Morphological and behavioral alterations in zebrafish larvae after exposure to contaminated river sediments collected in different weather conditions.

Wastewater treatment plants (WWTPs) are the primary source of micropollutants in aquatic ecosystems. Many micropollutants tend to bind to sediments and persist until remobilizion by bioturbation or flood events. Advanced effluent treatment by ozonation has been proven to eliminate most micropollutants. The present study characterizes sediments' toxic potential regarding zebrafish embryo development, which highly complex nervous system is vulnerable to exposure to neurotoxic substances. Furthermore, behavioral changes can be induced even at low pollutant concentrations and do not cause acute toxicity. The study area includes stretches of the main waterbody, the Wurm River (sampling sites W1-W5), and its tributary the Haarbach River (sampling sites H1, and H2) in North-Rhine Westphalia, Germany. Both waterbodies serve as recipients of WWTPs' effluents. The effluent entering the Haarbach River is conventionally treated, while the Wurm River receives ozonated effluent from the Aachen-Soers WWTP. Seven sampling sites up- and downstream of the WWTPs were investigated in June of two subsequent years. The first sampling campaign in 2017 was characterized by prolonged dry weather. The second sampling campaign in 2018 occurred after prolonged rain events and the release of the rainwater overflow basin. Direct exposure of zebrafish embryos to native sediments using the sediment contact test represented an ecologically realistic scenario and showed no acute sublethal effects. Exposure of the zebrafish embryo to freeze-dried sediments representing the ecotoxicological status of sediments during flood events unfolded acute sublethal toxicity. Behavioral studies with zebrafish larvae were an essential part of environmental neurotoxicity testing. Zebrafish larvae exposed to sediments' concentrations causing no acute effects led to behavioral changes signalizing neurotoxic substances in sediments. Polyaromatic hydrocarbons, polychlorinated biphenyls, and nitroaromatic compounds were identified as potential toxicity drivers, whereby the rainwater overflow basin served as a possible source of pollution. Mixture toxicity, effect-directed analysis, and further sediment monitoring are needed.

Endocrine disrupting chemicals entering European rivers: Occurrence and adverse mixture effects in treated wastewater.

In the present study on endocrine disrupting chemicals (EDCs) in treated wastewater, we used chemical and effect-based tools to analyse 56 wastewater treatment plant (WWTP) effluents from 15 European countries. The main objectives were (i) to compare three different receptor-based estrogenicity assays (ERα-GeneBLAzer, p-YES, ERα-CALUX®), and (ii) to investigate a combined approach of chemical target analysis and receptor-based testing for estrogenicity, glucocorticogenic activity, androgenicity and progestagenic activity (ERα-, GR-, AR- and PR-GeneBLAzer assays, respectively) in treated wastewater. A total of 56 steroids and phenols were detected at concentrations ranging from 25 pg/L (estriol, E3) up to 2.4 µg/L (cortisone). WWTP effluents, which passed an advanced treatment via ozonation or via activated carbon, were found to be less contaminated, in terms of lower or no detection of steroids and phenols, as well as hormone receptor-mediated effects. This result was confirmed by the effect screening, including the three ERα-bioassays. In the GeneBLAzer assays, ERα-activity was detected in 82 %, and GR-activity in 73 % of the samples, while AR- and PR-activity were only measured in 14 % and 21 % of the samples, respectively. 17ß-estradiol was confirmed as the estrogen dominating the observed estrogenic mixture effect and triamcinolone acetonide was the dominant driver of glucocorticogenic activity. The comparison of bioanalytical equivalent concentrations (BEQ) predicted from the detected concentrations and the relative effect potency (BEQchem) with measured BEQ (BEQbio) demonstrated good correlations of chemical target analysis and receptor-based testing results with deviations mostly within a factor of 10. Bioassay-specific effect-based trigger values (EBTs) from the literature, but also newly calculated EBTs based on previously proposed derivation options, were applied and allowed a preliminary assessment of the water quality of the tested WWTP effluent samples. Overall, this study demonstrates the high potential of linking chemical with effect-based analysis in water quality assessment with regard to EDC contamination.

Extensive rain events have a more substantial impact than advanced effluent treatment on the endocrine-disrupting activity in an effluent-dominated small river.

