Chronic toxicity of chloroform to Japanese medaka fish.

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.

Chronic toxicity of bromodichloromethane to the Japanese medaka (Oryzias latipes).

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.

Biomarkers of immunotoxicity in fish: from the lab to the ocean.

Historically, host immunocompetence has been monitored using a battery of immune parameters. Recently, many of these same assays have been employed as biomarkers for predicting chemical-induced immunotoxicity in wildlife species. In this laboratory, assays measuring immunopathology, immune cell function, and host resistance against bacteria have been used successfully to assess immunotoxicity in laboratory-reared Japanese medaka (Oryzias latipes) and in feral fish populations. As an example of the latter, smallmouth bass collected from a PCB-contaminated site demonstrated significantly reduced phagocyte function and antioxidant activity compared to reference site fish. Taken together, these studies along with those from other investigators demonstrate the usefulness of immune assays as indicators to predict the toxicological risk associated with 'real-world' polluted aquatic environments.

Mammalian immunoassays for predicting the toxicity of malathion in a laboratory fish model.

This study describes the use of a panel of immune assays, originally developed by the National Toxicology Program for assessing xenobiotic-induced immunotoxicity in mice, to quantify the effects of sublethal malathion exposure on the immune responses of fish. For this study, Japanese medaka (Oryzias latipes) were exposed subchronically to the organophosphate pesticide malathion in a series of two experiments. In the first set of studies, fish were exposed for 7 or 14 d to untreated well water (i.e., controls) or to waterborne malathion at 0.2 or 0.8 mg/L. Following exposure, fish from each group were sacrificed and their kidneys (primary organ of leukopoiesis in fish and equivalent to mammalian bone marrow) were used to provide cells for assessing any malathion-induced effects upon nonspecific and acquired immune defense mechanisms. Effects upon humoral-mediated immunity were determined by enumerating antibody plaque-forming cell (PFC) numbers from a subset of fish exposed to malathion for 14 d and then injected intraperitoneally (ip) with sheep erythrocytes (sRBC). Results of these studies demonstrated that while malathion exposure had no significant effect upon hematocrit/leukocrit values or upon mitogen-stimulated T-cell lymphoproliferation, PFC numbers in the kidney of exposed fish were significantly reduced (compared to control fish) in a dose-dependent manner. In addition, total recoverable kidney cell numbers and viability, as well as superoxide anion production by kidney phagocytes, were reduced slightly (compared to control values) in fish exposed for 14 d to the highest malathion concentration tested. In the second set of experiments, medaka exposed for up to 21 d to either 0.1 or 0.3 mg malathion/L were challenged ip with an LD50 dose of the bacterial fish pathogen Yersinia ruckeri. Results from these infectivity studies demonstrated that exposure to either malathion concentration, for 14 or 21 d reduced host resistance against Yersinia infection. Taken together, these findings demonstrate the applicability of mammalian immune assays for predicting malathion-induced immunosuppression in a teleost fish, as well as the potential utility of a small laboratory fish to serve as an alternate model for mammals in immunotoxicological studies.

Bioavailability of TNT residues in composts of TNT-contaminated soil.

Composting is being explored as a means to remediate 2,4,6-trinitrotoluene (TNT) contaminated soils. This process appears to modify TNT and to bind it to organic matter. The health hazards associated with dusts generated from such materials cannot be predicted without knowing if the association between TNT residues and compost particulate is stable in biological systems. To address this question, single doses of [14C]-TNT, soil spiked with [14C]-TNT, or compost generated with [14C]-TNT-spiked soils were administered to rats by intratracheal instillation. The appearance of 14C in urine and tissues was taken as an indication of the bioavailability of TNT residues from compost particles. In rats instilled with neat [14C]-TNT, about 35% of the 14C dose appeared in urine within 3 d. The 14C excreted in urine by these rats decreased rapidly thereafter, and was undetectable by 4 wk after treatment. Similar results were obtained with soil-treated rats. In contrast, after treatment with [14C]-TNT-labeled compost, only 2.3% of the total 14C dose appeared in urine during the first 3 d. Low levels of 14C continued to be excreted in urine from compost-treated rats for more than 6 mo, and the total amount of 14C in urine was comparable to that in TNT-treated animals. Determination of the radiolabel in tissues showed that 14C accumulated in the kidneys of rats treated with labeled compost but not in rats treated with [14C]-TNT or [14C]-TNT-spiked soil. These results indicate that the association between TNT and particulate matter in compost is not stable when introduced into the lungs. Accumulation of 14C in kidneys suggests the presence of a unique TNT residue in compost-treated rats. The rate of excretion and tissue disposition of 14C in rats treated with TNT-spiked soil indicate that TNT in soil is freely available in the lungs.

Experimental transmission of eastern equine encephalitis virus by strains of Aedes albopictus and A. taeniorhynchus (Diptera: Culicidae).

The vector competence of Aedes taeniorhynchus (Wiedemann) and four strains of Aedes albopictus (Skuse) was assessed for eastern equine encephalitis (EEE) virus isolated from Ae. albopictus collected in Polk County, Florida. Both species became infected with and transmitted EEE virus by bite after feeding on 1-d-old chicks that had been inoculated with EEE virus (viremia = 10(10.1) plaque-forming units [PFU] per ml of blood). However, when fed on an older chick with a lower viremia (viremia = 10(6.1) PFU per ml of blood), Ae. albopictus was significantly more susceptible to infection (90%, n = 61) than was Ae. taeniorhynchus (15%, n = 40). Transmission was also significantly more efficient by Ae. albopictus (36%, n = 44), than by Ae. taeniorhynchus (0%, n = 14). These data, combined with the recent isolation of EEE virus from Ae. albopictus and its opportunistic feeding behavior, indicate that Ae. albopictus could function as a bridge vector between the enzootic Culiseta melanura (Coq.)-avian cycle and susceptible mammalian hosts.

