Journal Article Citations and Abstracts

Swimming performance of the estuarine sheepshead minnow, Cyprinodon variegatus, was measured in a stamina tunnel at the end of life-cycle toxicity tests with the organophosphate pesticides EPN (265 days) and Guthion (219 days). Effects of acetylcholinesterase (AChE) inhibition were also determined. These measures were compared to survival, growth and reproduction data obtained in the life-cycle toxicity test. Significant effects on swimming stamina were detected in fish exposed to 2.2 µg EPN/l (57% that of control fish) and in fish exposed to 4.1 µg EPN/l (46% of controls). Survival and growth were reduced only in 7.9 µg EPN/l. Swimming performance was not affected by Guthion concentrations up to 0.5 µg/l, a concentration affecting reproduction. AChE was significantly inhibited by all tested concentrations of EPN, 0.25 to 7.9 µg/l, and Guthion, 0.06 to 0.50 µg/l. Swimming stamina may be an important endpoint to include with measurements of effects on survival, growth and reproduction in chronic toxicity tests with fish, but the relation of AChE inhibition to these endpoints is uncertain.

Reish, Donald J., Philip S. Oshida, Frank G. Wilkes, Alan J. Mearns, Thomas C. Ginn and Robert Scott Carr. 1984. Fate and Effects of Pollutants: Effects on Saltwater Organisms. EPA-600/J-84-108. J. Water Pollut. Control Fed. 56(6):758-774. (ERL,GB 414).

This literature review summarizes current data on the effects of pesticides and metals on marine organisms, aquatic environmental research methods, bioaccumulation of pollutants by estuarine and marine organisms and in water/sediment residues and biota. Results of studies of the environmental effects of dredging, dumping, wastewater sludge and complete effluents are reported. The occurrence of diseases and tissue abnormalities in fish is discussed, as well as research related to marine oil pollution.

Couch, J.A. 1984. Histopathology and Enlargement of the Pituitary of a Teleost Exposed to the Herbicide Trifluralin. EPA-600/J-82-152. J. Fish Dis. 7(2):157-163. (ERL,GB 438). (Avail. from NTIS, Springfield, VA: PB84-175306)

Pituitaries of sheepshead minnows, Cyprinodon variegatus, exposed for 19 months to 1-5 µg/l trifluralin in the laboratory exhibited enlargement, pseudocysts, congestion of blood vessels and oedema. Most of the fish with enlarged pituitaries also had induced diffuse and/or focal vertebral hyperostosis and other dysplastic vertebral changes. Several speculative mechanistic paths are suggested for the mode of effect of trifluralin on the vertebral and pituitary tissues. Abnormal enlargement or variation in sizes of pituitary from fish of the same age, along with histopathological findings may be related to the environmental quality of the fish's habitat, and provide indications of stress within individuals resulting from toxicant exposure.

Middaugh, Douglas P. and Michael J. Hemmer. 1984. Spawning of the Tidewater Silverside, Menidia peninsulae (Goode and Bean), in Response to Tidal and Lighting Schedules in the Laboratory. Estuaries. 7(2):139-148. (ERL,GB 441).

Tidewater silverside, Menidia peninsulae were maintained in 1.3 m diameter holding tanks in identical laboratory recirculating systems. During two weeks under constant conditions (a current velocity of 8 cm s-1 and continous illumination, 24 L:O D) there was a low relative frequency of arrhythmic spawning. In the subsequent two-week period, fish in one pair of tanks were maintained under the singular influence of twice daily decreases in current velocity from 8 to 0 cm s-1 at 0600-0700 and 1800-1900, under continous illumination. The relative frequency of spawning remained low and there was no evidence of a daily spawning rhythm. However, the mean number of eggs per spawn increased substantially. Fish in the second holding system were subjected to diel light cycle of 13 L:11 D with a constant current velocity of 8 cm s-1 for two weeks. The relative frequency of spawning remained low and there was no indication of spawning rhythmicity; moreover, there was only a slight increase in the mean number of eggs per spawn. During the third two-week period, fish in the first pair of tanks were provided a 13 L:11 D diel light cycle, in conjunction with preexisting twice daily decreases in current velocity; those in the second pair of tanks were provided twice daily decreases in current velocity in conjunction with the preexisting 13 L:11 D light cycle. Under the combined influence of decreases in current velocity and a diel light cycle, there was a marked increase in the relative frequency of spawning in both pairs of tanks. Fish manifested a discernible spawning periodicity, spawns typically occurred between 1800 and 2400; the mean number of eggs per spawn also increased. When fish were returned to constant conditions, current velocity 8 cm s-1 and 24 L:0 D for two weeks, the frequency of spawning decreased and there was no indication of a spawning periodicity. Results of another experiment (decreased current velocities at 1200-1300 and 2400-0100 with 13 L:11 D light cycle) indicated gradual expression of a tidal spawning rhythm during nighttime, 2000-0359. Our laboratory results indicate that M. peninsulae is predominantly a nocturnal spawner and that spawning coincides with decreased current velocities.

Courtney, Lee A. and John A. Couch. 1984. Usefulness of Cyprinodon variegatus and Fundulus grandis in Carcinogenicity Testing: Advantages and Special Problems. EPA-600/J-84-063. Natl. Cancer Inst. Monogr. 65:83-96. (ERL,GB 442). (Avail. from NTIS, Springfield, VA: PB84-226513)

Cyprinodon variegatus and Fundulus grandis, two species of cyprinodontid fishes extensively studied and used in toxicological and biological investigations, are compared as laboratory test animals. Their ecology, general biology, and suitability for various types of experimentation are examined. A laboratory system for exposure of these species at critical life stages (embryos, fry, juveniles) to suspect carcinogens is described. A discussion of the use, findings, and potential of these species in oncological studies and carcinogen assays is presented, particularly in regard to responses to 3 known or suspect carcinogens (trifluralin, benzidine, and aflatoxin). Finally, advantages and disadvantages or special problems in using the species as carcinogen test animals are reviewed.

