-old Mysidopsis bahia was 2.41 mg L-1. By partially purifying destruxins from the neutral extract, it was shown that destruxins alone were not responsible for the observed toxicity in mysids. The neutral extract was fetotoxic to developing grass shrimp, Palaemonetes pugio, and frog, Xenopus laevis, embryos; the LC50 values were 52 and 32 mg L-1, respectively. Eye spot abnormalities were observed in shrimp and frog embryos exposed to the neutral extract. In extract-exposed frog embryos, moderate to severe cranial, facial, and gut malformations were also observed. The neutral extract was toxic to juvenile mosquito fish, Gambusia affinis, at an LC50 value of 141 mg L-1. Adult female G. affinis surviving a 24-hour exposure to 200 µg ml-1 of the neutral extract produced healthy broods. After three months, no mortalities or adverse effects were observed in adult G. affinis fed a diet partially composed of a freeze-dried M. anisopliae culture. The neutral extract did not show mutagenicity in the Ames test using strains TA98 and TA100 with and without metabolic activation by rat liver S9. By partially purifying destruxins from the neutral extract, it was shown that destruxins alone were not responsible for the observed toxicity in mysids. Significant (p 0.05) mortalities or malformations.

Butler, P.A. 1971. Influence of Pesticides on Marine Ecosystems. Proc. R. Soc. Lond. B Biol. Sci. 177:321-329. (ERL,GB 129).

A bioassay programme undertaken in 1958 has evaluated the toxicity of about 240 pesticides to estuarine fauna. Studies indicate that chronic levels of sublethal amounts of pesticides may have more damaging effects than transitory changes due to acutely toxic levels of pollution. The first five years of a programme monitoring the incidence of synthetic pesticide residues in populations of North American shellfish has been completed. The results demonstrate the ubiquity of DDT and its metabolites. Levels of contamination, however, are not high enough to indicate a human health problem. The run-off of surface waters from agricultural districts is indicated as the chief source of this type of pollution; municipal and industrial wastes, and the control of noxious insects are regionally important sources. Observations of laboratory populations experimentally contaminated with DDT indicate, by extrapolation, that pesticide pollution is causing significant changes in mortality, growth rates, or resistance to disease in some marine populations.

Butler, P.A., L.E. Andren, G.J. Bonde, A.B. Jernelov and D.J. Reish. 1972. Test, Monitoring and Indicator Organisms. In: Guide to Marine Pollution. Edward D. Goldberg, Editor. Gordon and Breach, London. Pp. 146-159. (ERL,GB 148).

This material has been prepared to enable the investigator to select bioassay organisms which will be most useful for the detection and evaluation of pollution. Ideally, the selected species or community of different species would reflect not only the presence or absence of specific pollutants but also relative pollution levels and their periodic fluctuations, and perhaps identify factors other than chemical that contribute to environmental degradation. The species selected should be of value in circumscribed geographic locations as well as in larger water areas. Such an ideal type does not exist, of course, and bioassay organisms may be grouped functionally into two general categories as either monitoring or indicator types. The demarcation between these two is not always sharp and there are numerous exceptions to the following generalized definitions.

Butler, Philip A. 1972. DDT in Estuarine Molluscs. BioScience. 22(12):690-691. (ERL,GB 155A).

One segment of the National Monitoring Program has been concerned with organochlorine pollution in estuaries. A summary of the data collected in 1965-68 was published in 1969 (BioScience, 19:389). That report discussed the usefulness of pelecypod molluscs (oysters, clams, mussels) as bioassay tools because of their sensitivity to these pollutants at parts-per-trillion levels and because tissue residues were quickly flushed away when the pollution was interrupted for periods as short as 2 weeks. This monitoring program was terminated in June 1972. A manascript discussing the results and including the analytical data of the 8095 samples has been submitted for publication to the Pesticides Monitoring Journal. The overall conclusions of this program are of sufficient general interest to warrant this brief comment.

Lewis, M.A., D.E. Weber and R.S. Stanley. 2000. Wetland Plant Seedlings as Indicators of Near-Coastal Sediment Quality: Interspecific Variation. EPA/600/J-00/. Mar. Environ. Res. 50(1-5):535-540. (ERL,GB 1068). (PRIMO 10)

The toxicities of whole sediments collected from an urbanized Florida bayou-estuary were determined for the epibenthic mysid, Mysidopsis bahia, and the infaunal amphipod, Ampelisca abdita. In addition, the phytotoxicities of the same sediments were evaluated using rooted macrophytes, Scirpus robustus Pursh and Spartina alterniflora Loisel. The results of the 24 bioassays conducted for 7-28 days, were compared for interspecific differences. Toxicity to the benthic invertebrate species was predicted to be likely in all cases, based on comparison to effects-based sediment quality assessment guidelines. However, acute toxicity was uncommon and occurred at only one of the six sampling stations. In contrast, several sediments were either significantly phytostimulatory or phytoinhibitory (P<0.05) relative to a reference estuarine sediment. The results show the importance of considering interspecific variation and phytoassessment in contaminated sediment evaluations. Phytoassessment, the focus of this research, provided useful information that would have been missing if only the animal test species were used. For this reason, the phytotoxicity data base needs to be increased to better evaluate the relevance of most current contaminated sediment evaluations for coastal areas and also to determine the realism of proposed numerical sediment quality assessment guidelines and criteria.

Hansen, D.J., P.R. Parrish and J. Forester. 1974. Aroclor 1016: Toxicity to and Uptake by Estuarine Animals. Environ. Res. 7(3):363-372. (ERL,GB 172).

Bioassays were conducted to determine the acute toxicities of the polychlorinated biphenyl (PCB) Aroclor 1016 in flowing sea water to American oysters (Crassostrea virginica), brown shrimp (Penaeus aztecus), grass shrimp (Palaemonetes pugio), and pinfish (Lagodon rhomboides), and to determine its chronic toxicity to, and uptake and retention by pinfish. Acute 96-hour EC50's or LC50's were: oysters, 10.2 u/liter; brown shrimp, 10.5 ug/liter; grass shrimp, 12.5 ug/liter. The PCB was not toxic to pinfish at 100 ug/liter for 96 hours, but significant mortality occurred when pinfish were exposed to 32 ug/liter of Aroclor 1016 for 42 days. Pinfish exposed to 1 ug/liter for 56 days accumulated the chemical with maximum concentrations attained in whole-fish by 21 to 28 days. Maximum whole-body residue (wet-weight) was 17,000 X the normal concentration in test water. Tissue alterations, such as severe vacuolation in the pancreatic exocrine tissue surrounding the portal veins, occurred in pinfish exposed to 32 ug/liter of Aroclor 1016 for 42 days.

Schimmel, Steven C., David J. Hansen and Jerrold Forester. 1974. Effects of Aroclor 1254 on Laboratory-Reared Embryos and Fry of Sheepshead Minnows (Cyprinodon variegatus). Trans. Am. Fish. Soc. 103(3):582-586. (ERL,GB 175).

Eggs of the sheepshead minnow (Cyprinodon variegatus) were artificially fertilized and maintained at temperatures from 15° to 35°C and in salinities from 0 to 35 o/oo to determine efficient culture conditions. Fertilization was not affected by temperature or salinity ranges chosen, but hatching success was greatest (x2; a=0.01) at a temperature range of 24° to 35°C and a salinity range of 15 to 30 o/oo. Artificially fertilized sheepshead minnow eggs were exposed to logarithmic concentrations of Aroclor® 1254 (10.0 to 0.1 µg/liter) in seawater averaging 30°C and 24 o/oo in a flow-through bioassay. Fertilization was not affected but significantly fewer embryos developed in the 10.0 µg/liter concentration, and fewer fry survived in concentrations greater than 0.1 µg/liter. Fry were more susceptible to Aroclor® 1254 than were embryos, juveniles, or adults.

Borthwick, Patrick W., Marlin E. Tagatz and Jerrold Forester. 1975. Gravity-Flow Column to Provide Pesticide-Laden Water for Aquatic Bioassays. Bull. Environ. Contam. Toxicol. 13(2):183-187. (ERL,GB 189).

Concentrations of mirex among individual tanks in each test were not statistically different at the 5-percent significance level; whereas, differences in mirex concentrations in tank water among experiments were significant. Paired comparisons indicated statistical differences between the first and second, and the second and third experiments, but not between the first and third experiment. These differences in mean mirex concentrations in tank water may have been caused by seasonal variations in water temparature. Fluctuations in the mirex concentrations within individual tanks were not significant. In its present state of development, the described gravity-flow column is being utilized in seasonal tests to deliver mirex-laden water to determine toxicity and uptake of mirex by several animal species in an artificial estuarine ecosystem.

Hemmer, Michael J., Becky L. Hemmer, Chris J. Bowman, Kevin J. Kroll, Leroy C. Folmar, Dragoslav Marcovich, Marilynn D. Hoglund and Nancy D. Denslow. 2001. Effects of p-Nonylphenol, Methoxychlor and Endosulfan on Vitellogenin Induction and Expression in the Sheepshead Minnow, Cyprinodon variegatus. Environ. Toxicol. Chem. 20(2):336-343. (ERL,GB 1098).

Temporal and dose-response relationships of vitellogenin (VTG) mRNA induction and subsequent plasma VTG accumulation were established for sheepshead minnows (Cyprinodon variegatus) treated with p-nonylphenol (an alkylphenol) and the organochlorine pesticides methoxychlor and endosulfan. Thirty-two adult male fish per treatment were continuously exposed to measured concentrations of 0.64, 5.4, 11.8, 23.3 and 42.7 µg/L p-nonylphenol; 1.1, 2.5, 5.6, 12.1 and 18.4 µg/L methoxychlor; and in two separate tests, 15.9, 36.3, 68.8, 162, 277, 403, 590 and 788 ng/L endosulfan using an intermittent flow-through dosing apparatus. Separate triethylene glycol (50 µl/L) and 17b-estradiol (65.1 ng/L) treatments served as the negative and positive controls, respectively. Four fish were randomly sampled from each test concentration on days 2, 5, 13, 21, 25, and 42 of exposure and levels of hepatic VTG mRNA induction and serum VTG accumulation were determined for each individual. Overall, fish exposed to p-nonylphenol or methoxychlor demonstrated a rapid, dose-dependent synthesis of VTG mRNA up to day 5 of exposure, followed by a relatively constant dose-dependent expression through day 42. Both chemicals showed a dose-dependent increase in plasma VTG over the entire time course of exposure, with significantly elevated VTG levels by the fifth day of exposure to p- nonylphenol at concentrations of 5.4 µg/L or greater and to methoxychlor at concentrations of 2.5 µg/L or greater. Exposure to 0.64 µg/L p-nonylphenol resulted in highly variable plasma VTG levels of less than 6 mg/ml. Exposures with endosulfan failed to induce measurable levels of either hepatic VTG mRNA or serum VTG at the chemical concentrations tested. Our results demonstrate that the sheepshead minnow bioassay is a suitable estuarine/marine teleost model for in vivo screening of potentially estrogenic substances.

Lewis, Michael A., Steven S. Foss, Peggy S. Harris, Roman S. Stanley and James C. Moore. 2001. Sediment Chemical Contamination and Toxicity Associated with a Coastal Golf Course Complex. Environ. Toxicol. Chem. 20(7):1390-1398. (ERL,GB 1099).

The increasing density of golf courses represents a potential source of sediment contamination to nearby coastal areas, the chemical and biological magnitude of which is almost unknown. The objective of this study was to determine the concentrations of contaminants and toxicities of sediments impacted by a coastal golf course complex. Sediment contaminant concentrations were determined at least twice during the two-year study period at 14 sampling stations. In addition, a combination of acute and chronic bioassays were conducted exposing four invertebrate test species to whole sediments and associated pore waters. Overall, the Florida, USA, golf course complex had a measurable impact on sediment chemical quality, particularly in near-field areas. Higher concentrations of several trace metals and organochlorine pesticides were detected in many golf course-associated sediments compared to reference areas; however, concentrations decreased seaward and only a few, primarily chlorinated pesticides, exceeded proposed sediment quality guidelines. Chromium, zinc and mercury were detected more frequently than other trace metals. The DDT and associated metabolites, dieldrin and chlordane were the more commonly detected organic contaminants. Acute toxicity was uncommon and occurred consistently for sediment collected from one coastal location. In contrast, chronic toxicity occurred at several study sites based on the response of Mysidopsis bahia. It was concluded that the impact of golf course runoff on sediment quality may be subtle and sensitive biological assessment methods, such as chronic toxicity tests, will be needed to determine adverse effects.

Lewis, M.A., D.E Weber, R.S. Stanley and J.C. Moore. 2001. Relevance of Rooted Vascular Plants as Indicators of Estuarine Sediment Quality. Arch. Environ. Contam. Toxicol. 40(1):25-34. (ERL,GB 1100).

Toxicity assessments and numerical quality assessment guidelines for estuarine sediments are rarely based on information for aquatic plants. The effect of this lack of information on contaminated sediment evaluations is largely unknown. For this reason, the toxicities of whole sediments collected from 15 sites in three urbanized Florida bayou-estuaries were determined for the benthic invertebrates, Mysidopsis bahia, and Ampelisca abdita and the plants Scirpus robustus Pursh (saltmarsh bulrush) and Spartina alterniflora Loisel (saltmarsh cordgrass). The results of the bioassays, conducted for 7 to 28 days, were compared for interspecific differences and to effects-based, sediment quality assessment guidelines. A variety of inorganic and organic analytes were detected in the estuarine sediments and concentrations of as many as 7 analytes exceeded the sediment guidelines at the 15 sampling locations. Toxicity occurred at 2 of the 15 sampling stations based on invertebrate survival. Twelve of the 15 sediments had either a significant stimulatory or inhibitory effect on early seedling growth relative to a reference sediment (P<0.05) The phytoresponse was specific to the location, test species and plant tissue. There was no consistent trend between the sensitivities of the plants and invertebrates exposed to the sediments collected from the same sites. Of the 12 sediments that significantly affected seedling growth, 10 were not acutely toxic to the invertebrates. Consequently, the plant test species provided information that would have been missing if only animal test species were used. For this reason, the phytotoxicity data base needs to be expanded for contaminated sediments to further evaluate interspecific sensitivities, and to provide perspective on the environmental relevancy of proposed sediment quality criteria and effects-based assessment guidelines for which this information is usually missing. However, additional test method development and field validation are needed to support this effort, which includes the identification of sensitive plant test species, response parameters, and the chemical and physical sediment factors that influence plant growth.

Folmar, Leroy C., Michael J. Hemmer, Nancy D. Denslow, Kevin Kroll, Jian Chen, Ann Cheek, Harold Richman, Hillary Meredith and E. Gordon Grau. 2002. Comparison of the Estrogenic Potencies of Estradiol, Ethynylestradiol, Diethylstilbestrol, Nonylphenol and Methoxychlor In Vivo and In Vitro. EPA/600/J-00/470. Aquat. Toxicol. 60(1-2):101-110. (ERL,GB 1123).

Five natural, pharmaceutical, or xenobiotic chemicals (17b-estradiol (E2), ethynylestradiol (EE2), diethystilbestrol (DES), methoxychlor (MXC), nonylphenol(NP)) were tested in two in vitro assays (yeast estrogen screen [YES], MCF-7 breast tumor cell proliferation (E-screen)], and compared with previously reported results from two in vivomale sheepshead minnow vitellogenin (VTG) production studies. The purpose of this investigation was to determine how accurately the two in vitro assays predicted responses observed in live animals. EC50 values for all five chemicals were approximately one order of magnitude less sensitive in the YES assay than in the MCF-7 assay. Based on the EC50 values, DES was 1.1 (YES) to 2.5 (MCF-7) times more potent in these receptor binding assays than was E2, while EE2 was slightly less potent than E2 in the YES assay (0.7) and nearly twice as potent (1.9) as E2 in the MCF-7 assay. EE2 and DES were of approximately equal potency in the 13-day sheepshead minnow VTG production bioassay. Both MXC and NP were 107 times less potent than E2 in the YES assay, MXC was 105 times less estrogenic than E2 in the MCF-7 assay, while both were approximately 100 times less potent than E2 in the live animal bioassay. The in vitro tests were substantially less sensitive (at least 1000 times) than the sheepshead minnow VTG assay for estimating estrogenic potency of the two xenobiotic chemicals, which suggests that in vitro-based, large-scale screening programs could potentially result in many false negative evaluations.

Murrell, Michael C., Roman S. Stanley, Emile M. Lores, Guy T. DiDonato, Lisa M. Smith and David A. Flemer. 2002. Evidence That Phosphorus Limits Phytoplankton Growth in a Gulf of Mexico Estuary: Pensacola Bay, FL, USA. EPA/600/J-01/437. Bull. Mar. Sci. 70(1):155-167. (ERL,GB 1131).

Nutrient limitation bioassays were conducted on six dates from November 1998 to September 1999 at two sites, including oligohaline (Upper Bay) and mesohaline regions (Lower Bay), in Pensacola Bay, FL. Phytoplankton growth responses (measured as changes in chlorophyll a concentration) to inorganic nitrogen (N) and phosphorus (P) additions were monitored for three days. The results showed that, in 8 of 12 experiments, phytoplankton growth was stimulated by P additions in comparison with N-amended and un-amended treatments. Nitrogen additions alone did not stimulate growth over P additions in any experiment. The spatial patterns suggest that potential for P limitation was similar in Upper and Lower Bays.. The four experiments with statistically non-significant results were all conducted during winter and spring, suggesting a lower potential for nutrient limitation during the cooler months when nutrient demand (i.e. productivity) is typically low and nutrient supply (i.e. freshwater runoff) is typically high. This study adds to a small but growing literature suggesting that P limitation of phytoplankton growth may be relatively common in warm temperate estuarine systems such as those along the Gulf of Mexico coast.

McKenney, C.L., Jr., G.M. Cripe, S.S. Foss, S.R. Tuberty and M. Hoglund. 2004. Comparative Embryonic and Larval Developmental Responses of Estuarine Shrimp (Palaemonetes pugio) to the Juvenile Hormone Agonist, Fenoxycarb. Arch. Environ. Contam. Toxicol. 47(4):463-470. (ERL,GB 1166).

Grass shrimp (Palaemonetes pugio) were reared separately through both embryonic and total larval development during exposure to fenoxycarb at measured concentrations of <2.2 to 888 µg L-1. A fenoxycarb concentration of 888 µg L-1 significantly (p<0.05) inhibited embryonic development to larval hatching and extended the embryonic developmental period from 11.9 to 12.7 days. Exposure to fenoxycarb concentrations less than or equal to 502 µg L-1 had no significant (p>0.05) effect on complete embryonic development. Significantly fewer shrimp successfully metamorphosed to postlarvae while exposed through complete larval development to fenoxycarb concentrations greater than or equal to 4 µg L-1. Larval development of grass shrimp was, therefore, greater than two orders of magnitude more sensitive to this juvenile hormone agonist than was embryonic development. Viability of larvae developing in fenoxycarb was concentration dependent. Development beyond third zoeal stage was significantly inhibited at fenoxycarb concentrations greater than or equal to 190 µg L-1, whereas development beyond fourth zoeal stage was inhibited by greater than or equal to 45 µg L-1. Fenoxycarb exposure of developing larvae did not alter either the duration of total larval development or the total number of larval stages before metamorphosis. Rearing of fenoxycarb-exposed embryos through larval development without further exposure had no significant effect on number of larval stages, larval development rate, or metamorphic success of larvae. Similarities in the sensitivity of grass shrimp larvae and mosquito larvae to fenoxycarb suggests that the use of a bioassay protocol measuring the metamorphic success of crustacean larvae would be a valuable adjunct to the hazard assessment of newly developed pesticides which target endocrine control of metamorphosis in insects and possibly other endocrine-disrupting xenobiotics as well.

Vogelbein, Wolfgang K., Vincent J. Lovko, Jeffrey D. Shields, Kimberly S. Reece, Patrice L. Mason, Leonard W. Haas and Calvin C. Walker. 2002. Pfiesteria shumwayae Kills Fish by Micropredation Not Ecotoxin Secretion. EPA/600/J-02/378. Nature. 418(6901):967-970. (ERL,GB 1170).

Pfiesteria piscicida and P.shumwayae reportedly secrete potent exotoxins thought to cause fish lesion events, acute fish kills and human disease in mid-Atlantic USA estuaries. However, Pfiesteria toxins have never been isolated or characterized. We investigated mechanisms by which P. shumwayae kills fish using three different approaches. Here we show that larval fish bioassays conducted in tissue culture plates fitted with polycarbonate membrane inserts exhibited mortality (100%) only in treatments where fish and dinospores were in physical contact. No mortalities occurred in treatments where the membrane prevented contact between dinospores and fish. Using differential centrifugation and filtration of water from a fish-killing culture, we produced "dinoflagellate", "bacteria" and "cell-free" fractions. Larval fish bioassays of these fractions resulted in mortalities (60-100% in less than 24 h) only in fractions containing live dinospores ("whole water", dinoflagellate"), with no mortalities in "cell-free" or "bacteria"-enriched fractions. Video-micrography and electron microscopy show dinospores swarming toward and attaching to skin, actively feeding, and rapidly denuding fish of epidermis. We show here that our cultures of actively fish-killing P. shumwayae do not secrete potent exotoxins; rather, fish mortality results from micropredatory feeding.

Salinas, K. A., S. L. Edenborn, A. J. Sexstone and J. B. Kotcon. 2007. Bacterial Preferences of the Bacterivorous Soil Nematode Cephalobus brevicauda (Cephalobidae): Effect of Bacterial Type and Size. Pedobiologia. 51(1):55-64. (ERL,GB 1272). (Cephalobidae)

Cell size and type may affect availability of bacteria for consumption by bacterivorous nematodes in the soil and in culture. This study explored the feeding preferences of the bacterivorous soil nematode Cephalobus brevicauda (Cephalobidae) by comparing bacteria isolated directly from soil, from commercial bacterial biological control agents, and from in vitro soil nematode cultures. The 16S rRNA sequences of bacterial isolates were compared to known sequences to identify phylogenetic affiliations. Food preference of the nematode was observed by counting the number of C. brevicauda that were attracted to a bacterial food source in a bioassay. Additionally, the reproductive output of single females reared on the bacteria was recorded. An attraction index established that C. brevicauda preferred to feed upon gram-negative, small-volume bacteria, however these bacteria did not support maximum reproductive success. Bacterial biocontrol agents had no negative effects on reproduction, but C. brevicauda always preferred other bacteria to Dipel®, (Bacillus thuringiensis ssp. kurstaki) in the food preference assay.

Juhl, Andrew R. and Michael C. Murrell. 2008. Nutrient Limitation of Phytoplankton Growth and Physiology in a Subtropical Estuary (Pensacola Bay, Florida). Bull. Mar. Sci. 82(1):59-82. (ERL,GB 1309).

