Municipal wastewater treatment plants (WWTPs) regularly release complex chemical milieus into the environment in the form of treated wastewater. Compounds, including pharmaceuticals, antibiotics and hormones have been detected in treated wastewater and may retain their bioactive properties beyond the treatment system. Because WWTPs typically discharge concentrated effluents into lakes and streams, fish living downstream of effluent discharges are at risk of physiological and reproductive disruption from exposure to bioactive compounds in WWTP effluents.

Recent field investigations demonstrated reproductive disruption in white sucker fish (Catostomus commersoni) residing downstream of the City of Boulder's WWTP. Observed biochemical and histological alterations were suggestive of endocrine disruption by endocrine-active compounds in the effluent. In the follow-up investigation presented here, I employed isolated hepatic and gonadal tissues of the adult male fathead minnow (Pimephales promelas) in an in vitro biological system to test whether there is a direct causative relationship between the WWTP effluent and the reported reproductive disruption at the tissue-level. Vitellogenin release from the liver and spermatogonial proliferation in the gonad were analyzed as endpoints.

Via reconstitution of powdered incubation medium, isolated liver and testis tissue were exposed to distilled water (control), upstream water from Boulder Creek (reference), and to 50% effluent (fractionated with distilled water) and 100% WWTP effluent. In three separate validation experiments, 100% effluent exerted estrogen-like action by inducing vitellogenin release from the liver and reducing spermatogonial proliferation in the testis compared to control and reference. Effluent fractionated to 50% elicited the same estrogen-like effects in one of the three validation experiments.

A mixed design study aimed at characterizing treatment effects across time (daily sampling for 5 days) revealed that vitellogenin release increased over time for all treatments, but only treatment with 100% effluent was statistically significant. Additionally, maximal vitellogenin release for 100% effluent occurred after 24-hours incubation time, and at 72 hours for reference and 50% effluent.

A dose-response assay employing 17β-estradiol over four orders of magnitude (0.0001 to 0.1 µM) resulted in a dose-dependent increase in vitellogenin release at the highest three doses, and a dose-dependent decrease in spermatogonial proliferation with all four doses of the hormone. The dose-response assay was used to estimate the half-maximal effective concentration (EC ₅₀) of the wastewater for the two endpoints. The EC₅₀ of the effluent with regard to vitellogenin release was estimated to be 2.2 x 10^-5 µM (∼6.01 ng/L), whereas the EC₅₀ of the effluent with regard to spermatogonia proliferation was estimated to be 0.059 µM (∼16.10 µg/L).

The results of these experiments add in vitro data to mounting evidence that fish exposed to bioactive WWTP effluents are at risk of reproductive and physiological disruption.