Product Citations and Abstracts

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.

Grass shrimp are one of the more widely distributed estuarine benthic organisms along the Gulf of Mexico and Atlantic coasts, but they were used infrequently in contaminated sediment assessments. Early-life stages of caridean grass shrimp, Palaemonetes pugio (Holthuis), were used in this baseline survey as indicators of sediment pore water quality for several Florida coastal areas affected by nonpoint and point sources of contamination. Static toxicity tests were conducted using 24 well culture plates assessing embryo and larval survival after 12 days exposure to pore water (interstitial water). Acute toxicity was observed in 28% of pore waters (15 of 53 samples) suggesting that sediment contamination was not widespread in the study area based on the response of this species. When toxicity was observed, grass shrimp pore water toxicity test was usually a more sensitive indicator of contamination than solid-phase tests conducted with the epibenthic invertebrate, Mysidopsis bahia. P. pugio embryos were more sensitive than larval form and response of different brood stocks were similar (P>0.05) when exposed to same pore waters. Based on all considerations, this early-life stage toxicity test shows promise being useful detecting pore water toxicity. This conclusion is based on availability of a test methodology which requires minimal space and effort, and the test species widespread geographical distribution. However, there are several remaining research needs, including the important issues of its sensitivity relative to that for other pore water test species as well as for species used in solid-phase toxicity tests.

Couch, John A., Lee A. Courtney and Steven S. Foss. 1981. Laboratory Evaluation of Marine Fishes as Carcinogen Assay Subjects. In: Phyletic Approaches to Cancer. EPA-600/D-80-039. Clyde J. Dawe, Editor. Japan Scientific Society Press, Tokyo. Pp. 125-139. (ERL,GB 171). (Avail. from NTIS, Springfield, VA: PB82-205824)

The U.S. Environmental Protection Agency (EPA) and the National Cancer Inst. (NCI) have major responsibilities for determining the fate and risks of carcinogenic agents in the natural environment. Under the auspices of EPA/NCI, the Carcinogen Research Team at the U.S. EPA Lab, Gulf Breeze, has a major role in investigating the fate, effects, and risks of carcinogenic agents in the aquatic portion of the biosphere. In regard to this role, there is a need for practical, experimental exposure systems for the short term, and long term exposure of fishes and invertebrates in order to evaluate their responses to environmentally significant carcinogens. We have designed and tested an adaptable, aquatic laboratory system for flowing water or static water assays of carcinogenic or suspect carcinogenic agents against marine fishes. We report here the design, results of long term tests, and the future uses of the system for determining the risks of carcinogenic agents in the aquatic environment, and as a system complementary to mammalian assay systems, but which permits the phylogenetic expansion of carcinogen assay methodology. A pilot test of the described system has been completed. This test utilized flowing filtered, estuarine water, controlled water temperature, controlled photo period, controlled nutrition of test species, oxygen concentration monitoring, and various life cycle stages of the test fish, the sheepshead minnow, Cyprinodon variegatus, and the suspect carcinogenic herbicide, trifluralin. Continuous exposures to 1 to 5 µg/l trifluralin were conducted with zygote, thru embryogenesis to adult stages of the fish.

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.

Couch, John A., Lee A. Courtney, James T. Winstead and Steven S. Foss. 1979. American Oyster (Crassostrea virginica) as an Indicator of Carcinogens in the Aquatic Environment. In: Animals as Monitors of Environmental Pollutants. EPA-600/J-79-080. National Academy of Sciences, Washington, DC. Pp. 65-84. (ERL,GB 338). (Avail. from NTIS, Springfield, VA: PB80-18524)

The American oyster (C. virginica) was used as the experimental animal for chronic exposure to 3-methylcholanthrene (3-MC) and benzo[a]pyrene (BP) in an exposure system in which the carcinogens can be continuously injected into free flowing water at fixed rates ranging from 1 to 5 µg/l. Experiments designed to determine uptake and distribution of H3MC and H3BP showed that these are concentrated in oyster tissues in direct proportion to the dosage of carcinogen injected into the system. Residual concentrations as high as 84.4 µg/kg of MC and 36.4 µg/kg of BP were present in oysters as long as 6 months following exposure. Autoradiography showed intense localization of H3BP in distal portions of the tubules of the digestive gland and to a lesser extent in the gonadal tissues. Aryl hydrocarbon hydroxylase (AHH) activity was present in homogenates of hepatopancreas after 5.5 months of exposure to the carcinogens, in contrast to control animals in which AHH activity was quite low. In eight oysters exposed to MC, an infiltration of cells believed to be of hematopoietic origin was encountered in the mantle. Some appear to be identical in type to those which constitute sarcoma-like lesions encountered in feral oyster populations. However, it would be premature at this stage to assign any etiological significance to the experimental findings.