Phytoplankton nutrient limitation was studied in a sub-estuary of lower Pensacola Bay using several techniques. Nutrient—addition bioassays indicated year-round nutrient limitation, in contrast to seasonal patterns often described for higher-latitude estuaries. Although an earlier study found frequent P-limitation in Pensacola Bay, N-limitation dominated during this study, despite dissolved inorganic N:P ratios consistently above Redfield proportions. However, combined N and P additions enhanced growth more than single nutrients, indicating incipient co-limitation. Variability of in-situ alkaline phosphatase (Pase) activity was not clearly related to the inorganic nutrient concentrations or nutrient ratios. However, changes in Pase activity after nutrient additions were consistent within the bioassays. Despite evidence for nutrient limitation, the maximum quantum yield of photosystem II photochemistry (Fv/Fm) was generally high, indicating that nutrient limitation was not severe enough to reduce photosynthetic efficiency. Techniques like Pase activity and Fv/Fm measure the physiological state of the in situ phytoplankton community, while nutrient-addition bioassays test whether community production and yield could change with additional nutrient inputs. This distinction explains why phytoplankton in this environment evidenced nutrient limitation without apparent physiological impairment. From this perspective, the results indicate that Pensacola Bay would be very sensitive to increased N and P loads, causing strongly increased phytoplankton production and yield (i.e. eutrophication) with potentially negative ecosystem consequences.

Schimmel, Steven C. and David J. Hansen. 1975. Sheepshead Minnow (Cyprinodon variegatus): An Estuarine Fish Suitable for Chronic (Entire Life-Cycle) Bioassays. In: Proc. 28th Annu. Conf. Southeast. Assoc. Game Fish Comm. Pp. 392-398. (ERL,GB 205).

The sheepshead minnow (Cyprinodon variegatus) an estuarine fish of the Atlantic and Gulf coasts, is suitable for both partial chronic and chronic (egg-to-egg) bioassays. The fish is easily held at high population densities in the laboratory and, at about 30°C, produces numerous eggs. The average 30-day survival of the fish from fertile egg to fry is 75%. Generation time for this species is short (3-4 months) and its small adult size (male average standard length = 48mm) provides for relatively inexpensive bioassays. This killifish's susceptibility to organochlorine toxicants is similar to that of other estuarine fishes tested and thus should produce significant information on the effects of these toxicants on the estuarine community.

Hansen, David J., Steven C. Schimmel and Jerrold Forester. 1975. Effects of Aroclor 1016 on Embryos, Fry, Juveniles, and Adults of Sheepshead Minnows (Cyprinodon variegatus). Trans. Am. Fish. Soc. 104(3):584-588. (ERL,GB 206).

We investigated the toxicity of Aroclor 1016 to, and uptake by, fry and juvenile and adult sheepshead minnows (Cyprinodon variegatus) in intermittent-flow bioassays lasting 28 days. Survival of eggs, of fry hatched from them, and of juvenile and adult fish apparently was not affected by 0.1, 0.32, 1.0, 3.2, or 10 µg/liter of Aroclor 1016 added to aquaria, but 32 and 100 µg/liter killed newly hatched fry and juvenile and adult fish. Sheepshead minnows accumulated the chemical in proportion to its concentration in the test water. Fry contained 2,500 to 8,100 X the concentration of Aroclor 1016 added to the test water, adults 4,700 to 14,000 X, and juveniles 10,000 to 34,000 X. As much as 77 µg/g of Aroclor 1016 in eggs from exposed adults apparently did not affect survival of embryos and fry.

Schoor, W. Peter. 1974. Theoretical Model and Solubility Characteristics of Aroclor 1254 in Water: Problems Associated with Low-Solubility Compounds in Aquatic Toxicity Tests. EPA-660/3-74-013. U.S. Environmental Protection Agency, Cincinnati, OH. 30 p. (ERL,GB 208). (Avail. from NTIS, Springfield, VA: PB-240 550)

A theoretical model of the behavior of substances having low water-solubility is presented and discussed with respect to aqueous bioassay. Ultracentrifugal techniques were used in an attempt to study size distributions of Aroclor® 1254 aggregates in aqueous emulsions. Results indicate strong adsorption from emulsion by surfaces and a water-solubility at 20°C of less than 0.1 µg l-1 in distilled water and approximately 40% of that value in water containing 30 g l-1 NaCl. Implications with regard to aqueous bioassay are discussed.

Schoor, W.P. 1975. Theoretical Model and Solubility Characteristics of Aroclor 1254 in Water: Problems Associated with Low-Solubility Compounds in Aquatic Toxicity Tests. Water Res. 9(11):937-944. (ERL,GB 208A).

A theoretical model of the behavior of substances having low water-solubility is presented and discussed with respect to aqueous bioassay. Ultracentrifugal techniques were used in an attempt to study size distributions of Aroclor® 1254 aggregates in aqueous emulsions. Results indicate strong adsorption from emulsion by surfaces and a water-solubility at 20° C of less than 0.1 µg/l in distilled water and approximately 40% of that value in water containing 30 g/l NaCl. Implications with regard to aqueous bioassay are discussed.

Bahner, Lowell H. and DelWayne R. Nimmo. 1975. Salinity Controller for Flow-Through Bioassays. Trans. Am. Fish. Soc. 104(2):388-389. (ERL,GB 214).

An electro-mechanical device has been constructed to monitor and dilute seawater to a constant salinity for flowing-water bioassays. It has been used successfully in pesticide bioassays and requires little maintenance.

Schimmel, Steven C., Patrick R. Parrish, David J. Hansen, James M. Patrick, Jr. and Jerrold Forester. 1975. Endrin: Effects on Several Estuarine Organisms. In: Proc. 28th Annu. Conf. Southeast. Assoc. Game Fish Comm. Pp. 187-194. (ERL,GB 218).

Acute (96-hour) bioassays were performed with endrin and the following estuarine organisms: American oyster (Crassostrea virginica), pink shrimp (Penaeus duorarum), grass shrimp (Palaemonetes pugio), sailfin molly (Poecilia latipinna) and sheepshead minnow (Cyprinodon variegatus). Endrin was acutely toxic to all organisms tested, except oysters, whose shell growth was appreciably inhibited by 56 µg/l (parts per billion) of the chemical. Pink shrimp were the most sensitive animal tested, but significant numbers of both species of shrimps and fishes died when exposed to concentrations of one µg/l or less. In a separate test, embryos and fry of the sheepshead minnow were exposed to concentrations of endrin ranging from 0.046 to 1.0 µg/l (nominal) for 33 days in an intermittant-flow bioassay. Embryos were not affected by the concentrations to which they were exposed, but the estimated LC50 (probit analysis, a=.05) of fry was 0.27 µg/l.

Schimmel, Steven C. and David J. Hansen. 1975. Automatic Brine Shrimp Feeder for Aquatic Bioassays. J. Fish. Res. Board Can. 32(2):314-316. (ERL,GB 224).

An electrically operated brine shrimp feeder is described. The device may be set to cycle 1-12 times each day for tests in fish and invertebrate culture and bioassay. Major advantages of the feeder are that it is readily adapted to flow-through bioassay and culture apparatuses which require that equal quantities of food be delivered to animals in two or more test aquaria and that the number of feedings be recorded. The components, all readily available, cost approximately $190.

Middaugh, Douglas P. and John M. Dean. 1977. Comparative Sensitivity of Eggs, Larvae and Adults of the Estuarine Teleosts, Fundulus heteroclitus and Menidia menidia to Cadmium. Bull. Environ. Contam. Toxicol. 17(6):645-652. (ERL,GB 231).

The present study measured the sensitivity of two common species of estuarine fish, the mummichog, Fundulus heteroclitus, and the Atlantic silverside, Menidia menidia, to cadmium at specific stages in their life histories. Developing eggs were tested because of the proven sensitivity of this life stage to cadmium toxicity in marine fish. Three age groups of larvae, 1-, 7- and 14-days old were tested to determine if changes in sensitivity occur during the first few weeks after emergence. Bioassays were also conducted with adults for comparison with data from egg and larval bioassays. The duration of tests was limited to 48 hrs so that comparisons of sensitivity to cadmium could be made for specific developmental stages (ages) of each species.

Bahner, L.H., C.D. Craft and D.R. Nimmo. 1975. Saltwater Flow-Through Bioassay Method with Controlled Temperature and Salinity. Prog. Fish-Cult. 37(3):126-129. (ERL,GB 239).

For several years, researchers at the Gulf Breeze Environmental Research Laboratory have been refining techniques for the flow-through bioassay, a testing method in which a continuous supply of natural seawater flows through experimental tanks. The flow-through bioassay offers many advantages over static exposure methods. Continuously flowing seawater simulates more closely the natural estuarine or marine environment, substantially reducing problems associated with static methods such as poor mixing of toxicants, death of experimental animals from anoxia, adsorption of toxicant to sediments and to walls of exposure tanks, and excess growth of microorganisms. Temperature and salinity affect bioassays. For example, in a freshwater study, the combination of the pesticide dieldrin and thermal changes reduced survival of the darter, Etheostoma nigrum. Temperature and salinity stress increased mortality in fiddler crabs, Uca pugilator, exposed to mercury. Similarly, pink shrimp, Penaeus duorarum, previously exposed to sublethal concentrations of the polychlorinated biphenyl Aroclor® 1254, died when the salinity was gradually lowered from 20 o/oo to approximately 12 o/oo. If results of toxicity tests are to be confirmed, identical test conditions must be repeated. It is important, therefore, to control temperature and salinity in a flow-through bioassay. Finally, the ability to control temperature and salinity facilitates studies of toxicant and environmental stress interactions. Our flow-through system has been used extensively in bioassays with the pink shrimp, Penaeus duorarum, as well as grass shrimp, Palaemonetes vulgaris and P. pugio. The pink shrimp is a valuable commercial species and both pink and grass shrimp are integral parts of both estuarine and marine food webs. Although the method described here deals with shrimp, this flow-through bioassay method, with minor modifications, is readily adaptable to a wide variety of estuarine and marine macroinvertebrates. The cost of this system for a laboratory with flowing seawater would be approximately $1,500, an amount within the means of many research budgets.

Bahner, Lowell H. and DelWayne R. Nimmo. 1976. Precision Live-Feeder for Flow-Through Larval Culture or Food Chain Bioassays. Prog. Fish-Cult. 38(1):51-52. (ERL,GB 246).

This report describes an inexpensive automatic feeder that features precise timing of a wide choice of food delivery periods and time intervals between food delivery. The feeder can also control simultaneous delivery of a variety of foods, and is compatible for use in flow-through water systems. Although several automatic feeders have been described for culturing fish and crustaceans (Benoit et al. 1969; Serfling et al. 1974; Schimmel and Hansen 1975), we believe that ours is more versatile.

Couch, John, George Gardner, John C. Harshbarger, M.R. Tripp and Paul P. Yevich. 1974. Histological and Physiological Evaluations in Some Marine Fauna. In: Marine Bioassays. Marine Technology Society, Washington, DC. Pp. 156-173. (ERL,GB 250).

The development of pathology, as applied to aquatic toxicology, depends heavily on the knowledge of normal histology and physiology if anomalies, due to pollutants or disease, are to be accurately defined. However, at present, knowledge of normal morphology or metabolic activities is either incomplete or lacking for most marine or coastal organisms. Obviously, well-coordinated efforts will be required to characterize normal ranges and interpret the morphological or physiological responses of aquatic organisms to various factors including pollutants. Most laboratories cannot justify well-defined pathological units, although the need often arises through governmental enforcement organizations and other activities. The techniques and examples thus presented are intended to offer some means of obtaining evaluations of aquatic organisms' well-being or disorders.

Atema, Jelle, C.C. Coutant, P. Decoursey, D. Hansen, J.S. Kittredge, J.J. Magnuson, D. Miller, B.L. Olla, M.J. Schneider and W.B. Vernberg. 1974. Behavioral Bioassays. In: Marine Bioassays. Marine Technology Society, Washington, DC. Pp. 1-31. (ERL,GB 251).

Until fairly recently, water quality bioassay techniques have been limited to observations of the lethal concentrations of a pollutant. Such measures as LD50 , TLm50, and LC50 were, and still are, commonly used to assess the acute effect of a pollutant, usually based on the mortality of adult organisms. While this approach was probably inevitable as a step in the evolution of both the philosophy and techniques of bioassaying, it has become abundantly clear that this concept has serious limitations as a measure of the effects of a pollutant on the environment. The need for additional, more comprehensive measures of organismic response to contaminants has stimulated the search for new testing techniques in a variety of disciplines, including behavior. The most important advantage to using behavior as a tool to measure stress is that the results of behavioral tests often lend themselves to a direct interpretation regarding environmental quality as related to possible consequences at the population and ecosystem levels. Also complex biochemical and physiological responses of an organism may be reflected in rather easily observable acts. Although behavioral measures may suffer in regard to quantification because of the high degree of inherent variation, they are highly sensitive to stress. The general aim of the workshop was to explore various aspects of applying behavioral measures to bioassay. Although in a few instances the use of behavioral bioassays has reached the standard test stage, the state of the art is still very young. Consequently, the scope of the discussion was intended as a beginning toward integrating a variety of basic research techniques into logical steps towards developing standard tests.

Bahner, Lowell H. and DelWayne R. Nimmo. 1975. Methods to Assess Effects of Combinations of Toxicants, Salinity and Temperature on Estuarine Animals. In: Trace Elements in Environmental Health, IX: Proceedings. D.D. Hemphill, Editor. University of Missouri, Columbia, MO. Pp. 169-177. (ERL,GB 259).

Aquatic species are exposed to toxicants singly, but more often in combinations, under varying environmental regimes. Consequently, an experimental flowing-water bioassay system was developed that controls salinity and temperature while testing toxicants either singly or in combination. Obvious advantages of this control were that rates of toxicant accumulation, translocation, loss or acute and chronic toxicity to animals could be better assessed and repeated. Our bioassays were conducted with pink shrimp (Penaeus duorarum) exposed to the following toxicant combinations: cadmium-malathion, cadmium-methoxychlor, cadmium-methoxychlor-Aroclor® 1254 and a complex industrial waste which contained both inorganic and organic constituents. The toxicities of the pesticide-metal combinations, when compared to that of each constituent singly, appeared to be independent of each other.

Schimmel, Steven C., James M. Patrick, Jr. and Jerrold Forester. 1976. Heptachlor: Uptake, Depuration, Retention, and Metabolism by Spot, Leiostomus xanthurus. J. Toxicol. Environ. Health. 2(1):169-178. (ERL,GB 264).

The estuarine fish, spot (Leiostomus xanthurus), was exposed to 0.27, 0.52, 1.01, 1.99, and 3.87 µg/liter technical grade heptachlor (65% heptachlor, 22% trans-chlordane, 2% cis-chlordane, 2% nonachlor, and 9% unidentified compounds) for 24 days in a flowthrough bioassay, followed by 28 days in heptachlor-free seawater. Concentrations of heptachlor, heptachlor epoxide, and trans- and cis -chlordane in edible tissues were monitored at day 3 and weekly thereafter throughout the bioassay and at the end of the postexposure period. All four chemicals were accumulated by spot. Maximum concentrations of heptachlor were observed on day 3; maximum concentrations of the other three compounds were observed on day 17. The average bioconcentration factors for heptachlor and trans-chlordane were 3,600 and 4,600 respectively. Only 10% or less of the maximum concentrations of heptachlor, heptachlor epoxide, and trans-chlordane accumulated during the exposure period remained after 28 days in pesticide-free seawater; an average of 35% of the cis-chlordane remained. Relative total amounts of heptachlor and cis-chlordane changed during the exposure and postexposure periods. Nearly all of the heptachlor was eliminated or metabolized to its epoxide. Cis-chlordane, which averaged 4-7% of the total residues (chlordanes and heptachlors) in edible tissues during the exposure, increased to 18-23% of the total residues by the end of the postexposure period.

Schimmel, Steven C., James M. Patrick, Jr. and Jerrold Forester. 1976. Heptachlor: Toxicity to and Uptake by Several Estuarine Organisms. J. Toxicol. Environ. Health. 1(6):955-965. (ERL,GB 265).

Technical-grade heptachlor (65% heptachlor, 22% trans-chlordane, 2% cis -chlordane, and 2% nonachlor) was tested in 96-hr bioassays to determine its toxicity to estuarine animals. The test organisms and the 96-hr LC50 or EC50s (based on measured concentrations in water) are as follows: American oyster (Crassostrea virginica), 1.5 µg/liter; pink shrimp (Penaeus duorarum), 0.11 µg/ liter; grass shrimp (Palaemonetes vulgaris), 1.06 µg/liter; sheepshead minnow (Cyprinodon variegatus), 3.68 µg/liter; pinfish (Lagodon rhomboides), 3.77 µg/ liter; and spot (Leiostomus xanthurus), 0.85 µg/liter. Analytical-grade heptachlor (99.8% heptachlor) and heptachlor epoxide (99%) were also studied. The analytical-grade heptachlor 96-hr LC50 for pink shrimp and spot was 0.03 µg/ liter and 0.86 µg/liter, respectively, while that for pink shrimp exposed to heptachlor epoxide was 0.04 µg/liter. Heptachlor was accumulated and some metabolized to its epoxide by all animals tested. Fish and oysters accumulated heptachlor in their tissues 2,800-21,300 times the measured concentration in water; shrimp, only 200-700 times.

Hansen, David J. 1976. Techniques to Assess the Effects of Toxic Organics on Marine Organisms. In: Water Quality Criteria Research of the U.S. Environmental Protection Agency. EPA-600/3-76-079. U.S. Environmental Protection Agency, Environmental Research Laboratory, Corvallis, OR. Pp. 63-76. (ERL,GB 266). (Avail. from NTIS, Springfield, VA: PB-257 091)

Acute static or flow-through bioassays generally have been used to set marine water quality standards, but few new bioassay techniques are available to determine long-term effects of one or more toxicants on survival, growth and reproduction of individual species of mollusks, arthropods or fish and on communities of estuarine organisms. Not only has the duration of bioassays increased from 96 hours or less to periods of from one month to two years, but the complexity has increased as well. Effects of toxicants on the entire life-cycle of an oviparous estuarine fish, Cyprinodon variegatus, can now be studied; one bioassay with endrin has been completed. This fish typically develops from an embryo to maturity in 10 weeks, with about 70% survival overall. Females produce an average of eight eggs per day and fertilization success exceeds 90%. Effects of a polychlorinated biphenyl, Aroclor® 1254, and a pesticide, toxaphene, on developing communities of estuarine animals have been investigated. These studies provide data for prediction of pollution-induced shifts in composition of estuarine animal communities.

Schoor, W.P. and S.M. Newman. 1976. Effect of Mirex on the Burrowing Activity of the Lugworm (Arenicola cristata). Trans. Am. Fish. Soc. 105(6):700-703. (ERL,GB 268).

An inexpensive bioassay system was developed to estimate pollutant effects on a benthic animal. Mirex, a fire ant toxicant, was taken into the substrate by the burrowing and feeding activity of the lugworm, Arenicola cristata, and significantly affected this activity. Mirex was present in the adult worm as well as in its juvenile stage.

Schimmel, Steven C., James M. Patrick, Jr. and Jerrold Forester. 1977. Uptake and Toxicity of Toxaphene in Several Estuarine Organisms. EPA-600/J-77-066. Arch. Environ. Contam. Toxicol. 5(3):353-367. (ERL,GB 269). (Avail. from NTIS, Springfield, VA: PB-277 156)

The organochlorine insecticide, toxaphene, was tested in flow-through bioassays to evaluate its toxicity to estuarine organisms. The organisms tested and their respective 96-hr LC50s (based on measured concentrations) are: pink shrimp (Penaeus duorarum), 1.4 µg/L; grass shrimp (Palaemonetes pugio), 4.4 µg/L; sheepshead minnow (Cyprinodon variegatus), 1.1 µg/L; and pinfish (Lagodon rhomboides), 0.5 µg/L. Toxaphene concentration estimated to reduce shell deposition in American oysters (Crassostrea virginica) by 50% (EC50) was 16 µg/ L. Concentration factors (concentration of toxaphene in tissues divided by concentration measured in water) for fishes and oysters in 96 hr ranged from 3,100 to 20,600 and for shrimp, from 400 to 1,200. Individuals from various ontogenetic stages of longnose killifish (Fundulus similis) were exposed to toxaphene for 28 days in flow-through bioassays. Toxaphene was toxic to embryos, fry, juveniles, and adult fish, but fertilization of ova in static tests was not affected by the concentrations tested (0.32 to 10 µg/L). The 28-day measured LC50s for all stages ranged from 0.9 to 1.4 µg/L. Toxaphene was accumulated in ova and other body tissues of the longnose killifish; concentration factors in ova were 1,000 to 5,500, and in whole-body tissues, 4,200 to 60,000.

Nimmo, DelWayne R. and Lowell H. Bahner. 1976. Metals, Pesticides and PCBs: Toxicities to Shrimp Singly and in Combination. In: Estuarine Processes, Vol. 1: Uses, Stresses, and Adaptation to the Estuary. Martin W. Wiley, Editor. Academic Press, Inc., New York, NY. Pp. 523-531. (ERL,GB 271).

The objective of this study was to assess potential deleterious effects of certain toxicants, singly and in combination, to penaeid shrimp. In nature, these shrimp are exposed to combinations of toxicants from industrial and municipal outfalls, from agricultural runoff or from dredge-and-fill operations. The combined toxicities of methoxychlor and cadmium to penaeid shrimp, Penaeus duorarum, where either independent or additive, and varied with the method(s) of bioassay. Conclusions were based on the results of 10-, 25- and 30-day bioassays conducted with the toxicants added singly or in combination to flowing water of constant salinity and temperature. Cadmium, but not methoxychlor, was accumulated by shrimp and methoxychlor appears to influence the processes of accumulation or loss of cadmium from tissues of shrimp.

Hansen, David J. 1975. PCB's: Effects on and Accumulation by Estuarine Organisms. In: Proceedings of the National Conference on Polychlorinated Biphenyls. EPA-560/6-75-004. U.S. Environmental Protection Agency, Cincinnati, OH. Pp. 282-283. (ERL,GB 277).

Effects of PCB's on and accumulation by estuarine organisms were studied in laboratory bioassays. Aroclors 1016, 1242, and 1254 were acutely toxic to certain estuarine organisms at concentrations greater than 10 µg/l, but these bioassays underestimated toxicities of PCB's as shown by data from exposures that lasted longer than 2 weeks. Concentrations that were lethal to selected invertebrates and fishes in chronic exposures ranged from 0.1 to 5 µg/l. Reproduction of sheepshead minnows was impaired by concentrations of Aroclor 1254 in their eggs >5 µg/l, but this was not observed in eggs that contained up to 77 µg/g of Aroclor 1016. Bioaccumulation of PCB's in estuarine organisms generally exceeded 104 times the concentration in water in laboratory studies and 105 times in the estuary.

Hansen, David J. 1978. Impact of Pesticides on the Marine Environment. In: First American-Soviet Symposium on the Biological Effects of Pollution on Marine Organisms. EPA-600/9-78-007. Thomas W. Duke and Anatoliy I. Simonov, Editors. U.S. Environmental Protection Agency, Environmental Research Laboratory, Gulf Breeze, FL. Pp. 126-137. (ERL,GB 279). (Avail. from NTIS, Springfield, VA: PB-285 923)

The impact of pesticides on the marine environment can be assessed by monitoring their occurrence in the marine environment and by evaluating their toxic effects in laboratory bioassays. Acute static and flow-through bioassays generally have been used to set marine water quality criteria, but bioassay techniques now can determine effects of long-term exposure to one or more toxicants on survival, growth, and reproduction of individual species of mollusks, arthropods and fishes and effects on communities of estuarine organisms in the laboratory. Bioassays have been lengthened from 96 hours or less to between one month and two years, and their complexity has also been broadened. Effects of toxicants on the entire life cycle of an oviparous estuarine fish, Cyprinodon variegatus, can now be studied, and bioassays have been completed with endrin and heptachlor. Preliminary experiments using this fish revealed that they typically develop from an embryo to maturity in 10 to 14 weeks, with about 70% survival in the laboratory. Females produce an average of eight eggs per day and fertilization success exceeds 90%. Effects of a polychlorinated biphenyl, Aroclor® 1254, and of a pesticide, toxaphene, on developing communities of estuarine animals have been investigated. These studies provided data for predicting pollution-induced shifts in composition of estuarine animal communities.

