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Chemical contamination of wastewater is a problem of great environmental concern, as it poses a hazard to both the ecosystem and to human health. In this study, we have performed a bioanalytical evaluation of the presence and removal efficiency for bioactive chemicals in wastewater treatment plants (WWTPs), using in vitro assays for toxicity endpoints of high relevance for human health. Water samples were collected at the inlet and outlet of five Swedish WWTPs, all adopting a treatment technology including pretreatment, primary treatment (sedimenation), seconday treatment (biological processes), post-sedimentation, and sludge handling. The water samples were analyzed for cytotoxicity, estrogenicity, androgenicity, aryl hydrocarbon receptor (AhR) activity, oxidative stress response (Nrf2) and the ability to activate NFĸB (nuclear factor kappa-light-chain-enhancer of activated B cells) signaling. We observed clear androgenic and estrogenic activities in all inlet samples. Androgenic and estrogenic activities were also observed in all outlet samples, but the activities were lower than the respective inlet sample. AhR activity was observed in all samples, with higher activities in the inlet samples compared to the outlet samples. The removal efficiency was found to be high for androgenic (>99% for two plants and 50-60% for two plants) and estrogenic (>90% for most plants) compounds, while the removal efficiency for AhR-inducing compounds was 50-60% for most plants and 16% for one plant.

Waste water treatment facilities are a major sources of organic micropollutants (MPs) in surface water. In this study, surface water samples were collected from seven sites along a river system in Uppsala, Sweden, during four seasons and evaluated based on the occurrence of MPs in the samples and bioactivity using in vitro bioassays. The sampling sites were differentially impacted by on-site sewage treatment facilities (OSSFs), small scale, and large scale waste water treatment plants (WWTPs). The bioassays used included activation of aryl hydrocarbon receptor (AhR), estrogen receptor (ER), nuclear factor kappa-light-chain-enhancer of activated B cells (NFkB), nuclear factor erythroid 2-related factor 2 (Nrf2), and androgen receptor (AR). Occurrence of 80 MPs, were analyzed using liquid chromatography coupled to tandem mass spectrometry. Most water samples induced AhR activity, and all sampling sites showed a similar profile regarding this activity. With the exception of one water sample, we did not detect any NFkB, Nrf2 or AR activity of the water samples. The exception was a sample impacted by OSSFs, which showed an activity in multiple bioassays, but the activity could not be explained by the occurrence of target MPs. The occurrence of MPs showed a spatial trend, with the highest number and amount of MPs detected in the samples collected downstream of the WWTPs, where up to 47 MPs were detected in one single sample. A seasonal variation was observed with highest levels of MPs and highest AhR activities in samples collected in June and September 2015. However, neither the seasonal activity nor the on-site activity could be explained by the measured MPs, suggesting unknown contributory agents in the water.