Examination Of Environmental Estrogen By Gobbid Fish (Odontobutis Potamophila) In Taihu Lake

Taihu Lake was the biggest freshwater lake in the Yangtze Delta and the source of water supply for Shanghai and east Zhejiang Province. The pollution happened there has taken more and more attentions of the public. The abundant wildness fish was the most effective test animal resources for the environment test there.Gobbid fish (Odontobutis potamophila (Günther)) was one kind of raptatorial benthonic fish in Taihu Lake. They occupied the niche of top predator in the ecosystem and has an effect of bioaccumulation to the contaminants in the water. Lots of research for the physiology, growth and reproduction of the Gobbid fish had already been done before and that was the reason for us to choose it as the bioassay animal in the lab research. Methods were developed to detect environmental estrogens in the lab and also in the Taihu Lake in order to provide base date for the endocrine-disrupting contaminants (EDCs) there as well as other research.Serum vitellogenin (Vtg) in male or immature fish has been used as a sensitive biomarker for studies of environmental estrogens in aquatic environments and the enzyme-linked immunosorbent assay (ELISA) was always suggested. The purification of the Vtg was complex and low yield, so that antiserum against Vtg was hard to prepare. That troubled the method to be popular. For that reason. Lipovitellin (LV), which was the purpose protein for Vtg, was used to prepare the antiserum instead of Vtg for the ELISA.LV was purified from Gobbid fish egg by AKTA Purifier-10 with a DEAE-Cellulose DE52 column and Vtg was purified from Gobbid fish serum with Sephacryl S-300 column. The molecular weight of LV is 440kD and that of Vtg is 556.5kD. Lipovitellin can be dissociated into three major subunits and the molecular weight of them was 105kD, 41.18kD and 31.30kD. Vtg had several subunits. The molecular weight of the major subunit is 110 kD .Although there were difference between LV and Vtg, the ant-LV antiserum showed clear specificity to Vtg. That indicated that the use of LV as antigen and standards was reliable for Vtg detecting in ELISA. The working range, which had a linear performance characteristics (R²>0.95) was 160ng/mL to 1280ng/mL and the lowest detectable concentration was 40ng/mL in ELISA.The serum vitellogenin concentrations in male gobbid fish was checked with the indirect competitive ELISA after exposed to 17β-estradiol (E₂) and 4-nonlyphenol (NP). Result shows that there was a significantly increase which was reliable with expose dosage. The lowest effectual concentration for E₂ was 0.001mg/kg (E₂/fish-weight) and for NP was 50mg/kg (NP/fish-weight).The serum vitellogenin of male gobbid fish sampled from three study sites around the Taihu Lake, which were named Dong Shan, Wang Ting and Zhou Tie, were checked with the indirect competitive ELISA. Positive examples were founded at Dong Shan and Zhou Tie in the investigation. A whole-year investigation was taken at Dong Shan, Taihu Lake and 50% samples caught in March and 35% samples caught in April were found with a positive reaction. The result prompted that environmental estrogens had been existed on that region.Results for acute toxicology test, which used NP and benzo[a]pyrene (BaP), showed that BaP could cause an increase in male fish serum E₂ concentrations and restrain NP's inducement effect to serum vitellogenin increase. Besides that, an increase in hepatic EROD activities of the male fish and a decrease in hepatic SOD activities were observed after exposed to BaP. The lowest effectual concentration for all these testes was 2mg/kg BaP (BaP/fish-weight) plus 200mg/kg NP (NP/fish-weight).