Walsh, Gerald E., Kenneth M. Duke and Robert B. Foster. 1982. Algae and Crustaceans as Indicators of Bioactivity of Industrial Wastes. EPA-600/J-81-112. Water Res. 16(6):879-883. (ERL,GB 280). (Avail. from NTIS, Springfield, VA: PB82-236423)

Freshwater (Selenastrum capricornutum) and estuarine (Skeletonema costatum) algae were exposed to liquid wastes from 10 industrial sites in laboratory bioassays. All wastes affected algal growth, either by stimulation only or by stimulation at low concentrations and inhibition at high concentrations. Generally, S. capricornutum and Sk. costatum responded similarly to each waste: SC20's (concentrations that stimulated growth by 20%) were between 0.01 and 20.0% waste; EC50's (concentrations that inhibited growth by 50%), between 5.1 and 85.5% waste. Since toxicity to S. capricornutum was usually lost by the sixth or seventh day of exposure in all wastes except one, it is recommended that algal tests be carried out for 4 days. Both algal species were more sensitive to the wastes than were Daphnia magna (freshwater) and Mysidopsis bahia (estuarine). Only three wastes were toxic to D. magna and two were toxic to M. bahia. SC20 and EC50 values are used to calculate the 7-day, 10-year flow rate of the receiving stream required for dilution of effluents to non-toxic concentrations.

Hansen, David J. 1976. Survey of Efforts to Abate Water Pollution in Fiscal 1973 by North American Agencies: Report of the 1974 Water Quality Committee American Fisheries Society. Fisheries. 1(1):15-21. (ERL,GB 285).

Natural resource agencies in Canada and the United States were asked to complete a questionnaire regarding their FY-73 manpower, funds, activities, and attitudes toward pollution abatement and research to develop water quality criteria for aquatic life. Respondents included 53 state agencies, 25 U.S. Federal agencies, and 13 Canadian agencies. Summarized results indicate that environmental protection activities generally comprise only a modest share of an agency's budget. However, the funds and manpower spent on research to develop water quality criteria were comparatively small and accomplished mainly in federal laboratories. Most reported bioassay activities were of short duration and predominantly used freshwater fish. Respondent attitudes toward bioassay methods varied greatly. Whether warranted or not, a majority of respondents would prefer to license pollution bioanalysts and would favor having AFS sanction specific bioassay methods. Specialized waste treatment facilities were absent at most laboratories and most laboratories discharged waste water without treatment or sent their effluent to municipal sewage plants. Typically, about two years lapsed between data analysis and publication in technical journal articles. Research efforts that did not produce publications averaged 67% for states, 25% for U.S. Federal, and 41% for Canadian agencies. Editorial assistance seemed to be associated with a higher conversion of research effort into scientific articles for public comsumption.

Nimmo, D.R., R.A. Rigby, L.H. Bahner and J.M. Sheppard. 1978. Acute and Chronic Effects of Cadmium on the Estuarine Mysid, Mysidopsis bahia. EPA-600/J-78-071. Bull. Environ. Contam. Toxicol. 19(1):80-85. (ERL,GB 287). (Avail. from NTIS, Springfield, VA: PB-290 039)

Mysids, small shrimp-like crustacea, proved to be a practical bioassay animal for investigating the effects of cadmium in seawater and may serve this purpose for other pollutants. In the laboratory under flow-through test conditions, the mysid, Mysidopsis bahia, was more sensitive to cadmium than other crustaceans tested. LC50 values were 15.5 µg/l within 96 hrs and 11.3 µg/l during a 17-day life-cycle, whereas LC50's for other selected crustaceans were between 120 and 720 µg/l. Results of life-cycle bioassays can aid in the establishment of water quality criteria for marine and estuarine organisms.

Schimmel, Steven C., James M. Patrick, Jr. and Jerrold Forester. 1977. Toxicity and Bioconcentration of BHC and Lindane in Selected Estuarine Animals. EPA-600/J-77-070. Arch. Environ. Contam. Toxicol. 6(2/3):355-363. (ERL,GB 288). (Avail. from NTIS, Springfield, VA: PB-277 150)

Flow-through, 96-hr bioassays were conducted to determine the acute toxicity of technical BHC and lindane to several estuarine animals. Test animals and their respective 96-hr lindane LC50 values were: mysid (Mysidopsis bahia), 6.3 µg/L; pink shrimp (Penaeus duorarum), 0.17 µg/L; grass shrimp (Palaemonetes pugio), 4.4 µg/L; sheepshead minnow (Cyprinodon variegatus), 104 µg/L; and pinfish (Lagodon rhomboides), 30.6 µg/L. The 96-hr LC50 values for pink shrimp and pinfish exposed to BHC were 0.34 and 86.4 µg/L, respectively. Two BHC bioconcentration studies were conducted with the oyster, Crassostrea virginica, and pinfish. After 28 days exposure, oysters bioconcentrated an average of 218 X the BHC measured in exposure water, while pinfish bioconcentrated 130 X in their edible tissues and 617 X in offal. After one week in BHC-free sea water, no detectable residues were measured in oysters or pinfish.

Walsh, Gerald E., Karen Ainsworth and Alfred J. Wilson. 1977. Toxicity and Uptake of Kepone in Marine Unicellular Algae. Chesapeake Sci. 18(2):222-223. (ERL,GB 292).

Four species of marine unicellular algae were exposed to Kepone in laboratory bioassays. EC50 values after seven days' growth, in mg/liter (ppm), were: Chlorococcum sp., 0.35; Dunaliella tertiolecta, 0.58; Nitzschia sp., 0.60; Thalassiosira pseudonana, 0.60. When exposed to 100 µg/liter (ppb) Kepone for 24 hr, residues associated with the algae, in mg/kg (ppm) wet weight, were: Chlorococcum sp., 80; D. tertiolecta, 23; Nitzschia sp., 41; T. pseudonana, 52.

Schimmel, Steven C. and Alfred J. Wilson, Jr. 1977. Acute Toxicity of Kepone to Four Estuarine Animals. Chesapeake Sci. 18(2):224-227. (ERL,GB 293).

Recent contamination of the James River Estuary, Virginia, with Kepone prompted acute flow-through bioassays to determine the 96-hour toxicity of the insecticide to four estuarine species native to that ecosystem. The species and their 96-hour LC50 values were: grass shrimp (Palaemonetes pugio), 121 µg/ liter; blue crab (Callinectes sapidus) >210 µg/liter; sheepshead minnow (Cyprinodon variegatus),, 69.5 µg/liter; and spot (Leiostomus xanthurus) 6.6 µg/ liter. Surviving animals were analyzed for Kepone. Average bioconcentration factors (the concentration of Kepone in tissues divided by the concentration of Kepone measured in seawater) were: grass shrimp, 698; blue crab, 8.1; sheepshead minnow, 1,548; and spot, 1,221.

Bahner, Lowell H., Alfred J. Wilson, Jr., James M. Sheppard, James M. Patrick, Jr., Larry R. Goodman and Gerald E. Walsh. 1977. Kepone Bioconcentration, Accumulation, Loss, and Transfer Through Estuarine Food Chains. EPA-600/J-77-074. Chesapeake Sci. 18(3):299-308. (ERL,GB 294). (Avail. from NTIS, Springfield, VA: PB-277 183)

Accumulation, transfer, and loss of Kepone in estuarine organisms were studied in laboratory bioassays. Kepone was bioconcentrated by oysters (Crassostrea virginica), Mysids (Mysidopsis bahia), grass shrimp (Palaemonetes pugio), sheepshead minnows (Cyprinodon variegatus), and spot (Leiostomus xanthurus), from concentrations as low as 0.023 µg/l seawater. Bioconcentration factors ranged from 10 to 340 in static exposures and 900 to 13,500 in flow-through bioassays, and were dependent on species and exposure duration. Depuration of Kepone from oysters in Kepone-free water was rapid (35% loss in 24 hours); however, depuration of Kepone was slow in crustaceans and fish, with tissue concentrations decreasing 30-50% in 24-28 days. Oysters, fed Chlorococcum containing approximately 34 µg Kepone/g wet weight, attained 0.21 µg Kepone/g (wet tissue) in 14 days, but when fed Kepone-free plankton, depurated Kepone to below detectable concentrations (< .02 µg/g) within 10 days. Spot obtained Kepone when fed live mysids that had grazed on Kepone-laden brine shrimp. Kepone residues (1.05 µg/g wet tissue) in these fish approached the concentration of their food (1.23 µg/g wet tissue); at the lower concentration tested, Kepone concentrations below detection limits (less than .2 µg/g) in prey accumulated in the predator to detectable concentrations (0.02 µg/g) within 30 days. Bioaccumulation factors (concentration of Kepone in predator/concentration in prey) at 30 days were equal (0.85 spot/mysid; 0.53 mysid/brine shrimp) in the high and low concentrations tested. The initial bioconcentration of Kepone from water by plankton was the dominant source of Kepone to each member of this food chain, but significant (>85%) quantities of Kepone transferred from prey to predatory fish.

Goodman, Larry R., David J. Hansen, John A. Couch and Jerrold Forester. 1978. Effects of Heptachlor and Toxaphene on Laboratory-Reared Embryos and Fry of the Sheepshead Minnow. In: Proc. 30th Annu. Conf. Southeast. Assoc. Game Fish Comm., Oct. 24-27, 1976, Jackson, MS. EPA-600/J-76-086. Wilmer Rogers, Editor. Southeastern Association of Game and Fish Commissioners, Frankfort, KY. Pp. 192-202. (ERL,GB 297). (Avail. from NTIS, Springfield, VA: PB-290 072)

Flow-through seawater bioassays of 28-days duration were conducted with the organochlorine pesticides heptachlor and toxaphene to determine their toxicity to and bioconcentration by embryos and fry of the sheepshead minnow (Cyprinodon variegatus). At technical heptachlor measured concentrations of 4.3, 3.5, 2.2, 2.0, and 1.2 µg/l (ppb), test animal survival was 1, 5, 61, 79, and 88% respectively. At toxaphene measured concentrations of 2.5, 1.1, 0.6, 0.3, and 0.2 µg/l, test animal survival was 10, 85, 79, 88, and 80% respectively. Average standard length of fry continuously exposed from fertilization to heptachlor concentrations of 4.3 and 3.5 µg/l was significantly reduced (a =0.01). Concentration factors (concentration in fish/measured concentration in water) for heptachlor averaged 3,600 and for trans-chlordane averaged 8,600. Heptachlor epoxide and cis-chlordane were also present in the fish. Concentration factors for toxaphene in fry averaged 9,800. Various histopathological characteristics not seen in control fish were observed in the liver, kidney, pancreas, and intestine of the few fish that survived 4.3 and 3.5 µg/l of heptachlor.

Couch, John A. and Lee Courtney. 1977. Interaction of Chemical Pollutants and Virus in a Crustacean: A Novel Bioassay System. EPA-600/J-77-140. Ann. N.Y. Acad. Sci. 298:497-504. (ERL,GB 300). (Avail. from NTIS, Springfield, VA: PB-290 036)

A large group of shrimp, 23.3% of which had light patent Baculovirus infections, was divided equally into two groups. One group was exposed to the chemical stressor Aroclor® 1254 (a polychlorinated biphenyl) at 0.7 ppb for 35 days in flowing seawater. The other group was maintained as a control group in flowing seawater. Viral prevalence in exposed shrimp samples increased with time at a significantly greater rate than did viral frequency in control shrimp. Viral prevalence in Aroclor-exposed shrimp survivors was 75% after 35 days, whereas in control shrimp, only 45.7% had patent viral infections. This finding suggests an interaction among chemical stressor (Aroclor® 1254), host, and virus. The nature or mechanism of this interaction has not been defined, but the shrimp-virus system shows promise for future bioassays of influence of low concentrations of pollutants on natural pathogen-host interactions.

Middaugh, Douglas P., John A. Couch and Allan M. Crane. 1977. Responses of Early Life History Stages of the Striped Bass, Morone saxatilis to Chlorination. Chesapeake Sci. 18(1):141-153. (ERL,GB 304). (Avail. from NTIS, Springfield, VA: PB-269 932)

The toxicity of total residual chlorination (TRC) to early life stages of the striped bass, Morone saxatilis, was determined using percent embryo hatchability, incipient LC50 bioassays, histopathology, and avoidance responses. Beginning 8 to 9 hours after fertilization, developing embryos were exposed continuously to TRC in flowing water at 1.0-3.0 ppt salinity and 18 ± 1 C. Fifty-six percent of the control group (no TRC exposure) hatched. None of the embryos exposed to a measured TRC concentration of 0.21 mg/l hatched. Only 3.5 percent of the embryos exposed to 0.07 mg/l TRC and 23 percent of those exposed to 0.01 mg/l TRC hatched. Incipient LC50 bioassays were used to determine the sensitivity of 2-, 12- and 30-day-old striped bass to concentrations of TRC in flowing water (1.0-3.0 ppt salinity at 18 ± 1 C). The estimated incipient LC50 was 0.04 mg/l TRC for 2-day-old prolarvae, 0.07 mg/l for 12-day-old larvae and 0.04 mg/l for 30-day-old juveniles. Histological examination of 30-day-old juveniles which survived exposure in the incipient LC50 bioassay indicated gill and pseudobranch damage for fish exposed to 0.21 to 2.36 mg/l TRC. Statistical analysis of avoidance tests conducted at 1.0-3.0 ppt salinity and 18 ± 1 C with 24-day-old larvae showed significant (X2, p < 0.05) and reproducible avoidance responses to measured TRC concentrations of 0.79-0.82 mg/l and 0.29-0.32 mg/l. No avoidance was indicated at TRC concentrations of 0.16-0.18 mg/l.

Hansen, David J., Alfred J. Wilson, DelWayne R. Nimmo, Steven C. Schimmel and Lowell H. Bahner. 1976. Kepone: Hazard to Aquatic Organisms. Science. 193(4253):528. (ERL,GB 307).

We would like to document our concern about the hazard of Kepone to aquatic organisms in the James River and the Chesapeake Bay. On-site tests of organisms taken from the James River showed significantly high Kepone concentrations. These tests revealed that concentrations in edible tissues of most fresh and estuarine fin- and shellfish commonly ranged from 0.1 to more than 1 microgram per gram. These concentrations exceeded allowable health limits for commercial and sport fisheries and forced closure of the river to some commercial and sport fishing. This year Kepone concentrations have increased in anadromous fishes as they spent more time in the river. Further, after laboratory exposures, we found that Kepone, like other chlorinated insecticides, is highly bioaccumulative and persists in estuarine organisms. Oysters, grass shrimp, and fishes have bioconcentrated Kepone from 425 to 20,000 times the concentration in the surrounding water. Therefore, action levels for edible seafood in force might be reached by as little as 5 parts of Kepone per trillion parts of water (nanograms per liter). In Kepone-free water, oysters can depurate about 90% of the accumulated Kepone in 4 days, but fish may require more than 3 weeks to lose 30 to 50%. Five weeks after fertilization of sheepshead minnow eggs containing Kepone, the juvenile fish retained as much as 46% of the Kepone present in the eggs. Kepone can be accumulated by fish to concentrations that exceed those in their food. Kepone is acutely toxic to estuarine organisms, but long-term bioassays reveal that the hazard to these organisms is greatly underestimated by the 96-hour tests. The concentrations in micrograms per liter, estimated to be lethal to 50% of the test animals in 96 hours (LC50), were 6.6 for spot, 70 for sheepshead minnows, 10 for an estuarine mysid, 121 for grass shrimp, and more than 210 for blue crabs. Kepone was lethal to adult sheepshead minnows exposed to 0.8 microgram per liter for 28 days. A significant number of embryos from adults exposed to 1.8 micrograms per liter were abnormal and died. When embryos were exposed to 0.08 microgram of Kepone per liter of water, 36 days later, resulting juvenile fish were shorter than control fish and some exhibited scoliosis. Mysid shrimp exposed for 20 days to about 0.2 microgram per liter produced fewer progeny: with greater concentrations, their growth and survival were reduced. We are concerned because all concentrations tested thus far in long-term exposures of sheepshead minnows and mysids have reduced survival, reproduction, or growth.

Hansen, D.J., D.R. Nimmo, S.C. Schimmel, G.E. Walsh and A.J. Wilson, Jr. 1977. Effects of Kepone on Estuarine Organisms. In: Recent Advances in Fish Toxicology, a Symposium. EPA-600/3-77-085. U.S. Environmental Protection Agency, Environmental Research Laboratory, Corvallis, OR. Pp. 20-30. (ERL,GB 311).

Laboratory toxicity tests were conducted to determine the effects and accumulations of Kepone in estuarine algae, mollusks, crustaceans, and fishes. Nominal Kepone concentrations calculated to decrease algal growth by 50% in static bioassays lasting seven days were: 350 µg/l, Chlorococcum sp.; 580 µg/l, Dunaliella tertiolecta; 600 µg/l, Nitzschia sp.; and 600 µg/l, Thalassiosira pseudonana. Measured Kepone concentrations calculated to cause 50% mortality in flowing-seawater toxicity tests lasting 96 hours were: 10 µg/l for the mysid shrimp (Mysidopsis bahia); 120 µg/l for the grass shrimp (Palaemonetes pugio); >210 µg/l for the blue crab (Callinectes sapidus); 70 µg/l for the sheepshead minnow (Cyprinodon variegatus); and 6.6 µg/l for the spot (Leiostomus xanthurus). Bioconcentration factors (concentration in whole animals divided by concentration measured in water) in these tests were greatest for fishes (950 to 1,900) and less for grass shrimp (420 to 930). Survival, growth, and reproduction of mysids and sheepshead minnows were decreased in chronic bioassays lasting 14 to 64 days. Growth of mysids and sheepshead minnows in the chronic bioassay averaged 5,200 (range, 3,100-7,000) for adults exposed for 28 days and 7,200 (3,600-20,000) for juveniles exposed for 36 days. The chronic toxicity and bioconcentration potential of Kepone are more important factors than its acute toxicity in laboratory evaluations of environmental hazard. Therefore, these factors should be considered when attempting to assess present impacts and to limit future impacts of this insecticide on the aquatic environment.

Coppage, David L. 1976. Bioassays as Indicators of Pollution Effects. In: Proceedings of the International Symposium on Marine Pollution Research. EPA-600/9-76-032. Samuel P. Meyers, Editor. U.S. Environmental Protection Agency, Environmental Research Laboratory, Gulf Breeze, FL. Pp. 133-139. (ERL,GB 314). (Avail. from NTIS, Springfield, VA: PB-267 601)

The objective of marine bioassay usually is to determine a pollutant's (a) concentration harmful to organisms, (b) persistence and degradability, (c) rate of accumulation and loss in organisms consumed by other organisms, and (d) mode of action on organisms. These data are used to establish water quality standards. Although it is not practical to study all species that may be affected by a pollutant, a variety of marine or estuarine species may be tested to estimate which taxonomic groups are most sensitive. Findings may result in a recommendation that a particular pollutant not be allowed to exceed certain concentrations for a particular period, and restriction of use and disposal may result. When it is shown that monitoring of concentrations in water or organisms is not adequate to indicate effects, additional standards may be recommended, such as in situ measurement of persistent and cumulative enzyme inhibition in organisms caused by metabolites that are not chemically measurable in organisms. The bioassays used at the U.S. Environmental Protection Agency, Environmental Research Laboratory, Gulf Breeze, Florida, may be regarded as three interrelated types: macroscopic, microscopic, and metabolic (fig. 1). I will briefly review some of the bioassays and results as examples of their variety and applications.

Bourquin, A.W., R.L. Garnas, P.H. Pritchard, F.G. Wilkes, C.R. Cripe and N.I. Rubinstein. 1979. Interdependent Microcosms for the Assessment of Pollutants in the Marine Environment. Int. J. Environ. Stud. 13(2):131-140. (ERL,GB 348).

Laboratory microcosms are described for assessing the fate and effects of pollutants in marine and estuarine environments. These systems focus on specific ecosystem processes and interactions and are interdependent in that the results of all are necessary for a complete description of a pollutant's environmental impact. The following individual systems are described using methyl parathion as the pollutant: Environmental Fate Screening System; Eco-Core System; Continuous Flow Systems; Aquatic Gradient Avoidance Response System; Benthic Bioassay System.

Middaugh, D.P., A.M. Crane and J.A. Couch. 1977. Toxicity of Chlorine to Juvenile Spot, Leiostomus xanthurus. EPA-600/J-78-080. Water Res. 11(12):1089-1096. (ERL,GB 364). (Avail. from NTIS, Springfield, VA: PB-290 976)

The sensitivity of juvenile spot, Leiostomus xanthurus, to total residual chlorine (TRC) in flowing sea-water was investigated. Incipient LC50 bioassays, histopathology, avoidance tests and the combined effect of thermal stress and TRC were used to assess sensitivity. Estimated incipient LC50 values were 0.12 mg1-1 TRC at 10 degrees C and 0.06 mg1-1 TRC at 15 degrees C. Histological examination of spot used in the incipient LC50 bioassay at 15 degrees C and sacrificed while alive indicated pseudobranch and gill damage occurred in individuals exposed to a measured TRC concentration of 1.57 mg1-1. Spot exposed to lower concentrations of TRC, 0.02-0.06 mg1-1 at 15 degrees C and sacrificed alive showed no consistent tissue damage.Spot demonstrated temperature dependent avoidance responses to TRC. At 10 degrees C, a concentration of 0.18 mg1-1 was required for significant (X2; P is less than 0.05) avoidance; at 15 and 20 degrees C, spot showed significant avoidance of TRC concentrations as low as 0.05 mg1-1. Simultaneous exposure of spot to thermal stress (5, 10 or 13 degrees C above the acclimation temperature of 15 degrees C) at measured TRC concentrations of 0.05-0.07 and 0.34-0.52 mg1-1 demonstrated a significant, (X2 with Yates correction, P is less than 0.05) increase in sensitivity to TRC with increased temperature and exposure times for some of the groups tested.

Walsh, Gerald E. and Lowell H. Bahner. 1979. Assessment of Textile Waste Toxicity with Marine Bioassays. In: Symposium Proceedings: Textile Industry Technology (December 1978, Williamsburg, Va). EPA-600/2-79-104. U.S. Environmental Protection Agency, Research Triangle Park, NC. Pp. 251-271. (ERL,GB 376).