Schoor, W. Peter. 1984. Benzo[a]pyrene Metabolism in Marine Fish and Some Analytical Aspects of Its Metabolites. EPA-600/J-84-061. Natl. Cancer Inst. Monogr. 65:391-396. (ERL,GB 454). (Avail. from NTIS, Springfield, VA: PB84-2266497)

In interspecies comparisons of benzo[a]pyrene metabolism, standardized procedures in the analysis of the metabolites must be established. Technical problems arising in metabolite analysis are discussed, and data are presented showing the activities of the mixed function oxygenase and transferase systems in phenobarbital- and 3-methylcholanthrene-induced mullet and killifish.

Couch, J.A., S.M. Martin, G. Tompkins and J. Kinney. 1984. Simple System for the Preliminary Evaluation of Infectivity and Pathogenesis of Insect Virus in a Nontarget Estuarine Shrimp. EPA-600/J-84-134. J. Invertebr. Pathol. 43(3):351-357. (ERL,GB 460). (Avail. from NTIS, Springfield, VA: PB85-124618)

Biological control agents (biorationals) are increasingly important in pest control concepts. Certain insect viruses, particularly the baculoviruses (nuclear polyhedrosis viruses), are considered to have potential as biological pesticides and could be used widely in the environment. Therefore, test animals must be selected and methods and laboratory systems developed to evaluate the safety of these agents to nontarget species. A simple laboratory system has been designed and used to determine risks of infectivity and pathogenicity of an insect Baculovirus, originally isolated from the Alfalfa looper, Autographa cailfornica, to a nontarget arthropod, the grass shrimp, Palaemonetes vulgaris, by dietary exposure. This laboratory method also permits evaluation of other microbial biorationals against nontarget aquatic species, and provides an inexpensive standardized procedure of safety testing. Results from this study indicated that histopathological, ultrastructural and serological methods used provided no evidence that experimental exposure to the virus in our test system caused viral infection or related pathogenicity in the grass shrimp.

Takita, Toru, Douglas P. Middaugh and John M. Dean. 1984. Predation of a Spawning Atherinid Fish, Menidia menidia, by Avian and Aquatic Predators. EPA/600/J-84/266. Jpn. J. Ecol. 34(4):431-437. (ERL,GB 465).

Observations were made of predation on Atlantic silversides, Menidia menidia, during spawning runs in the intertidal zone of the North Edisto River estuary, South Carolina. Several fishes and avian predators captured M. menidia. Snowy egrets, Egretta thula, and Great egrets, Casmerodius albus, were the most dominant avian predators. We found specific differences in each mode and actual predatory pressure for the two wading birds. Before the onset of spawning runs, Snowy egrets often made hovering catches which were inefficient, while Great egrets always made standing catches, striking at M. menidia from a standing position. Once a spawning run began, Snowy egrets ceased hovering and both Great and Snowy egrets frequently struck at fish from a standing position. Because of inefficiency in catching prey prior to a spawning run, Snowy egrets always remained unsatiated after a run had ended. A Great egret was satiated after consuming 114 fish (42% of its body weight) during a run. Intraspecific disturbances were often observed in both Snowy and Great egrets. Interspecific interaction did not present a serious problem to the subordinate species, Snowy egret.

Dawe, Clyde J. and John A. Couch. 1984. Debate: Mouse Versus Minnow: The Future of Fish in Carcinogenicity Testing. EPA-600/D-83-039. Natl. Cancer Inst. Monogr. 65:223-235. (ERL,GB 472).

The text is part of a debate and advocates the use of small fish species as laboratory test organisms in detecting and determining potency and activity of suspected carcinogens. The usefulness of fish species is compared and contrasted with the usefulness of the more conventional assay species, such as rodents.

Rubinstein, Norman I., W.T. Gilliam and N.R. Gregory. 1984. Dietary Accumulation of PCBs from a Contaminated Sediment Source by a Demersal Fish (Leiostomus xanthurus). EPA/600/J-84/267. Aquat. Toxicol. 5(4):331-342. (ERL,GB 485).

Accumulation and dietary transfer of PCBs from contaminated harbor sediments were studied in a laboratory food chain consisting of sediments, polychaetes (Nereis virens) and a predatory fish (Leiostomus xanthurus). The study was conducted in two phases to distinguish dietary uptake from PCB accumulation resulting from sediment exposure alone. In phase I fish and polychaetes were separately exposed to field-collected PCB contaiminated sediments (5.2 µg/g dry weight as Aroclor 1242 and 1254) in flow-through sea-water systems for 40 days to allow organisms to attain steady state concentrations. In phase II the dietary fraction of PCB accumulation was determined by selectively feeding exposed and control groups of fish polychaetes having a known PCB body burden. In addition the effect of direct sediment contact on PCB accumulation by L. xanthurus was investigated. Results demonstrate that contaminated sediments can serve as a source of PCBs for uptake and trophic transfer in marine systems. Fish exposed to PCB-contaminated sediments and fed a daily diet of polychaetes from the same sediment accumulated more than twice the PCB whole-body residues than fish exposed to similar conditions but fed uncontaminated polychaetes. The dietary contribution of PCBs accounted for 53% of the total body burden measured in fish fed for 20 days, and this percentage appeared to be increasing. Results also indicate that fish isolated from direct contact with PCB-contaminated sediment do not significantly (p < = 0.05) Accumulate PCB residues when compared with fish allowed contact with sediment.

Middaugh, Douglas P., Richard G. Domey and Geoffrey I. Scott. 1984. Reproductive Rhythmicity of the Atlantic Silverside. EPA-600/J-84-191. Trans. Am. Fish. Soc. 113(4):472-478. (ERL,GB 491).

