The in vitro estrogen receptor (ER) reporter gene assay has long been used to measure estrogenic activity in wastewater. In a previous study, we demonstrated that the assay represents net estrogenic activity in the balance between estrogenic and antiestrogenic activities in wastewater. However, it remained unclear whether the net estrogenic activity measured by the in vitro ERα reporter gene assay can predict the in vivo estrogenic effect of wastewater. To determine this, we measured the following: estrogenic and antiestrogenic activities of wastewater and reclaimed water by the in vitro ERα reporter gene assay, expression of vitellogenin-1 (vtg1) and choriogenin-H (chgH) in male medaka (Oryzias latipes) by quantitative real-time PCR, and estrone, 17β-estradiol, estriol, and 17α-ethynylestradiol concentrations chemically to predict estrogenic activity. The net estrogenic activity measured by the in vitro medaka ERα reporter gene assay predicted the in vivo vtg1/chgH expression in male medaka more accurately than the concentrations of estrogens. These results also mean that in vivo vtg1/chgH expression in male medaka is determined by the balance between estrogenic and antiestrogenic activities. The in vitro medaka ERα reporter gene assay also predicted in vivo vtg1/chgH expression on male medaka better than the human ERα reporter gene assay.

†Research Center for Environmental Quality Management Graduate School of Engineering Kyoto University 1 2 Yumihama Otsu Shiga 520 0811 Japan

†Research Center for Environmental Quality Management Graduate School of Engineering Kyoto University 1 2 Yumihama Otsu Shiga 520 0811 Japan §University of South Bohemia in Ceske Budejovice Faculty of Fisheries and Protection of Waters South Bohemian Research Center of Aquaculture and Biodiversity of Hydrocenoses Research Institute of Fish Culture and Hydrobiology Zátiší 728 2 389 25 Vodňany Czech Republic

‡Public Works Research Institute 1 6 Minamihara Tsukuba Ibaraki 305 8516 Japan

‡Public Works Research Institute 1 6 Minamihara Tsukuba Ibaraki 305 8516 Japan ∥Environment and Bio Group Korea Institute of Science and Technology Europe 66123 Saarbrücken Germany

⊥Okazaki Institute for Integrative Bioscience National Institute for Basic Biology National Institutes of Natural Sciences and Department of Basic Biology Faculty of Life Science Graduate University for Advanced Studies SOKENDAI Higashiyama 5 1 Myodaiji Okazaki Aichi 444 8787 Japan

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