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Surface waters can contain a diverse range of organic pollutants, including pesticides, pharmaceuticals and industrial compounds. While bioassays have been used for water quality monitoring, there is limited knowledge regarding the effects of individual micropollutants and their relationship to the overall mixture effect in water samples. In this study, a battery of in vitro bioassays based on human and fish cell lines and whole organism assays using bacteria, algae, daphnids and fish embryos was assembled for use in water quality monitoring. The selection of bioassays was guided by the principles of adverse outcome pathways in order to cover relevant steps in toxicity pathways known to be triggered by environmental water samples. The effects of 34 water pollutants, which were selected based on hazard quotients, available environmental quality standards and mode of action information, were fingerprinted in the bioassay test battery. There was a relatively good agreement between the experimental results and available literature effect data. The majority of the chemicals were active in the assays indicative of apical effects, while fewer chemicals had a response in the specific reporter gene assays, but these effects were typically triggered at lower concentrations. The single chemical effect data were used to improve published mixture toxicity modeling of water samples from the Danube River. While there was a slight increase in the fraction of the bioanalytical equivalents explained for the Danube River samples, for some endpoints less than 1% of the observed effect could be explained by the studied chemicals. The new mixture models essentially confirmed previous findings from many studies monitoring water quality using both chemical analysis and bioanalytical tools. In short, our results indicate that many more chemicals contribute to the biological effect than those that are typically quantified by chemical monitoring programs or those regulated by environmental quality standards. This study not only demonstrates the utility of fingerprinting single chemicals for an improved understanding of the biological effect of pollutants, but also highlights the need to apply bioassays for water quality monitoring in order to prevent underestimation of the overall biological effect.

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Surface water can contain countless organic micropollutants, and targeted chemical analysis alone may only detect a small fraction of the chemicals present. Consequently, bioanalytical tools can be applied complementary to chemical analysis to detect the effects of complex chemical mixtures. In this study, bioassays indicative of activation of the aryl hydrocarbon receptor (AhR), activation of the pregnane X receptor (PXR), activation of the estrogen receptor (ER), adaptive stress responses to oxidative stress (Nrf2), genotoxicity (p53) and inflammation (NF-κB) and the fish embryo toxicity test were applied along with chemical analysis to water extracts from the Danube River. Mixture-toxicity modeling was applied to determine the contribution of detected chemicals to the biological effect. Effect concentrations for between 0 to 13 detected chemicals could be found in the literature for the different bioassays. Detected chemicals explained less than 0.2% of the biological effect in the PXR activation, adaptive stress response, and fish embryo toxicity assays, while five chemicals explained up to 80% of ER activation, and three chemicals explained up to 71% of AhR activation. This study highlights the importance of fingerprinting the effects of detected chemicals.

Rain events may impact the chemical pollution burden in rivers. Forty-four small streams in Germany were profiled during several rain events for the presence of 395 chemicals and five types of mixture effects in in vitro bioassays (cytotoxicity, activation of the estrogen, aryl hydrocarbon and peroxisome proliferator-activated receptors and oxidative stress response). While these streams were selected to cover a wide range of agricultural impacts, in addition to the expected pesticides, wastewater-derived chemicals and chemicals typical for street run-off were detected. The unexpectedly high estrogenic effects in many samples indicated impact by wastewater or overflow of combined sewer systems. The 128 water samples exhibited a high diversity of chemical and effect patterns, even for different rain events at the same site. The detected 290 chemicals explained only a small fraction (<8 %) of the measured effects. The experimental effects of designed mixtures of detected chemicals that were expected to dominate the mixture effects of detected chemicals were consistent with predictions for concentration addition by a factor of two for 94 % of the mixtures.Overall, the burden of chemicals and effects were much higher than previously detected in surface water during dry weather with the effects often exceeding effect-based trigger values. In-vitro bioassay test batteryEndocrine effects • ER Metabolism • AhR • PPARy Adaptive stress response: • Oxidative stress Chemical analysis Sampling triggered by rain events Mixture models

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There is limited knowledge regarding the adverse effects of wastewater-derived microplastics, particularly fibers, on aquatic biota. In this study, we examined the acute (48 h) and chronic (8 d) effects of microplastic polyester fibers and polyethylene (PE) beads on freshwater zooplankton Ceriodaphnia dubia. We also assessed the acute response of C. dubia to a binary mixture of microplastic beads and fibers for the first time. Acute exposure to fibers and PE beads both showed a dose-dependent effect on survival. An equitoxic binary mixture of beads and fibers resulted in a toxic unit of 1.85 indicating less than additive effects. Chronic exposure to lower concentrations did not significantly affect survival of C. dubia, but a dose-dependent effect on growth and reproduction was observed. Fibers showed greater adverse effects than PE beads. While ingestion of fibers was not observed, scanning electron microscopy showed carapace and antenna deformities after exposure to fibers, with no deformities observed after exposure to PE beads. While much of the current research has focused on microplastic beads, our study shows that microplastic fibers pose a greater risk to C. dubia, with reduced reproductive output observed at concentrations within an order of magnitude of reported environmental levels.