Metabolite-Driven Estrogenic Activity in Freshwater Fish Associated with Sewage Treatment Plant Effluents Revealed by...

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-KBluc 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 (CnHnOn) 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-EQchem 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.