Renewed interest in the use of Metarhizium anisopliae and its toxins for insect 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 the Ames test. The average LC50 of the neutral extract obtained in static, acute 96-h tests conducted with ≤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-h exposure to 200 μg ml−1 of the neutral extract produced healthy broods. After 3 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. Significant (p  ≤ 0.05) mortalities were obtained when embryos of grass shrimp and inland silverside fish, Menidia beryllina, were exposed to the same lot of M. anisopliae conidiospores. Exposure of frog embryos to M. anisopliae conidiospores did not cause significant (p > 0.05) mortalities or malformations.

Well-differentiated hepatocellular carcinomas (HCC's) induced in the sheepshead minnow Cyprinodon variegatus w ~t h N-nitrosodiethylamine perm~tted light microscop~cal and ultrastructural comparisons of normal hepatocytes and adjacent HCC cells.Normal hepatocytes contained typical organelles with their intracellular distribution similar to that described for other teleosts.These cells revealed a strong compartmentalization of organelles consisting of restricted perinuclear cytoplasm containing rough endoplasmic reticulum (RER) and mitochondria; most of the rest of the cell's cytoplasm volume was filled with P-glycogen particles.Adjacent HCC cells at the border of the neoplasm interdigitated (invaded) between normal hepatocytes and replaced normal hepatocytes close to the HCC edge and adjacent to the bile canaliculi.Within the HCC, cells had little intercellular space between them and atypical bile canaliculi were found occasionally between adjacent hepatocytes.The HCC cells appeared to differ mainly in a quantitative, rather than in a qualitative fashion, and in the distribut~on of organelles from normal hepatocytes.HCC cells were not compartmentalized; their organelles (RER and mitochondria) were distributed throughout the cytoplasm, and only small amounts of glycogen remained diffusely distnbuted or in small masses or foci.Mitochondrial intermembrane myelin bodies (MMB's) were found frequently in the HCC cells but less frequently in the normal hepatocytes.Some cells at the edge of the HCCs appeared to be intermediate between inner HCC cells and normal hepatocytes in terms of abundance and distribution of organelles and glycogen.The possible significance of the MMB's and intermediate, edge cells is discussed, as well as the need to better characterize features of well-differentiated hepatic neoplasms in order to assure their proper inclusion in neoplasm incidence/prevalence data in fish carcinogenesis assays and field studies.

The origins and morphogenesis of the membranous labyrinth (ML) in Baculov~rus penaei (BP) infected cells of penaeid shrimps (Crustacea:Decapoda) are described. The \IL is usually a highly ordered, complex system of membranes arranged as a labyrinth or in concentric form in the cytoplasm near or against the nuclear envelope. The ML originates from dilated Golgi and endoplasmic reticulum vesicles, and from the outer nuclear envelope. It grows apparently from proliferation of the cellular membranes of these systems, and its developn~ent may be correlated with the stages of BP infection. It is hypothesized that, because of the close parallel and concurrent developnlent of the ML and virus reproduction, and other evidence, the ML is virus-~nduced and -controlled and may play at least 3 roles In the virus reproductive strategy: (1) provides a conduit or transport system for viral precursors from cytoplasm to nucleus; (2 ) provides ~ncreasc!d membrane surface and volume for increased ATPase activity (related to energy demand for virus reproduction and transport of viral products); and (3) provides a mechanism for cell collapse and release of virus and occlusion bodies at the end of virus reproduction period. Possible experimental methods with which to determine the functional role of the ML are discussed and implications for such a system for nuclear. DNA, viruses are considered.

Embryos of gulf killifish Fundulus grandis were injected with N-methyl-N'-nitro-Nnitrosoguanidine (MNNG).Retention of carcinogen in the egg immediately after injection was highly variable.Hatching percentages of MNNG-injected and uninjected embryos were similar, but survival of MNNG-injected fish during the first week after hatching was less than half of the survival of controls.One of 3 MNNG-exposed fish examined after 20 wk had a pancreatic acinar cell carcinoma, a type of neoplasm seldom found in fish.No neoplasms were recorded in controls nor in MNNG-exposed fish examined after 34 wk.The neoplasm was invasive and had a high mitotic index, but no metastases were found.The acinar cell origin of the tumor was confirmed by ultrastructural examination of tissue reprocessed from paraffin into epoxy resin.

American oysters Crassostrea virginica exposed to high concentrations (600 mg I-') of nnitrosodiethylamine (DENA) during winter (February to May) showed significant enhancement of an epizootic apicomplexan parasite, Perkinsus rnarinus.The parasite reproduced and caused atypical lesions in exposed oysters in water temperatures at its lower range (20°C).The reasons for this enhancement are not clear but may reflect damage to the oysters' nonspecific, cellular defense mechanisms by the DENA without concomitant negative effects on the parasite.

Microbial pest control agents (MPCAsviruses, bacteria, protozoa, 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 (College@), 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.

