Mixtures of Estrogenic Contaminants in Bile of Fish Exposed to Wastewater Treatment Works Effluents

Most effluents from wastewater treatment works (WwTWs) contain estrogenic chemicals that include steroidal estrogens and xenoestrogens. We investigated the nature of mixtures of estrogenic contaminants taken up by two species of fish exposed to two WwTWs effluents. Sexually immature rainbow trout, Oncorhynchus mykiss, and sexually mature roach, Rutilus rutilus, were exposed to tap water, river water, or one of two estrogenic WwTWs effluents for up to 10 days, when the fish were sacrificed and tissues removed for chemical analysis. Estrogenic contaminants in the bile and gonads were hydrolyzed, concentrated by solid-phase extraction, and fractionated by RP-HPLC. Active fractions were detected and quantified using a yeast estrogen receptor transcription screen (YES assay) and the identities of estrogenic components in the fractions determined by GC-MS. Bile from rainbow trout exposed to either tap water or river water contained low amounts of 17beta-estradiol (E2) and estrone (E1) with a total estrogenic activity (mean+/-standard error) of 10+/-5 and 31+/-9 ng of E2 equivalents/mL (ng of E2eq/mL) for male and female fish, respectively. In effluent-exposed trout the total estrogen content of bile was considerably higher with the following composition and concentrations (ng of E2eq/mL) of individual estrogens: E2 (male, 591+/-125; female, 710+/-207), E1 (male, 338+/-75; female, 469+/-164), ethinylestradiol, EE2 (male, 32+/-2; female, 40+/-6), nonylphenol (NP) and short-chain NP polyethoxylates (male, 21+/-4; female, 22+/-3). An additional estrogenic compound, 17beta-dihydroequilenin (DHQ), was identified for the first time in effluent-exposed fish, and was present in trout bile at concentrations of (male) 40+/-9 and (female) 30+/-5 ng of E2 eq/mL. DHQ, E2, E1, and EE2, but not NP or NP polyethoxylates, were also detected in bile of effluent-exposed roach, and the concentrations of all these steroidal estrogens in ng of E2eq/mL were lower in male (E2, 62+/-2; E1, 35+/-11; EE2, 10+/-2; DHQ, 1+/-1) compared with female (E2, 740+/-197; E1, 197+/-37; EE2, 40+/-6; DHQ, 8+/-2) roach. The synthetic estrogen EE2 was also detected in the testes and ovaries of effluent-exposed roach. This study shows that a mixture of estrogenic contaminants present in WwTWs effluents bioconcentrate in fish tissues, resulting in the induction of vitellogenin, and are likely to contribute to feminizing effects in wild fish living in U.K. rivers. The composition of the mixture of estrogenic contaminants in the bile is species dependent and may determine the susceptibility of fish to the effects of exposure to estrogenic effluents.