The reproductive periodicity of the Atlantic silverside Menidia menidia was studied at two locations on the North Edisto River estuary in South Carolina during March-July 1976-1978. Spawning runs occurred in the upper intertidal zone and coincided precisely with daytime high tides. Time-series analysis of daily changes in the intensity of spawning runs revealed a fortnightly reproductive periodicity and indicated that the observed reproductive rhythmicity in Atlantic silversides may be mediated by a high tide-sunrise cue that also occurs at fortnightly intervals. During the 1976 and 1977 reproductive seasons, there were highly significant correlations (P < 0.01) among the male gonadal index, the female gonadal index, and the occurrence of intermediate, maturing, and hydrated-egg stages of sexual development in females. The percentage of females with hydrated eggs was greatest on days when a high tide occurred within 1 hour after sunrise.

Schoor, W. Peter and Meera Srivastava. 1984. Position-Specific Induction of Benzo(a)pyrene Metabolism by 3-Methylcholanthrene and Phenobarbital in Mullet (Mugil cephalus), a Marine Fish. EPA-600/J-84-079. Comp. Biochem. Physiol. C. Comp. Pharmacol. 78C(2):391-396. (ERL,GB 499).

1. Mullet (Mugil cephalus), a marine fish, were treated with 3-methylcholanthrene and phenobarbital by i.p. injection and the effects on the metabolism of benzo(a)pyrene by isolated liver microsomes were evaluated. 2. The most significant effect found in treated fish was an increase in the oxidation of benzo(a)pyrene at the bay region, a position-specific increase which was not reflected in an increase in the total activity. 3. Comparison of metabolite patterns of the dihydrodiols of benzo(a)pyrene in the presence and absence of trichloropropene oxide showed the predicted inhibitions were observed in control as well as treated animals. 4. No significant differences in metabolite patterns were found between the 3-methylcholanthrene- and phenobarbital-treated fish in either presence or absence of trichloropropene oxide. 5. Comparison is made to published data on similar position-specific effects observed in rats.

Couch, J.A. and W.J. Hargis, Jr. 1984. Aquatic Animals in Toxicity Testing. EPA-600/J-84-199. J. Am. Coll. Toxicol. 3(6):331-336. (ERL,GB 501).

Aquatic animals provide useful models for toxicological evaluations that bridge the gap between real world and laboratory problems. Select aquatic organisms are adaptable to laboratory experimentation in areas such as acute toxicity testing and chronic sublethal risks evaluation, including such phenomena as carcinogenesis, mutagenesis, and teratogenesis. General and specific examples of how aquatic animals are useful to toxicologists, as well as theoretical bases for their use, are discussed in this paper.

McKenney, Charles L., Jr. and Dana Beth Hamaker. 1984. Effects of Fenvalerate on Larval Development of Palaemonetes pugio (Holthuis) and on Larval Metabolism During Osmotic Stress. EPA-600/J-84-198. Aquat. Toxicol. 5(4):343-355. (ERL,GB 502). (Avail. from NTIS, Springfield, VA: PB85-157055)

Larvae of the estuarine grass shrimp, Palaemonetes pugio, were reared in the laboratory from hatch through metamorphosis under optimal salinity conditions (20 o/oo) in a range of lethal and sublethal concentrations of the pyrethroid insecticide, fenvalerate. A nominal concentration of 3.2 ng fenvalerate/l significantly reduced the percentage of larvae that completed metamorphosis. Oxygen consumption rates were significantly higher for larvae exposed to this lethal concentration for only 24 h. Exposure to a sublethal concentration of 1.6 ng fenvalerate/l prolonged the duration of complete larval development. Acute osmotic stress modified the metabolism of larval P. pugio reared in sublethal fenvalerate concentrations of 0.1 and 0.2 ng/l and these metabolic responses varied with stage of development. After 8 days of exposure to fenvalerate, oxygen consumption rates were elevated when larvae were exposed acutely to hypoosmotic stress (10 o/oo S). Metabolic responses of premetamorphic larvae to hyperosmotic stress (30 o/oo S) were also modified by sublethal fenvalerate exposure. Alterations in metabolic-salinity patterns of larval grass shrimp developing under sublethal concentrations of fenvalerate suggest reduction in the ecological fitness of this sensitive life stage by limiting their capacity to adapt to the fluctuating salinity conditions of estuarine waters.

Spain, J.C., P.A. Van Veld, C.A. Monti, P.H. Pritchard and C.R. Cripe. 1984. Comparison of p-Nitrophenol Biodegradation in Field and Laboratory Test Systems. EPA-600/J-84-218. Appl. Environ. Microbiol. 48(5):944-950. (ERL,GB 506).

Acclimation of microbial communities exposed to p-nitrophenol (PNP) was measured in laboratory test systems and in a freshwater pond. Laboratory tests were conducted in shake flasks with water, shake flasks with water and sediment, eco-cores, and two sizes of microcosm. The sediment and water samples used in the laboratory experiments were obtained from the pond. After a 6-day acclimation period, PNP was biodegraded rapidly in the pond. When the pond was treated with PNP a second time, biodegradation began immediately. The acclimation periods in laboratory test systems that contained sediment were similar to that in the pond. The acclimation period was threefold longer in shake flasks without sediment. PNP was biodegraded more slowly by microbial communities acclimated in the laboratory than it was in the pond, and the rate of biodegradation varied wih the type of test. The number of bacteria able to mineralize PNP increased by 3 orders of magnitude in the pond during the acclimation period. Similar increases accompanied acclimation in the laboratory systems.

Walker, William W., C.R. Cripe, P.H. Pritchard and A.W. Bourquin. 1984. Dibutylphthalate Degradation in Estuarine and Freshwater Sites. EPA-600/J-84-219. Chemosphere. 13(12):1283-1294. (ERL,GB 509).

Biotic and abiotic degradation of di-n-butylphthalate (DBP) in water and sediment/water systems from six different sites was investigated under laboratory conditions. DBP disappearance was rapid in all microbially active systems and substantially reduced under sterile conditions. Adaptation of microbial populations to degrade DBP was indicated in six of nine evaluations conducted. The presence of sediment significantly increased biodegradation rates in five of the six sites.