Marine bioassays were conducted with textile wastes on an alga (Skeletonema costatum), the grass shrimp (Palaemonetes pugio), and the sheepshead minnow (Cyprinodon variegatus). Effects measured were inhibition or stimulation of algal growth and death of animals after 96-h exposure. All wastes affected growth of S. costatum, whereas only five were acutely toxic to the animals. Grass shrimp were more sensitive than juvenile sheepshead minnows: the range of the LC50 was 12.8 to 34.5 percent waste for the shrimp compared to 37.5 to 69.5 percent for the minnow. Four wastes stimulated growth of algae (SC20=0.50 to 2.25 percent); four inhibited growth (EC50=1.5 to 84 percent); six were both stimulatory at low concentrations (SC20=1.5 to 21.75 percent) and inhibitory at high concentrations (EC50=50 to 93 percent). The SC20, a term introduced here, is the calculated concentration of waste that would stimulate growth by 20 percent above the control. Algae must be considered very useful for assessment of possible effects of textile wastes on aquatic systems because they responded to all wastes tested. It is suggested that bioassays be used to assess the potential impact of whole waste on aquatic systems; and after chemical analysis of the waste, bioassays be performed on components that may be bioactive. If the whole waste or any of its components has an effect, then that waste must be considered for application to treatment technology.

Walsh, Gerald E. and Shelley V. Alexander. 1980. Marine Algal Bioassay Method: Results with Pesticides and Industrial Wastes. EPA-600/J-80-024. Water Air Soil Pollut. 13(1):45-55. (ERL,GB 385). (Avail. from NTIS, Springfield, VA: PB80-200033)

A simple marine algal bioassay method is described for short- and long-term studies on pesticides and industrial wastes. It can be used for rapid screening of a variety of substances with single-species and multiple-species tests and gives relative toxicities of the pollutants tested. Algae are grown in optically matched culture tubes that fit directly into a spectrophotometer, allowing population density to be estimated by absorbance without removal of samples. 96 h EC50 values for some pesticides and the diatom Skeletonema costatum are: EPN, 340 µg l-1; carbophenothion, 109 µg l-1; DEF, 366 µg l-1; ethoprop, 8.4 mg l-1; methyl parathion, 5.3 mg l-1; and phorate, 1.3 ug l-1. Presence of the chelator EDTA in medium had no effect on toxicity of carbaryl to S. costatum, Nitzschia angularum, Chlorococcum sp. and Chlorella sp. Liquid industrial wastes either stimulated growth, inhibited growth, or stimulated growth at low concentrations but inhibited it at higher concentrations. In mixed-species studies with the herbicide neburon, presence of a resistant species protected the sensitive species. Liquid industrial waste from a paper products plant caused changes in relative numbers, as compared to controls, when S. costatum and Porphyridium cruentum were grown together.

Rubinstein, Norman I., Charles N. D'Asaro, Charnell Sommers and Frank G. Wilkes. 1980. Effects of Contaminated Sediments on Representative Estuarine Species and Developing Benthic Communities. In: Contaminants and Sediments, Vol. 1: Transport, Fate and Case Studies. EPA-600/J-80-154. Robert A. Baker, Editor. Ann Arbor Science Publishers, Ann Arbor, MI. Pp. 445-461. (ERL,GB 406). (Avail. from NTIS, Springfield, VA: PB81-129447)

Bioassay techniques developed to examine acute and sublethal effects of dredged sediments on marine life are described. Results are reported for laboratory tests conducted to determine sublethal and acute effects of Kepone-sorbed sediment and dredged spoil material from the James River and the Houston Ship Channel on the mysid shrimp, Mysidopsis bahia; oyster, Crassostrea virginica; and polychaete, Arenicola cristata. Criteria established for the study include (1) survival of mysids; (2) shell deposition and bioaccumulation of contaminants; (3) substrate-reworking and bioaccumulation by lugworms; (4) resiliency of macrofaunal organisms that settled onto test sediments from planktonic larvae.

Borthwick, Patrick W. and James M. Patrick. 1982. Use of Aquatic Toxicology and Quantitative Chemistry to Estimate Environmental Deactivation of Marine-Grade Creosote in Seawater. Environ. Toxicol. Chem. 1(4):281-288. (ERL,GB 421).

The acute toxicity of marine-grade creosote, expressed as the 96-h LC50, is 0.018 mg/l for mysids (Mysidopsis bahia, Molenock), .024 mg/l for pink shrimp (Penaeus durorum, Burkenroad), and 0.72 mg/l for sheepshead minnows (Cyprinodon variegatus, Lacepede). The 96-h EC50 (shell deposition) for Eastern oysters (Crassostrea virginica, Gmelin) is 0.71 mg/l. Mysid bioassays and chemical analyses estimate the half-life (<1 week) for marine-grade creosote in seawater.

Walsh, G.E. and R.G. Merrill. 1984. Algal Bioassays of Industrial and Energy Process Effluents. In: Algae as Ecological Indicators. EPA-600/D-82-141. L. Elliot Shubert, Editor. Academic Press, Inc., New York, NY. Pp. 329-360. (ERL,GB 427). (Avail. from NTIS, Springfield, VA: PB82-154444)

This review presents laboratory data and their interpretation with regard to effects of pollutants on marine and freshwater unicellular algae. Stimulation and inhibition of growth are considered to be equally undesirable. It is suggested that a search for new species for use in toxicity tests be made because the widely-used green alga, Selenastrum capricornutum, may not be one of the most sensitive freshwater species. Several algal species should be used to test each toxicant because there is not a single most-sensitive species and the conditions under which species are tested affects response. The batch method is the most commonly used exposure system for defining relative effects of pollutants, but mixed-species continuous culture presents a more realistic approach to estimation of effects in natural systems. Algae are more sensitive to industrial and energy-process wastes than animals. They respond to growth stimulants and growth inhibitors, and the stimulation response occurs at concentrations much lower than those that inhibit growth.

Rubinstein, N.I., E. Lores and N.R. Gregory. 1983. Accumulation of PCB, Mercury and Cadmium by Nereis virens, Mercenaria mercenaria and Palaemonetes pugio from Contaminated Harbor Sediments. Aquat. Toxicol. 3(3):249-260. (ERL,GB 452).

Accumulation of polychlorinated biphenyls (PCBs), mercury, and cadmium by sandworms (Nereis virens), hard clams (Mercenaria mercenaria) and grass shrimp (Palaemonetes pugio) exposed to contaminated sediments from four sites in New York Harbor was studied for a 100-day period. Of the three contaminants monitored, only PCBs were found to bioaccumulate above background (control) concentrations. Small increases in PCB body burden were detected in M. mercenaria and P. pugio, whereas higher concentrations were measured in N. virens. Uptake was affected by the organic content of the sediment. Bioaccumulation factors (concentration in tissue/concentration in sediment) for N. virens ranged from 1.59 in a low organic sediment to 0.15 in a high organic sediment. Results from this study support the contention that sediment concentration alone does not reflect bioavailability and that toxicity tests (bioassays) and field monitoring remain the most direct method for estimating bioaccumulation potential of sediment-bound contaminants.

Couch, J.A. and S.S. Foss. 1990. Potential Impact of Microbial Insecticides on the Estuarine and Marine Environments. In: Safety of Microbial Insecticides. EPA/600/D-87/331. Marshall Laird, Lawrence A. Lacy and Elizabeth W. Davidson, Editors. CRC Press, Boca Raton, FL. Pp. 85-97. (ERL,GB 622). (Avail. from NTIS, Springfield, VA: PB88-125448)

This chapter presents an overview of safety tests of microbial insecticides to estuarine and marine organisms that have been performed to date. Approaches and experimental design, species of MPCAs tested, systems used, and endpoints and results evaluated for determiniation of risks of MPCAs to nontarget marine species are reviewed. The review order is by exemplary microbial agents, as follows: viruses, bacteria, fungi and protozoa. The studies presented describe relative simple procedures for exposing single species nontarget hosts to MPCAs. They incorporate a positive control bioassay to confirm the infectivity of the MPCA and are relatively inexpensive and reliable. Studies are usually based on the null hypothesis that infection and relative effects of nontarget host will not occur. To date, this null hypothesis has not been rejected based on results obtained following experimental exposure of nontarget estuarine species to relatively high concentrations of MPCAs.

Bortone, Stephen A., William P. Davis and Charles M. Bundrick. 1989. Morphological and Behavioral Characters in Mosquitofish as Potential Bioindication of Exposure to Kraft Mill Effluent. EPA/600/J-89/537. Bull. Environ. Contam. Toxicol. 43(3):370-377. (ERL,GB 659). (Avail. from NTIS, Springfield, VA: PB91-199893)

Although the specific chemicals or factors actually responsible for induction of arrhenoidy among mosquitofish have not yet been identified, it is known that a wide variety of potential compounds occur as by-products from the processing of wood pulp (Keith 1976). Purpose of study was to investigate the morphological and behavioral responses of mosquitofish environmentally exposed to kraft mill effluent (KME) and to evaluate the potential of these responses as bioassay endpoints. A method to quantify the morphological or behavioral responses of mosquitofish should provide an in situ bioindicator to assess impact of KME discharge on receiving water biota.

Anderson, B.S., D.P. Middaugh, J.W. Hunt and S.L. Turpen. 1991. Copper Toxicity to Sperm, Embryos, and Larvae of Topsmelt Atherinops affinis, with Notes on Induced Spawning. EPA/600/J-91/343. Mar. Environ. Res. 31(1):17-35. (ERL,GB 707).

Topsmelt, Atherinops affinis, were induced to spawn repeatedly in the laboratory using a combination of environmental cues. Temperature 'spikes' appeared to be the most important factor to induce spawning. Egg production peaked four days after a 2°C increase in water temperature, and declined thereafter. A series of toxicity tests compared the relative sensitivity of topsmelt sperm, embryos, and larvae to copper chloride in static bioassay experiments. Of the three developmental stages compared, sperm were more sensitive than embryos, and embryos were more sensitive than larvae. The mean EC50 from four separate 48-h fertilization experiments was 109 µg copper liter-1. The mean EC50 from three, 12-d embryo development tests was 142-147 µg copper liter-1, depending on the endpoint used. The mean LC50 from three, 96-h larval mortality tests was 238 µg copper liter-1. Topsmelt are amenable to laboratory culture and are a promising eastern Pacific toxicity test species.

Hawkins, William E., John W. Fournie, Takatoshi Ishikawa and William W. Walker. 1996. Germ Cell Neoplasms in Medaka. J. Aquat. Anim. Health. 8(2):120-129. (ERL,GB 752).

Twenty-six cases of germ cell neoplasms were identified from approximately 10,000 specimens of Japanese medaka Oryzias latipes used in carcinogenesis bioassays and examined histologically. The neoplasms resembled spermatocytic seminomas and occurred in both female and male specimens. Most were confined to the peritoneal cavity but some infiltrated adjacent organs, including the kidney. The presence of neoplastic cells in the heart, gill capillaries, and orbital cavity suggested that some neoplasms had spread by way of the cardiovascular system. Histologically, the components of neoplasms resembled the cellular stages of spermatogenesis. Primary spermatocytes, secondary spermatocytes, and spermatids, but not mature spermatozoa, could be recognized. The cellular patterns of the neoplasms were solid, lobular, or a combination of those. Mitotic figures were not frequently seen in the lesions. Many of the neolasms in males contained scattered cells that resembled oocytes. The occurrence of the neoplasms appeared related to age but not to chemical exposure, because they were seen in both controls and in fish exposed to a variety of carcinogenic and noncarcinogenic chemicals. Multiple cases of the neoplasm occurred in fish maintained in the same aquarium or from the same cohort, suggesting that culture practices, genetic factors, or a virus may affect the development of the lesions.

Middaugh, D.P., S.M. Resnick, S.E. Lantz, C.S. Heard and J.G. Mueller. 1993. Toxicological Assessment of Biodegraded Pentachlorophenol: Microtox(R) and Fish Embryos. EPA/600/J-93/058. Arch. Environ. Contam. Toxicol. 24(2):165-172. (ERL,GB 777). (Avail. from NTIS, Springfield, VA: PB93-168920)

A Gram-negative bacterium, Pseudomonas vesicularis, (strain SR3) was isolated from soil at a former wood treatment plant in north central Florida. The ability of this bacterium to degrade pentachlorophenol (PCP) was confirmed by growing cells in a basic salt medium in which PCP was the only source of carbon and energy. Degradation from a measured concentration of 39-40 ug PCP/mL to 0.0006 ug PCP/mL was observed within 120 hrs of incubation in the presence of PCP induced cells of P. vesicularis. The initial cell density in these cultures was 6 x 10 to the sixth power cfu/mL. Microtox(R) 5 min EC50 toxicity tests revealed that aqueous solutions of PCP, measured concentrations 39-40 ug/mL were toxic but that final biodegraded samples, 0.0006 ug PCP/mL were nontoxic. However, bioassays with embryonic inland silversides, Menidia beryllina, showed that the biodegraded samples were embryotoxic or teratogenic. Water containing PCP at concentrations up to 30 times higher than measured in the final biodegraded samples were less toxic/teratogenic. These results indicate that while biodegradation of PCP was nearly complete, intermediate metabolites of the degradation process were toxic or teratogenic. Thus, the M. beryllina bioassay allows extremely sensitive assessment of toxicity associated with biodegraded environmental pollutants and may be a useful criterion for determining whether bioremediated water or soil is safe for discharge back into the environment.

Connolly, John P. and Richard B. Coffin. 1995. Model of Carbon Cycling in the Planktonic Food Web. EPA/600/J-96/081. J. Environ. Engineering. 121(10):682-690. (ERL,GB 865).

A mathematical model of carbon fluxes through the heterotrophic microbial food web is developed from a synthesis of laboratory and field research. The basis of the model is the segregation of organic carbon into lability classes that are defined by bioassay experiments. Bacteria, phytoplankton, three trophic levels of zooplankton, and dissolved organic carbon (DOC) and particulate organic carbon (POC) are modeled. The descriptions of bacterial growth and utilization of the various classes of substrate were treated as "universal constants" in the application of the model to three distinct ecosystems, ranging from oligotrophic to highly eutrophic. The successful application of the model to these diverse ecosystems supports the basic validity of the description of the microbial food web and the dynamics of carbon flux. The model indicates that the dynamics of bacteria and protozoan zooplankton production govern the rates of oxidiation of carbon entering the water column. Explicit consideration of these groups would improve the capability of eutrophication models to predict dissolved oxygen dynamics, particularly when projecting responses to loading changes.

Heppell, Scott A., Nancy D. Denslow, Leroy C. Folmar and Craig V. Sullivan. 1995. Universal Assay of Vitellogenin as a Biomarker for Environmental Estrogens. EPA/600/J-96/079. Environ. Health Perspect. 103(Suppl 7):9-15. (ERL,GB 913).

Vitellogenin (VTG), the serum phospholipoglycoprotein precursor to egg-yolk, is potentially an ideal biomarker for environmental estrogens. This study was undertaken to develop antibodies against conserved regions on the vitellogenin molecule, antibodies that could form the basis for establishing bioassays to detect estrogen exposure in any oviparous vertebrate. We developed monoclonal antibodies (mAbs) generated against purified rainbow trout (Oncorhynchus mykiss) VTG, and selected for the property of specifically recognizing VTG purified from two phylogenetically distant vetebrates, trout and striped bass (Morone saxatilis). Results of enzyme-linked immunosorbant assay and Western blotting indicated that these mAbs specifically recognize purified VTG and VTG or other estrogen-inducible proteins in plasma or serum from representative species of 4 vertebrate classes (fish, amphibians, reptiles and birds). All of the mAbs generated were IgM class. A polyclonal antiserum was raised against a synthetic consensus peptide representing the conserved N-terminal amino acid sequence of VTG. The results of Western blotting indicate that this antiserum specifically recognizes VTG in plasma or serum from teleost fish of diverse families. It was used to detect VTG in Western blots of serum from brown bullhead (Ameiurus nebulosus) with cancer (hepatocellular and cholangio-carcinoma) collected from a contaminated industrial site outside of their normal vitellogenic season. Our results indicate that it is feasible to generate antibodies capable of recognizing VTG without regard to species and that development of a "universal" VTG assay is an achievable goal.

Vogelbein, W.K., D.E. Zwerner, M.A. Unger, C.L. Smith and J.W. Fournie. 1997. Hepatic and Extrahepatic Neoplasms in a Teleost Fish from a Polycyclic Aromatic Hydrocarbon Contaminated Habitat in Chesapeake Bay, USA. In: Spontaneous Animal Tumors: A Survey. Proceedings of the First World Conference on Spontaneous Animal Tumors, Genoa, Italy. Lorenzo Rossi, Ralph Richardson, and John Harshbarger, Editors. Press Point di Abbiategrasso, Milano, Italy. Pp. 55-63. (ERL,GB 932).

The mummichog, Fundulus heteroclitus, is one of the most abundant teleost fishes inhabiting salt marshes of the Atlantic coast of North America. Although the geographic range of this small cyprinodontid extends from Nova Scotia, Canada, to northern Florida, USA, local subpopulations are thought to be largely non-migratory. This, and it's adaptability to laboratory conditions, has led to the increasing use of this species as a bioassay organism in aquatic toxicology and as a sentinel of environmental quality. We recently reported high prevalences of liver neoplasms and associated lesions in mummichog inhabiting a polycyclic aromatic hydrocarbon (PAH) contaminated environment in Virginia, USA. This site is adjacent to a wood treatment facility that has treated railroad ties, pilings, and telephone poles with creosote and pentachlorophenol since the late 1920s. In 1991, this facility was placed into the U.S. Environmental Protection Agency's (EPA) Superfund Program and wood treatment operations were suspended. However, the property continues to be used to store timbers, and the adjacent aquatic habitat remains heavily contaminated. In addition to the hepatocellular neoplasms, mummichog from this site also exhibit elevated prevalences of neoplasms derived from biliary, exocrine pancreatic, vascular, lymphoid, and renal tissues. The extremely high PAH concentrations in sediments at this site (e.g., 2200 mg/kg dry wt), the diversity of the tumorigenic response, and the absence of neoplasms in fish from a mildly contaminated habitat only about 0.5 km away have suggested a chemical etiology for these lesions. Histopathology has recently gained acceptance in several U.S. pollution monitoring programs, and morphologic characterizations of tissue alterations in fish from polluted habitats have been given a high priority. The objective of this communication is to report histology and prevalence data for the hepatic and extra-hepatic proliferative lesions and neoplasms observed in mummichog from the above described creosote-contaminated environment.

Gormly, Nia M., Samuel Singer and Fred J. Genthner. 1996. Nontarget Testing of Microbial Pest Control Agents Using Larvae of the Coot Clam, Mulinia lateralis. Dis. Aquat. Org. 26:229-235. (ERL,GB 939).

A short-term (48 h) chemical toxicity test using larvae of the coot clam, Mulinia lateralis, was modified to evaluate potential toxicity and pathogenicity of microbial pest control agents. M. lateralis larvae, at the straight-hinged stage of development, were exposed to various microbial pest control agents including: a mosquito larvacide, Bacillus thuringiensis var. israelensis (Bti); a molluscicidal strain of Bacillus alvei; a viral pathogen of the gypsy moth, Lymantria dispar nuclear polyhedrosis virus (LdNPV); and a broad host-range fungal insect pathogen, Metarhizium anisopliae. Mortalities significantly higher than heat-killed controls were obtained with Bti at a 10-4 dilution of a commercial preparation, and with LdNPV at an occlusion body density of 1 x 106ml-1. Sodium dodecyl sulfate (SDS) and the water-soluble fraction of number 2 fuel oil (WSFoil) were also tested to provide a measure of comparison, sensitivity and precision. SDS, toxic at an LC50 of 6.3mg l-1, had a mean coefficient of variation of 23%. The clam larval bioassay was very sensitive to WSFoil; exposures resulted in an LC50 <10% v/v. Because of its precision, sensitivity and simplicity, the M. lateralis larval test has the potential to be useful for assessing nontarget effects that microbial pest control agents may have on nontarget bivalves.

Rayburn, J.R. and W.S. Fisher. 1997. Developmental Toxicity of Three Carrier Solvents Using Embryos of the Grass Shrimp, Palaemonetes pugio. Arch. Environ. Contam. Toxicol. 33(2):217-221. (ERL,GB 980).

Embryos of the grass shrimp (Palaemonetes pugio) have shown sensitivity to the water soluble fraction of number 2 fuel oil. To determine the possible use of carrier solvents in grass shrimp bioassays, detailed concentration-response experiments for ethanol (EtOH), dimethylsulfoxide (DMSO), and acetone were performed and LC50 values were obtained using two test methods. The 4-day assay included development prior to time of hatch through the time of hatch, a critical life stage of these embryos. The 12-d assay included development from tissue cap stage embryos (late gastrula) through 2-days post-hatch. The average 4-day LC50s for Et0H, DMS0 and acetone were 12.07, 22.57, and 6.78 g/L, whereas the average 12-day LC50s were 3.63, 12.33, and 6.94 g/L, respectively. The coefficient of variation for each test was less than 25.2%. Based on concentration-response curves, the maximum allowable limit of Et0H, DMS0 and acetone to be used as a carrier in the grass shrimp embryo toxicity studies should be <1, <6, and <4 g/L, respectively.

Genthner, Fred J., James T. Winstead, Jeanne E. Gillet, Amy L. Van Fleet, John J. Viel, Erin E. Genevese and Samuel Singer. 1997. Effects of a Molluscicidal Strain of Bacillus alvei on Digestive Tubules of Zebra Mussels, Dreissena polymorpha. J. Invertebr. Pathol. 69(3):289-291. (ERL,GB 981).

The zebra mussel, Dreissena polymorpha, an invading exotic pest, has been a great concern in North America. Control of the zebra mussel through physical or chemical means has numerous drawbacks and has met with limited success (Kovalak et al., 1993). Microbes have been considered as possible alternative means of control (Molly and Griffin, 1992). Genovese and Singer (1994) found several strains of Bacillus sp. of morphological group II that possessed toxic activity toward zebra mussels. In the present study, we conducted bioassays with adult zebra mussels and dilutions of a culture of B. alvei 2771. Histopathological analysis was performed to identify affected tissues. These results suggest exposure of zebra mussels of B. alvei 2771 caused death and that the digestive gland was adversely affected prior to death.

Rakocinski, Chet F., Steven S. Brown, Gary R. Gaston, Richard W. Heard, William W. Walker and J. Kevin Summers. 1997. Macrobenthic Responses to Natural and Contaminant-Related Gradients in Northern Gulf of Mexico Estuaries. Ecol. Appl. 7(4):1278-1298. (ERL,GB 995).

Effects of pollution on biotic integrity are difficult to identify when correlations occur between environmental gradients and contaminant effects, as they do in estuaries. In this broad-scale study, we used canonical correspondence analysis (CCA) to distinguish influences of natural and contaminant-related gradients on macrobenthic community structure among 319 sites from estuaries throughout the northern Gulf of Mexico. Natural gradients in salinity, depth, and sediment composition obscured the detection of macrobenthic responses to sediment contamination. After adjusting for natural environmental variability, however, partial CCA revealed important macrobenthic variation in relation to sediment contamination. A rotated principal component analysis (PCA) distinguished five composite environmental factors, each largely reflecting contaminant or natural variation. Two complex gradients in sediment contamination identified by the PCA diverged in partial CCA space and correlated with different macrobenthic indicator taxa. Contaminant gradients represented variation in two different classes of sediment contaminants: trace metals and organic chemicals. Dispersion patterns of CCA site coordinates enabled cross validation of implied contamination-related variation in community function and the utility of several interpretive or management metrics. Trophic diversity decreased with sediment contamination, linking shifts in macrobenthic community function and community structure along contaminant gradients. The CCA model complemented on earlier benthic index developed from these data to examine biotic integrity, but the benthic index could not discern macrobenthic responses to the different contaminant gradients. Neither was the benthic index useful for showing transitions in macrobenthic community structure commensurate with different levels of contamination. Ampelisca amphipod sediment bioassays were inadequate for identifying contaminant effects on biotic integrity, whereas Mysidopsis mysid sediment bioassays conservatively reflected sediment contamination and associated macrobenthic indicators.

