Japanese medaka (Oryzias latipes) were continually exposed in a flow-through diluter system for 9 months to measured chloroform concentrations of 0.017, 0.151, or 1.463 mg/L. Parameters evaluated were hepatocarcinogenicity, hepatocellular proliferation, hematology, and intrahepatic chloroform concentration. Histopathology was evaluated at 6 and 9 months. Chloroform was not hepatocarcinogenic to the medaka at the concentrations tested. Chronic toxicity was evidenced at these time points by statistically significant (alpha = 0.05) levels of gallbladder lesions and bile duct abnormalities in medaka treated with 1.463 mg/L chloroform. We assessed hepatocellular proliferation by exposing test fish to 5-bromo-2'-deoxyuridine in the aquarium water for 72 hr after 4 and 20 days of chloroform exposure; we then quantified area-labeling indices of the livers using computer-assisted image analysis. We observed no treatment-related increases in cellular proliferation. We analyzed cells in circulating blood in medaka after 6 months of chloroform exposure. Hematocrit, leukocrit, cell viability, and cell counts of treated fish were not significantly different from those of control fish. Using gas chromatography (GC), we evaluated intrahepatic concentrations of chloroform in fish after 9 months of exposure. Livers from the 0.151 and 1.463 mg/L chloroform-treated fish had detectable amounts of chloroform, but these levels were always lower than the aquaria concentrations of chloroform. Thus, it appeared that chloroform did not bioaccumulate in the liver. Unidentified presumptive metabolite peaks were found in the GC tracings of these fish livers.

Japanese medaka (Oryzias latipes) were continually exposed in a flow-through diluter system for 9 months to measured bromodichloromethane (BDCM) concentrations of 0.018, 0.143, or 1.424 mg/L. Parameters evaluated were hepatocarcinogenicity, hepatocellular proliferation, hematology, and intrahepatic BDCM concentration. BDCM was not hepatocarcinogenic to medaka at the concentrations tested. Chronic toxicity was evidenced at 6 and 9 months by statistically significant (alpha = 0.05) levels of gallbladder lesions and bile duct abnormalities in medaka treated with 1.424 mg/L BDCM. Hepatocellular proliferation was assessed after 1, 4, and 20 days of BDCM exposure. Treatment-related increases or decreases in cellular proliferation were not observed at any time point. Hematocrit, leukocrit, cell viability, and cell counts of treated fish after 9 months of BDCM exposure were not significantly different from control fish. Intrahepatic concentrations were evaluated by gas chromatography after 9 months of BDCM exposure. Fish livers from all three BDCM treatments had detectable amounts of BDCM, with median intrahepatic concentrations of 1.02, 2.89, and 21.25 mg BDCM/kg fish liver in the low, middle, and high concentrations, respectively. Medaka chronic toxicity effects of statistically significant gallbladder and bile duct abnormalities occurred at 1.424 mg/L BDCM, well above median drinking water levels.

Japanese medaka fish ( Oryzias latipes) were used to develop an in vivo method to assess hepatocellular proliferation in a nonmammalian model. Proliferative responses were assessed in medaka at 7, 17, 24, and 94 days after a 48-hour exposure to 10 or 100 mg/L diethylnitrosamine (DEN). Subgroups of medaka were exposed to 50 or 75 mg/L of 5-bromo-2'-deoxyuridine (BrdU) in water for 72 hours, sacrificed, and then processed for immunohistochemical staining. Proliferative indices of BrdU-labeled hepatocytes were quantified and compared using both count and area measurements. There was a significant increase ( p < 0.05) in hepatocellular proliferation in the 100 mg/L DEN-treated fish as compared to controls and 10 mg/L DEN-treated fish for the first 3 time points. Hepatocarcinogenicit y was evaluated 26 weeks post-DEN exposure. There was a significant increase ( p < 0.0001) in hepatocellular neoplasms in 100 mg/L DEN-treated fish compared to other fish. Effective BrdU-labeling of S-phase hepatocytes in medaka was achieved by adding BrdU to the aquarium water, and an increase in hepatocellular proliferation using this method was detected 7 days after exposure to a carcinogenic concentration of DEN. Additionally, the new method of area measurement indices of proliferation were as precise as count indices (R 2 ≥0.92).