Fournie, John W., Steven S. Foss and John A. Couch. 1988. Multispecies System for Evaluation of Infectivity and Pathogenicity of Microbial Pest Control Agents in Nontarget Aquatic Species. EPA/600/J-88/284. Dis. Aquat. Org. 5(1):63-70. (ERL,GB 640). (Avail. from NTIS, Springfield, VA: PB89-209654)

Microbial pest control agents (MPCAs-viruses, bacteria, protozoa, and fungi) are being used as biological pesticides and herbicides. Many of these agents are considered potential MPCAs and could be used widely in the environment. Therefore, test animals must be selected and laboratory systems developed to evaluate safety of such agents to nontarget species. A simple, multispecies laboratory system has been designed and used to determine risks of infectivity and pathogenicity of Colletotrichum gloeosporioides (Collego®), a registered postemergent mycoherbicide, to nontarget freshwater and estuarine plant and animal species. Test organisms included a freshwater and an estuarine fish, crustacean, bivalve mollusc, and plant. These multispecies systems also permit evaluation of other MPCAs against nontarget aquatic species and provide a standardized procedure for safety testing. Results from this study have shown these multispecies test systems to be viable, inexpensive, and reliable. Histopathological methods used indicated no evidence that experimental exposure to the fungal MPCA in our test system caused infection or related pathogenicity in the selected nontarget species.

Fournie, John W., Steven S. Foss, Lee A. Courtney and Albert H. Undeen. 1990. Testing of Insect Microsporidians (Microspora:Nosematidae) in Nontarget Aquatic Species. EPA/600/J-90/376. Dis. Aquat. Org. 8:137-144. (ERL,GB 680). (Avail. from NTIS, Springfield, VA: PB91-163899)

This paper reports results of tests with the mosquito microsporidian Nosema algerae and the orthopteran microsporidian N. locustae on nontarget aquatic organisms. Organisms tested were the freshwater grass shrimp Palaemonetes kadiakensis, the estuarine grass shrimp P. pugio, the marine rotifer Brachionus plicatilis, and the inland silverside Menidia beryllina. These organisms were exposed by intrahemocoelic injection, gavage, or ingestion. Infections did not develop in either the freshwater grass shrimp or the estuarine grass shrimp that were gavaged with N. algerae spores. However, infections did develop in both species of grass shrimp after intrahemocoelic injections with N. algerae spores. Infected tissues included the gills, antennal gland, eyes, skeletal muscle, heart, and gonads. Proof of infection was demonstrated ultrastructurally by the presence of mature spores and developmental stages in infected tissues. Infections did not develop in P. pugio following intrahemocoelic injections of N. locustae spores. N. algerae infections did not develop in P. pugio following intrahemocoelic injections of N. locustae spores. N. algerae infections did not develop in the marine rotifer after ingestion of spores or in inland silversides fed marine rotifers containing ingested spores.

Genthner, Fred J., Douglas P. Middaugh and Steven S. Foss. 1992. Development of Test Methods to Determine Fate of Microbial Pest Control Agents and Their Effects on Nontarget Aquatic Animals. In: Proceedings of the 2nd International Symposium on the Biosafety Results of Field Tests of Genetically Modified Plants and Microorganisms, May 11-14, 1992, Goslar, Germany. R. Casper and J. Landsmann, Editors. Biologische Bundesanstalt fur Land- und Forstwirtschaft, Braunschweig, Germany. Pp. 107-115. (ERL,GB 789).

Two test methods are described. The first involved the design and field validation of a fully contained test system. This system was used to assess potential adverse effects of microbial pest control agents (MPCAs) on a nontarget invertebrate and determine fates of the MPCAs in the animal. American oysters, Crassostrea virginica, were exposed to various MPCA types represented by the vegetative bacterium, Pseudomonas fluorescens, gram-positive bacterial spores of Bacillus sphaericus, and fungal spores of Colletotrichum gloeosporioides. Each MPCA cleared from the oysters at a different rate. None of the microbes caused infection, pathogenesis or toxicity. Spore germination was not observed in oyster tissue. In the second method, developing embryos of the inland silverside fish, Menidia beryllina, were exposed to spores of the insect pathogenic fungus, Beauveria bassiana. Fungal growth on the chorion surface as well as embryo rupture and death were observed. Spores treated with a dispersant (biological detergent, Triton X-100) showed significantly less binding (p less than or equal to 0.01) to embryos than untreated spores. Neither detergent-treated or heat-killed spores caused significant adverse effects.

Genthner, Fred J., Steven S. Foss, Robert P. Campbell and John W. Fournie. 1993. Fate and Survival of Microbial Pest Control Agents in Nontarget Aquatic Organisms. EPA/600/J-94/112. Dis. Aquat. Org. 16(2):157-162. (ERL,GB 808). (Avail. from NTIS, Springfield, VA: PB94-155512)

