Quantifying the antiestrogen activity of wastewater treatment plant effluent using the yeast estrogen screen

The yeast estrogen screen (YES) assay was used to measure both estrogenic and antiestrogenic activity of wastewater treatment plant (WWTP) effluent for the purpose of developing a method to quantify antiestrogenic activity. Wastewater treatment plant effluent samples were concentrated by solid-phase extraction (SPE) and serially diluted. Five microliters of each dilution plus 195 microl of assay medium was placed in well plates and tested for estrogenic substances. Antiestrogen activity in WWTP effluent samples was indirectly measured by an effluent-volume-dependent suppression of the beta-galactosidase activity induced by an estradiol (E2) standard. Antiestrogens and estrogens were quantified by median inhibition concentration (IC50) and median effective concentration (EC50) statistics, respectively, and were expressed in terms of effluent volume (prior to concentration by SPE). Antiestrogen IC50 and estrogen EC50 values, calculated by standard linear regression methods, averaged 25.6 microl and 22.1 microl effluent, respectively. Taken together, these values suggest that antiestrogens were responsible for approximately a 50% reduction in estrogen-induced activity in WWTP effluent. Therefore, measurements of estrogenicity by the YES assay in WWTP effluent that typically contains a mixture of estrogenic and antiestrogenic substances should be considered net estrogenic activity. The potential for false-positive antiestrogen activity was addressed by assays of beta-galactosidase activity in effluent, by measurements of yeast cell turbidity, and by stirred cell ultrafiltration for removal of solid-phase coextracted organic substances.