A workshop titled "Using Sentinel Species Data to Address the Potential Human Health Effects of Chemicals in the Environment," sponsored by the U.S. Army Center for Environmental Health Research, the National Center for Environmental Assessment of the EPA, and the Agency for Toxic Substances and Disease Registry, was held to consider the use of sentinel and surrogate animal species data for evaluating the potential human health effects of chemicals in the environment. The workshop took a broad view of the sentinel species concept, and included mammalian and nonmammalian species, companion animals, food animals, fish, amphibians, and other wildlife. Sentinel species data included observations of wild animals in field situations as well as experimental animal data. Workshop participants identified potential applications for sentinel species data derived from monitoring programs or serendipitous observations and explored the potential use of such information in human health hazard and risk assessments and for evaluating causes or mechanisms of effect. Although it is unlikely that sentinel species data will be used as the sole determinative factor in evaluating human health concerns, such data can be useful as for additional weight of evidence in a risk assessment, for providing early warning of situations requiring further study, or for monitoring the course of remedial activities. Attention was given to the factors impeding the application of sentinel species approaches and their acceptance in the scientific and regulatory communities. Workshop participants identified a number of critical research needs and opportunities for interagency collaboration that could help advance the use of sentinel species approaches.

Japanese medaka (Oryzias latipes) were used to evaluate the carcinogenicity of a complex groundwater that contained 5 U.S. Environmental Protection Agency priority pollutant heavy metals and 13 chlorinated aliphatic hydrocarbons. A test protocol that used 10 mg/L diethylnitrosamine (DEN) prior to groundwater exposure was designed to assess both initiation and promotion. The fish were exposed continuously for 9 mo with 0, 1, 5, or 25% groundwater, by volume, with either West Branch of Canal Creek water (Aberdeen Proving Ground-Edgewood Area, Aberdeen Proving Ground, MD) or dechlorinated tap water as the diluent, while concurrent controls were run in the laboratory. Incidental findings included various neoplasms in the nares, ovary, skeletal muscle, skin, swim bladder, testis, thymus, and thyroid. Factors evaluated during statistical analyses of fish neoplasm prevalence included diluent type, groundwater percentage, fish gender, and DEN initiation. Liver neoplasm prevalence was higher in DEN-initiated fish and was frequently higher in males. Concentrations of up to 25% groundwater, by volume, showed no evidence of being a complete carcinogen and showed no consistent, conclusive evidence of being a promoter.

Trichloroethylene (TCE) was found as a contaminant in the well supplying water to an aquatic testing laboratory. The groundwater was routinely screened by a commercial laboratory for volatile and semivolatile compounds, metals, herbicides, pesticides, and polychlorinated biphenyls using U.S. Environmental Protection Agency methods. Although TCE was the only reportable peak on the gas chromatograph, with average concentrations of 0.200 mg/l, other small peaks were also present, indicating the possibility that the contamination was not limited to TCE alone. A chronic 6-month carcinogenicity assay was conducted on-site in a biomonitoring trailer, using the Japanese medaka fish (Oryzias latipes) in an initiation-promotion protocol, with diethylnitrosamine (DEN) as the initiator and the TCE-contaminated groundwater as a promoter. Study results indicated no evidence of carcinogenic potential of the groundwater without initiation. There was, however, a tumor-promotional effect of the groundwater after DEN initiation. A follow-up laboratory study was conducted using reagent grade TCE added to carbon-filtered groundwater to simulate TCE concentrations comparable to those found in the contaminated groundwater. Study results indicated no promotional effects of TCE. These studies emphasize the necessity for on-site bioassays to assess potential environmental hazards. In this instance, chemical analysis of the groundwater identified TCE as the only reportable contaminant, but other compounds present below reportable limits were noted and may have had a synergistic effect on tumor promotion observed with the groundwater exposure. Laboratory toxicity testing of single compounds can produce toxicity data specific to that compound for that species but cannot take into account the possible toxic effects of mixtures of compounds.

