David E. Hinton

The Toxicology of Fishes

Ecological Risk Assessments (ERA) are performed to evaluate the likelihood of adverse ecological effects occurring as a result of exposure to physical or chemical stressors. These stressors are defined as any biological, physical, or chemical factor that causes adverse responses in the environment. An ERA evaluates any potential harm that human activities have on living organisms within ecosystems. Within the framework of an Ecological Risk Assessment, scientific information is developed, organized and presented so that it is relevant to environmental decisions.General Principles Introduction, R.T. Di Giulio and D.E. Hinton Bioavailability of Chemical Contaminants in Aquatic Systems, R.J. Erickson, J.W. Nichols, P.M. Cook, and G.T. Ankley Toxicokinetics in Fishes, K.M. Kleinow, J.W. Nichols, W.L. Hayton, J.M. McKim, and M.G. Barron Biotransformation in Fishes, D. Schlenk, M. Celander, E.P. Gallagher, S. George, M. James, S.W. Kullman, P. van den Hurk, and K. Willett Receptor-Mediated Mechanisms of Toxicity, M.E. Hahn and E.V. Hestermann Reactive Oxygen Species and Oxidative Stress, R.T. Di Giulio and J.N. Meyer Key Target Systems and Organismal Effects Liver Toxicity, D.E. Hinton, H. Segner, D.W.T. Au, S.W. Kullman, and R.C. Hardman The Osmoregulatory System, S.E. Wendelaar Bonga and R.A.C. Lock Toxic Responses of the Fish Nervous System, S.P. Bradbury, R.W. Carlson, T.R. Henry, S. Padilla, and J. Cowden The Endocrine System, P. Thomas The Immune System of Fish: A Target Organ of Toxicity, E. Carlson and J.T. Zelikoff Chemical Carcinogenesis in Fishes, J.M. Rotchell, M.R. Miller, D.E. Hinton, R.T. Di Giulio, and G.K. Ostrander Toxicity Resistance, P.A. Van Veld and D.E. Nacci Methodologies and Applications Exposure Assessment and Modeling in the Aquatic Environment, D. Mackay and L. Milford Fish Toxicity Studies, G.M. Rand Biomarkers, D. Schlenk, R. Handy, S. Steinert, M.H. Depledge, and W. Benson Aquatic Ecosystems for Ecotoxicological Research: Considerations in Design Analysis for Fish, T.W. La Point, J.H. Kennedy, J.K. Stanley, and P. Balci Ecological Risk Assessment, D.R. Mount and T.R. Henry Case Studies Mining Impacts on Fish in the Clark Fork River, Montana: A Field Ecotoxicology Case Study, S.N. Luoma, J.N. Moore, A. Farag, T.H. Hillman, D.J. Cain, and M. Hornberger Toxicology of Synthetic Pyrethroid Insecticides in Fish: A Case Study, J.R. Coats Reproductive Impairment of Great Lakes Lake Trout by Dioxin-Like Chemicals, D.E. Tillitt, P.M. Cook, J.P. Giesy, W. Heideman, and R.E. Peterson The Effects of Polycyclic Aromatic Hydrocarbons in Fish from Puget Sound, Washington, L.L. Johnson, M.R. Arkoosh, C.F. Bravo, T.K. Collier, M.M. Krahn, J.P. Meador, M.S. Myers, W.L. Reichert, and J.E. Stein Effects of the Exxon Valdez Oil Spill on Pacific Herring in Prince William Sound, Alaska, G.D. Marty Case Study: Pulp and Paper Mill Impacts, M.G. Dube, K.R. Munkittrick, and L.M. Hewitt Estrogenic Effects of Treated Sewage Effluent on Fish: Steroids and Surfactants in English Rivers, C.R. Tyler, E.J. Routledge, and R. van Aerle Index

The Fish Embryo Toxicity Test as an Animal Alternative Method in Hazard and Risk Assessment and Scientific Research