Foss, S.S. and J.R. Rayburn. 1997. Effects of Culture Duration on Toxicity of Ethanol to Developing Embryos of the Grass Shrimp, Palaemonetes pugio. Bull. Environ. Contam. Toxicol. 59(3):467-471. (ERL,GB 998).

Embryos of the grass shrimp, Palaemonetes pugio, have been utilized by several investigators for determining the toxicity and/or infectivity of chemical and microbial pest control agents. Fisher and Foss (1993) determined the toxicity of #2 fuel oil and two oil dispersants using methodology adapted from Wilson (1985) and Middaugh et al. (1988). Similar tests were used to examine the toxicity of metabolic products of fossil fuel biodegradation (Chapman et al. 1995). Genthner et al. (1994) identified the effects of a fungal microbial pest control agent, Beauveria bassiana, on embryos of P. pugio. To refine and characterize the grass shrimp test, Rayburn et al. (1996) examined the effects of reduced durations of exposure, sample numbers (N), and test solution volumes on LC50 values for water soluble fractions (WSF) of #2 fuel oil. Rayburn and Fisher (In press) defined the 12 d grass shrimp development toxicity test as the Shrimp Embryo Teratogenesis Assay-Palaemonid (SETAP). Sensitivity to WSF of number 2 fuel oil, fractions of oil biodegradation metabolites and the ability to infect embryos of P. pugio indicate the potential usefulness of this bioassay in determining developmental toxicity of single chemicals and chemical mixtures to an estuarine crustacean.

D'Asaro, Charles N. 1982. Cycling of Xenobiotics Through Marine and Estuarine Sediments. EPA-600/3-82-074. U.S. Environmental Protection Agency, Environmental Research Laboratory, Gulf Breeze, FL. 40 p. (Avail. from NTIS, Springfield, VA: PB82-239252)

The results of five broadly defined projects are reported: cycling of xenobiotics was studied with a photo-bioassay system, that used time-lapse photography to evaluate effects of Kepone and sodium pentachlorophenate on feeding activity of the lugworm, Arenicola cristata. Radio-labeled methyl parathion was used to demonstrate fate and effect in microcosms inhabited by lugworms. Uptake and depuration of chrysene by lugworms were evaluated in a flow-through system. A toxic sediment bioassay system was developed to provide a means to test effects of dredge spoil. The system included microcosms that held mysid shrimp, Mysidopsis bahia; oysters, Crassostrea virginica; and lugworms, Arenicola cristata. Criteria of effect were survival of mysids, shell deposition and bioaccumulation by oysters, substrate reworking and bioaccumulation by lugworms, and settlement of zooplankton. Kepone-sorbed sediment and dredge spoil from James River and Houston Ship Channel were tested for 28 days. Long-term tests (100 days), with the same systems, were used to evaluate effects of a specific drilling mud from an active exploratory platform. Predator-prey tests of sublethal effects of xenobiotics demonstrated effect in one-prey and two-prey systems. The effects of methyl parathion on predator-prey relationships between grass shrimp, Palaemonetes pugio; juvenile sheephead minnows, Cyprinodon variegatus; and gulf killifish, Fundulus grandis, were demonstrated. The effects of methyl parathion on the predator prey relationships between Palaemonetes pugio, and pinfish, Lagodon rhomoboides, were also demonstrated. A method that could be used to evaluate effects xenobiotics on predator-prey relationships between cryptically shaded flounder and pinfish prey was developed. Evaluation of sublethal effects, such as avoidance of pollution gradients, was studied in a trough-type avoidance-response system. The system recorded responses automatically and independently of an observer. Tests with pinfish demonstrated that they will avoid chlorine-produced oxidants. The system was modified to demonstrate toxicant-induced changes in cyclic burrowing activity by pink shrimp, Penaeus duorarum, exposed to methyl parathion. Usefulness of small-scale microcosms was evaluated by developing methods to culture polychaetes and crustaceans. Various aspects of the biology of selected species were studied.

Koenig, Christopher C., Daniel C. Abel, Courtney W. Klingensmith and Michael B. Maddock. 1982. Usefulness of the Self-Fertilizing Cyprinodontid Fish, Rivulus marmoratus as an Experimental Animal in Studies Involving Carcinogenesis, Teratogenesis and Mutagenesis. EPA-600/3-82-075. U.S. Environmental Protection Agency, Environmental Research Laboratory, Gulf Breeze, FL.. 129 p. (Avail. from NTIS, Springfield, VA: PB82-249194)

Rivulus marmoratus is a naturally self-fertilizing cyprinodontid fish inhabiting mangrove marshes throughout the caribbean. As a result of internal self-fertilization this oviparous species is composed of a number of isogenic, homozygous lines (clones), several of which have been identified by histocompatibility experiments and maintained in laboratory culture for over 30 years. Simplified culture and handling methods are given and data are presented on the reproduction, growth and development of rivulus under laboratory culture as a prelude to the evaluation of its potential as a bioassay animal. Several types of bioassays were run and evaluated using rivulus: behavioral, carcinogenicity,teratogenicity, toxicity, and mutagenicity. Advantages and disadvantages of using rivulus for such bioassays are discussed. Behaviorally, rivulus is capable of detecting and avoiding water contaminated with H2S. They respond (EC50 = 123.6 ppb H2S) by leaping from the water and remaining emergent for various periods of time while respiring cutaneously. Hepatocellular carcinoma among other pathologic changes were observed in livers of rivulus a year after exposure of adults and larvae to diethylnitrosamine (45, 30, and 15 ppm in water) for 5 weeks and 12 weeks, respectively. No pathologic changes were found in embryos exposed similarly. High rates of various skeletal malformations resulted in offspring of adults exposed to dibutyl phthalate (DBP) and 2,3,4,6-tetrachlorophenol (TECP) at concentrations of 20, 10 and 5% (DBP - 0.740, 0.370, 0.185 mg/l; TECP - 0.220, 0.110, 0.055 mg/l) of the larval 96-hour LC50. No dose-response relationships of skeletal malformations were found for similar exposures to pentachlorophenol, 2,3,5-trichlorophenol or bromoform, however, chronic exposure of developing hatchlings to TECP resulted in fin and gill erosion and chronic exposure to bromoform produced dorsal fin abnormalities. As part of a mutagenesis bioassay 14 enzyme systems representing 28 loci were screened for the three laboratory clones and one wild-caught clone but no electrophoretic differences were found. Attempts to culture rivulus cells failed. Also, the karyotype of rivulus is not suitable for short-term cyctogenetic assays such as the sister chromatid exchange (SCE) assay. Alternatively, however, the toadfish (Opsanus tau) possesses a suitable karyotype for SCE analysis and we have been successful in culturing toadfish cells to fourth passage. Increased rates of SCE were obtained when toadfish cells were exposed in vitro to the mutagen ethyl methanesulfonate but not bromoform. Another set of experiments is presented which involves characterization of the nature of the toadfish cytochrome P450 system.

Borthwick, Patrick W. and Gerald E. Walsh. 1981. Initial Toxicological Assessment of Ambush, Bolero, Bux, Dursban, Fentrifanil, Larvin, and Pydrin: Static Acute Toxicity Tests with Selected Estuarine Algae, Invertebrates, and Fish. EPA-600/4-81-076. U.S. Environmental Protection Agency, Environmental Research Laboratory, Gulf Breeze, FL. 9 p. (Avail. from NTIS, Springfield, VA: PB82-137654)

Selected static toxicity tests were conducted with Ambush, Bolero, Bux, Dursban, Fentrifanil, Larvin, and Pydrin to determine the sensitivity of species representing four major phyla. Algal bioassays were conducted with marine algae to determine the concentration of pesticide that would inhibit population growth by 50% in 96 h. Static toxicity tests with mollusk larvae estimated the concentration of pesticide that would cause 50% of the exposed larvae to develop abnormally in 48 h. Static acute lethality tests with crustaceans and fish determined the concentration of pesticide that is lethal to 50% of the test organisms during a 96-h exposure

Capuzzo, Judith M. and Jennifer G. Smith Derby. 1982. Drilling Fluid Effects to Developmental Stages of the American Lobster. EPA-600/4-82-039. U.S. Environmental Protection Agency, Environmental Research Laboratory, Gulf Breeze, FL. 52 p. (Avail. from NTIS, Springfield, VA: PB82-220740)

Laboratory experiments were conducted to evaluate the impact of drilling operations for oil exploration on populations of the American lobster (Homarus americanus). The effects of used, whole drilling fluids on the larval stages of the lobster were assessed in continuous flow bioassay experiments. The five drilling fluids tested were shown to vary markedly in their toxicity, with LC-50 values ranging from 74 ppm to >500 ppm. Sublethal exposures to drilling fluids resulted in reductions in growth rates, molting frequencies, respiration rates, feeding rates, and growth efficiencies. Reduced O:N ratios and increased protein:lipid ratios demonstrated a change in the energetics of the larval lobsters as a result of drilling fluid exposure. Results show that it is primarily the chemical and not the physical features of drilling fluids that were responsible for the detrimental effects observed. The drilling fluids tested that had a diesel component were more toxic than those without this component, although direct correlations between percent diesel and relative toxicity could not be made. The phenol and metal content of the drilling fluids may have also contributed to their toxicity. Field dispersion studies on drilling fluids are discussed in terms of potential impact areas.

EPA Ocean Disposal Bioassay Working Group. 1976. Bioassay Procedures for the Ocean Disposal Permit Program. EPA-600/9-76-010. U.S. Environmental Protection Agency, Environmental Research Laboratory, Gulf Breeze, FL. 96 p. (Avail. from NTIS, Springfield, VA: PB-278 631)

The bioassay procedures given in this manual were developed to provide tests for conducting toxicity evaluations of waste materials considered for ocean disposal under EPA's Ocean Disposal Permit Program. Nine bioassay procedures are described; three of which are considered 'special' and are not recommended for routine use. The procedures specify the use of various organisms representing several trophic levels. Both flow-through and static tests are included. Methods given vary in their utility and complexity of performance. The procedures are not intended to be considered 'standard methods', but are to be used as reference methods or official methods dependent on the judgement of the EPA Regional Administrator responsible for the management of the permit program.

Meyers, Samuel P., Editor. 1976. Proceedings of the International Symposium on Marine Pollution Research, Jan. 27-29, 1976, Gulf Breeze, FL. EPA-600/9-76-032. U.S. Environmental Protection Agency, Environmental Research Laboratory, Gulf Breeze, FL. 171 p. (Avail. from NTIS, Springfield, VA: PB-267 601)

The participants in the International Symposium on Marine Pollution Research expressed concern about the continual and increasing pollution of the marine environment, in particular the potential adverse effect of marine pollution on the production of protein from the sea. Participants discussed broad issues relating to international marine pollution and some specific areas of potential cooperation. Broad issues and specific areas of potential research are listed in order of discussions, not necessarily in order of priority. Broad issues: (1) identification of pollution problems--each country must identify and prioritize marine pollution problems within their boundaries, only then can international problems be discussed and prioritized. (2) Training and technology transfer--training of non-professionals and professionals in the field of marine pollution must be accomplished on a worldwide basis. Training also can be accomplished through the transfer of technical information from one country to another. This can be accomplished through communications: individual to individual, workshops with scientific personnel from many countries, seminars and other technology transfer methods. (3) Monitoring--monitoring should be conducted only with a clear and concise purpose for the use of the monitoring data and a knowledge of the mechanisms in which the monitored pollutants were identified and quantitated. (4) Impact on biological systems--criteria must be developed for determining the impact of pollutants on biological systems. (5) Water quality criteria--quantitative and qualitative information has been developed at national and international levels on water quality criteria for ocean and coastal waters. This information should be made available to all interested countries and could be used as a starting point for countries who have not yet developed such criteria. (6) Education--the importance and necessity of scientific education must be emphasized. It is particularly important to educate laymen, both government authorities and the public who may not have adequate technical backgrounds to understand the complexities of marine pollution and its impact on global systems. (7) Communication of techniques--this is a special aspect of technology transfer that should be stressed in relation to the compilation of methods manuals for such items as (a) sampling, (b) chemical analysis, and (c) bioassays so that data collected from different nations can be compared.

EPA Ocean Disposal Bioassay Working Group. 1978. Bioassay Procedures for the Ocean Disposal Permit Program. EPA-600/9-78-010. U.S. Environmental Protection Agency, Environmental Research Laboratory, Gulf Breeze, FL. 121 p. (Avail. from NTIS, Springfield, VA: PB-278 631)

The bioassay procedures given in this manual were developed to provide tests for conducting toxicity evaluations of waste materials considered for ocean disposal under EPA's Ocean Disposal Permit Program. The procedures specify the use of various organisms representing several trophic levels. Both flow-through and static tests are included. Methods given vary in their utility and complexity of performance. The procedures are not intended to be considered 'standard methods,' but, depending on the judgement of the EPA Regional Administrator responsible for the managing of the permit program, are to be used as reference methods or official methods.

Lee, Jong H., Colin E. Nash and Joseph R. Sylvester. 1975. Effects of Mirex and Methoxychlor on Striped Mullet, Mugil cephalus L. EPA-660/3-75-015. U.S. Environmental Protection Agency, National Environmental Research Laboratory, Corvallis, OR. 24 p. (Avail. from NTIS, Springfield, VA: PB-241 635)

The effects of two chlorinated insecticides, mirex and methoxychlor, on striped mullet, Mugil cephalus L., were studied. Test concentrations of both insecticides used were 0.01, 0.1, 1.0 and 10.0 ppm in dynamic bioassay. Young juveniles were more susceptible to mirex exposure than older juveniles or adults. No mortalities occurred in older juveniles and adults exposed to mirex for 96 hours. For young juveniles, mortalities were highest in concentrations of 0.1 and 1.0 ppm and were less in concentrations of 0.01 and 10.0 ppm. Significant amounts of mirex residues were accumulated in the body tissues of the test fish; concentrations increased with increased environmental concentrations. Methoxychlor was more toxic to mullet than mirex. Mortalities were greater than 90% over a 96-hour period for all life stages studied at concentrations of 0.1, 1.0 and 10.0 ppm. Mortality at a concentration of 0.01 was 5.1% or less for 96 hours. Relative to mirex, small amounts of methoxychlor residues accumulated in the tissues of the test fish. Results of the experiments on eggs and larvae were inconclusive. Egg survival was slightly better in mirex than in methoxychlor over a 96-hour period. Larval survival was generally better in mirex than methoxychlor.

Scott, G.I., M.H. Fulton, M.C. Crosby, P.B. Key, J.W. Daugomah, J.T. Waldren, E.D. Strozier, C.J. Louden, G.T. Chandler, T.F. Bidleman, K.L. Jackson, T.W. Hampton, T. Huffman, A. Shulz and M. Bradford. 1994. Agricultural Insecticide Runoff Effects on Estuarine Organisms: Correlating Laboratory and Field Toxicity Tests, Ecophysiology Bioassays, and Ecotoxicological Biomonitoring. EPA/600/R-94/004. U.S. Environmental Protection Agency, Environmental Research Laboratory, Gulf Breeze, FL. 288 p. (Avail. from NTIS, Springfield, VA: PB94-160678)

This study compared in situ, field and laboratory toxicity testing results for several insecticides (azinphosmethyl - an organophosphate; endosulfan - an organochlorine, and fenvalerate - a synthetic pyrethroid) with ecotoxicological biomonitoring results from the macropelagic, estuarine tidal creek community in pristine habitats and in areas receiving significant insecticide runoff from agriculture. Field studies were conducted over a four-year period (1985-88) at several coastal field sites on Wadmalaw (Leadenwah Creek) and Johns (unnamed tidal creek near Kiawah Island) Island, coastal sea islands located just south of Charleston, South Carolina. Results indicated that laboratory and field toxicity testing and biomonitoring methodologies should be integrated to provide holistic environmental risk assessments for pesticides. Laboratory toxicity tests provide the initial bench mark for estimating toxic effects. In situ, field toxicity tests provide a mechanism to validate initial laboratory tests and expand their design to test differences in formulations, life history stages, pulsed versus continuous dose, salinity interactions, and pesticide mixtures for more realistic estimations of effects of field exposures. Application of this method in the environmental risk assessment for three classes of pesticides (organochlorines-endosulfan, pyrethroids-fenvalerate, and organophosphates-azinphosmethyl) has been demonstrated in assessing the effects of nonpoint source agricultural runoff on sensitive estuarine tidal creek fauna in South Carolina. Over a three year period of study, the integration of this approach has provided significant data to assist environmental regulators trying to control recurrent problems of agricultural runoff effects in Leadenwah Creek and other areas of the state. Future studies should be expanded to broaden our understanding of the usefulness of this integrated approach in better assessing pesticide runoff in other aquatic ecosystems throughout the U.S.

Fulton, Michael H., Geoffrey I. Scott, Peter B. Key, G. Tom Chandler, Robert F. Van Dolah and Phillip P. Maier. 1999. Comparative Toxicity Testing of Selected Benthic and Epibenthic Organisms for the Development of Sediment Quality Test Protocols. EPA/600/R-99/085. U.S. Environmental Protection Agency, National Health and Environmental Effects Research Laboratory, Gulf Ecology Division, Gulf Breeze, FL. 52 p.

Sediment contamination has resulted in the need to develop an appropriate suite of toxicity tests to assess ecotoxicological impacts on estuarine ecosystems. Existing Environmental Protection Agency (EPA) protocols recommend a number of test organisms, including amphipods, polychaetes, molluscs, crustaceans and fish for use in sediment toxicity tests. While this suite of test animals represents a diverse group of fauna, many of the species recommended by the EPA are not indigenous to all geographic regions of the United States, particularly the Gulf of Mexico and South Atlantic. As a result, environmental risk assessment based on these organisms may not adequately protect ecosystem health in the Gulf of Mexico. Ideally, appropriate test organisms to evaluate sediment toxicity should include species indigenous to the Gulf of Mexico that are representative of a variety of faunal classes and feeding types. Additionally, the toxicity test endpoints should include both lethal (mortality) and sublethal (reproduction, growth, physiological impairment) effects and they should be sensitive to either porewater and/or whole sediment exposures for all major classes of chemical contaminants (trace metals, polycyclic aromatic hydrocarbons [PAHs], pesticides). Finally, test species should be easy to collect and maintain in the laboratory. This study examined the relative sensitivity of a variety of test organisms, broadly distributed throughout the southeastern United States and the Gulf of Mexico to several classes of chemical contaminants in both whole sediment and aqueous/porewater exposures. Additionally, several rapid screening assays were compared with these more traditional toxicity evaluations. The three model contaminants selected for study were cadmium (an inorganic toxicant), DDT (a persistent organochlorine pesticide) and fluoranthene (a polycyclic aromatic hydrocarbon [PAH]). These compounds represent contaminants frequently measured in sediments throughout the Gulf of Mexico. Overall, the juvenile clam was the most sensitive species tested in this study from an acute toxicity standpoint. The grass shrimp and the two amphipod species were generally similar in sensitivity to each of the three compounds. The copepod assay, although relatively insensitive in terms of adult mortality, was capable of detecting sublethal effects at contaminant concentrations below those which caused mortality in the other more sensitive species. Both the juvenile clam assay and the copepod partial life cycle test have the potential to serve as sensitive indicators of potential sediment-associated toxicity which might not be detected using standard acute toxicity bioassays. The differing species sensitivities observed with the different classes of chemical contaminants in this study suggest that a multiple species approach may be more appropriate for a holistic ecological risk assessment of sediment contamination. The "Crustacean Triad" (copepods, amphipods and grass shrimp) provide a battery of tests which predict toxicity to epibenthic and benthic crustaceans with known sensitivity to a variety of chemical contaminants and represent the base of the food chain for most recreationally and commercially important finfish species that utilize estuarine nursery grounds. The addition of the juvenile clam assay provides a herbivorous filter feeder with the ability to bioconcentrate pollutants and which is extremely sensitive in the size range tested (>212<350 µm). Field studies in South Carolina have indicated that sites with high sediment contaminant levels have degraded benthos, with significant effects observed in crustaceans and molluscs. These findings support our laboratory results and suggest that an integrated battery of assays may be most appropriate for estimating field effects.

Lewis, Michael. 2000. Using Grass Shrimp as an Indicator of Pore Water Toxicity. Contaminated Sediment News. 24:7-8. (ERL,GB R713).

No scientific consensus currently exists on a suite of test species needed to determine if a near-coastal sediment is toxic. To provide some insight on this issue, scientists at the ORD's Gulf Ecology Division recently completed a baseline study evaluating the usefulness of the grass shrimp, Palaemonetes pugio Holthius (Fig. 1), as an indicator of pore water toxicity. Grass shrimp are one of the more widely distributed and abundant benthic organisms in shallow estuaries along the Gulf of Mexico and Atlantic coasts but they have been used infrequently in contaminated sediment assessments. In this survey, early life stages of P. pugio were exposed to pore waters obtained from sediments collected from 37 Florida coastal areas affected by coastal golf courses, urbanization, agriculture and wastewater discharges. An overview of the 12 static bioassay procedure for P. pugio used in this coastal survey is presented in Table 1. One to three females possessing embryos at the tissue cap stage (2-3 days after oviposition) were selected from a laboratory culture to provide the test organisms. Twenty-four embryos were exposed to each pore water individually in wells of disposable plastic culture plates. Each well contained 2 ml of undiluted (100%) pore water. Filtered natural seawater (10 ppt and 20 ppt salinity) served as controls. The grass shrimp were examined on test days 10, 11, and 12 for mortality and morbidity. In a few cases, additional sediment bioassays were conducted using duplicate samples of the sediments from which the pore waters were extracted and used in the shrimp bioassays. Acute bioassays were conducted with the epibenthic invertebrate, Mysidopsis bahia, and whole sediments (solid phase) to provide some information on the effect of the test media on the sediment toxicity evaluations. The 7-day bioassays were static; a total of 50 organisms were exposed to three replicate sediment samples collected from each site. Most pore waters, regardless of the collection site, were not acutely toxic to grass shrimp embryos. Of the 53 bioassays conducted with this species, mortality above control levels was observed in 15 tests (28% of total). The average survival in these tests was 27 (1 standard deviation =±27)% relative to 94(±7)% in the corresponding seawater controls. The embryonic life stage demonstrated the greatest sensitivity to pore waters. The response of different brood stocks were statistically similar P=0.05) when exposed to the same pore waters. The whole sediments were not acutely toxic to juvenile M. bahia. Survival ranged between 97 and 100%. Consequently, the pore water bioassays with grass shrimp embryos were a more sensitive indicator of sediment quality. Based on all considerations, it was concluded that the grass shrimp early-life stage test shows promise as being useful to detect pore water toxicity. This is based on the availability of a test methodology that requires minimal space and effort, and the test species geographical distribution and year-round availability. However, the important issue of its sensitivity remains to be determined relative to that for other pore water test species as well as for those used in solid-phase bioassays. For this reason, the use of grass shrimp is encouraged for contaminated sediment evaluations conducted with its geographic range in order to establish a sufficient background data base on which to more definitively judge its value in contaminated sediment toxicity asssessments.