Groups of estuarine sheepshead minnows (Cyprinodon variegatus) were exposed to approximately 57 mg N-nitrosodiethylamine [(DENA) CAS: 55-18-5]/liter for 5-6 weeks. Exposure was stopped and the fish were then transferred to clean, flowing seawater. Induced liver lesions were studied in periodic samples of fish taken during the next 140 weeks of holding. Lesions found following exposure were early altered basophilic and eosinophilic foci, oval cell hyperplasia, clear cell foci, neoplastic nodules, hepatocellular carcinomas, cholangiolar carcinomas, possible pericytomas originating in liver, hemangiopericytomas, spongiosis hepatis, and cholangiofibrosis. The relative prevalence of these lesions was given. Most of these lesions morphologically were compared to their counterpart lesions in the rat. Certain lesions in our fish such as hepatocellular carcinomas, cholangiolar carcinomas, pericytomas, hemangiopericytomas, spongiosis hepatis, and cholangiofibrosis have apparent similar cellular origins and morphogenesis to those lesions in rats and perhaps in other mammals. Spongiosis hepatis in the sheepshead minnow apparently arises from perisinusoidal cells and may be a neoplasm of this cell type. The general similarity of response to DENA in sheepshead minnows and rats suggests that this fish has promise as a model subject for studying some hepatocarcinogens and as a sentinel organism for detecting hepatocarcinogens in contaminated coastal waters.

This study of 3 northern US Gulf Coast estuaries (Pensacola and Escambia Bays in Northwest Florida; Mobile Bay, Alabama; Pascagoula Harbor, Mississippi Sound, Mississippi) investigates: (1) frequencies of known or new diseases, including neoplasms, in oysters and fishes at sites among the estuaries; (2) general relations between disease frequency and human activity in influencing disease prevalences in fish and shellfish populations in coastal regions characterized by varying degrees of human impact.One offshore control station was collected quarterly (1979)(1980) for fishdisease prevalence in comparison with the estuarine stations.Monthly samples of fishes and oysters were collected for disease analyses and diagnoses from August 1978 through August 1980.Sampling stations near heavily industrialized sites in Pascagoula Harbor yielded a higher frequency of diseased fishes and oysters compared to sampling stations in the other estuaries.Even though there were significant differences among disease prevalences in the 3 estuaries, the total prevalence of diseases in fishes and oysters suggested no grossly evident epizootics of pollutant-related disease at the time of study.However, further in-depth study of certain oyster and fish disorders encountered is recommended.Though far from pristine, the estuaries studied were not severely unhealthy environments as evidenced by scattered and relatively low disease prevalences.However, because the Gulf Coastal Plain will probably be the fastest growing region in the USA in the next decade, this study assists in providing base-line data for studies that should be made periodically on the health status of coastal biota in relation to the expected increase in human impact.* ; 2 3 5' G l n z.U). -Couch: Diseases of oysters and fishes in Gulf of Mexiko estuaries ' There was no June 1980 sample Indicates no significant difference (p = 0.05) among estuaries based on a chi-square statistic

Abstract. Pituitaries of sheepshead minnows, Cyprinodon uariegatus, 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 an enlarged pituitary 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 pituitaries 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.

Abstract. Sheepshead minnows, Cyprinodon variegatus Lacépède, exposed to 5.5 to 31 μg/1 of the herbicide trifluralin, throughout their first 28 days of life, developed a heretofore undescribed vertebral dysplasia. This dysplasia consisted of semi‐symmetrical hypertrophy of vertebrae (three to 20 times normal), characterized by foci of osteoblast and fibroblasts actively laying down bone and bone precursors. Effects of the abnormal vertebral development were dorsal vertebral growth into the neural canal, ventral compression of renal ducts, and longitudinal fusion of vertebrae. Pish, exposed for 51 days to 16.6 μg/1 trifluralin and thereafter depurated for 41 days, showed no increase in vertebral dysplasia during depuration; however, residual spinal column damage was evident. Serum calcium concentrations were elevated in adult fish exposed for 4 days to 16.6 μg/1 trifluralin. Fluorosis or mimicry of hypervitaminosis A are considered possible mechanisms for the osseous effect, but are not considered to be the only possible causes. The highly predictable nature of this disorder in experimental exposures strengthens the probability that young fish may serve as experimental models for determining effects of chemicals on early vertebrate ontogeny, particularly in regard to skeletal development.

Scoliosis in fish is caused by several diverse agents that possibly act on the central nervous system, neuromuscular junctions, or ionic metabolism. The organochlorine pesticide Kepone induces scoliosis in the sheepshead minnow. Some effects associated with Kepone-induced scoliosis in these fish are disruption of myotomal patterns, inter- and intramuscular hemorrhage, fractured centra of vertebrae, and death. The histological syndrome of Kepone poisoning in fish and the clinical syndrome in humans suggest that the nervous system is a primary target for Kepone and that scoliosis is a secondary effect of Kepone poisoning in fish.

Pathologic black gills of pink shrimp, Penaeus duorarum, exposed to 763 μg/l of cadmium chloride for 15 days were studied with transmission electron microscopy and were compared with normal gills of control pink shrimp. Local as well as extensive areas of cell death and necrosis were found in the distal gill filaments of black gills from cadmium-exposed shrimp. It is proposed that necrosis of specialized epithelial cells and septum cells in black gill filaments and contiguous nonblack gill tissue could cause osmoregulatory, detoxifying, and respiratory dysfunction in crustacea, particularly in individuals undergoing environmental stress such as salinity fluctuation.

Minchinia nelsoni, which Haskin, Stauber, and Mackin found in connective tissue and-digestive diverticula of the American oyster, is characterized by a distinct peripheral endosome in its plasmodial and spore nuclei and by spores of unique dimension (7.5 by 5.4 micro) This species is believed to be the etiologic agent, formerly reported as "MSX," responsible for extensive oyster mortalities along the Middle Atlantic Coast.