The natural steroids estradiol-17beta (E2) and estrone (E1) and the synthetic steroid ethynylestradiol-17alpha (EE2) have frequently been measured in waters receiving domestic effluents. All of these steroids bind to the estrogen receptor(s) and have been shown to elicit a range of estrogenic responses in fish at environmentally relevant concentrations. At present, however, no relative potency estimates have been derived for either the individual steroidal estrogens or their mixtures in vivo. In this study the estrogenic activity of E2, E1, and EE2, and the combination effects of a mixture of E2 and EE2 (equi-potent fixed-ratio mixture), were assessed using vitellogenin induction in a 14-day in vivo juvenile rainbow trout screening assay. Median effective concentrations, relative to E2, for induction of vitellogenin were determined from the concentration-response curves and the relative estrogenic potencies of each of the test chemicals calculated. Median effective concentrations were between 19 and 26 ng L(-1) for E2, 60 ng L(-1) for E1, and between 0.95 and 1.8 ng L(-1) for EE2, implying that EE2 was approximately 11 to 27 times more potent than E2, while E2 was 2.3 to 3.2 times more potent than E1. The median effective concentration, relative to E2, for the binary mixture of E2 and EE2 was 15 ng L(-1) (comprising 14.4 ng L(-1) E2 and 0.6 ng L(-1) EE2). Using the model of concentration addition it was shown that this activity of the binary mixture could be predicted from the activity of the individual chemicals. The ability of each individual steroid to contribute to the overall effect of a mixture, even at individual no-effect concentrations, combined with the high estrogenic potency of the steroids, particularly the synthetic steroid EE2, emphasizes the need to consider the total estrogenic load of these chemicals in our waterways.

Abstract-A fish full life-cycle (FFLC) study was conducted for 17 using the fathead minnow, Pirnephales promelas. Newly fertilized embryos «24 h old) were exposed to fi ve concentrations of EEl (0.2, 1.0, 4.0, 16, and 64 ng/L nominal) in continuous flow-through conditions for 305 d at 25 ::' : 1°C. Exposure concentrations were verified by 14e-EE2 radiochemistry, supported by radioimmunoassay, and mean measured values were~70% of nominal. For the F o adult phase until 301 d posthatch, the no-observed-effect concentrations (NOEes) for growth, survival, and reproduction (as egg production) were all~1.0 ng/L. The NOEC values for F 1 embryo hatching success and larval sll1'vival (at 28 d posthatch) were both~LO ng/L. While statistically detectable changes in F 1 growth were evident at 0.2 nglL, these were not considered to be biologically significant when compared with historical control data. Male fish exposed to EE z at 4.0 ng/L failed to develop normal secondary sexual characteristics; on the other haud, assumed females exposed to this level of EE z were able to breed when paired with males that had not been exposed to EE,. Histology of F o control, 0.2-, and l-ng/L exposed fish at 56 d posthatch indicated an approximate female-to-male (F:M) sex ratio of 50:50 (with no ovatestes observed in the control), while fish exposed to EE z at 4.0 ng/L for 56 d posthatch had a F: M sex ratio of 84:5 (with ovatestes in 11% of fish). After 172 d posthatch, no testicular tissue was observed in any fish exposed to EE z at 4.0 ng/L. At the same time point, plasma vitellogenin levels were significantly higher in fish exposed to EE z at 16 ngl 1. A lack of sexual differentiation oceurred in males at concentrations~4.0 ng/L. Taking into account these data, the overall noobserved-adverse-effect concentration was considered to be 1.0 ng/L.