This study tested the hypotheses that (1) exposure to treated Water Reclamation Plant (WRP) effluent will induce biological effects in exposed fish that are consistent with environmental estrogen (EE) exposure; and (2) seasonal differences in effluent composition will moderate biological effects. We conducted seven on-site exposures using a mobile laboratory. Total estrogenicity of effluents was 10- to 20-fold higher during spring than in fall. Common EEs including steroid estrogens, alkylphenols, and bisphenol-A were ubiquitous. An unusual spike in total estrogenicity identified a combined sewer overflow event. Fathead minnows (Pimephales promelas) responded to exposure with subtle changes in vitellogenin concentrations and secondary sex characteristics. An opportunity to assess a common carp (Cyprinus carpio) population permanently sustained inside the Stickney WRP revealed pronounced exposure effects, but also the resilience of biological organisms even under long-term exposure. In contrast to other studies, no histopathological changes were found. The mobile exposure laboratory proved capable of maintaining U.S. Environmental Protection Agency-recommended exposure conditions while providing flexibility for rapid deployment at multiple sites with minimal operational disruption. Further studies using this platform hold promise to resolve the convoluted interactions between complex effluents and inherent biological complexity.