A fractionation system, combined with an in vitro assay for detecting estrogenic activity, was developed in order to isolate and identify the major estrogenic chemicals present in seven sewage-treatment works (STW) effluents, receiving primarily domestic effluent, discharging into British rivers. Three sterols were isolated from estrogenic fractions of sewage extracts; these were the natural hormones 17 -estradiol and estrone and the synthetic hormone 17R-ethynylestradiol. 17 -Estradiol and estrone were present in all the effluents at measured concentrations ranging from 1 ng/L to almost 50 and 80 ng/L, respectively. The concentration of 17R-ethynylestradiol was generally below the limit of detection but was positively identified in three of the effluent samples at concentrations ranging from 0.2 to 7.0 ng/L. These data suggest that natural and synthetic hormones may be responsible for the observed induction of vitellogenin synthesis in male fish placed downstream of effluent discharges from STWs that receive mainly domestic inputs.

Abstract-An estrogen-inducible screen was developed in yeast (Saccharomyces cerevisiae) in order to assess whether surfactants and their major degradation products are estrogenic. The DNA sequence of the human estrogen receptor (hER) was integrated into the yeast genome, which also contained expression plasmids carrying estrogen-responsive sequences (ERE) controlling the expression of the reporter gene lac-Z (encoding the enzyme ␤-galactosidase). Thus, in the presence of estrogens, ␤-galactosidase is synthesized and secreted into the medium, where it causes a color change from yellow to red. This recombinant strain was used to determine whether representatives of major surfactant classes and some of their principal degradation products possess estrogenic activity. The results were compared to the effects of the main natural estrogen 17␤-estradiol. None of the parent surfactants tested possessed estrogenic activity. However, one class of surfactants, the alkylphenol polyethoxylates, degrade to persistent metabolites that were weakly estrogenic. Another group of degradation products, the sulfophenyl carboxylates, which are derived from the biodegradation of linear alkylbenzene sulfonates, do not appear to possess estrogenic activity.

The occurrence of certain natural and synthetic steroidal estrogens in the final effluent from STW has been demonstrated. 17β-Estradiol and estrone were present at concentrations in the tens of nanograms per liter range, and the synthetic estrogen 17R-ethynylestradiol was also identified, albeit in the low nanogram per liter range. The findings from subsequent in vivo tank trial experiments, in which adult male rainbow trout (Oncorhynchus mykiss) and adult roach (Rutilus rutilus) were exposed for 21 days via the water to environmentally relevant concentrations of 17β-estradiol and estrone are presented. In addition, the response of adult male and female roach following exposure to 17β-estradiol (1, 10, and 100 ng/L) was compared to the response to the alkylphenolic xenoestrogen, 4-tert-octylphenol (1, 10 and 100 µg/L). Plasma levels of vitellogenin were determined using previously validated radioimmunoassays in order to measure the estrogenic response of the fish to the varying concentrations of the compounds tested. The results indicate that environmentally relevant concentrations of natural steroidal estrogens are sufficient to account for the levels of vitellogenin synthesis observed in caged male fish placed downstream of certain STW effluent discharges in British rivers.