Effects of ammonium sulfate on stress physiology and innate immunity of Western mosquitofish (Gambusia affinis).

Fertilizers increase yield of crops but may have unintended negative effects on fish as a byproduct of runoff into bodies of freshwater. The objective of this study was to determine if environmentally relevant concentrations of an ammonium fertilizer impacts stress and innate immunity in Western mosquitofish (Gambusia affinis). The mosquitofish were exposed to different concentrations of ammonium sulfate fertilizer: 0 ppm, 40 ppm, and 80 ppm. To test the effects of ammonium sulfate on stress physiology, cortisol released into water by individual fish was collected after 1 week of exposure and again after 2 weeks of exposure and quantified with an enzyme immunoassay. Cortisol levels in the 0-ppm group were not significantly different over the course of the study, but we found a significant increase in cortisol levels in the fish exposed to 40 ppm and 80 ppm. We found reduced survival in fish from the 40 ppm and 80 ppm of ammonium sulfate groups compared with the 0-ppm group. We also used blood samples to complete a lysozyme assay as a measure of innate immune defense. Higher concentrations of ammonium sulfate correlated with significantly lower lysozyme activity in the fish. Overall, our results suggest that relatively low amounts of ammonium sulfate runoff into bodies of water are likely to have negative sublethal and lethal effects on small fishes.

Toxic and genotoxic effects of the 2,4-dichlorophenoxyacetic acid (2,4-D)-based herbicide on the Neotropical fish Cnesterodon decemmaculatus.

Acute toxicity and genotoxicity of the 54.8% 2,4-D-based commercial herbicide DMA® were assayed on Cnesterodon decemmaculatus (Pisces, Poeciliidae). Whereas lethal effect was used as the end point for mortality, frequency of micronuclei (MNs), other nuclear abnormalities and primary DNA damage evaluated by the single cell gel electrophoresis (SCGE) assay were employed as end points for genotoxicity. Mortality studies demonstrated an LC50 96 h value of 1008 mg/L (range, 929-1070) of 2,4-D. Behavioral changes, e.g., gathering at the bottom of the aquarium, slowness in motion, slow reaction and abnormal swimming were observed. Exposure to 2,4-D within the 252-756 mg/L range increased the frequency of MNs in fish exposed for both 48 and 96 h. Whereas blebbed nuclei were induced in treatments lasting for 48 and 96 h, notched nuclei were only induced in fish exposed for 96 h. Regardless of both concentration and exposure time, 2,4-D did not induce lobed nuclei and binucleated erythrocytes. In addition, we found that exposure to 2,4-D within the 252-756 mg/L range increased the genetic damage index in treatments lasting for either 48 and 96 h. The results represent the first experimental evidence of the lethal and several sublethal effects, including behavioral alterations and two genotoxic properties namely the induction of MNs and primary DNA strand breaks, exerted by 2,4-D on an endemic organism as C. decemmaculatus.

Evaluation of the genotoxicity of a herbicide formulation containing 3,6-dichloro-2-metoxybenzoic acid (dicamba) in circulating blood cells of the tropical fish Cnesterodon decemmaculatus.

Acute toxicity and genotoxicity of the dicamba-based commercial herbicide formulation Banvel(®) were evaluated on Cnesterodon decemmaculatus (Pisces, Poeciliidae) exposed under laboratory conditions. A lethal effect was used as the end point for mortality, whereas frequency of micronuclei (MNs) and DNA single-strand breaks evaluated by the single cell gel electrophoresis assay were employed as end points for genotoxicity. Mortality studies revealed an LC50 96 h value of 1639 mg/L (range, 1471-1808) of dicamba. Furthermore, behavioral changes, e.g., gathering at the bottom of the aquarium, slowness in motion, abnormal swimming, and slow reaction, were observed. Whereas increased frequency of MNs was observed when 1229 mg/L dicamba was assayed for 48 h, no induction of MNs was observed in fish exposed to the herbicide for 96 h, regardless of the concentration of dicamba. Furthermore, other nuclear abnormalities, i.e., binucleated cells and lobed and notched nuclei, were induced in fish exposed for 48 h but not 96 h. Increase in the genetic damage index was observed in those treatments (lasting for both 48 and 96 h) within the 410-1229 mg/L dicamba concentration-range. This study represents the first evidence of acute lethal and sublethal effects exerted by dicamba on a piscine species native to Argentina. The results could indicate that dicamba-based formulation Banvel(®) is the less toxic emerging pollutant reported so far for C. decemmaculatus. Finally, our findings highlight the properties of this herbicide that jeopardize nontarget living species exposed to this agrochemical.

Exploring fish bioassay of textile dye wastewaters and their selected constituents in terms of mortality and erythrocyte disorders.

Acute (4 day) and short-term (7 day) toxicity studies (at 1/5th and 1/10th of LC(50)) of textile dye wastewaters and their selected ingredients (azo dye methyl red and heavy metals Cd, Cu, Ni and Zn) were made on a freshwater fish Gambusia affinis under laboratory conditions. LC(50) value was found to be the lowest in four cases, and the EC(50) value for reduction in erythrocyte counts in the remaining four tests. Thus, the reduction in erythrocyte counts to the 50% level was similar in sensitivity to fish mortality. The short-term toxicity studies revealed significant disorders in erythrocyte morphology (poikilocytosis) and its counts to be the better indices for toxicity monitoring in the absence of fish mortality.