Pritchard, P.H. 1984. Fate and Effects of Pollutants: Fate of Environmental Pollutants. J. Water Pollut. Control Fed. 56(6):718-725. (ERL,GB 514).

The author presents a review of the use of microcosms and field studies to verify predictions of fate and transport of toxic chemicals. Microcosms are laboratory systems that simulate portions of the field and allow its complexity to be represented in the laboratory.

Kendall, J.J., Jr., E.N. Powell, S.J. Connor, T.J. Bright and C.E. Zastrow. 1984. Importance of Monitoring Metabolic Recovery in the Coral Acropora cervicornis After Short-Term Exposure to Drilling Muds: Calcification Rate and Protein Concentration. EPA-600/J-84-040. Coral Reefs. 2:215-225. (ERL,GB X008).

The effect of used drilling muds on coral health was examined by monitoring changes in calcification rate and soluble tissue protein concentration in the coral Acropora cervicornis. Exposure to 25 ppm (v/v) of one mud for 24 h reduced calcification rate in the growing tips by as much as 62%. In recovery experiments, corals were exposed to drilling muds for 24 h; some of them were allowed to recover in clean seawater for 48 h. After the 24-hour exposure, calcification rates were significantly less than those of the controls. After a 48-hour recovery period, calcification rates returned to control levels for one mud but were still significantly below control levels for another. The results indicate that the capacity for recovery after exposure cannot be predicted from the results of experiments on exposure only. Recovery capacity must be independently verified for all studies on the effects of short-term exposure to drilling muds.

Martin, Billy J., Rudolph D. Ellender, Susan A. Hillebert and Mitchell M. Guess. 1984. Primary Cell Cultures from the Teleost, Cyprinodon variegatus: Culture Establishment and Application in Carcinogen Exposure Studies. EPA/600/J-84/271. Natl. Cancer Inst. Monogr. 65:175-178. (ERL,GB X260).

Methods were developed for aseptic maintenance of Cyprinodon variegatus fry for extended periods. Preliminary studies indicated that under optimum conditions sterile embryos develop normally for a sufficient time to function as carcinogen/teratogen assay systems. An embryo-primary cell culture technique was developed that incorporates, in a single system, certain characteristics of both intact embryos and primary cell cultures and allows simultaneous observation of the effects of carcinogens on the whole organism and primary cell monolayers. The effective use of these systems provides one the opportunity to study the effects of carcinogens on teleosts at the cellular and organismic level.

Meador, C. Brent, Robert L. Middlebrooks and Billy J. Martin. 1984. Serological Alterations in Carcinogen-Exposed Teleosts: Procedures for Preparation and Analysis of Samples from Small Fish. EPA/600/J-84/269. Natl. Cancer Inst. Monogr. 65:211-216. (ERL,GB X261).

To study the effects of environmental carcinogens on the immune system of Cyprinodon variegatus, we had to miniaturize or modify standard immunological procedures due to the small size of the fish. Modifications in standard bleeding procedures allowed collection of sufficient serum for most serological procedures. Serum electrophoresis showed considerable variaton between exposed and unexposed fish as did qualitative immunoelectrophoresis techniques. We successfully adapted a bacteriophage neutralization procedure for use with the C. variegatus system to measure antiviral antibodies. The presence of antibody-forming cells in spleen suspensions from fish immunized with human type 0 erythrocytes was demonstrated by a modified immune rosette procedure. A capillary tube procedure was developed for separation of leukocytes from erythrocytes in blood from C. variegatus.

Hendricks, Jerry D., Theodore R. Meyers, John L. Casteel, Joseph E. Nixon, Patricia M. Loveland and George S. Bailey. 1984. Rainbow Trout Embryos: Advantages and Limitations for Carcinogenesis Research. EPA-600/J-84-238. Natl. Cancer Inst. Monogr. 65:129-137. (ERL,GB X279).

Rainbow trout embryos are sensitive to the initiation of neoplasms in various tissues by brief exposures to solutions of water-soluble carcinogens. This characteristic was first demonstrated with the sparingly soluble liver carcinogen, aflatoxin B1 (AFB1). A 30-minute exposure of 21-day-old embryos (embryos hatch in 24-25 days at 12 degrees C) to a 0.5-ppm aqueous solution of AFB1 will result in approximately 65% of the survivors having at least 1 liver tumor, 1 year after treatment. The embryos are responsive to both AFB1 dose and the length of exposure and become increasingly sensitive with increased embryonic age. We have used rainbow trout embryos to demonstrate the hepatocarcinogenicity of other aflatoxin metabolites and precursors; aflatoxicol, aflatoxin G1, versicolorin A, and sterigmatocystin. In addition to mycotoxins, trout embryos are sensitive to several nitrosamine hepatocarcinogens including: dimethylnitrosamine, diethylnitrosamine, nitrosopyrrolidine, and 2,6-dimethylnitrosomorpholine. However, with the highly water-soluble nitrosamines, longer exposure times (up to 24 hr) are required. It is generally accepted that each of the above-named carcinogens requires metabolic activitation to the ultimate carcinogenic form. This provides indirect evidence that the trout embryo is capable of cytochrome P-450-mediated metabolism. Finally, trout embryos are sensitive to the direct-acting carcinogen, n-methyl-n'nitro-n-nitrosoguanidine. This compound produces tumors of the liver, stomach, kidney, and swim bladder, and a pronounced female-to-male sex reversal. Results to date have shown that the trout embryo is a sensitive, convenient, and economical whole animal model system with many distinct advantages for carcinogen testing and research. The major limitation has been the difficulty with exposure of the embryos to adequate doses of highly water-insoluble compounds. Alternate exposure techniques will be required for alleviation of this problem.