Animal alternatives research has historically focused on human safety assessments and has only recently been extended to environmental testing. This is particularly for those assays that involve the use of fish. A number of alternatives are being pursued by the scientific community including the fish embryo toxicity (FET) test, a proposed replacement alternative to the acute fish test. Discussion of the FET methodology and its application in environmental assessments on a global level was needed. With this emerging issue in mind, the ILSI Health and Environmental Sciences Institute (HESI) and the European Centre for Ecotoxicology and Toxicology of Chemicals (ECETOC) held an International Workshop on the Application of the Fish Embryo Test as an Animal Alternative Method in Hazard and Risk Assessment and Scientific Research in March, 2008. The workshop included approximately 40 scientists and regulators representing government, industry, academia, and non-governmental organizations from North America, Europe, and Asia. The goal was to review the state of the science regarding the investigation of fish embryonic tests, pain and distress in fish, emerging approaches utilizing fish embryos, and the use of fish embryo toxicity test data in various types of environmental assessments (e.g., hazard, risk, effluent, and classification and labeling of chemicals). Some specific key outcomes included agreement that risk assessors need fish data for decision-making, that extending the FET to include eluethereombryos was desirable, that relevant endpoints are being used, and that additional endpoints could facilitate additional uses beyond acute toxicity testing. The FET was, however, not yet considered validated sensu OECD. An important action step will be to provide guidance on how all fish tests can be used to assess chemical hazard and to harmonize the diverse terminology used in test guidelines adopted over the past decades. Use of the FET in context of effluent assessments was considered and it is not known if fish embryos are sufficiently sensitive for consideration as a surrogate to the sub-chronic 7-day larval fish growth and survival test used in the United States, for example. Addressing these needs by via workshops, research, and additional data reviews were identified for future action by scientists and regulators.

Histopathologic biomarkers in feral freshwater fish populations exposed to different types of contaminant stress

Histopathologic alterations of gill, liver, and spleen were studied in feral fish from three freshwater ecosystems that experience different types of contaminant stress. East Fork Poplar Creek (EFPC), a third-order stream in East Tennessee, receives point source discharges of mixed contaminants from a nuclear weapons facility located near its headwater. The Pigeon River (PR), a high-gradient fifth order stream, is impacted by bleached kraft mill effluent (BKME). Hartwell Reservoir (HR), a US Army Corp of Engineers impoundment of the Savannah River, contains high levels of PCBs in sediment and biota. Brushy Fork Creek (BFC), the Little River (LR), and the Tugaloo River (TR) are relatively free of contaminants, and served as reference sites for the three respective ecosystems of this study. Certain organ and tissue lesions, detected microscopically, were common to fish from both reference and contaminated sites. These included parasites, inflammation, glycogen deficiency, macrophage aggregates (MA), and diffuse fatty change in the liver; parasites and MA in the spleen; and parasites, secondary lamellar fusion, and variable epithelial cell hyperplasia in the gills. Lesions found only in fish from contaminated sites were: (1) cholangiomas in liver of redbreast sunfish (Lepomis auratus) collected from EFPC; (2) amphophilic and eosinophilic foci of cellular alteration, diffuse biliary preductular and ductular hyperplasia with islands of hyperplastic basophilic hepatocytes, and two metastatic thyroidal carcinomas in spleen of redbreast sunfish from PR; (3) severe lipidosis, vacuolated and basophilic foci in largemouth bass (Micropterus salmoides) from HR; (4) splenic lymphoid cell depletion and vascular congestion, with necrosis of reticuloendothelial cells in fish collected from EFPC and HR; (5) hyperplasia of mucous and chloride cells, deformed branchial cartilage, severe and diffuse aneurysms of lamellae, and edema at the base of the secondary lamellae were in gill of fish from all three sites. The finding of specific lesions only in fish from contaminated sites suggests a contaminant etiology. Histopathology biomarker lesions identified in this study are similar to those observed in laboratory exposures of fishes to specific pollutants. Further refinement of these biomarker approaches will be discussed in light of multiple stressors and their effects.