Bahner, L.H., A.J. Wilson, J.M. Sheppard, J.M. Patrick, L.R. Goodman and G.E. Walsh. 1978. Kepone Accumulation and Food Chain Transfer. In: Proceedings of the Kepone Seminar II. EPA-903/9-78-011. U.S. Environmental Protection Agency, Region III, Philadelphia, PA. Pp. 294-329. (ERL,GB X006).

Accumulation, transfer, and loss of Kepone in estuarine organisms were studied in laboratory bioassays. Kepone was bioconcentrated by oysters (Crassostrea virginica), mysids (Mysidopsis bahia), grass shrimp (Palaemonetes pugio), sheepshead minnows (Cyprinodon variegatus), and spot (Leiostomus xanthurus), from concentrations as low as 0.023 ug/l seawater. Bioconcentration factors ranged from 10 to 340 in static exposures and 900 to 13,500 in flow-through bioassays and were dependent on species and exposure duration. Depuration of Kepone from oysters in Kepone-free water was rapid (35% loss in 24 hours); however, depuration of kepone was slow in crustaceans and fish, with tissue concentrations decreasing 30-50% in 24-28 days. Oysters, fed Chlorococcum containing approximately 34 ug Kepone/g wet weight, attained 0.21 ug Kepone/g (wet tissue) in 14 days, but when fed Kepone-free plankton, depurated Kepone to below detectable concentrations (less than .02 ug/g) within 10 days. Spot obtained Kepone when fed live mysids that had grazed on Kepone-laden brine shrimp. Kepone residues (1.05 ug/g wet tissue) in these fish approached the concentration of their food (1.23 ug/g wet tissue); at the lower concentration tested, Kepone concentrations below detection limits (less than .2 ug/g) in prey accumulated in the predator to detectable concentrations (0.02 ug/g) within 30 days. Bioaccumulation factors (concentration of Kepone in predator/concentration in prey) at 30 days were equal (0.85 spot/mysid; 0.53 mysid/brine shrimp) in the high and low concentrations tested.

Borthwick, Patrick W. 1978. Methods for Acute Static Toxicity Tests with Mysid Shrimp (Mysidopsis bahia). In: Bioassay Procedures for Ocean Disposal Permit Program. EPA-600/9-78-010. U.S. Environmental Protection Agency, Environmental Research Laboratory, Gulf Breeze, FL. Pp. 61-63. (ERL,GB X013).

Mysidopsis bahia is a shrimp-like estuarine crustacean that has been shown to be very sensitive to toxic substances and used successfully in acute static toxicity tests with complex wastes. M. bahia is recommended as a test species due to its sensitivity, short life-cycle, small size, and practical culture methods. Results from toxicity tests with mysids can be used to estimate the impact of ocean-dumped materials on other salt water crustaceans.

Butler, P.A. and J.I. Lowe. 1978. Flowing Sea Water Toxicity Test Using Oysters (Crassostrea virginica). In: Bioassay Procedures for the Ocean Disposal Permit Program. EPA-600/9-78-010. U.S. Environmental Protection Agency, Environmental Research Laboratory, Gulf Breeze, FL. Pp. 25-27. (ERL,GB X019).

The following test procedure is included as a 'special bioassay' for evaluating short-term effects of specific wastes on marine mollusks. It is recommended only for use with the commercial eastern oyster, Crassostrea virginica, and requires flowing unfiltered, natural sea water. This test should be used only with materials which can be dissolved in water or other solvents. The test has proven valuable at ERL, Gulf Breeze, where is has been used for several years to evaluate the effect of insecticides, herbicides, and other toxic organics on oysters.

Butler, P.A. and J.I. Lowe. 1976. Flowing Sea Water Toxicity Test Using Oysters (Crassostrea virginica). In: Bioassay Procedures for the Ocean Disposal Permit Program. EPA-600/9-76-010. U.S. Environmental Protection Agency, Environmental Research Laboratory, Gulf Breeze, FL. Pp. 81-83. (ERL,GB X020).

The following test procedure is included as a 'special bioassay' for use in evaluating short-term effects of specific wastes on marine molluscs. It is recommended only for use with the commercial eastern oyster, Crassostrea virginica, and requires flowing unfiltered, natural sea water. This test should be used only with materials which can be dissolved in water or other solvents and then metered into test aquaria. The test has proved to be a valuable bioassay procedure at the Gulf Breeze Environmental Research Laboratory (EPA) where it has been used for several years to evaluate the effects of insecticides, herbicides, and other toxic organics on oysters.

Conklin, Philip J. and K. Ranga Rao. 1978. Toxicity of Sodium Pentachlorophenate (Na-PCP) to the Grass Shrimp, Palaemonetes pugio, at Different Stages of the Molt Cycle. Bull. Environ. Contam. Toxicol. 20(2):275-279. (ERL,GB X021).

Pentachlorophenol (PCP) and its salt, sodium pentachlorophenate (Na-PCP) are widely used as biocides. Previous investigations indicate that adult crustaceans are more tolerant than fish to PCP and Na-PCP. The toxicity data for crustaceans are based on short term (usually 96 hours or less) bioassays on individuals whose physiological status in relation to the molt cycle was unknown. Since changes in the permeability of cuticles are expected to occur in relation to the cyclic shedding, secretion and hardening of the exoskeleton in crustaceans, it is important to evaluate the toxicity of pesticides at known stages of the molt cycle. Although there are reports of an apparent increase in the sensitivity of adult crustaceans during or soon after molting (ecdysis) to toxicants such as copper, Aroclor 1254, and Methoxychlor, there have been no toxicological evaluations in relation to the different stages of the molt cycle. The crustacean molt cycle is divided into five major stages (A through E) and several substages which can be identified by distinct morphological criteria. The aim of this investigation was to evaluate the toxicity of Na-PCP to the grass shrimp, Palaemonetes pugio, at specific stages of the molt cycle using standard 96 hour bioassays.

U.S. Environmental Protection Agency Technical Work Group. 1974. Physical, Chemical and Biological Considerations with Respect to the Dupont Permit for Ocean Dumping of Industrial Wastes. U.S. Environmental Protection Agency, Office of Health and Ecological Effects, Washington, DC. 44 p. (ERL,GB X033).

Executive summary: the working group, as a result of the examination of technical information contained in the hearing testimony, finds that there is inadequate scientific data upon which to make an informed judgment of the probable environmental effects of the proposed activity. The quality and quantity of data relating to circulation of water masses in the Gulf of Mexico and to the transport of discharged materials is not adequate to estimate probable effect. Although the chemical nature of the Dupont wastes is qualitatively and quantitatively known, many uncertainties exist about the behavior of the waste in the marine environment and its fate and transport in the Gulf of Mexico. While many of the waste constituents are considered to be of minor environmental consequence because they occur in trace amounts, others, such as antimony, occur in large amounts and must be presumed to be environmentally harmful until definitive data are developed. Available biological data suggest that the composite waste may be of low relative toxicity with respect to acute (short-term) bioassays. Nevertheless, the information base is inadequate to estimate chronic (long-term) effects or possible accumulation of materials in Gulf species or in the food chain.

Conklin, Philip J. and K. Ranga Rao. 1978. Toxicity of Sodium Pentachlorophenate to the Grass Shrimp, Palaemonetes pugio, in Relation to the Molt Cycle. In: Pentachlorophenol: Chemistry, Pharmacology, and Environmental Toxicology. K. Ranga Rao, Editor. Plenum Press, New York, NY. Pp. 181-192. (ERL,GB X040).

The toxicity of sodium pentachlorophenate (Na-PCP) to the grass shrimp, Palaemonetes pugio, was evaluated at different stages of the molt cycle. In 96-hour bioassays, the shrimp in later stages of the proecdysial period exhibited a greater sensitivity to Na-PCP than that exhibited by shrimp in the intermolt and early proecdysial stages of the molt cycle. The shrimp in later proecdysial stages generally molted (underwent ecdysis) during the 96-hour test period and died shortly after ecdysis. The 96-hour LC50 value obtained for these shrimp (0.436 ppm) is the lowest of all the LC50 values reported previously for adult crustaceans and is comparable to those for fish and larval crustaceans. The increased sensitivity to Na-PCP during the early postecdysial period was also apparent in a long-term (66 days) test. The observed postecdysial mortality of shrimp exposed to 1.0 ppm Na-PCP was not dependent on the duration of exposure of shrimp to Na-PCP during the proecdysial period. Studies with 14C-PCP indicate that an abrupt increase in the uptake of PCP during the period shortly after ecdysis may cause increased mortalities during this period.

Coppage, David. 1976. Fish Brain Acetylcholinesterase Inhibition Assay. In: Bioassay Procedures for the Ocean Disposal Permit Program. EPA-600/9-76-010. U.S. Environmental Protection Agency, Environmental Research Laboratory, Gulf Breeze, FL. Pp. 91-95. (ERL,GB X042).

The purpose of this procedure is to provide a method for determining the effect of materials to be dumped in the ocean on acetylcholinesterase (AChE) in fish brains. This is a test for nerve poisons which disrupt nerve impulse transmission by inhibiting AChE, the enzyme that modulates levels of the neurotransmitter acetylcholine. This procedure is not necessary for materials that contain no AChE inhibiting poisons. It has been shown that brain-AChE of fishes is inhabited by in vivo exposure to organophosphate and carbamate pesticides under laboratory conditions. Furthermore, environmental water pollution by these pesticides has been monitored by measuring AChE activity in fish brains. Coppage defined the conditions necessary for obtaining reliable and reproducible data in the laboratory AChE assays and reported in vitro effects of four pesticides on AChE acitivity in brains of sheepshead minnows (Cyprinodon variegatus). Coppage and Matthews further refined assay techniques and reported acute effects of in vivo exposure to organophosphate pesticides oncholinesterases of four estuarine fishes and a shrimp.

Hansen, David J. and Steven C. Schimmel. 1976. Chronic Bioassay Using Sheepshead Minnows (Cyprinodon variegatus). In: Bioassay Procedures for the Ocean Disposal Permit Program. EPA-600/9-76-010. U.S. Environmental Protection Agency, Environmental Research Laboratory, Gulf Breeze, FL. Pp. 84-90. (ERL,GB X045).

The purpose of this procedure is to provide a method of determining the effect of a material on survival of sheepshead minnow embryos and fry, their growth to adulthood, and spawning success. Spawning success is measured by observing the ability of pairs of fish to spawn naturally, their fecundity, fertilization success and survival of embryos and fry. This test has several limitations and should not be considered valid for assessing toxicity of all types of materials. Sheepshead minnows are tolerant of low oxygen and a wide range of temperature and salinity and bioassays using this fish may underestimate the toxicity of materials that are toxic because they alter these environmental conditions. Materials tested should be ones that mix well with water. Insoluble or highly turbid materials mix poorly, and their toxicity may be under or overestimated.

Potomac Research Incorporated. 1978. Marine Oil Pollution Research Study Final Report 1 June 1978. Potomac Research Incorporated, Panama City, FL. 52 p. (ERL,GB X063).

This report documents the biological bioassay work done on Stage I, a stationary Navy research platform located 12 miles off the coast of Panama City, Florida, for the Naval Coastal Systems Center (NCSC) in conjunction with an interagency agreement between NCSC and the Environmental Protection Agency, Environmental Research Laboratory (ERL), Gulf Breeze, Florida. The report summarizes the results of the community studies, short term bioassays and polychaete identification. Conclusions are based upon the compiled information.

Cross, F.A. and T.W. Duke. 1974. Contaminant Bioassays: Contamination of Marine Resources for Human Consumption. In: Marine Bioassays. Marine Technology Society, Washington, DC. Pp. 32-108. (ERL,GB X064).

Our panel dealt with research an monitoring needs for current and potential residue problems in marine organisms although long-term effects of contaminants on marine ecosystems also were considered. The panel was divided into three groups to consider petroelum hydrocarbons, toxic metals and synthetic oganic compounds. Although each group started with the same general outline, special needs and research problems with specific contaminants resulted in three reports which differ considerably in emphasis.

Erickson, Stanton J. 1972. Toxicity of Copper to Thalassiosira pseudonana in Unenriched Inshore Seawater. J. Phycol. 8(4):318-323. (ERL,GB X069).

Toxicity of copper to T. pseudonana (formerly Cyclotella nana, clone 13-1) was examined in inshore seawater using a 96-hr bioassay methods. Raw unenriched seawater was filtered through a 0.22-u membrane filter and then pasteurized for 30 min at 60 C. Following this treatment, samples contained 0.68-1.14 ug Cu/liter. Copper was added as the chloride in 5-ug increments over the range of 5 to 30 ug/liter (about 0.1-0.5 uM). Population densities, mean cell volume, and 14C bicarbonate uptake were measured. Population growth and 14C uptake by T. pseudonana displayed inhibition over the entire range of added copper. Growth rate constant (k) of T. pseudonana decreased with increasing copper concentration and during the course of growth at each concentration. Correspondingly, mean cell volumes increased with copper concentration and time. Copper toxicity varied in different water samples. The presence of decomposed natural plankton and detritus decreased toxicity. In the absence of enrichment, bacteria had little effect on copper toxicity. Results were influenced by glassware treatment, collection and storage of seawater, and absence of enrichments.

Hansen, David J., Steven C. Schimmel, DelWayne Nimmo, Jack I. Lowe, Patrick R. Parrish and William H. Peltier. 1976. Continuous-Flow Method for Acute Toxicity Tests Using Fish and Macroinvertebrates. In: Bioassay Procedures for the Ocean Disposal Permit Program. EPA-600/9-76-010. U.S. Environmental Protection Agency, Environmental Research Laboratory, Gulf Breeze, FL. Pp. 69-80. (ERL,GB X080).

Continuous-flow (often referred to as 'flow-through') bioassays have definite advantages over static tests in evaluating certain types of wastes to be disposed of at sea. They are desirable in testing waste chemicals that have high biochemical oxygen demands, and are unstable or volatile. Many test species of fish and macroinvertebrates have high rates of metabolism and are difficult to maintain in jars or tanks of standing sea water. Continuous-flow bioassays, conducted under proper conditions, provide for well-oxygenated test solutions, nonfluctuating concentrations of the toxicant, and continual removal of metabolic wastes of the test organisms. This method provides general procedures for conducting a 96-hour, flow-through bioassay on marine fish and macroinvertebrates such as shrimp and crabs. Evaluation of different types of waste will, no doubt, require some modification of these procedures.

Hansen, David J., Steven C. Schimmel, DelWayne Nimmo, Jack I. Lowe, Patrick R. Parrish and William H. Peltier. 1978. Flow-Through Methods for Acute Toxicity Tests Using Fishes and Macroinvertebrates. In: Bioassay Procedures for the Ocean Disposal Permit Program. EPA-600/9-78-010. U.S. Environmental Protection Agency, Environmental Research Laboratory, Gulf Breeze, FL. Pp. 97-106. (ERL,GB X081).

Continuous-flow (often referred to as 'flow-through') bioassays have definite advantages over static tests in evaluating certain types of wastes to be disposed of at sea, particularly in testing waste chemicals that have high biochemical oxygen demands, and are unstable or volatile. Many test species of fish and macroinvertebrates have high rates of metabolism and are difficult to maintain in jars or tanks of standing sea water. Continuous-flow bioassays, conducted under proper conditions, provide for well-oxygenated test solutions, nonfluctuating concentrations of the toxicant, and continual removal of metabolic wastes of the test organisms. This method provides general procedures for conducting a 96-hour, flow-through bioassay on marine fish and macroinvertebrates such as shrimp and crabs. Evaluation of different types of waste will require some modification of these procedures.

Hansen, David J., Steven C. Schimmel, DelWayne Nimmo, Jack I. Lowe, Patrick R. Parrish and William H. Peltier. 1978. Static Method for Acute Toxicity Tests Using Fish and Macroinvertebrates. In: Bioassay Procedures for the Ocean Disposal Permit Program. EPA-600/9-78-010. U.S. Environmental Protection Agency, Environmental Research Laboratory, Gulf Breeze, FL. Pp. 89-96. (ERL,GB X082).

Hansen, D.J., P.R. Parrish, S.C. Schimmel and L.R. Goodman. 1978. Life-Cycle Toxicity Test Using Sheepshead Minnows (Cyprinodon variegatus). In: Bioassay Procedures for the Ocean Disposal Permit Program. EPA-600/9-78-010. U.S. Environmental Protection Agency, Environmental Research Laboratory, Gulf Breeze, FL. Pp. 109-117. (ERL,GB X083).

This procedure provides a method to determine the effect of continuous exposure of a toxic material on sheepshead minnow embryos and fry: their survival and growth to adulthood, and spawning success. Spawning success is measured by the ability of fish to spawn naturally, number of eggs spawned, fertilization success, and survival of embryos and fry. The experiment requires from 4 to 6 months. The primary advantage of this test is that results, when compared with those of acute tests with this species, can be used to calculate an application factor. This factor, used to assess relative chronic hazards of materials, is important in establishing water quality criteria. This test has several limitations and should not be considered valid in assessing toxicity of all materials. Sheepshead minnows can tolerate low dissolved oxygen and wide ranges of temperature and salinity. Therefore, toxicity tests using this fish may underestimate the toxicity of materials that alter these environmental conditions. Materials tested should mix well with water. Insoluble or highly turbid materials mix poorly, and their toxicity may be under- or overestimated.

Hansen, David J. 1978. Laboratory Culture of Sheepshead Minnows (Cyprinodon variegatus). In: Bioassay Procedures for the Ocean Disposal Permit Program. EPA-600/9-78-010. U.S. Environmental Protection Agency, Environmental Research Laboratory, Gulf Breeze, FL. Pp. 107-108. (ERL,GB X084).

Sheepshead minnows can be readily cultured in the laboratory in aquaria with under-substrate filters or in aquaria receiving flowing salt water. The following discussion presents culture techniques used successfully at the U.S. Environmental Protection Agency, Environmental Research Laboratory, Gulf Breeze, Florida.

Nimmo, D.R., T.L. Hamaker and C.A. Sommers. 1978. Culturing the Mysid (Mysidopsis bahia) in Flowing Sea Water or a Static System. In: Bioassay Procedures for the Ocean Disposal Permit Program. EPA-600/9-78-010. U.S. Environmental Protection Agency, Environmental Research Laboratory, Gulf Breeze, FL. Pp. 59-60. (ERL,GB X107).

Many freshwater but few estuarine or marine animals have been found practical for life-cycle toxicity tests. Life cycles of certain marine species are complex: many require an estuarine existence as larvae or juveniles, followed by adult migration to deeper waters offshore to reproduce. Culture and maintenance of estuarine and marine species entail elaborate and expensive equipment with temperature or salinity controls, anticorrosion surfaces, and if necessary, special filtration systems. We have cultured the bay mysid, Mysidopsis bahia, for life-cycle toxicity tests at ERL, Gulf Breeze, in (1) flowing sea water and (2) a recirculating aquarium. Both methods are described below; however, the re-circulating method is appropriate for laboratories not equipped with flowing seawater.

Nimmo, D.R., T.L. Hamaker and C.A. Sommers. 1978. Entire Life Cycle Toxicity Test Using Mysids (Mysidopsis bahia) in Flowing Water. In: Bioassay Procedures for the Ocean Disposal Permit Program. EPA-600/9-78-010. U.S. Environmental Protection Agency, Environmental Research Laboratory, Gulf Breeze, FL. Pp. 64-68. (ERL,GB X108).

The purpose of this method is to determine effects of continuous exposure of a pollutant on the survival, growth, and behavior of this crustacean through a life cycle. Among the advantages of using this species in toxicity tests are: (1) ease of culture and maintenance; (2) short generation time (14-17 days depending on the temperature); and reproduction data based on actual count (of juveniles) rather than estimates. Further, this species is representative of many intermediates in estuarine food webs. Data on toxicity, reproduction, and growth, using a modification of the procedure described here, have been published. Mysidopsis bahia is an estuarine species and three reports in the literature suggest that its range is from Galveston, Texas to Miami, Florida. We have successfully captured mysids from small shallow ponds fed by salt water from Santa Rosa Sound near Pensacola, Florida. A small fish net, used by tropical fish retailers, or a 3-4 foot push net with very small mesh, is sufficient to capture the adult mysid shrimp.

Rao, K. Ranga, Philip J. Conklin and Anita C. Brannon. 1978. Inhibition of Limb Regeneration in the Grass Shrimp, Palaemonetes pugio, by Sodium Pentachlorophenate. In: Pentachlorophenol: Chemistry, Pharmacology, and Environmental Toxicology. K. Ranga Rao, Editor. Plenum Press, New York, NY. Pp. 193-203. (ERL,GB X116).

The initiation and progress of regeneration following the removal of the left fifth pereiopod were studied using the grass shrimp, Palaemonetes pugio. The regeneration patterns of 400 shrimp subjected to various treatments revealed that sodium pentachlorophenate (Na-PCP) affects the initiation and progress of limb regeneration. Depending on the concentration used, Na-PCP caused either a complete inhibition of regeneration, a delay of initiation of limb bud development, or a reduction of limb bud growth without altering the intermolt duration. By comparing the regeneration indices (R values) of control and experimental shrimp noted on specified days preceding ecdysis and on the day following ecdysis it was possible to determine the extent (%) of inhibition of regeneration in shrimp exposed to Na-PCP. EC50 values were computed using probit analysis. For example, the R values of shrimp nine days after limb removal yielded the following EC50 values with 95% confidence intervals shown in parenthesis: unfed shrimp, 0.473 ppm Na-PCP (0.306-0.670); fed shrimp, 0.565 ppm (0.452-0.706). The EC50 values based on postecdysial R values were: unfed shrimp, 0.615 ppm Na-PCP (0.451-0.852); fed shrimp, 0.637 ppm (0.485-0.850). The inhibitory effects of Na-PCP were more pronounced on the initial phases of limb regeneration (involving wound healing, cell division and dedifferentiation) than on the later phases of regeneration (involving further differentiation and cellular enlargement). Crustacean limb regeneration can be used as a sensitive bioassay for studying the effects of chemical pollutants.

Rubinstein, Norman I. 1979. Benthic Bioassay Using Time-Lapse Photography to Measure the Effect of Toxicants on the Feeding Behavior of Lugworms (Polychaeta: Arenicolidae). In: Marine Pollution: Functional Responses. EPA-600/J-79-097. W.B. Vernberg, A. Calabrese, F. Thurberg, and F.J. Vernberg, Editors. Academic Pres, Inc., New York, NY. Pp. 341-351. (ERL,GB X120).