Schultz, R. Jack and Mary E. Schultz. 1984. Characteristics of a Fish Colony of Poeciliopsis and Its Use in Carcinogenicity Studies with 7,12-Dimethylbenz[a]anthracene and Diethylnitrosamine. EPA/600/J-84/268. Natl. Cancer Inst. Monogr. 65:5-13. (ERL,GB X311).

The viviparous fish Poeciliopsis from north-western Mexico is characterized according to its potential in cancer research. A colony of these fishes started in 1961 includes inbred strains of P. lucida (M61-9, M61-31, M61-35, and S68-4), P. monacha (S68-4 and S68-5), and P. viriosa (M65-23), which have been demonstrated by electrophoresis and tissue graft analysis to be homozygous. All-female species of hybrid origin are represented by 12 clones which, although highly heterozygous, are genetically identical or isogenic and readily accept within-clone tissue transplants. Two studies are cited in which hepatic tumors were chemically induced. In one, liver neoplasms were induced in P. lucida and P. monacha by repeated short-term exposures to an aqueous suspension of 5 ppm 7,12-dimethylbenz[a]anthracene. In the other, dose responses to the carcinogen diethylnitrosamine (DENA) were compared among 4 inbred strains, 3 hybrids from crosses between inbred strains, and 2 wild stocks of P. lucida. The incidence of hepatic tumors gradually increased from 0 to 93% depending on the concentration of the compound and the number of exposures, but no significant difference was found among the inbred strains, their hybrids, or the wild stocks. Hepatic tumors, initially induced with DENA have been successfully transplanted into the muscle tissue and abdominal cavity of members of the same strain.

Hendricks, Jerry D., Theodore R. Meyers and Dennis W. Shelton. 1984. Histological Progression of Hepatic Neoplasia in Rainbow Trout (Salmo gairdneri). EPA-600/J-84-237. Natl. Cancer Inst. Monogr. 65:321-336. (ERL,GB X353).

The histological progression of hepatic neoplasia has not been as systematically studied in rainbow trout as it has been in rodents. Two putative preneoplastic lesions have been identified, the eosinophilic focus and basophilic focus, but whether these correspond to similar lesions in rodent livers is not known. Preneoplastic liver lesions in rodents have been extensively characterized histochemically, but adaptation of these techniques to trout livers has not always been successful. Eosinophilic foci consist of hypertrophied cells, enlarged atypical nuclei, and dense glycogen-free cytoplasm. Mitotic figures are also occasionally seen. Usually, these foci have been infiltrated and at least partially destroyed by inflammatory cells, largely lymphocytes. In some liver sections, eosinophilic foci are intact and occasionally an eosinophilic-basophilic transformation can be seen. However, most often basophilic foci appear independently, surrounded by normal heptatocytes, with no indication of a prior eosinophilic stage. The cells of basophilic foci are similar to those of carcinomas: intensely basophilic, mitotically active, devoid of glycogen, and grouped into cords several cells in thickness. These nodules may appropriately be referred to as carcinomas in situ, because the only distinguishing characteristic is the size of the lesion. Attempts at differentiation between benign and malignant liver lesions appear arbitrary. We believe the best classification of the neoplastic liver lesion in trout is a hepatocellular carcinoma because the potential for malignant behavior always exists and, with sufficient time, can often be histologically demonstrated. We have also described our experience with the characteristics of other liver lesions associated with hepatocarcinogenesis.

Melius, Paul. 1984. Comparative Benzo(a)pyrene Metabolite Patterns in Fish and Rodents. Natl. Cancer Inst. Monogr. 65:387-390. (ERL,GB X365).

Benzo(a)pyrene is converted to 3-hydroxybenzo(a)pyrene, 9-hydroxybenzo(a)pyrene, benzo(a)pyrene-4,5-dihydrodiol, benzo(a)pyrene-7,8-dihydrodiol, benzo(a)pyrene-9,10-dihydrodiol and benzo(a)pyrene quinones by post mitochondrial supernatant or microsomes in such fish as the rainbow trout, flounder, salmon, mullet, little skate, Fundulus grandis, and sea catfish. It is also now well-established that many fish convert benzo(a)pyrene to potent mutagenic metabolites as has been demonstrated with the Ames test, especially when the fish are induced with Aroclor 1254 or 3-methylcholanthrene. The metabolite patterns obtained at different substrate concentration levels indicate that the metabolism is more complex at low concentrations when metabolites are recycled in the in vitro system.

Wiernicki, C. 1984. Assimilation Efficiency by Procambarus clarkii Fed Elodea (Egera densa) and Its Products of Decomposition. EPA-600/J-84-004. Aquaculture. 36(3):203-215. (ERL,GB X372).

Elodea cut into two initial particle sizes (2.0 cm sections and 3.0 mm(2) fragments) underwent microbial decomposition for periods of 0, 15, 33, and 45 days, and then was fed to four size classes of crayfish (2.0, 3.5, 5.0, and 9.0 cm). Assimilation efficiency was determined by Conover's method. All four size classes of crayfish assimilated 15-day detritus more efficiently than 0-day detritus. Two-cm crayfish assimilated 33-day detritus more efficiently than 15- or 0-day detritus. In all other cases, assimilation efficiency was reduced. Radiotracer techniques utilizing 14C were used to determine the percentage of carbon assimilated in the form of plant fragments compared with the percentage assimilated in the form of microorganisms. Results indicate an inverse relationship between crayfish size and the percentage of carbon assimilated in the form of microorganisms.

Bookhout, C.G., R.J. Monroe, R.B. Forward, Jr. and J.D. Costlow, Jr. 1984. Effects of Hexavalent Chromium on Development of Crabs, Rhithropanopeus harrisii and Callinectes sapidus. Water Air Soil Pollut. 21:199-216. (ERL,GB X374).

