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The assessment of water quality is crucial for safeguarding drinking water resources and ecosystem integrity. To this end, sample preparation and extraction is critically important, especially when investigating emerging contaminants and the toxicity of water samples. As extraction methods are rarely optimised for bioassays but rather adopted from chemical analysis, this may result in a misrepresentation of the actual toxicity.In this study, surface water, groundwater, hospital and municipal wastewater were used to characterise the impacts of common sample preparation techniques (acidification, filtration and solid phase extraction (SPE)) on the outcomes of eleven in vitro bioassays. The latter covered endocrine activity (reporter gene assays for estrogen, androgen, aryl-hydrocarbon, retinoic acid, retinoid X, vitamin D, thyroid receptor), mutagenicity (Ames fluctuation test), genotoxicity (umu test) and cytotoxicity. Water samples extracted using different SPE sorbents (Oasis HLB, Supelco ENVI-Carb+, Telos C18/ENV) at acidic and neutral pH were compared for their performance in recovering biological effects.Acidification, commonly used for stabilisation, significantly altered the endocrine activity and toxicity of most (waste)water samples. Sample filtration did not affect the majority of endpoints but in certain cases affected the (anti-)estrogenic and dioxin-like activities. SPE extracts (10.4× final concentration), including WWTP effluents, induced significant endocrine effects that were not detected in aqueous samples (0.63× final concentration), such as estrogenic, (anti-)androgenic and dioxin-like activities. When ranking the SPE methods using multivariate Pareto optimisation an extraction with Telos C18/ENV at pH 7 was most effective in recovering toxicity. At the same time, these extracts were highly cytotoxic masking the endpoint under investigation. Compared to that, extraction at pH 2.5 enriched less cytotoxicity.In summary, our study demonstrates that sample preparation and extraction critically affect the outcome of bioassays when assessing the toxicity of water samples. Depending on the

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Anthropogenic micropollutants and transformation products (TPs) negatively affect aquatic ecosystems and water resources. Wastewater treatment plants (WWTP) represent major point sources for (micro)pollutants and TPs in urban water cycles. The aim of the current study was to assess the removal of micropollutants and toxicity during conventional and advanced wastewater treatment. Using wild-type and transgenic Caenorhabditis elegans, the endpoint reproduction, growth, and cytochrome P450 (CYP) 35A3 induction (via cyp-35A3::GFP) were assessed. Samples were collected at four WWTPs and a receiving surface water. One WWTP included the advanced treatments: ozonation followed by granular activated carbon (GAC) or biological filtration (BF), respectively. Relevant micropollutants and WWTP parameters (n = 111) were included. Significant reproductive toxicity was detected for one WWTP effluent (31-83% reduced brood size). Three of four effluents significantly promoted the growth of C. elegans larvae (49-55% increased lengths). This effect was also observed for the GAC (34-41%) and BF (30%) post-treatments. Markedly, significant cyp-35A3::GFP induction was detected for one effluent before and after ozonation, being more pronounced for the ozonated samples (5- and 7.4-fold above controls). While the advanced treatments decreased the concentrations of most micropollutants, the observed effects may be attributed to effects of residual target compounds and/or compounds not included in the target chemical analysis. This highlights the need for an integrated assessment of (advanced) wastewater treatment covering both biological and chemical parameters.