Metabolite-Driven Estrogenic Activity in Freshwater Fish Associated with Sewage Treatment Plant Effluents Revealed by Effect-Directed Analysis and Nontarget Screening

Major estrogen receptor (ER) agonists were identified in freshwater fish from the Gapcheon River, South Korea, using effect-directed analysis and nontarget screening (NTS). T47D-KB luc bioassays showed strong ER-mediated activity in polar fractions of crucian carp muscle and egg extracts. Well-known ER agonists, such as estrone, 17β-estradiol (E2), estriol, and 17α-ethinylestradiol, explained only 27 and 11% of the observed ER-mediated activity in crucian carp muscle and eggs, respectively, with E2 as the dominant contributor. LC-QTOFMS-based NTS data were processed using a six-step prioritization workflow based on molecular formulas (C n H n O n ) and mass ranges (250–320 Da), yielding 14 candidates. Of these, 7 of 11 available standards were newly identified as ER agonists, primarily estrogen metabolites and anabolic agents. Inclusion of these compounds, particularly the E2-derived metabolites epiestriol and 17-epiestriol, increased the explained activity to 37% in muscle and 40% in eggs of crucian carp, with 17-epiestriol contributing up to 27%. Matrix-specific differences in E2-EQ chem composition were observed, with E2 and epiestriol dominating in sewage treatment plant effluent, E2 in river water, and 17-epiestriol in fish and sediments. Epiestriol is likely formed during sewage treatment, whereas 17-epiestriol is produced in sediments and subsequently transferred to fish. These results show that metabolite-derived compounds retain ER-mediated activity and can contribute substantially to environmental ER-mediated activity, representing an important class of previously unrecognized ER-active compounds.