Survival of Rhithropanopeus harrisii larvae from hatching to first crab stage occurred in Na2CrO4 concentrations from 1.1 to 29.1 ppm. Estimated LC50 for complete zoeal development was 17.8 ppm Na2CrO4 and it was 13.7 ppm for development to first crab stage. A concentration of 1.1 ppm Na2CrO4 was nontoxic, while NaCrO4 concentrations of 7.2 and 14.5 ppm were sublethal and concentrations of 29.1 to 58.1 ppm were acutely toxic. Low concentrations of Na2CrO4 caused an increase in swimming speed and high concentrations caused a decline. Survival of Callinectes sapidus larvae occurred in Na2CrO4 concentrations from 1.1 to 4.7 ppm. The LC50 for development to first crab stage was estimated to be 1.0 ppm Na2CrO4. The total Cr in sodium chromate is 32% by weight (Tacey, 1981), hence, the total Cr concentrations tested were 32% of the Cr salts given above. Statistical analyses of the data on survival, duration and mortality of larvae are presented.

Parker, Jeffrey H., Janet S. Nickels, Robert F. Martz, Michael J. Gehron, Norman L. Richards and David C. White. 1984. Effect of Well-Drilling Fluids on the Physiological Status and Microbial Infection of the Reef Building Coral Montastrea annularis. EPA-600/J-84-236. Arch. Environ. Contam. Toxicol. 13(1):113-118. (ERL,GB X382).

The reef building coral Montastrea annularis was exposed continuously to suspensions of oil and gas-well drilling fluids at concentrations of 0.1, 0.01, and 0.001 ul/ml in flowing seawater at the U.S. Naval Stage I platform (30 degrees 7.5' N, 85 degrees 46.3' W). After 6 weeks exposure, coral fragments of 12 to 65 cm(2) surface area were broken off, rinsed in seawater, and extracted in a one-phase chloroform-methanol-buffer and returned to the laboratory. In the laboratory, the extraction was completed and the phases separated. The lipids were fractionated using silicic acid and thin layer chromatography. Total phospholipid, triglyceride glycerol, total extractable fatty acids, triglyceride fatty acids as well as the ester fatty alcohols showed no consistent changes with exposure to the drilling fluids. Changes in free amino acid concentrations were extracted as well as significant decreases in the recoverable diacyl phospholipid. Significant increases in plasmalogen phospholipids appeared with exposure. Increases in plasmalogen phospholipids are consistent with infection by anaerobic fermenting bacteria which can indicate disease. This evidence suggests that biochemical indicators of infection with anaerobic bacteria may be useful as sensitive markers for pollution-induced changes in reef building corals and thus for monitoring the health of coral reefs.

Doughtie, Daniel G. and K. Ranga Rao. 1984. Histopathological and Ultrastructural Changes in the Antennal Gland, Midgut, Hepatopancreas, and Gill of Grass Shrimp Following Exposure to Hexavalent Chromium. EPA-600/J-84-010. J. Invertebr. Pathol. 43(1):89-108. (ERL,GB X400).

Grass shrimp, Palaemonetes pugio, were exposed for one month to subacute concentrations of hexavalent chromium (0.5, 1.0, 2.0, 4.0 ppm) after which the gills, midgut, hepatopancreas, and antennal glands were examined for histopathological and ultrastructural changes. Pathological changes were greatest in the antennal glands, followed by hepatopancreas, gills, and midgut. Severe changes occurred in some shrimp, even at 0.5 ppm chromium. Cells of all tissues frequently had both swollen mitochondria and rough endoplasmic reticulum. Small, spherical or ring-like intranuclear inclusions, possibly indicative of cellular hyperactivity or manifestions of chromium and/or protein complexes, were most prevalent in the hepatopancreas and antennal glands but also occurred in the midgut and gills. Other major degenerative changes in the antennal glands were restricted to the labyrinth and included diminution of basal plasmalemmal infoldings and cytoplasmic density, nuclear hypertrophy followed by widespread nuclear pyknosis and epithelial desquamation. In severely altered hepatopancreas hypertrophy was indicated for the basal laminae, nuclei, possibly for the nucleoli. There was an apparent reduction in mitotic events and many observed mitotic nuclei were abnormal. Abnormal midgut hypertrophy was present in only eight of twenty examined shrimp, exposed to 0.5 and 1.0 ppm chromium. Further, the gills of only 10 of the 40 examined chromium-exposed shrimp possessed abnormal features detectable with light microscopy. Ultrastructural analysis of the latter indicated an increase in lysosomes and a decrease in cytoplasmic density. In addition, there was a pronounced diminution in the degree of lamellar, subcuticular plasmalemmal infolding. This latter feature is postulated to be a mechanism for the regulation of chromium influx. Possible explanations for most observed alterations in the above tissues are proposed.

Rao, K. Ranga and Daniel G. Doughtie. 1984. Histopathological Changes in Grass Shrimp Exposed to Chromium, Pentachlorophenol and Dithiocarbamates. EPA/600/J-84/270. Mar. Environ. Res. 14:371-395. (ERL,GB X435).

