Treated wastewater effluent is a mixture of different estrogenically active compounds (EACs), which can induce an endocrine response in exposed individuals. Furthermore, their presence as a mixture can lead to synergistic and additive effects that can surpass what would result from exposure to the individual compound. Previous studies have shown that fish populations are susceptible to environmentally relevant concentrations of these contaminants in treated effluent and exposure can result in disruption of several reproduction-related endpoints. Estrogenic effects have been observed in organisms that inhabit effluent-dominated rivers, where treated wastewater effluent and urban runoff make up greater than 50% of the stream flow composition. This study sought to assess the estrogenic impact that wastewater effluent discharge into the Santa Ana River, an effluent-dominated system in Southern California, by evaluating the biological and chemical activity associated with feminization throughout reaches of the river that receive effluent input. River extracts were collected by grab sample in summer, fall, and winter to evaluate “worst-case scenarios,” including base-flow and stormwater runoff conditions. River exposure effects were evaluated by examining relative vitellogenin (vtg) expression of Japanese Medaka (Oryzias latipes) exposed to collected water and Yellow Bullhead Catfish (Ameirus natalis) collected from the river.
Estrogenic activity, which was expressed in 17-β-estradiol equivalence (EEQ), did not vary between the summer (0.94 ± 0.1 ng/L; mean ± SEM) and fall (0.87 ± 0.11 ng/L; mean ± SEM) sampling events. Japanese medaka exposed for 14-days to fall river extracts exhibited a slight increase in vtg mRNA expression at sites that water extracts exceeding 1 ng/L EEQ determined by cell bioassays. Extracts taken following a significant rain event in January elicited significant estrogen receptor activity (4.2 ± 1.1 ng/L; mean ± SEM) that was above values at base-flow conditions. Targeted LC-MS/MS analysis of these samples correlated well with in vitro EEQs (r = 0.76, p = 0.03, Pearson Correlation). Yellow Bullhead Catfish male’s vtg expression was significantly different than females and sex ratios were unaltered, indicating that feminization of endogenous bullhead populations was not occurring. Overall, these results suggest that treatment process upgrades have had a significant positive impact on effluent estrogenic activity into the Santa Ana River.