Selenium biotransformations into proteinaceous forms by foodweb organisms of selenium-laden drainage waters in California

Selenium contamination represents one of the few clear cases where environmental pollution has led to devastation of wildlife populations, most notably in agricultural drainage evaporation and power plant coal-fly ash receiving ponds. Complex biogeochemistry, in particular extensive biotransformations and foodchain transfer, governs Se ecotoxicology and toxicology, for which the mechanism(s) are still elusive. However, total waterborne Se concentration has been widely used as a criterion for regulating and mitigating Se risk in aquatic ecosystems, which does not account for Se biogeochemistry and its site-dependence. There is a need for more reliable indicator(s) that encompass Se ecotoxicity and/or toxicity. Selenomethionine warrants special attention since it simulates Se toxicosis of wildlife in laboratory feeding studies. While low in free selenomethionine, microphytes isolated from Se-laden agricultural evaporation ponds were abundant in proteinaceous selenomethionine. This prompted a more extensive survey of Se speciation in foodchain organisms including microphytes, macroinvertebrates, fish, and bird embryos residing mainly in the agricultural drainage systems of the San Joaquin Valley, California. Total Se in biomass, water-soluble fractions, and protein-rich fractions were measured along with GC-MS analysis of proteinaceous selenomethionine. In all foodchain organisms, water-soluble Se constituted the major fraction of total biomass Se, while proteinaceous Se was a substantial, if not dominant, fraction of the water-soluble Se. In turn, proteinaceous selenomethionine comprised an important fraction of proteinaceous Se. In terms of total biomass Se, an average 1400-fold of Se biomagnification from water to microphytes was observed while subsequent transfer from microphytes to macroinvertebrates exhibited an average of only 1.9-fold. The latter transfer was more consistent and greater in extent for proteinaceous Se and proteinaceous selenomethionine, which is consistent with their importance in foodchain transfer. Proteinaceous Se in the omnivorous carp (Cyprinus carpio) liver also demonstrated a relation to ovarian lesions, while deformed stilt (Himantopus mexicanus) embryo was more abundant in proteinaceous selenomethionine than were normal embryos. Although limited in the number of organisms surveyed, these findings provide an impetus for further field and laboratory feeding studies to substantiate the hypothesis that proteinaceous selenomethionine underlies Se ecotoxicity, which may in turn prove to be a reliable indicator of Se risk in aquatic ecosystems.

In vitro modulation of 17-β-estradiol-induced vitellogenin synthesis: Effects of cytochrome P4501A1 inducing compounds on rainbow trout (Oncorhynchus mykiss) liver cells

Cytochrome P4501A1 (CYP1A1) induction, particularly in liver, is a useful marker of exposure of fish to polycyclic- and halogenated-aromatic hydrocarbons. However, the relationship between toxicity and CYP1A1 induction in fish is uncertain. Some compounds that induce CYP1A1 are antiestrogenic in mammalian bioassay, and this effect is linked to aryl hydrocarbon (Ah) receptor and/or increased catabolism of 17-β-estradiol. Liver of fish synthesizes estrogen-inducible egg yolk precursor protein vitellogenin (Vg) which is critical for oocyte maturation and ovarian development. To determine if CYP1A1-associated endocrine modulation could occur in fish liver, primary cultures of rainbow trout liver cells were co-administered 17-β-estradiol and CYP1A1 inducing compounds or mixtures. Protein synthesis and enzyme activity of cells were optimal when cultured in a modified HEPES buffered Medium 199 at 15 °C. Vg and albumin (Alb), estimated by ELISA measurement of concentration in the media 48 h after treatment, formed the basis for the test. Equivalent viability (mitochondrial dehydrogenase activity) and secretory functional capacity (Alb synthesis) were estimated and correlated with other results. In descending order, 2,3,4,7,8-pentachlorodibenzofuran (10−12 to 10−8 M) > 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) ≊ 2,3,7,8-tetrachlorodibenzofuran (10−11 to 10−8 M) > β-naphthoflavone (10−7 to 10−6 M) inhibited Vg synthesis in 17-β-estradiol treated liver cells. Potency of inhibition directly related to strength as an inducer of CYP1A1 protein. At 10−8 M, PCB congeners 77, 126, 156 did not inhibit Vg synthesis and induced no to moderate levels of CYP1A1 protein or EROD activity. At 10−8 M, congener 114, a weak EROD inducer, potentiated Vg synthesis relative to cells treated with 17-β-estradiol alone. The results of this study increase our understanding of the consequences of hepatic CYP1A1 induction, forewarn of reproductive impairment of sexually maturing fishes exposed to CYP1A1 inducing compounds and argue for further, more detailed in vivo investigation.