This report deals with the histopathological/ultrastructural changes in various tissues of grass shrimp (Palaemonetes pugio) exposed to hexavalent chromium, pentachlorophenol (PCP) and two dithiocarbamate formulations: Aquatreat DNM-30 15 percent sodium dimethyl dithiocarbamate plus 15 percent disodium ethylene bis-dithiocarbamate) and Busan-85 (50 percent potassium dimethyl dithiocarbamate). The pathological alterations induced by dithiocarbamates and PCP were most severe and first evident in the gills. Of the biocides tested, the dithiocarbamates caused the most extensive branchial damage--the so-called black gill syndrome, involving early melanization and eventual lamellar truncation. Hexavalent chromium, on the other hand, did not induce marked changes in the gills, but it caused invasive melanized cuticular lesions (particularly at the articulations of the pereiopods, pleopods and abdominal segments). Additionally, chromium caused apparent labyrinth hypoactivity in the antennal glands, whereas the dithiocarbamates seemed to induce labyrinth hyperactivity. Hepatopancreatic histopathology was more severe in shrimp exposed to chromium and PCP than in dithiocarbamate-exposed shrimp. The apparent mitotic activity in the hepatopancreas was increased in Aquatreat-exposed shrimp and suppressed in chromium-exposed shrimp; many mitotic figures in the latter case were abnormal. All four compounds caused varying degrees of midgut epithelial hypertrophy, cytoplasmic vacuolization and diminution of basal tubular endoplasmic reticulum, but only PCP caused widespread rupture of midgut epithelial cell apices. Additional notable ultrastructural anomalies included: mitochondrial compartmentalization in PCP-exposed shrimp; mitochondrial fusion in dithiocarbamate-exposed shrimp; and unusual intranuclear inclusions in different tissues of chromium-exposed shrimp. These pathological changes point to differences in the mechanisms of pollutant toxicity and indicate their potential use in the biological monitoring of aquatic pollutants.

Powell, E.N., S.J. Connor, J.J. Kendall, C.E. Zastrow and T.J. Bright. 1984. Recovery by the Coral Acropora cervicornis After Drilling Mud Exposure. The Free Amino Acid Pool. EPA-600/J-84-017. Arch. Environ. Contam. Toxicol. 13(2):243-258. (ERL,GB X462).

Corals were exposed to drilling mud for 24 hr and then allowed to recover for 48 hr in clean seawater. Depending on the concentration and the mud used, exposure produced either an increase or decrease in free amino acid (FAA) pool size. Aspartate was affected to a greater degree than other amino acids. No clear instance of recovery could be ascertained after 48 hr in clean seawater. In several cases, corals, apparently unaffected by a 24 hr exposure, nevertheless suffered significant changes in the FAA pool during the 48 hr recovery period. Thus, the degree of toxicity of the drilling mud could not be accurately predicted from the 24 hr exposure data. In many cases, the choice of normalizing parameter determined whether two sets of data were significantly different or not. Accurate effects assessment depends on a comparison of normalization to confirm statistical results.

Meyers, Theodore R. and Jerry D. Hendricks. 1984. Limited Epizootic of Neuroblastoma in Coho Salmon Reared in Chlorinated-Dechlorinated Water. EPA-600/J-84-003. J. Natl. Cancer Inst. 72(2):299-310. (ERL,GB X468).

During the 1976-77 brood year, approximately 12 cases of neuroblastoma were observed in a captive group of 100,000 fingerling coho salmon (Oncorhynchus kisutch) reared in a commercial hatchery. The tumors were large, occurring in the skeletal muscle near the dorsal fin causing conspicuous bulging of the overlying integument. Tumors examined from 3 fish each consisted of neuroblasts in trabecular patterns interspersed by glial fibrillar material and linear cavities resembling central neural canals lined by ependyma-like cells. Ganglion-like cells also were apparent morphologically and by special stain. Cancer of the tumor was characterized by an abundance of mitotic figures with occasional abnormal divisions, local invasion of normal tissues, and potentially metastatic tumor cell aggregates in organ vasculature. The etiology of this tumor may have been related to mutagenic-carcinogenic halogenated compounds possibly formed in the hatchery water supply during continuous chlorination of incoming river water.

Conklin, Philip J. and K. Ranga Rao. 1984. Comparative Toxicity of Offshore and Oil-Added Drilling Muds to Larvae of the Grass Shrimp Palaemonetes intermedius. EPA-600/J-84-189. Arch. Environ. Contam. Toxicol. 13(6):685-690. (ERL,GB X471).

Offshore drilling fluids (muds) varied widely in their toxicity to grass shrimp (Palaemonetes intermedius) larvae. The 96-hr LC50s for the eleven drilling muds tested ranged from 142 to more than 100,000 ppm (ul/l). There was a significant correlation between oil content of the drilling muds and their toxicity. Furthermore, addition of diesel oil (No. 2 fuel oil) or mineral oil to an offshore drilling mud having a low oil content or to an oil-free synthetic drilling mud led to a marked increase in the toxicity of these muds. Thus, much of the toxicity of the offshore drilling muds tested can be attributed to the oil content.

Koenig, Christopher C. and Michael P. Chasar. 1984. Usefulness of the Hermaphroditic Marine Fish, Rivulus marmoratus, in Carcinogenicity Testing. Natl. Cancer Inst. Monogr. 65:15-33. (ERL,GB X474).

Hepatocellular carcinoma and other pathological changes were induced in the self-fertilizing hermaphroditic fish Rivulus marmoratus by aqueous exposure to 15, 30, and 45 ppm diethylnitrosamine (DENA). Fish exposed as larvae for 12 weeks and adults exposed for 5 weeks showed rates of hepatocellular carcinoma as high as 76.5 and 31.3 percent, respectively, when examined 1 year later. Fish exposed as embryos in various stages of development for 1 week to 10, 32, 100, 320, and 1,000 ppm DENA were without neoplasms when examined 1 year later. Other pathological changes of rivulus livers were cholangioma, adenofibrosis, and granuloma with incidences as high as 38.9, 88.9, and 61.1 percent, respectively. Simple rearing methods were developed and relevant biological data pertaining to reproduction, embryonic development, and subsequent growth of R. marmoratus are discussed. Advantages and disadvantages of the use of R. marmoratus as a carcinogenicity test animal are assessed.

Powell, E.N., J.J. Kendall, S.J. Connor, C.E. Zastrow and T.J. Bright. 1984. Effect of Eight Outer Continental Shelf Drilling Muds on the Calcification Rate and Free Amino Acid Pool of the Coral Acropora cervicornis. EPA/600/J-84/304. Bull. Environ. Contam. Toxicol. 33(3):362-372. (ERL,GB X477).

