Mature male medaka were continuously exposed to 0.005, 0.0-5 or 1.0 ppb of estradiol-17 beta (E2 or 0.1, 10 or 100 ppb of p-nonylphenol (NP) or bis-phenol-A (BPA). Female-specific proteins (Fsp) were induced in medaka exposed to 0.005 ppb of E2, 0.1 ppb of NP, or 10 ppb of BPA. Concentrations of 0.005 pbb of E2 and 0.1 ppb of NP corresponded to concentrations of these chemicals detected in river water in Japan. The abilities of the 3 chemicals to induce Fsp were E2 > NP > BPA. Embryonic medaka were exposed to E2, NP and BPA under conditions of static-renewal for 200-230 days until pre-maturity. Survival ratios of medaka exposed to E2 and NP declined in concentrations more than 25 ppb and 50 ppb, respectively. The groups of medaka exposed to E2 had individuals with testis-ova or abnormal gonad. There was no male in exposure to 1.0 ppb E2. When exposed to 100 ppb of NP or BPA, abnormal gonad was also detected. Abnormal anal fin (female-like) was observed in male exposed to 100 ppb of NP. The LC50 values for each of the 3 chemicals were much higher than the concentrations detected in water in the environment--the 3 chemicals were considered to have no lethal effect on medaka in aquatic environments. However, exposures to E2 or NP at environmental concentrations induced Fsp. BPA also had the ability to affect medaka as an environmental estrogen, although its extrogenic activity was weaker than that of E2 or NP.

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2,2-bis (4-hydroxyphenyl) propane or Bisphenol A (BPA), has been reported to behave as an endocrine disrupter below acute toxic levels, and is widely present in the water environment. Although BPA is easily chlorinated, very little is reported on the effect of chlorinated BPA to the aquatic organisms. In this study, the estrogenic activities of BPA and its chlorinated derivatives were evaluated by the induction of vitellogenin (VTG) in the serum of mature male Japanese medaka. In addition, the effect of sodium hypochlorite on the decomposition of BPA was tested. The relative potencies of estrogenic activities of chlorinated BPA descended in the order 3,3'-diCIBPA>BPA> or =3-CIBPA>3,3',5-triCIBPA, and no estrogenic activity was observed in 3,3',5,5'-tetraCIBPA. Lowest Observed Effect Concentration (LOEC) and No Observed Effect Concentration (NOEC) for both 3-CIBPA and 3,3'-diCIBPA were 500 microg/L and 200 microg/L, respectively. LOEC for 3,3',5-triCIBPA was >500 microg/L. When BPA was reacted with sodium hypochlorite (24 hours; residual chlorine at 1 ppm), however, complete decomposition of BPA and its chlorinated derivatives was observed. The decrease in BPA and its chlorinated derivatives paralleled the decrease in estrogenic potency evaluated by the induction of vitellogenin (VTG) in the serum of mature male Japanese medaka.

Mature male medaka were continually exposed to four chemicals, p-n-nonylphenol (p-n-NP), nonylphenol (p-NP), bisphenol-A (BPA) and 17beta-estradiol (E2) to evaluate their estrogenic activities in the laboratory. In order to understand the effect of the chlorination that is applied widely in water and wastewater treatment, the above chemicals were chlorinated and then exposed to mature male medaka. Furthermore, in the case of vitellogenin, a is a female specific protein induced by the exposure to test waters containing the above chemicals after 5 weeks, medaka was returned to uncontaminated tap water to determine whether male medaka have a self recovery function from the effect of estrogenic chemicals. Much greater vitellogenin compared to the background levels were induced in the male medaka by separate exposure to 100 microg/L of p-NP, 1,000 microg/L of BPA and 0.05 microg/L of E2. The levels of vitellogenin increased with increasing exposure periods. The relative potencies of these chemicals descended in the order of E2>>p-NP>BPA. Vitellogenin levels inducible by these chemicals were drastically reduced as a result of the chlorination for 24 hours. However, a moderate increase in hepatocyte somatic index (HSI) meant the hepatic fatness was observed as a result of chlorination. It is not clear at this stage whether or not the formation of chlorination byproducts is responsible for this moderate increase in HSI. The vitellogenin concentration of male medaka exposed to chemicals for 5 weeks decreased gradually after return to the uncontaminated water. However, the vitellogenin concentration did not return to the initial normal levels even after 5 weeks. A clear relationship between the serum vitellogenin concentration and the hepatic vitellogenin concentration was also found. Since quantitative analytical procedures for hepatic vitellogenin are easier than those of the serum vitellogenin, measuring the estrogenic effect using the measurement of vitellogenin in liver is recommended.