Wastewater treatment plants (WWTPs) remain an important primary source of emission for endocrine-disrupting compounds in the environment. As an advanced wastewater treatment process, ozonation is known to reduce endocrine-disrupting activity. However, it remains unclear to which extend improved wastewater treatment may reduce the endocrine-disrupting activity in the receiving water body. The present study investigated possible factors for the endocrine-disrupting activity in a small receiving water body, the Wurm River (North-Rhine Westphalia, Germany), up- and downstream of a local WWTP. The cell-based reporter gene CALUX® assay was applied to identify the endocrine-disrupting activity in the water, sediment, and suspended particulate matter. The water phase and the effluent sampling were primarily driven by applying the full-scale effluent ozonation (sampling campaigns in June 2017 and March 2019). In contrast, the sediment sampling aimed to compare the particle-bound endocrine-disrupting activity during dry (June 2017) and rainy summer (June 2018) seasons. The water phase showed low to moderate estrogenic/antiandrogenic activity. Advanced effluent treatment by ozonation led to a complete reduction of the endocrine-disrupting activity according to the limit of detection of the CALUX® assays. The suspended particulate matter originated from the water phase of the second sampling campaign revealed antiandrogenic activity only. Sediments at the sampling sites along the local WWTP revealed higher estrogenic and antiandrogenic activity after extensive rain events and were not affected by the ozonated effluent. Fluctuation patterns of the endocrine-disrupting activity in sediments were in line with fluctuated concentrations of polycyclic aromatic hydrocarbons. Rainwater overflow basin release was suggested as a vector for particle-bound and dissolved endocrine-disrupting activity in the receiving water body. The present study underlined the necessity for monitoring both water and sediment phases to achieve reliable profiling of the endocrine-disrupting activity. The receptor-mediated CALUX® assays were proven to be suitable for investigating the endocrine-disrupting activity distribution in different river compartments and WWTP effluents.

Improvement of wastewater and water quality via a full-scale ozonation plant? - A comprehensive analysis of the endocrine potential using effect-based methods.

Micropollutants (MPs), especially endocrine disrupting compounds (EDCs), are mainly released from WWTPs into surface water bodies and can subsequently lead to adverse effects in biota. Treatment with ozone proved to be a suitable method for eliminating such MPs. This method was implemented at the WWTP Aachen-Soers by commissioning the largest full-scale ozonation plant in Europe at the moment. Recently, effect-based methods (EBMs) have been successfully proved for compliance monitoring, e.g. estrogenic compounds. Therefore, the impact of ozone treatment on endocrine potential (agonistic and antagonistic) of treated wastewater was investigated using the ERα- and AR CALUX assays. Additionally, the impact on the receiving stream and a potential preload of the water body was assessed. Therefore, the current study could deal as a case study for small rivers being highly impacted by WWTPs. The estrogenic potential was nearly fully eliminated after ozone treatment. Contrary, the antagonistic (anti-estrogenic and anti-androgenic) potential did not show a clear elimination pattern after ozone treatment independent of the applied ozone dosage and control system. Therefore, further investigations are required regarding the antagonistic potential. Additionally, preloading of the receiving stream was found during the study period. One significant impact is a rain overflow basin (ROB) located upstream of the WWTP effluent. The highest endocrine potential was found after a ROB overflow (2.7 ng EEQ/L, 2.4 µg TMX-EQ/L, 104 µg FLU-EQ/L), suggesting that such runoff events after a heavy rainfall may act as a driver of endocrine loading to the water body. This manuscript contributes significantly to the basic understanding of the efficiency of eliminating the endocrine potential of ozone treatment by, e.g., showing that there is a further need for improving the removal efficiency of antagonistic potential. Moreover, it highlights the need to include other point sources, such as ROBs, to assess polluted surface waters comprehensively.

Assessing the genotoxic potential of freshwater sediments after extensive rain events - Lessons learned from a case study in an effluent-dominated river in Germany.

Wastewater treatment plant effluents and releases from rainwater overflow basins can contribute to the input of genotoxic micropollutants in aquatic ecosystems. Predominantly lipophilic genotoxic compounds tend to sorb to particulate matter, making sediment a source and a sink of pollution. Therefore, the present study aims to investigate the genotoxic potential of freshwater sediments (i) during the dry period and (ii) after extensive rain events by collecting sediment samples in one small anthropogenically impacted river in Germany up- and downstream of the local wastewater treatment plant. The Micronucleus and Ames fluctuation assays with Salmonella typhimurium strains TA98, TA100, YG1041, and YG1042 were used to assess the genotoxic potential of organic sediment extracts. For evaluation of possible genotoxicity drivers, target analysis for 168 chemical compounds was performed. No clastogenic effects were observed, while the genotoxic potential was observed at all sampling sites primarily driven by polycyclic aromatic hydrocarbons, nitroarenes, aromatic amines, and polycyclic heteroarenes. Freshwater sediments' genotoxic potential increased after extensive rain events due to sediment perturbation and the rainwater overflow basin release. In the present study, the rainwater overflow basin was a significant source for particle-bound pollutants from untreated wastewater, suggesting its role as a possible source of genotoxic potential. The present study showed high sensitivity and applicability of the bacterial Salmonella typhimurium strains YG1041 and YG1042 to organic sediment extracts to assess the different classes of genotoxic compounds. A combination of effect-based methods and a chemical analysis was shown as a suitable tool for a genotoxic assessment of freshwater sediments.