Nine muds were tested; eight from outer continental shelf (OCS) drilling operations; one was the NBS (National Bureau of Standards) standard mud. At the 25-ppm exposure level, none of the nine muds tested by us produced observable zooxanthellae loss, however substantial protein loss occurred in P4-exposed corals. Loss in P1- and P5-exposed corals could only be demonstrated statistically using skeletal weight as the normalizing parameter. Similarly, reduced calcification rates occurred in P4- and P1-exposed corals. Rates were reduced in P5-exposed corals as well, however most statistical comparisons were equivocal. The three muds, P4, P5, and P1, also were the only muds to cause significant changes in the relative composition of the FAA pool (Table 4). Serine-threonine levels decreased relative to aspartate-alanine-glutamate in all three cases. The remaining muds produced slight, occasionally significant reductions in the FAA pool at the 25-ppm exposure level, but most significant results were equivocal. In all cases although only certain amino acids showed significant changes, the effect could not be distinguished from a general, moderate decrease in pool size.

Wang, Yei-Shung, R.V. Subba-Rao and Martin Alexander. 1984. Effect of Substrate Concentration and Organic and Inorganic Compounds on the Occurrence and Rate of Mineralization and Cometabolism. EPA-600/J-84-200. Appl. Environ. Microbiol. 47(6):1195-1200. (ERL,GB X482). (Avail. from NTIS, Springfield, VA: AD-A144964)

Isopropyl N-phenylcarbamate (IPC) at 400 pg and 1 ug/ml was mineralized in samples of sewage, but only the lower concentration was mineralized in lake water samples in a 50-day period. IPC at 1 ug/ml disappeared from lake water, but it was converted to organic products. Mineralization of IPC at 400 pg/ml in lake water was enhanced by additions of inorganic nutrients or a mixture of nonchlorinated water pollutants but not by yeast extract or mixtures containing aromatic compounds or excretions of primary producers. The mineralization of 200 pg of 2,4-dichlorophenoxyacetate per ml of lake water was not affected by additions of low levels of yeast extract or compounds excreted by primary producers but was enhanced by low concentrations of mixtures of water pollutants. It is suggested that some chemicals that are found to be converted only to organic products, presumably by cometabolism, in tests using the concentrations commonly employed in laboratory evaluations may be mineralized at the lower concentrations prevailing in natural waters.

Clark, James R., Dave Devault, Robert J. Bowden and Joseph A. Weishaar. 1984. Contaminant Analysis of Fillets from Great Lakes Coho Salmon, 1980. J. Great Lakes Res. 10(1):38-47. (ERL,GB X484).

Analyses of coho salmon from each of the Great Lakes by a single laboratory produced residue data on the accumulation of environmental contaminants which have been banned, severely restricted, or are currently permitted in the basin. Coho salmon from Lake Superior contained only trace amounts or low levels of most toxic substances quantified; Lake Erie fish were contaminated with low levels of a number of pesticides and industrial compounds; relatively higher residues were detected in coho salmon from Lake Huron and Lake Michigan; and the highest concentrations for a number of compounds were found in fillets from coho from Lake Ontario. Contaminant concentrations in migratory coho salmon indicate open lake contaminant problems rather than point source or nearshore conditions. Tissue residues were less than USFDA action levels, used by many agencies in assessing the severity of fish contaminant problems. Only mirex concentrations in fish collected from Lake Ontario exceeded a USFDA action level. The data reported in this study generally agree with recent findings from individual state contaminant monitoring programs. Problems with varying analytical and sampling techniques preclude direct comparisons with previously published data of other studies.

Grizzle, John M., Paul Melius and D. Ralph Strength. 1984. Papillomas on Fish Exposed to Chlorinated Wastewater Effluent. EPA-600/J-84-197. J. Natl. Cancer Inst. 73(5):1133-1142. (ERL,GB X490).

The presence of carcinogenic and mutagenic chemical(s) in the effluent of a wastewater treatment plant was indicated by papillomas developing on caged black bullheads (Ictalurus melas), hepatic enzyme induction in exposed fish, and Ames test mutagenicity of organic extracts of the wastewater. Although virus-like particles have been reported in papillomas of several other fish species, no evidence was obtained for the presence of viruses in the black bullhead papillomas. Mutagenic and carcinogenic chemicals were not identified in the wastewater, but chlorination was implicated as a factor contributing to the induction of the papillomas. The prevalence of papillomas on wild black bullheads exposed to the effluent decreased from 73 to 23 percent after the amount of residual chlorine (CAS: 7782-50-5) in the effluent leaving the chlorine contact chamber was reduced from 1.3-3.1 mg/liter to 0.25-1.2 mg/liter.

Swain, Louis and Paul Melius. 1984. Characterization of Benzo(a)pyrene Metabolites Formed by 3-Methylcholanthrene-Induced Goldfish, Black Bullhead and Brown Bullhead. Comp. Biochem. Physiol. 79C(1):151-158. (ERL,GB X588).

To date, carcinogenic research with polycyclic aromatic hydrocarbons (PAH), such as benzo[a]pyrene, primarily has involved mammalian tissues. In this report, authors summarize studies on the metabolism of BaP in three fish (goldfish, Carassius auratus; black bullhead, Ictalurus melas; and brown bullhead, Ictalurus nebulus). Results are compared with those obtained for the rat. Results show that these exposures of goldfish, brown bullhead, and black bullhead increased the ability of their liver enzymes to convert BaP into carcinogenic and mutagenic intermediates.

Swain, Louis and Paul Melius. 1984. Comparison of Six Different Reversed-Phase HPLC Columns Used to Separate Benzo(a)pyrene Metabolites. LC. 2(9):670-676. (ERL,GB X589).

Benzo[a]pyrene and its metabolites are carcinogenic compounds that are difficult to separate because their structures are very similar. This paper evaluates the performance of six different reversed-phase (ODS) HPLC columns produced by four manufacturers to separate a synthetic mixture of BaP metabolites. Column lifetimes and the time required for each separation are compared.