A comparative study on characterization of textile wastewaters (untreated and treated) toxicity by chemical and biological tests.

Toxicity of textile wastewaters (untreated and treated) and their ingredient chemicals was quantified in terms of their chemical characteristics, fish (Gambusia affinis) mortality and end point growth responses of duckweed (Lemna aequinoctialis) in short-term bioassays. Other parameters of fish bioassay were erythrocyte morphology and its counts. Despite of a definite correlation between data of biological tests (LC/EC(50) values) with that of chemical tests, biological tests were found to be relatively more sensitive to both wastewaters and ingredient chemicals. Amongst all the examined parameters of test organisms, fish RBCs (morphology and counts) sensitivity to pollutants in the wastewaters was usually maximum and therefore, their study should be included in the routine fish bioassay. Other advantage of biological test such as on Lemna is even detection of eutrophic potential of wastewaters, as noted at their higher dilutions. The ingredient chemicals (major) contributing maximum toxicity to textile dye wastewater were, acids (HCl and H(2)SO(4)), alkali (Na(2)O SiO(2)), salt (NaNO(2)) and heavy metal (Cu), whereas dyes (4) were relatively less toxic.

A comparative study on the toxic effects of textile dye wastewaters (untreated and treated) on mortality and RBC of a freshwater fish Gambusia affinis (Baird and Gerard).

Comparative toxicological studies of textile dye wastewater (untreated and treated) on a freshwater fish, Gambusia affinis, revealed a marked reduction in mortality and cytotoxic effects on RBCs, measured as reduction in their counts and percent changes in their shape (poikilocytosis) and variation in their size (anisocytosis)}, after subjecting them to both physicochemical and biological treatments. On comparing the data of mortality and the cytotoxic effects on RBCs, we found poikilocytosis is a better indicator for toxicity measurement of both untreated as well as treated wastewater, especially at their lowest concentrations where percent mortality was found to be either nil or lowerer than the percentage of poikilocytic RBCs. Although percent reduction in RBC counts and changes in their size (anisocytosis) indicated toxic effects of wastewaters, but EC5o values for RBC counts were usually higher than those for poikilocytosis and mortality, and non-calculable for anisocytosis suggesting their lesser sensitivity to pollutants. In view of these findings, we recommend monitoring of toxic effects of wastewaters during fish bioassay on both mortality and variation in RBC shape.

Reproductive characteristics of male mosquitofish (Gambusia affinis) inhabiting a small southeastern U.S. river receiving treated domestic sewage effluent.

A population of western mosquitofish (Gambusia affinis) living below a wastewater treatment plant in the vicinity of Birmingham (AL, USA) was studied for evidence of exposure to estrogens. Mosquitofish are sexually dimorphic live-bearing fish. Males have an elongated and modified anal fin, called a gonopodium, used in mating. It has been hypothesized that exposure to estrogens and/or anti-androgens in treated wastewater might inhibit the androgen-dependent development of the gonopodium. The population in this study showed no evidence of having been exposed to endocrine-disrupting chemicals. The difference in adjusted (for overall fish size via analysis of covariance) mean gonopodium length between effluent-exposed and control populations was not significant. No detectable levels of vitellogenin were observed in the blood of any of the male mosquitofish, either from the effluent-exposed or the control population. Testes and livers were weighed and examined histologically. The fish exposed to treated wastewater effluent had significantly larger adjusted mean weights for both testis and liver, but no histological changes indicating exposure to estrogens were observed.

Estimation of nuclear DNA content by flow cytometry in fishes of the genus Xiphophorus.

1. By use of flow cytometry we measured nuclear DNA content in cells from 16 stocks representing 9 species of the genus Xiphophorus. 2. Significant differences were detected between certain stocks and species with respect to DNA content. 3. Male-female differences were apparent in 5 of 7 stocks in which males and females were studied. 4. Estimation of nuclear DNA content is of potential significance in connection with the genetics of sex determination and the study of taxonomic relationships.

Serum androgens during the annual reproductive cycle of the male mummichog, Fundulus heteroclitus.

A technique for the simultaneous detection of serum concentrations of testosterone (T), 17 beta-hydroxy-4-androstene-3,11-dione (11-KT), and 11 beta, 17 beta-dihydroxy-4-androsten-3-one (11 beta-HT) was established using a combination of high performance liquid chromatography separation and radioimmunoassay. In the mummichog, Fundulus heteroclitus, serum concentrations of T, 11-KT, and 11 beta-HT varied from 11.3, 10.1, and 27.9 ng/ml, respectively, during the breeding season to 0.2, 0.3, and 0.9 ng/ml, respectively, during the nonbreeding season. The changes in serum androgen levels, determined weekly, did not coincide with changes in the gonosomatic index or measurement of sperm/gram testes. Increases in serum androgen levels did coincide with the rise in sperm index, but the decreases in serum concentrations of androgens preceded the fall in sperm index by more than a month. A model is proposed to explain the reproductive cycle of male mummichogs.