The use of bioindicators for assessing the effects of pollutant stress on fish

Abstract The use of bioindicators in environmental pollution studies involves monitoring a suite of selected stress responses at several levels of biological organization in order to (1) assess the effects of sublethal stress on fish, (2) predict future trends (early warning indicators) and (3) obtain insights into causal relationships between stress and effects at the community and ecosystem level. We have successfully applied this approach with redbreast sunfish (Lepomis auritus) in freshwater systems receiving inputs of complex contaminant mixtures containing polychlorinated biphenyls (PCBs), hydrocarbons, heavy metals and chlorine. Indicators such as mixed-function oxidase (MFO) enzymes and DNA damage have provided direct evidence of toxicant exposure, while condition indices and indicators related to lipid biochemistry and histopathology have reflected impaired lipid metabolism, immune and reproductive system dysfunction, and reduced growth potential. At higher levels of organization, stress-mediated effects have included changes in the richness and biotic integrity of fish communities.

Diagnostic Criteria for Degenerative, Inflammatory, Proliferative Nonneoplastic and Neoplastic Liver Lesions in Medaka (Oryzias latipes): Consensus of a National Toxicology Program Pathology Working Group

Diagnostic criteria are presented for degenerative, inflammatory, nonneoplastic proliferative, and neoplastic lesions in the liver of medaka (Oryzias latipes), a small fish species frequently used in carcinogenesis studies. The criteria are the consensus of a Pathology Working Group (PWG) convened by the National Toxicology Program. The material examined by the PWG was from medaka exposed to N-nitrosodiethylamine for 28 days, removed to clean water, and sacrificed 4, 6, or 9 mo after initiation of exposure. Degenerative lesions included hepatocellular intracytoplasmic vacuolation, hepatocellular necrosis, spongiosis hepatis, hepatic cysts, and hepatocellular hyalinization. Inflammatory lesions consisted of granulomas, chronic inflammation, macrophage aggregates, and focal lymphocytic infiltration. Nonneoplastic proliferative lesions comprised foci of cellular alteration (basophilic focus, eosinophilic focus, vacuolated focus, and clear cell focus) and bile duct hyperplasia. Neoplastic lesions included hepatocellular adenoma, hepatocellular carcinoma, cholangioma, and cholangiocarcinoma. Two lesions composed mainly of spindle cells were noted, hemangiopericytoma and spindle cell proliferation. Rather than being an exhaustive treatment of medaka liver lesions, this report draws from the published literature on carcinogen-induced liver lesions in medaka and other fish species and attempts to consolidate lesion criteria into a simplified scheme that might be useful to pathologists and other researchers using medaka lesions for risk assessment or regulatory purposes.

Determining the sensitive developmental stages of intersex induction in medaka (Oryzias latipes) exposed to 17β-estradiol or testosterone

