Development of a sensitive E-screen assay for quantitative analysis of estrogenic activity in municipal sewage plant effluents

A simplified proliferation test with human estrogen receptor-positive MCF-7 breast cancer cells (E-screen assay) was optimized and validated for the s ensitive quantitative determination of total estrogenic activity in effluen t samples from municipal sewage plants. After solid phase extraction of 1 l sewage on either 0.2 g polystyrene copolymer (ENV +) or 1 g RP-C18 materia l and removal of the solvent, analysis of the extracts in the E-screen assa y could be performed without any clean-up step. This was even possible with untreated sewage. Parallel extraction of four sewage samples on both diffe rent solid phase materials gave comparable quantitative results in the E-sc reen. A blank sample did not induce cell proliferation. As additive behavio ur of the estrogenic response of single compounds was proven for two differ ent mixtures each containing three xenoestrogens, total estrogenic activity in the sewage samples, expressed as 17 beta-estradiol equivalent concentra tion (EEQ), could be calculated comparing the EC50 values of the samples wi th those of the positive control 17 beta-estradiol. The detection limit of the E-screen method was 0.05 pmol EEQ/l (0.014 ng EEQ/l), the limit of quan tification 0.25-0.5 pmol EEQ/l (0.07-0.14 ng EEQ/l). In total, extracts of nine effluent and one influent sample from five different municipal sewage plants in South Germany were analyzed in the E-screen. All samples strongly induced cell proliferation in a dose-dependent manner which was completely inhibited by coincubation with 5 nM of the estrogen receptor-antagonist IC I 182,780. The proliferative effect relative to the positive control 17 bet a-estradiol (RPE) was between 30 and 101%. 17 beta-Estradiol equivalent con centrations were between 2.5 and 25 ng/l indicating a significant input of estrogenic substances via sewage treatment plants into rivers. (C) 1999 Els evier Science B.V. All rights reserved.