A fully enclosed test system was developed both to assess potential adverse effects of microbial pest control agents on nontarget aquatic invertebrates and to monitor their fate and survival. Eastern oysters Crassotrea virginica were exposed to various microbial pest control agents including the vegetative bacterium Pseudomonas fluorescens, bacterial spores of Bacillus sphaericus, and fungal spores of Colletotrichum gloeosporioides f. sp. aeschynomene. After an exposure of 3 d for the fungus, 14 d for the bacterial spores, and 7 d for the vegetative bacterial cells, half the oysters were placed into a fully enclosed 60 l aquarium equipped with a recirculating water system which passed water through an ultraviolet-irradiation sterilizer at a rate of 1 l min-1. The remaining oysters were placed into wire cages maintained in Santa Rosa Sound, Pensacola, Florida, USA. Plate counts, performed on homogenized oyster tissues, revealed that all of the microbial pest control agents were found in oysters after exposure. Oysters depurated each microorganism at a different rate. None of the agents colonized the oysters. For all microbes tested, rates of clearance from the oysters in the enclosed UV light depuration system were similar to rates of clearance from the oysters in Santa Rosa Sound. Histological examination of oyster tissues detected P. fluorescens and B. sphaericus in the gut and C. gloeosporioides in the gill. Spore germination was not observed and no apparent signs of infectivity or pathogenicity were detected.

Genthner, Fred J., Steven S. Foss and William S. Fisher. 1994. Testing of the Insect Pest Control Fungus Beauveria bassiana in Grass Shrimp, Palaemonetes pugio. Dis. Aquat. Org. 20:49-57. (ERL,GB 879).

Embryos, larvae, and adult grass shrimp, Palaemonetes pugio, were exposed to spores of the insect-control fungus, Beauveria bassiana. Conidiospores attached to embryos held by gravid females and remained with the egg mass for at least 6 d. In the first experiment where individual developing embryos contained in test tubes were exposed to conidiospores of B. bassiana, 2 of the 75 embryos became infected and died and a third was abnormal at hatch. In Experiment 2, a repeat experiment, no adverse effects were observed. In subsequent experiments, attempts were made to increase the probability of infection or abnormal development for embryos exposed to B. bassiana. Strategies for these attempts included lowering the salinity, adding a carbon source for the fungus to the test water, or rendering embryos free of potentially protective symbiotic bacteria with an antibacterial agent. All attempts were unsuccessful in promoting infection or abnormal development. B. bassiana did not cause any adverse effects during shrimp larval development. In adult shrimp, lethal infections only occurred in specimens injected with either conidiospores or blastospores of B. bassiana.

Genthner, Fred J., Steven S. Foss and Patricia S. Glas. 1997. Virulence of Metarhizium anisoplliae to Embryos of the Grass Shrimp Palaemonetes pugio. J. Invertebr. Pathol. 69(2):157-164. (ERL,GB 959).

Experiments were performed in which developing embryos of the grass shrimp, Palaemonetes pugio, were exposed to conidiospores of the insect pathogenic fungus, Metarhizium anisopliae. Responses were variable, with significant (P is less than or equal to 0.05) adverse effects observed in five of six experiments conducted. Dead embryos and larvae with visible growth of M. anisopliae were observed in all experiments. Growth of M. anisopliae was occasionally observed on embryos and larvae prior to death. Delayed hatch was also observed. In one of the initial experiments, an increase in N-acetyl-b-D-glucosaminidase, EC 3.2.1.30 (NAGase), activity accompanied by an increase in virulence toward shrimp embryos was observed. Additional experiments in which conidiospores were produced on homogenized caterpillars suggested a positive correlation between virulence of M. anisopliae to P. pugio embryos and activity of spore-associated NAGase. Under these laboratory conditions M. anisopliae was an invasive pathogen of grass shrimp embryos, and the growth substrates on which their spores develop can influence the severity of effects on these nontarget arthropods.

Genthner, Fred J., Steven S. Foss and Patricia S. Glas. 1996. Virulence of Metarhizium anisopliae to Embryos of the Grass Shrimp Palaemonetes pugio. In: Biotechnology Risk Assessment: Proceedings of the Biotechnology Risk Assessment Symposium, June 6-8, 1995, Pensacola, Florida. Morris Levin, Chris Grim, and J. Scott Angle, Editors. University of Maryland Biotechnology Institute, College Park, Maryland. Pp. 193-205. (ERL,GB X858).

Experiments were performed in which developing embryos of the grass shrimp, Palaemonetes pugio, were exposed to conidiospores of the insect pathogenic fungus, Metar-hizium anisopliae. Responses were variable with significant (p less than or equal to 0.05) adverse effects observed in 5 out of 6 experiments conducted. Dead embryos and larvae with visible growth of Metarhizium anisopliae were observed in all experiments. Growth of Metarhizium anisopliae was occasionally observed on embryos and larvae prior to death. Delayed hatch was also observed. In one of the initial experiments an increase in N-acetyl-B-D-glucosaminidase. EC3.2.1.30, (NAGase) activity accompanied by an increase in virulence toward shrimp embryos was observed. Additional experiments in which conidiospores were produced on homogenized caterpillars suggested a positive correlation between virulence of M. anisopliae to P. pugio embryos and activity of spore-associated NAGase. M. anisopliae was an invasive pathogen of grass shrimp embryos, and the growth substrates on which their spores develop can 'condition' them for enhanced virulence toward nontargets.