Toxicological and ecotoxicological evaluation of the water quality in a large and eutrophic freshwater lake of China.

Effect-based methods (EBMs) are recommended as holistic approach for diagnosis and monitoring of water quality; however, the application of EBMs is still scare in China. In the present study, water quality of the freshwater lake Taihu (China) was investigated by EBMs. Different types of water samples were collected from three bays of the lake during 2015, 2016 and 2017. A battery of seven effect-based bioassays, including both specific and non-specific toxicity assays, was used. The bioassay battery was recently suggested based on joint activities of the EU project SOLUTIONS and the NORMAN network on emerging pollutants and is also under discussion for being implemented into monitoring activities in the context of the European Water Framework Directive (WFD). Adverse effects were observed towards the primary producer, primary consumer and fish, indicating the potential ecotoxicity of water in Taihu Lake. Mutagenic and estrogenic effects were found in the Ames fluctuation assay and ERα CALUX (Chemically Activated Luciferase Gene-eXpression) assay, respectively, highlighting the potential risks on human health. Algal growth inhibition and mutagenic effects can be observed during each of the three years. Acute toxicity towards Daphnia magna and estrogen receptor agonistic effects were found in at least one of the samples collected in 2016 and 2017, but not in 2015. The endpoints for fish toxicity in the Danio rerio fish embryo test included both lethal and additionally several sublethal effects (only for samples from 2017) and were not compared between years. Algal growth inhibition, fish embryo toxicity, mutagenic effect and estrogenicity were observed in each of the three bays, while Daphnia acute toxicity was only found in Zhushan Bay. Taking together, this study provides a big picture on the water quality of Taihu Lake. The battery of effect-based tools is promising to be a routine for water quality monitoring in China.

Comparison of in vitro test systems using bacterial and mammalian cells for genotoxicity assessment within the "health-related indication value (HRIV) concept.

In numerous cases, the German health-related indication value (HRIV) concept has proved its practicability for the assessment of drinking water relevant trace substances (Umweltbundesamt 2003). The HRIV is based on the toxicological profile of a substance. An open point of the HRIV concept has been the assignment of standardized test procedures to be used for the assessment. The level of the HRIV is at its lowest as soon as the genotoxicity of the substance is detected. As a single test on its own, it is not sufficient enough to assess the human toxicological relevance of a genotoxic effect or exclude it in the case of a negative result; a reasonable test battery was required, technically oriented towards the already harmonized international, hierarchical evaluation for toxicological assessment of chemicals. Therefore, an important aim of this project was to define a strategy for the genotoxicological assessment of anthropogenic trace substances. The basic test battery for genotoxicity of micropollutants in drinking water needs to fulfill several requirements. Although quick test results are needed for the determination of HRIV, a high degree of transferability to human genotoxicity should be ensured. Therefore, an in vitro genotoxicity test battery consisting of the Ames fluctuation test with two tester strains (ISO 11350), the umu test and the micronucleus test, or from the Ames test with five tester strains (OECD 471) and the micronucleus test is proposed. On the basis of selected test substances, it could be shown that the test battery leads to positive, indifferent, and negative results. Given indifferent results, the health authority and the water supplier must assume that it is a genotoxic substance. Genetically modified tester strains are being sensitive to different chemical classes by expression of selected mammalian key enzymes for example nitroreductase, acetyltransferase, and glutathione-S-transferase. These strains may provide valuable additional information and may give a first indication of the mechanism of action. To check this hypothesis, various additional strains expressing specific human-relevant enzymes were investigated. It could be shown that the additional use of genetically modified tester strains can enhance the detectable substance spectrum with the bacterial genotoxicological standard procedures or increase the sensitivity. The additional use provides orienting information at this level as a lot of data can be obtained quite quickly and with little effort. These indications of the mechanism of action should be however verified with a test system that uses mammalian cells, better human cells, to check their actual relevance. The selection of appropriate additional tester strains has to be defined from case to case depending on the molecular structure and also still requires some major expertise.