Certain environmentally persistent compounds can adversely affect reproduction by acting as steroid hormone agonists or antagonists. The goal of the present study was to determine the developmental stage most susceptible to exogenous hormone (estradiol and testosterone) exposure using a small teleost model. In the first (pilot study) of two experiments, medaka (Oryzias latipes), at varying developmental stages, were bath-exposed to 5 micrograms/l 17 beta-estradiol for 24 h. At 5 months of age, fecundity, fertility and embryo and larval viability (reproductive success) were investigated in control and exposed groups. Fish at 1, 1.5, 2 and 5.5 months of age were also sampled, processed and examined histologically for gonadal alteration. No significant differences in mortality, gonadal morphology, body weight, sex-ratio or time to maturity were seen between control and exposed fish. At 5 months, however, when exposure groups were compared to controls, significant differences were seen in reproductive success and viability of offspring. A second experiment exposed embryo stage 10, and 1-, 7- and 21-day-old larvae for 6 days to 15 micrograms/l 17 beta-estradiol or 100 micrograms/l testosterone. No significant differences were seen at 5 months in mortality, body weight, or time to sexual maturity. However, sex-ratios were significantly biased toward female in the stage 10, 1- and 7-day post-hatch estradiol exposure groups. No significant changes in sex-ratio were associated with testosterone exposure at any developmental stage. Further, intersex gonads were observed in fish from all groups exposed to 15 micrograms/l estradiol. Only those fish exposed as newly hatched fry or at 1 week post-hatch displayed intersex gonads following 100 micrograms/l testosterone exposure. Data from these experiments show that newly hatched fry are that life stage most sensitive to hormone exposure and the most appropriate to use in determining effects of known endocrine-disrupting compounds.

Cytological changes during progression of neoplasia in selected fish species

Abstract Cytological changes during progression of experimentally induced hepatic neoplasia in fishes were reviewed with emphasis on recent findings in Cyprinodon variegatus and Oryzias latipes. Hepatocytes are particularly sensitive to toxic changes during early phases of response to carcinogens reflecting both lethal and sublethal alterations. In these degenerative lesions, enzyme histochemical studies reveal marked deficiency of glucose-6-phosphate dehydrogenase, glucose-6-phosphatase and adenosine triphosphatase. Surviving hepatocytes are either enlarged, encircled by cells with small nucleus to cytoplasm ratios, and have altered nuclear morphology suggestive of an inability to divide, or are smaller, apparently rapidly dividing, and have basophilic cytoplasm. In both species, development of spongiosis hepatis occurred following cytotoxic phases. This lesion apparently provides abundant space for cellular remodeling during neoplastic progression leading to eventual multinodular change. Foci of altered hepatocytes included basophilic, eosinophilic (both species) and clear cell (Cyprinodon variegatus only). Enzyme alterations preceded other tinctorial, morphologic alterations and were seen in cells composing foci and tumors, suggesting lineage of phenotypic alteration. Cytologic changes within other resident cell populations during neoplastic progression were reviewed.

Histological and histochemical changes in the digestive tract of white sturgeon larvae during ontogeny

Ontogenetic changes in digestive tract histology and digestive enzyme histochemistry were investigated 11 to 36 days post-hatch in white sturgeon Acipenser transmontanus larvae. From initiation of exogenous feeding (12 days post-hatch), larvae were fed a commercial salmonid diet for the ensuing 24 days. The digestive system of white sturgeon displayed a high degree of morphologic organization and functionality at the onset of exogenous feeding. An enhancement of digestive capacities occurred with transition to active feeding. On day 2 of feeding, there was a clear increase of alkaline phosphatase, aminopeptidase M, dipeptidyl peptidase IV, and γ-glutamyl transpeptidase activity in the brush border of the spiral intestine. This strong activity is an apparent confirmation of the importance of this segment of the intestine for protein digestion and nutrient absorption. The functional development of the pyloric intestine occurred on day 4 and was concomitant with an increase in the activity of brush border and cytoplasmic enzymes such as acetylcholinesterase, dipeptidyl peptidase II, α- and β-galactosidases. The absence of acetylcholinesterase, lactase, nonspecific esterase, and weak activity of exopeptidases and alkaline phosphatase in the anterior intestine suggests that this segment of the intestine may be less important in nutrient absorption than the pyloric and spiral intestines. The observed quantitative and qualitative differences in enzyme activity along the intestine indicate a high degree of specialization of each segment for specific digestive and absorptive processes.