Experimental transmission of Venezuelan equine encephalomyelitis virus by a strain of Aedes albopictus (Diptera: Culicidae) from New Orleans, Louisiana.

Experimental studies were undertaken to ascertain the vector competence of a strain of Aedes albopictus (Skuse) collected in New Orleans, LA, (Gentilly strain) for an epizootic (Trinidad donkey) strain of Venezuelan equine encephalomyelitis (VEE) virus. This strain of Ae. albopictus was significantly more susceptible to infection with VEE virus than were any of the four strains tested previously, including two from North America and two from South America. Likewise, dissemination (148 of 180) (82%) and transmission (40 of 88) (45%) rates were significantly higher in the Gentilly strain than in any of the strains previously tested. Analysis of the results of the present study along with those of a previous study with a second alphavirus, chikungunya (CHIK) virus, indicated that, although all three strains of Ae. albopictus tested were more susceptible to VEE virus than to CHIK virus, susceptibility to infection and dissemination with one alphavirus appeared to be directly related to susceptibility to infection and dissemination with the other virus and may indicate shared receptor sites for these two alphaviruses in Ae. albopictus.

Transmission of Venezuelan equine encephalomyelitis virus by Aedes sollicitans and Aedes taeniorhynchus (Diptera: Culicidae).

Experimental studies compared the vector competence of Aedes sollicitans (Skuse) and Ae. taeniorhynchus (Wiedemann) collected on Assateague Island, Va., for an epizootic strain (Trinidad donkey) of Venezuelan equine encephalomyelitis (VEE) virus. Infection rates were significantly higher in Ae. sollicitans (101/107, 94%) than in Ae. taeniorhynchus (103/175, 59%), even though both species fed concurrently on the same infected hamsters. Similarly, dissemination and transmission rates were significantly higher in the Ae. sollicitans population tested. Although both Ae. taeniorhynchus and Ae. sollicitans are natural vectors of VEE virus, the latter species should be considered a more efficient vector of VEE epizootic strains, based on its greater susceptibility to infection and higher transmission rates.

Susceptibility of selected strains of Aedes aegypti and Aedes albopictus (Diptera: Culicidae) to chikungunya virus.

The relative susceptibility of selected strains of Aedes aegypti (L.) and Aedes albopictus (Skuse) fed on a viremic monkey to infection with chikungunya virus was determined. Infection rates were consistently higher in 10 strains of Ae. albopictus tested than in 7 strains of Ae. aegypti tested, regardless of the geographic location from which the strains originated or the dose of virus ingested. Similarly, virus dissemination rates were higher in the Ae. albopictus strains compared with the Ae. aegypti strains. For nearly all (11 of 12) strains tested of both species, groups of mosquitoes with one or more females with a disseminated infection transmitted virus by bite to weanling mice. Based on these studies, Ae. albopictus appears to be a more competent laboratory vector of chikungunya virus than does Ae. aegypti.

Transmission of Venezuelan equine encephalomyelitis virus by strains of Aedes albopictus (Diptera: Culicidae) collected in North and South America.

Experimental studies were undertaken to ascertain the vector potential of North American (Houston and Alsace) and South American (Sao Paulo and Santa Teresa) strains of Aedes albopictus (Skuse) for an epizootic (Trinidad donkey) strain of Venezuelan equine encephalomyelitis (VEE) virus. Infection rates were similar in all four strains of Ae. albopictus tested after ingestion of VEE virus from a viremic hamster. Virus disseminated from the midgut to the hemocoel in about 80% of infected mosquitoes, regardless of the dose ingested (10(4.6) to 10(5.7) plaque-forming units per mosquito) or the time of extrinsic incubation (7-35 d). Although all four strains of this mosquito transmitted VEE virus by bite to hamsters, transmission rates were significantly higher for the South American strains (24%, 40 of 170) than for the North American strains (5%, 9 of 165). Although VEE virus has never been isolated from Ae. albopictus, the introduction of this species into the Americas may allow it to serve as an amplification vector in areas where epizootic strains of VEE are found or introduced.

Transmission of Rift Valley fever virus by adult mosquitoes after ingestion of virus as larvae.

We studied the ability of Culex pipiens, Aedes circumluteolus, and Ae. mcintoshi, exposed as larvae to liver tissue from a Rift Valley fever (RVF) virus-infected hamster, to become infected and transstadially transmit virus to the adult and for the adults to transmit virus by bite to hamsters. After exposure as larvae, 9% (5/54) of adult Cx. pipiens and 8% (38/505) of the adult Ae. (Neomelaniconion) species were infected. All of the infected Cx. pipiens and about half of the infected Ae. circumluteolus and Ae. mcintoshi tested transmitted RVF virus by bite to hamsters. Transmission rates for mosquitoes orally infected as larvae were higher than those for mosquitoes orally infected as adults. Animals infected with RVF virus may abort or die in the vicinity of mosquito larvae breeding habitats and infected tissue from these animals may contaminate the water.