Contribution of known endocrine disrupting substances to the estrogenic activity in Tama River water samples from Japan using instrumental analysis and in vitro reporter gene assay

To quantitatively characterize the substances contributing to estrogenic activity in river water, in vitro bioassay using MVLN cells and instrumental analysis using liquid chromatograph–mass spectrometer (LC/MS) or liquid chromatograph–tandem mass spectrometer (LC/MS/MS) were applied to river water extracts taken from various locations in the Tama River, Japan. Tama River water samples were extracted using solid phase extraction and the crude extracts were fractionated by high-performance liquid chromatography (HPLC) into 10 fractions. The sixth fraction contained nonylphenol (NP) and octylphenol (OP) at concentrations in the range of 51.6–147 and 6.9–81.9 ng/L, respectively (concentrations corresponding to the original sample volumes). No estrogenic activity, expressed as 17β-estradiol equivalents (E2-EQB), however, was observed in this fraction (<0.6 ng-E2eq/L). Instrumentally determined estrogenic activity (E2-EQC), which is the concentrations of NP and OP multiplied by their corresponding relative potency, was below the detection limit of the MVLN cell bioassay. Estrogenic activities were detected only in HPLC fraction nos. 7, 8 and 9. Estrone (E1), estradiol (E2) and bisphenol A (BPA) were detected in these fractions. Estriol (E3) and ethynylestradiol (EE2) were not detected (<0.2 ng/L) in these fractions. The calculated E2-EQC for BPA was below the detection limit of bioassay. The E2-EQC for E1 and E2 were on the same order as the estrogenic activity determined by the bioassay (E2-EQB). The ratios of E2-EQC and E2-EQB for E1 and E2 in the three factions collectively (nos. 7–9) were 0.49–0.97 and 0.29–1.12, respectively. Above results indicated that the major causal substances to the estrogenic activity in the Tama River were E1 and E2.