A benthic assay was developed utilizing time-lapse photography to measure the feeding activity of a lugworm, Arenicola cristata. Automated 35 mm cameras were used to record formation of feeding funnels at 12-hour intervals. Substrate surface area reworked by lugworms held under identical conditions in separate aquaria was plotted against time to determine substrate reworking rates for each group. Rates were subjected to linear regression analysis and compared to demonstrate that no significant difference between slopes of the calculated lines existed. Therefore, a difference in slope when one group is exposed to a toxicant could provide a measure of effect on lugworm activity. Lugworms also were exposed to the pesticide, Kepone, and their rate of substrate reworking was compared with unexposed lugworms. Kepone was acutely toxic to lugworms at a concentration of 29.5 ug/l. A significant difference in substrate reworking rates was observed following exposure to concentrations as low as 2.8 ug/l Kepone in seawater. It is suggested that a behavioral response to toxicity testing provides a sensitive and realistic approach for evaluation of ecological impact of pollutants on the marine environment.

Rubinstein, Norman I. 1978. Effect of Sodium Pentachlorophenate on the Feeding Activity of the Lugworm, Arenicola cristata Stimpson. In: Pentachlorophenol: Chemistry, Pharmacology, and Environmental Toxicology. K. Ranga Rao, Editor. Plenum Press, New York, NY. Pp. 175-179. (ERL,GB X121).

A benthic bioassay utilizing time-lapse photography was used to measure the effect of four concentrations of Na-PCP (45, 80, 156 and 276 ug/l) on the feeding activity of Arenicola cristata. There was no marked effect on feeding activity at 45 ug/l. Na-Pcp significantly affected feeding activity at concentrations of 80, 156, and 276 ug/l. As the lugworm feeds, it mixes organic material and oxygenated water into the substrate. Inhibition of this activity could affect benthic community trophic structure and substrate-water column dynamics.

Tyler-Schroeder, Dana Beth. 1978. Culture of the Grass Shrimp (Palaemonetes pugio) in the Laboratory. In: Bioassay Procedures for the Ocean Disposal Permit Program. EPA-600/9-78-010. U.S. Environmental Protection Agency, Environmental Research Laboratory, Gulf Breeze, FL. Pp. 69-72. (ERL,GB X124).

The grass shrimp, Palaemonetes pugio, a useful organism in assessing toxicity of various materials, is (1) easily cultured in the laboratory, (2) sensitive to toxicants, and (3) can be exposed in flow-through systems throughout a life cycle. Culture and holding procedures for the grass shrimp are described below. Laboratory spawning of P. pugio was first described by Little. Deposition of eggs began five to eight weeks after initiation of a photo-period and temperature regime. Egg production is directly proportional to rostrum-telson length of females greater than 18 to 20 mm. Shrimp are sexed by examination of the second pleopod, but field data show a 50/50 ratio of sexes.

Tyler-Schroeder, Dana Beth. 1978. Entire Life-Cycle Toxicity Test Using Grass Shrimp (Palaemonetes pugio Holthuis). In: Bioassay Procedures for the Ocean Disposal Permit Program. EPA-600/9-78-010. U.S. Environmental Protection Agency, Environmental Research Laboratory, Gulf Breeze, FL. Pp. 83-88. (ERL,GB X125).

The purpose of this method is to assess toxicity of a material to all life stages of the grass shrimp in flow-through systems. This experiment determines effects on survival, growth, and reproduction (including number of females spawning, number of days before onset of spawning, number of eggs per female, and hatching success) of parental generation shrimp. Effects on survival, larval development, and growth are also determined for F1 generation shrimp. These tests must extend through an entire life-cycle of the shrimp--from juvenile stage of the parental generation, sexual maturation and reproduction, through hatching, larval development, and growth of the F1 generation to juvenile stage. Tests may terminate at this point, or exposures can be continued if necessary to determine effect on F1 reproduction and F2 larval development.

Tyler-Schroeder, Dana Beth. 1978. Static Bioassay Procedure Using Grass Shrimp (Palaemonetes Sp.) Larvae. In: Bioassay Procedures for the Ocean Disposal Permit Program. EPA-600/9-78-010. U.S. Environmental Protection Agency, Environmental Research Laboratory, Gulf Breeze, FL. Pp. 73-82. (ERL,GB X126).

Procedures for static 96-hour bioassays utilizing grass shrimp larvae, Palaemonetes sp., are outlined here. The grass shrimp is an obvious bioassay choice for several reasons. Three species of the genus, P. pugio, vulgaris, and intermedius, are common inhabitants of estuaries along the Gulf and Atlantic coasts of the United States. They are easy to collect and maintain in the laboratory. Field populations are usually quite large, allowing greater numbers to be brought into the laboratory for testing. By manipulating environmental conditions of temperature and photoperiod, it has been possible to induce spawning in the laboratory, opening the way to laboratory cultures of genetic uniformity. Developing larvae are also available throughout the year for testing with these methods.

Tyler-Schroeder, Dana Beth. 1976. Static Bioassay Procedure Using Grass Shrimp (Palaemonetes Sp.) Larvae. In: Bioassay Procedures for the Ocean Disposal Permit Program. EPA-600/9-76-010. U.S. Environmental Protection Agency, Environmental Research Laboratory, Gulf Breeze, FL. Pp. 50-60. (ERL,GB X127).

Procedures for static 96-hour bioassays utilizing grass shrimp larvae, Palaemonetes sp., are outlined here. The grass shrimp is an obvious bioassay choice for several reasons. Three species of the genus, P. pugio, vulgaris, and intermedius, are common inhabitants of estuaries along the Gulf and Atlantic coasts of the United States. They are easy to collect and maintain in the laboratory. Field populations are usually quite large, allowing greater numbers to be brought into the laboratory for testing. By manipulating environmental conditions of temperature and photoperiod, it has been possible to induce spawning in the laboratory, opening the way to laboratory cultures of genetic uniformity. Developing larvae are also available throughout the year for testing with these methods. Larval stages of the three species are hardy and easy to culture in the laboratory. Developmental stages have been described for all species, and salinity-temperature optima are known for the larval development of P. vulgaris. Developing larvae have demonstrated a susceptibilty to polychlorinated hydrocarbons greater than adults or juveniles.

Wilkes, F.G. and R.M. Engler. 1977. First Annual Report: Environmental Protection Agency/Corps of Engineers Technical Committee on Criteria for Dredged and Fill Material. U.S. Army Engineer Waterways Experiment Station, Environmental Effects Laboratory, Vicksburg, MS. 41 p. (ERL,GB X130).

A technical committee of scientists and engineers from research elements of the Environmental Protection Agency (EPA) and the U.S. Army Corps of Engineers Waterways Experiment Station (CE) was formed in late 1975 to act as a focal point for coordinating and disseminating agency research related to regulatory functions pursuant to Sections 404 and 103 of Public Laws 92-500 and 92-532, respectively. The committee is cochaired by the EPA and CE. A major goal of the Technical Committee is the development of a comprehensive manual for technical implementation of all technical phases of Public Laws 92-500 and 92-532. Other objectives of the Technical Committee are to recommend needed research priorities in order to implement fully Sections 404 and 103, establish joint projects and priorities, conduct joint program reviews, avoid duplication of effort, and exchange and disseminate research results. The Technical Committee will also review and evaluate interim testing procedures promulgated by the CE for immediate implementation by field units. The Technical Committee consists of six subcommittees cochaired by EPA and CE personnel: the Bioassay/Bioevaluation, Area Definitions, Contaminants, Physical Impacts, Mixing Zone, and Fill Material Subcommittees. These subcommittees will guide the development of interim implementation manuals for Public Laws 92-500 and 92-532 and will recommend research programs designed at generating information that would fill knowledge gaps in order to ultimately develop and publish a comprehensive Implementation Manual. A listing of all related EPA and CE ongoing research programs is presented by each subcommittee in order that there would be no duplication in recommending and assigning priorities for needed research programs. A listing of 16 research areas is presented with each area of research assigned on overall priority; projected costs and duration of study are also presented herein.

Walsh, Gerald E. and Richard L. Garnas. 1982. Effects of Liquid Industrial Wastes on Estuarine Algae, Plants, Crustaceans, and Fishes. In: Second US/USSR Symposium: Biological Aspects of Pollutant Effects on Marine Organisms, Terskhol, USSR, June 7, 1979. EPA-600/3-82-034. U.S. Environmental Protection Agency, Environmental Research Laboratory, Corvallis, OR. Pp. 112-122. (ERL,GB X160).

A chemical fractionation scheme using ion-exchange resins for separation of organic and inorganic constituents of complex industrial wastes has been developed for use with bioassays in order to identify the toxic components of such wastes. Grass shrimp (Palaemonetes pugio) and sheepshead minnows (Cyprinodon variegatus) were not as good indicators of possible effects of complex wastes on estuarine organisms as the diatom, Skeletonema costatum in bioassays performed at our laboratory. The diatom was affected by wastes in either of the following three ways: stimulation, inhibition, or simulation at low concentrations but inhibition at higher concentrations. Toxicity to algae correlated with toxicity to the mysid, Mysidopsis bahia. Chemical fractionation with appropriate biological testing provides a means of estimating a more complete potential effect of an effluent on receiving waters. Effects of growth stimulators, for example, can be masked by toxicants in whole waste. We conclude that chemical fractionation is required for comprehensive analysis of possible effects of complex industrial wastes on estuarine organisms, and that fractionation should be coupled to bioassays that use at least an alga and a crustacean as test organisms.

Mix, Michael C., Diane L. Bunting and D.T. Abbott. 1979. Preliminary Studies to Evaluate the Potential of Using Embryo and Larval Stages of the Goose Barnacle, Pollicipes polymerus for Marine Bioassays. In: Proceedings of the Second Biennial Crustacean Health Workshop, TAMU-SG-79-114. D.H. Lewis and J.K. Leong, Editors. Texas A & M University, College Station, TX. Pp. 361-381. (ERL,GB X166).

The general purpose of these studies was to determine if the developmental and larval stages of the goose barnacle, Pollicipes polymerus would be useful organisms to use as a marine bioassay system. Different culture systems and culturing conditions were developed and evaluated. We designed a simple, inexpensive system that can be used to culture fertile eggs under normal laboratory conditions (room temperature and ambient light-dark cycle). The culture medium consists of Instant Ocean sea salts mixed in tap water to a salinity of 28-30 parts per thousand. Antibiotic concentrations of 10 mg/l seawater of streptomycin sulfate and 10 mg/l seawater of penicillin G are then added to the artificial seawater. As a result of these and other studies, it seems likely that P. polymerus may be a valuable bioassay organism. More complete studies, necessary to gather large amounts of data for developing and statistically evaluating different criteria to determine deleterious effects, are required before a final judgement can be made about the future use of P. polymerus for bioassays.

Koenig, Christopher C. and Claudia McLean. 1980. Rivulus marmoratus: A Unique Fish Useful in Chronic Marine Bioassays of Halogenated Organics. In: Water Chlorination: Environmental Impact and Health Effects, Vol. 3. Robert L. Jolley, William A. Brungs, and Robert B. Cumming, Editors. Ann Arbor Science Publishers, Ann Arbor, MI. Pp. 827-833. (ERL,GB X208). (Avail. from NTIS, Springfield, VA: PB81-125692)

Results are reported for chronic marine bioassays exposing the cyprinodontid fish Rivulus marmoratus, to 2,3,4,6-tetrachlorophenol. Purpose of the study was to illustrate the value of R. marmoratus as a chronic bioassay animal. Results demonstrated the relative insensitivity of R. marmoratus to chronic toxic effects of the chemical, but pathological changes were observed. Fish at least two-months-old showed gill damage ranging from severe (at the highest exposure concentration) to moderate and slight (at the lowest concentration). Such pathological changes might not be seen in chronic laboratory experiments with organisms of greater sensitivities.

Davey, Earl W., Myra J. Morgan and Stanton J. Erickson. 1973. Biological Measurement of the Copper Complexation Capacity of Seawater. Limnol. Oceanogr. 18(6):993-997. (ERL,GB X270).

Copper titrations of known concentrations of chelators in artificial seawater illustrated that the growth depression of Thalassiosira pseudonana in response to copper could be used to quantitate the chelator levels to within plus or minus 5 per cent at concentrations down to 10 to the minus 7 power MEDTA. These results suggest that measured variations in T. pseudonana-copper bioassays to samples from Narragansett Bay and vicinity could be due to organic chelators in seawater.

Roberts, M.H., Jr. 1980. Flow-Through Toxicity Testing System for Molluscan Larvae as Applied to Halogen Toxicity in Estuarine Water. In: Aquatic Invertebrate Bioassays, ASTM STP 715. A.L. Buikema and John Cairns, Jr., Editors. American Society for Testing and Materials, Philadelphia, PA. Pp. 131-139. (ERL,GB X316).

The routine static toxicity test method for molluscan larvae is outlined. A new flow-through test method for molluscan larvae is presented with data from studies of halogen toxicity. The test involves containment of larvae in a mesh-covered chamber suspended in a small aquarium. The water in the basket is replenished every 7 min by raising and lowering the water level in the tank by means of an automatic tidal siphon. Improvements in the test system are under development.

Richardson, Leonard B., Dennis T. Burton and John C. Rhoderick. 1981. Toxicity of Bromate to Striped Bass Ichthyoplankton (Morone saxatilis) and Juvenile Spot (Leiostomus xanthurus). EPA-600/J-81-543. J. Toxicol. Environ. Health. 8(4):687-695. (ERL,GB X339).

Striped bass (Morone saxatilis) eggs (12 h after fertilization) and larvae (4 d after hatching) and juvenile spot (Leiostomus xanthurus) were exposed to a series of bromate concentrations for 4, 10, and 10 d, respectively, using static replacement bioassay techniques. Three-dimensional mortality response surfaces were constructed by computerized probit regression techniques. Newly hatched striped bass prolarvae were most sensitive to bromate and had a 96-h LC50 of 30.8 mg/l (as BrO3). Four-day-old striped bass larvae were less sensitive, with 2- to 10-d LC50s ranging from 605.0 to 92.6 mg/l BrO3, respectively. Juvenile spot were least sensitive, with 1- to 10-d LC50s ranging from 698.0 to 278.6 mg/l BrO3, respectively.

Richardson, Leonard B., Dennis T. Burton, Ronald M. Block and Ann M. Stavola. 1983. Lethal and Sublethal Exposure and Recovery Effects of Ozone-Produced Oxidants on Adult White Perch (Morone Gmelin). EPA-600/J-83-017. Water Res. 17(2):205-213. (ERL,GB X370). (Avail. from NTIS, Springfield, VA: PB83-223644)

Adult white perch (Morone americana), acclimated to 15 degrees C, were exposed to a series of ozone-produced oxidant (OPO) concentrations for 96 h using continuous flow bioassay techniques. Toxicity data were analyzed using both response surface modeling and standard probit regression. White perch were also exposed to a series of near and sublethal OPO concentrations, selected from the acute toxicity study, for 96 h and then placed in clean non-ozonated water for 14 days. Blood pH, hematocrit and gill histopathology were analyzed during exposure at 24, 48 and 96 h and after 4 days in the recovery period. Blood pH and hematocrit levels were analyzed statistically using standard ANOVA and multiple range tests. Histopathological effects were examined using both light microscopy and scanning electron microscopy. The 24-, 48- and 96-h LC50's were 0.38, 0.26 and 0.20 mg OPO 1(-1), respectively. Blood pH was significantly reduced at concentrations greater than or equal to 0.15 mg OPO 1(-1) but not at 0.10 mg 1(-1) or lower concentrations. Hematocrit significantly increased at concentrations greater than or equal to 0.10 mg OPO 1(-1). Histopathological examination revealed minimal effects on gill tissue at 0.01 mg OPO 1(-1), moderate epithelial sloughing and heavy mucus production at 0.05 mg OPO 1(-1) and extreme tissue damage at concentrations greater than or equal to 0.10 mg 1(-1). Results from both the acute toxicity and the exposure and recovery study were compared with the effects of chlorine-produced oxidants (CPO) obtained from the literature. Both OPO and CPO appear to have similar effects on adult white perch.

Lowe, Jack I., Paul D. Wilson and Richard B. Davison. 1970. Laboratory Bioassays. In: U.S. Fish Wildl. Serv. Circ 335. U.S. Dept. of the Interior, Washington, DC.. Pp. 20-23. (ERL,GB X446).

New pesticides are continually being developed for commercial use, and some of these compounds will be used in or near estuaries. Therefore, there is continuing need for determining both acute and chronic toxicity of these chemical pesticides to commercially valuable marine species. Laboratory bioassays have been made at this station since 1961. Several new chemicals received during the year were evaluated on shrimp, fish, and oysters. We devoted much of our effort to long-term bioassays involving the chronic exposure of selected marine species to low-level pesticide pollution.

Morrill, Joy F. 1965. Microbioassays. In: U.S. Fish Wildl. Serv. Circ 247. U.S. Dept. of the Interior, Washington, DC.. Pp. 7-8. (ERL,GB X447).

During fiscal year 1964-65, renovation of an existing building provided laboratory space for expanding the pesticide program to include culture and testing of marine micro-organisms. When determining the various effects pesticides may have on commercially important species, we must consider not only immediate effects upon a particular species, but also indirect effects on its food supply and possible effects on its embryological and larval development. The best way at present to evaluate such effects is to observe under laboratory conditions those plants and animals known to be involved in the intricacies of the food web.

Wolman, Allen A. 1965. Bioassay of Plankton. In: U.S. Fish Wildl. Serv. Circ 247. U.S. Dept. of the Interior, Washington, DC.. Pp. 8. (ERL,GB X448).

In earlier studies of the effects of pesticides on plankton, natural samples were exposed to serial dilutions of the pesticide in the presence of radioactive carbon (BaC140(3)). The amount of labelled carbon used by the phytoplankton could be accurately measured and indicated the toxicity of the pesticides. These tests lasted only 4 hr.--probably too short a period to be valid. Currently, similar samples are distributed among light and dark bottles, and the production of oxygen over a 24-hr. Period is measured to evaluate pesticide toxicity. By using appropriate dilutions of the pollutant, it is possible to estimate the amount causing a 50-percent decrease in the production of oxygen during the exposure period (EC50). We have screened a broad array of pesticides and have made certain generalizations. Curiously, more than half of the herbicides evaluated have EC50 values in excess of 1.0p.p.m., i.e., they are not very toxic. A few are extremely toxic--e.g., the EC50 of Ametryne is 0.0016 p.p.m. Some of the fungicides are equally harmful. Casoron, however, is a herbicide that stimulates oxygen production at moderate concentrations. The chlorinated hydrocarbons (dieldrin, DDT, etc.) have EC50 values in the range of 0.03 to 0.3 p.p.m. Even with these tests there is some question about what part of the plankton population is being affected. All future tests will be made with pure cultures of algae.

Livingston, Robert J. and Duane A. Meeter. 1985. Correspondence of Laboratory and Field Results: What are the Criteria for Verification?. In: Multispecies Toxicity Testing. EPA/600/D-85/223. John Cairns, Jr., Editor. Pergamon Press Inc., New York, NY. Pp. 76-88. (ERL,GB X479).

Verification of laboratory bioassay results in the field is a complex process which is currently being tested in a range of freshwater and marine habitats by the Florida State University Aquatic Study Group. Verification studies range from single-species bioassays to multispecies microcosms of soft-sediment benthic macroinvertebrates. The basic question involves whether laboratory results can be reasonably extrapolated to field conditions. The chief factors which complicate direct extrapolation include physical-chemical habitat features, reproduction and recruitment of populations, immigration-emigration, predation, and competition. A basic verification approach is to measure the field response of natural populations along an established gradient of contamination or disturbance and compare such effects with a series of bioassays using both indigenous and standard test organisms. Statistical models are being developed to test the comparability of laboratory and field data in the estimation of the effects of toxic substances on natural aquatic systems.

Connolly, John P. 1985. Predicting Single-Species Toxicity in Natural Water Systems. EPA/600/J-85/338. Environ. Toxicol. Chem. 4(4):573-582. (ERL,GB X505). (Avail. from NTIS, Springfield, VA: PB86-171618)

A methodology is proposed to predict single-species toxicity in natural waters by using laboratory bioassay data which relate effect to a tissue concentration of toxicant or to injury accumulation. Such relationships should be independent of test conditions and therefore transferable from lab to field. A mathematical model of uptake and elimination of toxicants by fish is used to relate exposure concentration to tissue concentration and then to effect. Prediction of toxicity in a laboratory test in which the exposure concentration varies in time is presented as a first step in testing the methodology. Survival time under time-varying exposure is calculated and compared to observed data. The model predicted survival times that were consistent with the observed times. Whole-body residue is used as the estimate of dose.

Derby, Jennifer G. Smith and Judith M. Capuzzo. 1985. Physiological and Behavioral Effects of Drilling Fluid on Marine Crustaceans. In: Wastes in the Ocean, Vol. IV: Energy Wastes in the Ocean. I.W. Duedall, D.R. Kester, P.K. Park, and B.H. Ketchum, Editors. John Wiley & Sons, New York, NY. Pp. 289-305. (ERL,GB X532).

Acute and chronic bioassays that tested used, whole drilling fluids indicate wide variation in toxicity; drilling fluids from a single well differed in toxicity depending on the well depth and drilling conditions when the fluid was collected. Sublethal effects of used, whole drilling fluids on various life stages of the lobster Homarus americanus suggested alterations in growth rates, respiration rates, biochemical composition, and chemosensory and behavioral responses with sublethal drilling-fluid exposures. The results demonstrated that larval, juvenile, and early post-ecdysial adult crustaceans are particularly sensitive and that much of the toxicity of drilling fluids resides in the water-soluble fraction. Bactericides and diesel oil were shown to be particularly toxic components of drilling fluids. In addition, metal components of drilling fluids were shown to be incorporated into the tissues of exposed crustaceans.

Zelibor, J.L., Jr., M. Tamplin and R.R. Colwell. 1987. Method for Measuring Bacterial Resistance to Metals Employing Epifluorescent Microscopy. J. Microbiol. Methods. 7:143-155. (ERL,GB X578). (Avail. from NTIS, Springfield, VA: PB88-219803)

Direct viable counting method has been developed which can be used to measure resistance of bacteria to metal (DVCMR bioassay). Results obtained using DVCMR were compared with classical cultural methods and proven superior. The direct viable counting method was modified by addition of Hepes buffer and heavy metals at increasing concentrations and used as a bioassay to evaluate the metal resistance patterns for pure cultures of bacteria. The percent resistance (%R) plotted against metal concentration used in the bioassay proved to be a sensitive measurement of the level of resistance of the strain. Regression analysis was used to determine the concentration of metal resulting in %R = 50, which permitted differentiation of resistance from sensitivity to the metal. Evaluation of test strains resistant to arsenic or manganese showed a parabolic curve, whereas sensitive strains demonstrated a logarithmic curve fit. The DVCMR bioassay method to determine %R was approximately twice as sensitive for detecting metal-resistant strains and 23 times for metal-sensitive strains, compared with cultural methods. It is concluded that the DVCMR bioassay may be better suited for use in microbial biogeochemistry, i.e., contamination monitoring and mineral prospecting, than existing cultural methods.

O'Connor, Joel S. and David A. Flemer. 1987. Monitoring, Research, and Management: Integration for Decisionmaking in Coastal Marine Environments. In: New Approaches to Monitoring Aquatic Ecosystems: ASTM STP 940. Terence P. Boyle, Editor. American Society for Testing and Materials, Philadelphia, PA. Pp. 70-90. (ERL,GB X605).

