To determine the level of estrogenic substances in an aquatic environment, four types of in vitro assay, namely, E-Screen, Ishikawa cell-ALP, YES and ELISA, and chemical analysis using GC/MS were carried out. Water samples were collected from coastal areas and rivers in Japan. Estrogenic substances were extracted by solid-phase extraction using a Sep-pak C18 cartridge. Higher levels of estrogenic substances were observed at sites close to sewage treatment plants as measured by in vitro assays. The 17β-estradiol equivalent concentration (E2 equiv.) determined by E-screen tended to be higher than those of the other assays, whereas YES showed a lower sensitivity than E-screen and Ishikawa cell ALP. According to the chemical analysis, the levels of estrone (E1) were higher than those of 17β-estradiol (E2). Although 17α-ethynylestradiol, nonylphenol, octylphenol and bisphenol A were detected in a few samples, their levels were low. E1 and E2 contributed to most parts of E2 equiv. in the water samples.