The toxic potential of ground water contaminated with several probable carcinogenic heavy metals and halogenated solvents was evaluated using an integrated environmental assessment approach. A number of assays, which included acute toxicity, short‐term chronic toxicity, genotoxicity, developmental toxicity and carcinogenicity, were used to assist in a hazard assessment. Comprehensive analytical chemistry was performed throughout the 9‐month exposure to document the chemical characteristics of the ground water. An initiation‐promotion protocol using a non‐neoplastic concentration of diethylnitrosamine as an initiator in a 9‐month chronic exposure of Japanese medaka ( Oryzias latipes ) was used to evaluate potential carcinogenicity of the ground water. The fish were exposed to groundwater concentrations of 1% and 10% ground water by volume. No significant lesions were found in the Japanese medaka exposed to groundwater concentrations at 1% or 10% ground water by volume. Likewise, no genotoxicity, developmental toxicity, acute toxicity or short‐term chronic toxicity, were found at concentrations of ≤10% ground water by volume. The negative results obtained in this study show that the potential hazard posed by low concentrations of a complex mixture containing suspect carcinogens may not be manifested at environmentally relevant concentrations.

Investigators representing such diverse disciplines as biochemistry, pathology, genetics and environmental toxicology currently employ fish models as routine and reliable bioassay testing systems for the detection of toxins and/or carcinogens in the environment, and more appropriately, as rapid indicators of carcinogenicity. The Japanese medaka, Oryzias latipes, has served as a popular fish model for carcinogenicity studies, many of which have included diethylnitrosamine (DENA), a site specific carcinogen for hepatic neoplasms. One of the more intriguing manifestations of nitrosamine exposure reported for both fish and rats is spongiosis hepatis, a hepatic lesion characterized by multilocular cyst-like complexes. Spongiosis hepatis has not been reported in medaka exposed as embryos to hepatic carcinogens and the single article indicating the presence of such a lesion in adult medaka is limited in cytological descriptions.

The use of specific species of fish as bioassay systems for carcinogenicity studies is a relatively new approach to the investigation of cellular transformation. As a result, there are considerable voids in our basic knowledge of the biochemical, cytochemical and ultrastructural manifestations of exposure to selective carcinogens. The Japanese medaka, Oryzias latipes, has served as a popular fish model for carcinogenicity studies, many of which have included diethylnitrosamine (DENA), a site-specific carcinogen for hepatic neoplasms. The primary objective of this investigation was to obtain ultrastructural information on the cytotoxic effects of a carcinogen demonstrated at a location other than the normal site of cellular transformation.Embryos of medaka were exposed to 3000 mg/L DENA for 48 hours, rinsed and held in clean water at 30°C. An additional group of embryos was maintained at 30°C in a tank of tap water and functioned as a control. At 50 weeks subsequent to the exposure, several fishes from each tank were removed, anaesthetized with tricaine methanesulfonate and the kidneys excised and processed for electron microscopy. Pronounced manifestations of cytotoxicity were evident in the epithelium of the proximal convoluted tubule.

Recent interest has been expressed in the phenomenon of tumor initiation and development in poikilothermic animals. A number of fish model systems have demonstrated sensitivity to a variety of known carcinogens. The medaka Oryzias latipes, a small aquarium fish native to Japan, represents one of the model systems which has been employed frequently in carcinogenicity studies. The primary objective of this investigation was to determine the ultrastructural manifestations of acute exposure of the carcinogen diethylnitrosamine to hepatic tissue of medaka. Diethylnitrosamine is aproven hepatic carcinogen for several species of small fish including medaka.A group of 14 day old fry was selected from a stock population and exposed to diethylnitrosamine at a concentration of 200 mg/L for 48 hours. The fish were rinsed and held in clean water at 25°C. An additional group of fry was maintained at 25°C in a tank of tap water and functioned as a control.

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