A rationale is presented for making research and monitoring interdependent to maximize the contributions of both activities to environmental management. Emphasis is placed upon better choices of temporal and spatial scales of marine assessments, thereby improving managerial guidance from monitoring and research. While appropriate scales are functions of particular environmental issues, the most useful scales 'in the mean' appear to be long-term (including truly historical) and regional. The likelihood in the near-term of only limited incremental advances in understanding ecosystem processes, with marginal improvements in predictablity, leads to an argument for more emphasis upon the use of managerially helpful, necessarily simple models. One such model is presented, to characterize the geographical prevalence of fin erosion in winter flounder (Pseudopleuronectes americanus), relative to the sources of plausible causes, from Canada to Delaware Bay. Changing emphasis from laboratory bioassays to field population-level effects is an important and essential step toward integrating ecosystem-level knowledge into the managerial and regulatory milieu. It is now possible to quantify the geographic and, at least recent, temporal associations among man's waste sources and some of their biological effects. Further elaboration of source-fate-effects understanding with the help of simple models (for example, indices) is often more useful to managers than is detailed, piecemeal quantification of seemingly intractable ecosystem dynamics.

Kirby-Smith, William W., Suzanne P. Thompson and Richard B. Forward. 1989. Use of Grass Shrimp (Palaemonetes pugio) Larvae in Field Bioassays of the Effects of Agricultural Runoff into Estuaries. In: Pesticides in Terrestrial and Aquatic Environments: Proceedings of a National Research Conference, May 11-12, 1989. EPA/600/D-91/117. Diana L. Weigmann, Editor. Virginia Water Resources Research Center, Virginia Polytechnic Institute & State University, Blacksburg, VA. Pp. 29-36. (ERL,GB X647). (Avail. from NTIS, Springfield, VA: PB91-199869)

Investigations of the effects of runoff from pesticide sprayed fields on survivorship of the larvae of the grass shrimp Palaemonetes pugio began in the summer of 1988. Gravid shrimp were held in cages in the surface waters of two estuarine creeks; one creek received runoff from fields recently sprayed with the pesticides permethrin and thiodicarb while the other creek received runoff from an unsprayed forested area. Larvae released from field-exposed shrimp were reared in the laboratory in runoff water from the farm, in runoff water from forest drainage and in control water. Permethrin was undetectable (less than 1 ng/sample) in all water except one of three replicates of farm runoff where traces (8 ng/sample) were found. Survivorship was high (75-94%) in all treatments except for larvae reared in farm runoff water where survivorship was low (27-35%) and differed significantly from all other treatments. In a second experiment, three weeks after pesticide application, survivorship was high (94-98%) in all treatments. Data from pesticide residue analysis are complete only for permethrin. We conclude that runoff from agricultural fields sprayed with pesticides applied in full accordance with labeled instructions can significantly reduce survivorship of larval grass shrimp although at this time the cause of mortality is unknown.

Chandler, G. Thomas. 1990. Effects of Sediment-Bound Residues of the Pyrethroid Insecticide Fenvalerate on Survival and Reproduction of Meiobenthic Copepods. EPA/600/J-90/093. Mar. Environ. Res. 29(1):65-76. (ERL,GB X684). (Avail. from NTIS, Springfield, VA: PB90-245572)

Pure, microcosm-cultured populations of benthic copepods were established from pristine or pesticide-impacted Spartina marsh creeks and used as efficient bioassay groups to assess lethal and sublethal effects of sediment-bound residues. Naturally-weathered sediments contaminated with the synthetic pyrethroid insecticide fenvalerate were collected by traps moored in a tidal creek receiving major pesticide-laced runoff from an agricultural watershed, and used as dosing material. Silty sediments with fenvalerate residues reaching 100 ppb were trapped and then diluted with uncontaminated sediments to achieve an exposure range of 0, 25, 50 and 100 ppb (i.e. no dilution). Despite a broad database showing extreme sensitivity to water-solubilized fenvalerate by many marine invertebrates and fishes, a 7-day exposure to sediment-bound residues as high as 100 ppb caused no significant mortality for any life stages (i.e. nauplii, copepodites or adults) of the benthic harpacticoid copepods Microarthridion littorale or Paronychocamptus wilsoni, and no mortality for adults of Enhydrosoma propinquum. However, sediment-bound residues as low as 25 ppb significantly depressed egg production (50-100% reduction) and mean clutch sizes (40-100% reduction) of fertile M. littorale and P. wilsoni. If sedimenting fenvalerate depresses copepod reproduction in the field, then lowered recruitment of new individuals will lead inevitably to a decline in population growth.

Brown, Edward J., Sol M. Resnick, Carl Rebstock, Huan V. Luong and Jon Lindstrom. 1991. UAF Radiorespirometric Protocol for Assessing Hydrocarbon Mineralization Potential in Environmental Samples. EPA/600/J-92/389. Biodegradation. 2:121-127. (ERL,GB X744). (Avail. from NTIS, Springfield, VA: PB93-121218)

Following the EXXON Valdez oil spill, a radiorespirometric protocol was developed at the University of Alaska Fairbanks (UAF) to assess the potential for microorganisms in coastal waters and sediments to degrade hydrocarbons. The use of bioremediation to assist in oil spill cleanup operations required microbial bioassays to establish that addition of nitrogen and phosphorus would enhance biodegradation. A technique assessing 1-14C-n-hexadecane mineralization in seawater or nutrient rich sediment suspensions was used for both of these measurements. Hydrocarbon-degradation potentials were determined by measuring mineralization associated with sediment microorganisms in sediment suspended in sterilized seawater and/or marine Bushnell-Hass broth. Production of 14CO2 and CO2 was easily detectable during the first 48 hours with added hexadecane levels ranging from 10 to 500 mg/l of suspension and dependent on the biomass of hydrocarbon degraders, the hydrocarbon-oxidation potential of the biomass and nutrient availability. In addition to assessment of the hydrocarbon-degrading potential of environmental samples, the radiorespirometric procedure and concomitant measurement of microbial biomass has utility as an indicator of hydrocarbon contamination of soils, aqueous sediments and water, and can also be used to evaluate the effectiveness of bioremediation treatments.

Mearns, Alan, Kenneth Doe, William Fisher, Rebecca Hoff, Kenneth Lee, Robert Siron, Cornelia Mueller and Albert Venosa. 1995. Toxicity Trends During an Oil Spill Bioremediation Experiment on a Sandy Shoreline in Delaware, USA. In: Proceedings of the Eighteenth Arctic and Marine Oilspill Program (AMOP) Technical Seminar. Volume 2. Environment Canada, Ottawa, Ontario. Pp. 1133-1145. (ERL,GB X809).

A 13-week, refereed, inter-agency toxicity testing program involving five bioassay methods was used to document the effectiveness of shoreline bioremediation to accelerate toxicity reduction of an oiled sandy shoreline at Fowler Beach, Delaware, USA. The study was part of an international oiling experiment using a randomized complete block design with repeated measures. Bioremediation - treatment with nutrients or nutrients and oil-degrading bacteria - did not accelerate toxicity reduction. Nor did treatment increase toxicity at weeks 0. 6 or 12-13. However, results of one high-frequency test suggested there may have been a substantial delay in toxicity reduction due to treatment during the first few weeks of treatment. All tests provided information but the most sensitive tests were the 10-day sediment amphipod and grass shrimp embryo bioassays. Standardized sediment and water toxicity tests can play a valuable role in evaluating the effectiveness and effects of oil spill shoreline countermeasures.

Klaine, Stephen J. and Michael A. Lewis. 1995. Algal and Plant Toxicity Testing. In: Handbook of Ecotoxicology. David J. Hoffman, Barnett A. Rattner, G. Allen Burton, Jr. and John Cairns, Jr., Editors. Lewis Publishers, Boca Raton, FL. Pp. 163-184. (ERL,GB X818).

The evaluation of the phytotoxicity of a chemical is an essential component of the ecological risk assessment of that compound. Primary producers form an essential trophic level of any ecosystem. Further, since all chemicals introduced into the environmental ultimately find their way into aquatic ecosystems, aquatic algal and plant toxicity evaluations are particularly critical. This chapter presents the current state of phytotoxicity testing, with particular attention paid to algal and vascular plant bioassays. The algal bioassay section focuses on test methods due to the relatively long history of algal toxicity testing. The vascular plant section focuses on the different plants used for bioassays and the various endpoints used in these bioassays.

Hughes, Patrick R. and H. Alan Wood. 1996. In Vivo Production, Stabilization, and Infectivity of Baculovirus Preoccluded Virions. Appl. Environ. Microbiol. 62(1):105-108. (ERL,GB X850).

Wild-type and polyhedrin-negative isolates of Autographa californica nuclear polyhedrosis virus were replicated in fifth-instar Trichoplusia ni larvae. Insect tissues infected with wild-type virus contained two types of virions that are highly infectious when ingested, those occluded in polyhedra and preoccluded virions. Tissue infected with the polyhedrin-negative virus contained only preoccluded virions. The relative potencies of the two types of infected tissue were determined by dose-mortality bioassays by using the neonate droplet feeding procedure. On a fresh weight basis, preparations of tissues infected with the polyhedrin-negative virus were approximately four times more potent than equivalent preparations of tissue infected with wild-type virus. Approximately half of the observed potency of the wild-type-virus preparations was due to polyhedra, and the remaining activity was due to preoccluded virions present in the tissue. The potency of the polyhedrin-negative preparations was not reduced significantly by lyophilization. The polyhedrin-negative isolate produced about 60% more infectious virus per unit of larval weight than did the wild-type isolate. The ability to produce large amounts of high-potency viral preparations in larvae and the convenience of being able to lyophilize the preparations for long-term storage shows promise for the use of preoccluded virus preparations as biopesticides.

Cifuentes, L.A., R.B. Coffin, L. Solorzano, W. Cardenas, J. Espinoza and R.R. Twilley. 1996. Isotopic and Elemental Variations of Carbon and Nitrogen in a Mangrove Estuary. Estuarine Coastal Shelf Sci. 43(6):781-800. (ERL,GB X868).

Variations in elemental and isotopic ratios of suspended particulate matter (SPM) were investigated in the Guayas River Estuary Ecosystem (GREE) that empties into the Gulf of Guayaquil, Ecuador. Detritus in the system was identified onthe basis of extremely high carbon:chlorophyll a ratios (>1000). This material had mean d13C of -26.4 ± 0.3, d15N of + 4.8 ±0.2, and (C:N)atomic of 14.1 ± 0.9. The isotopic data were comparable to measurements reported for fresh and degrading mangrove leaves, whereas the elemental ration was comparatively enriched in nitrogen. Isotope measurements of SPM throughout the GREE were more similar to values for riverine material and detritus compared with that for the coastal end-member. Values indicative of in situ produced algae, sewage and shrimp pond effluent were only found at selected sites. Bacterial bioassays, which were used to document potential sources of dissolved organic matter in the GREE, were isotopically similar to SPM. This correspondence coupled with the relatively low (C:N)a of SPM could be explained by bacterial immobolization of nitrogen onto detritus. Finally, tidal variations of (C:N)a and d13C at a brackish mangrove site were similar in magnitude to spatial variations encountered throughout the GREE. Based on these results, the authors caution that care must be taken when samples are taken for food-web studies in these systems.

Davis, T.R. and H.A. Wood. 1996. In Vitro Characterization of a Trichoplusia ni Single Nucleocapsid Nuclear Polyhedrosis Virus. J. Gen. Virol. 77(9):2303-2310. (ERL,GB X869).

A Trichoplusia ni single nucleocapsid nuclear polyhedrosis virus (TnSNPV) isolate was cloned and its replication studied in the BTI-Tn-5B1-4 insect cell line. The BTI-Tn-5B1-4 cells were highly susceptible to TnSNPV infection, with 99% of the cells containing viable polyhedra by 30 h post-inoculation. Viral DNA synthesis was detected by 9 h post-infection (p.i.). Infectious budded virus (BV) was first detected at 13 h p.i. and reached an average maximum titre of 3.875 x 106 p.f.u./ml 27 h p.i. A total of 25 BV structural proteins having apparent molecular masses ranging from 27.5 kDa to 86 kDa were identified. Using [35S]methionine pulse-labelling, 19 virus-induced proteins with molecular masses ranging from 27 kDa to 106 kDa were detected from 4 to 28 h p.i. Host cell protein synthesis continued throughout virus replication, although at gradually decreasing rates. Thirty-two structural proteins of occlusion-derived virus ranging in apparent molecular masses from 11 kDa to 98kDa were identified using silver staining procedures. Digestion of viral DNA with the restriction endonucleases EcoRI, HindIII and BamHI generated 31, 26 and 12 fragments, respectively. Estimates for the molecular mass of the TnSNPV genome ranged from 115.5 to 119.2 kbp. In bioassays performed with neonate T. ni larvae, the mean LD50s for the TnSNPV and Autographa californica MNPV were 1.5 (±0.3) and 11.0 (±4.0) polyhedra per larva, respectively.

Rasmussen, Lasse D., Ralph R. Turner and Tamar Barkay. 1997. Cell-Density-Dependent Sensitivity of a mer-lux Bioassay. Appl. Environ. Microbiol. 63(8):3291-3293. (ERL,GB X908).

The sensitivity of a previously described assay (O. Selifonova, R. Burlage, and T. Barkay. Appl. Environ. Microbiol. 59:3083-3090, 1993) for the detection of bioavailable inorganic mercury (Hg2+) by the activation of a mer-lux fusion was increased from nanomolar to picomolar concentrations by reducing biomass in the assays from 107 to 105 cells ml-1. The increase in sensitivity was due to a reduction in the number of cellular binding sites that may compete with the regulatory protein, MerR, for binding of the inducer, Hg2+. These results show that (i) the sensitivity of the mer-lux assay is sufficient for the detection of Hg2+ in most contaminated natural waters and (ii) mer-specified reactions, Hg2+ reduction and methylmercury degradation, can be induced in natural waters and may participate in the geochemical cycling of mercury.

Adams, G.G., P.L. Klerks, S.E. Belanger and D. Dantin. 1999. Effect of the Oil Dispersant Omni-Clean(R) on the Toxicity of Fuel Oil No. 2 in Two Bioassays with the Sheepshead Minnow Cyprinodon variegatus. Chemosphere.(12):2141-2157. (ERL,GB X935).

Bioassays (7-day early life stage and 96h acute bioassays) were conducted with the sheepshead minnow, Cyprinodon variegatus, to determine the toxicity of the dispersant Omni-Clean® by itself and in combination with fuel oil no. 2. Performance characteristics of both bioassay types were also compared. Bioassays used oil by itself, dispersant by itself, and oil and dispersant by itself, and oil and dispersant in various ratios. Omni-Clean® was less toxic than many other dispersants, and had a relatively small effect on individual biomass. Toxicities of the oil/dispersant combinations were generally higher than expected from the toxicities of the oil and dispersant by themselves, indicating a more-than-additive effect on toxicity. The comparison of performance characteristics between the 7-day and the 96-hour bioassays showed that the early life stage test is generally more sensitive, and has the added advantage of an additional and sensitive endpoint (fish biomass).

Steevens, J.A., M. Slattery, D. Schlenk, A. Aryl and W.H. Benson. 1999. Effects of Ultraviolet-B Light and Polyaromatic Hydrocarbon Exposure on Sea Urchin Development and Bacterial Bioluminescence. Mar. Environ. Res. 48(4/5):439-457. (ERL,GB X947).

Polycyclic aromatic hydrocarbons (PAHs) are relatively common contaminants of the Gulf of Mexico and may be activated to more toxic metabolites by ultraviolet-B (UV-B) light. A marine bacterial bioassay system (Vibrio fischeri) which focused on the reduction of luciferase-mediated bioluminescence was utilized as a measure of toxicity to concurrent exposure to anthracene, benzo[a]pyrene, naphthalene, and phenanthrene and UV-B light (l = 280-320nm). Inhibition of bacterial bioluminescence was observed following exposure to naphthalene (5 and 10 ppm) and phenanthrene (5 and 10 ppm) for 5 and 15 min. UV-B significantly (p< 0.05) decreased the average bacterial bioluminescence in all treatments by 23.8%, as compared to control. No significant decrease in bioluminescence was observed with anthracene and benzo[a]pyrene at concentrations up to 24 and 1.6 ppm, respectively. Anthracene and benzo[a]pyrene are relatively insoluble in water (<45 ppb); therefore, the bioavailability of the two PAHs may significantly influence the exposure and effects of anthracene and benzo[a]pyrene to V. fischeri. Results of bacterial bioluminescence experiments were compared to a sea urchin (Lytechinus variegatus) development assay. Development of sea urchin embryos to the four- and 32-cell stage was utilized as a measure of toxicity to the combined effects of UV-B light and the PAHs, benzo[a]pyrene and phenanthrene. Developing sea urchins demonstrated a significant (p <0.05) dose-dependent decrease in growth rate following exposure to benzo[a]pyrene (1,5,50, and 100 ppb) and phenanthrene (1,5,50, and 100 ppb), with UV-B light (5.8 µW/cm2). Interaction analysis demonstrates that concurrent exposure to UV-B and PAHs results in additive toxicological effects in both marine organisms tested. Results of the present study suggest that environmental conditions present in the Gulf of Mexico, including both UV-B light and PAH exposure, have the potential to cause adverse toxicological effects to marine organisms.

Folmar, L.C., M. Hemmer, R. Hemmer, C. Bowman, K. Kroll and N.D. Denslow. 2000. Comparative Estrogenicity of Estradiol, Ethynyl Estradiol and Diethylstilbestrol in an In Vivo, Male Sheepshead Minnow (Cyprinodon variegatus), Vitellogenin Bioassay. Aquat. Toxicol. 49(1-2):77-88. (ERL,GB X963).

An in vivo bioassay for vitellogenin (VTG) synthesis was developed to screen individual chemicals or mixtures of chemicals for potentially estrogenic effects in a marine teleost model. An enzyme-linked immunosorbent assay (ELISA) was used to quantitate VTG synthesis in male sheepshead minnows (Cyprinodon variegatus) exposed to five concentrations of the natural estrogen (17b -estradiol), a synthetic, steroidal pharmaceutical estrogen (17a-ethynyl estradiol), or a synthetic, non-steroidal, pharmaceutical estrogen (diethystilbestrol) for 16 days. At an exposure concentration of 20 ng/l, only diethystilbestrol elicited a vitellogenic response. At all test concentrations greater than 100 ng/l, VTG appeared in the plasma in a dose-dependent manner for the three estrogen treatments. Liver VTG mRNA measurements were also made, exhibiting no clear correlations between quantities, nor temporal appearance of the message and mature protein were apparent. This assay is short-term, relatively inexpensive, shows a direct response, and easily quantitated.

Lewis, Michael, David Weber, Roman Stanley and Barbara Albrecht. 2000. Treated Wastewater as a Source of Sediment Contamination in Gulf of Mexico Near-Coastal Areas: A Survey. Environ. Toxicol. Chem. 19(1):192-203. (ERL,GB X967).

The primary objective of this baseline survey was to provide some needed perspective on the magnitude of sediment contamination associated with wastewater outfalls discharged to Gulf of Mexico near-coastal areas. The chemical quality and toxicities of whole sediments and pore waters collected from three coastal rivers and four coastal bays receiving wastewaters were assessed during a 2-year period. Rooted plants, invertebrates, and fish were used to assess the acute and chronic toxicities of sediments associated with a total of 10 industrial, municipal, steam electric-power generation and forest product wastewater outfalls. Effects of bacterial bioluminescence, early seedling biomass, survival, reproduction, fertility, and growth were determined in bioassays ranging from 30 min to 28 d duration. Sediment chemical contamination was localized and decreased with increasing distance from the discharge areas. The major sediment contaminants, with few exceptions, were divalent trace metals, which increase, on average, by 69% below 8 of the 10 outfalls. However, only a few concentrations exceeded proposed threshold sediment quality assessment guidelines. Toxicity to either the plant or animal test species was observed occasionally below 7 of the 10 outfalls, but its detection was dependent on the type of bioassay and the frequency of use. Consequently, a suite of bioassays conducted on multiple occasions appears to be needed for toxicity assessments of sediments collected below wastewater discharges in the Gulf region to ensure relevancy of the results. This is particularly true for low to moderately contaminated sediments where acute toxicity is uncommon, which was the case in this study.

Morton, Michael G., Kenneth L. Dickson, William T. Waller, Miguel F. Acevedo, Foster L. Mayer, Jr. and Magdiel Ablan. 2000. Methodology for the Evaluation of Cumulative Episodic Exposure to Chemical Stressors in Aquatic Risk Assessment. Environ. Toxicol. Chem. 19(4-2):1213-1221. (ERL,GB X980).

An ecological risk assessment method was developed to evaluate the magnitude, duration, and episodic nature of chemical stressors on aquatic communities. The percent of an ecosystem's species at risk from a designated chemical exposure scenario is generated. In effects assessment, probabilistic extrapolation methods are used to generate temporal stressor concentration profiles. In risk characterization, area under the curve integration is performed on predicted exposure concentration profiles to calculate a cumulative exposure concentration (CEC) for the exposure event. A correction is made to account for the allowable exposure duration to the stressor ESC. Finally, the CEC is applied to the extrapolation model (curve) of the stressor to predict percent species at risk to the episodic exposure. The method may be used for either prospective or retrospective risk assessments. The results of a retrospective risk assessment performed on the Leadenwah Creek, South Carolina, USA, estuarine community are presented as a case study. The creek experienced periodic episodes of pesticide-contaminated agricultural runoff from 1986 through 1989. Although limited biological data were available for method validation, the risk estimates compared well with the Leadenwah Creek in situ bioassay results.

Hawkins, William E., William W. Walker, John W. Fournie, C. Steve Manning and Rena M. Krol. 2003. Use of the Japanese Medaka (Oryzias latipes) and Guppy (Poecilia reticulata) in Carcinogenesis Testing Under National Toxicology Program Protocols. EPA/600/J-03/391. Toxicol. Pathol. 31(Suppl):88-91. (ERL,GB X1034).

A need exists for whole animal toxicity, mutagenesis, and carcinogenesis models that are alternative to the traditional rodent test models and that are economical, sensitive, and scientifically acceptable. Among small fish models, the Japanese medaka (Oryzias latipes) is preeminent for investigating effects of carcinogenic and/or toxic waterborne hazards to humans. The guppy (Poecilia reticulata), although less widely used, is valuable as a comparison species. Both species are easy to maintain and handle in the laboratory and there is a large body of background information on their responsiveness to a range of classes of carcinogens. There are considerable data on the occurrence of background diseases and on spontaneous neoplastic lesions, both of which occur relatively rarely. With few modifications, the medaka and guppy are amenable to carcinogenicity testing under the rigid standards established by the National Toxicology Program (NTP) for rodent tests. The advantages of the small fish in carcinogenesis studies are best realized in long-term studies that involve environmentally realistic exposures. Studies to identify chronic effects can be conducted in about 12 months, near the life span of medaka in our laboratory. Practically, 9-month studies are optimal but shorter study cycles and a variety of exposure/growout and initiation/promotion scenarios are available. Studies on 3 compounds tested in medaka under NTP protocols are under review and preliminary analysis indicates that chronic carcinogenicity bioassays with medaka, guppy, and potentially with other small fish species are feasible and scientifically valid.