Linking in Vitro Effects and Detected Organic Micropollutants in Surface Water Using Mixture-Toxicity Modeling.

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.

Accumulation and distribution of multiwalled carbon nanotubes in zebrafish (Danio rerio).

No data on the bioaccumulation and distribution of multiwalled carbon nanotubes (MWCNTs) in aquatic vertebrates is available until now. We quantified uptake and elimination of dispersed radiolabeled MWCNTs ((14)C-MWCNT; 1 mg/L) by zebrafish (Danio rerio) over time. The influences of the feeding regime and presence of dissolved organic carbon (DOC) on accumulation of the nanomaterial were determined. The partitioning of radioactivity to different organs and tissues was measured in all experiments. A bioaccumulation factor of 16 L/kg fish wet weight was derived. MWCNTs quickly associated with the fish, and steady state was reached within 1 day. After transfer to clear medium, MWCNTs were quickly released to the water phase, but on average 5 mg of MWCNTs/kg fish dry weight remained associated with the fish. The nanomaterial mainly accumulated in the gut of all fish. Feeding led to lower internal concentrations due to facilitated elimination via the digestive tract. In the presence of DOC, 10-fold less was taken up by the fish after 48 h of exposure compared to without DOC. Quick adhesion to and detachment from superficial tissues were observed. Remarkably, little fractions of the internalized radioactivity were detected in the blood and muscle tissue of exposed fish. The part accumulated in these fish compartments remained constant during the elimination phase. Hence, biomagnification of MWCNTs in the food chain is possible and should be a subject of further research.

Demonstration of an aggregated biomarker response approach to assess the impact of point and diffuse contaminant sources in feral fish in a small river case study.

The assessment of the exposure of aquatic wildlife to complex environmental mixtures of chemicals originating from both point and diffuse sources and evaluating the potential impact thereof constitutes a significant step towards mitigating toxic pressure and the improvement of ecological status. In the current proof-of-concept study, we demonstrate the potential of a novel Aggregated Biomarker Response (ABR) approach involving a comprehensive set of biomarkers to identify complex exposure and impacts on wild brown trout (Salmo trutta fario). Our scenario used a small lowland river in Germany (Holtemme river in the Elbe river catchment) impacted by two wastewater treatment plants (WWTP) and diffuse agricultural runoff as a case study. The trout were collected along a pollution gradient (characterised in a parallel study) in the river. Compared to fish from the reference site upstream of the first WWTP, the trout collected downstream of the WWTPs showed a significant increase in micronucleus formation, phase I and II enzyme activities, and oxidative stress parameters in agreement with increasing exposure to various chemicals. By integrating single biomarker responses into an aggregated biomarker response, the two WWTPs' contribution to the observed toxicity could be clearly differentiated. The ABR results were supported by chemical analyses and whole transcriptome data, which revealed alterations of steroid biosynthesis and associated pathways, including an anti-androgenic effect, as some of the key drivers of the observed toxicity. Overall, this combined approach of in situ biomarker responses complemented with molecular pathway analysis allowed for a comprehensive ecotoxicological assessment of fish along the river. This study provides evidence for specific hazard potentials caused by mixtures of agricultural and WWTP derived chemicals at sublethal concentrations. Using aggregated biomarker responses combined with chemical analyses enabled an evidence-based ranking of sites with different degrees of pollution according to toxic stress and observed effects.

Disentangling multiple chemical and non-chemical stressors in a lotic ecosystem using a longitudinal approach.

Meeting ecological and water quality standards in lotic ecosystems is often failed due to multiple stressors. However, disentangling stressor effects and identifying relevant stressor-effect-relationships in complex environmental settings remain major challenges. By combining state-of-the-art methods from ecotoxicology and aquatic ecosystem analysis, we aimed here to disentangle the effects of multiple chemical and non-chemical stressors along a longitudinal land use gradient in a third-order river in Germany. We distinguished and evaluated four dominant stressor categories along this gradient: (1) Hydromorphological alterations: Flow diversity and substrate diversity correlated with the EU-Water Framework Directive based indicators for the quality element macroinvertebrates, which deteriorated at the transition from near-natural reference sites to urban sites. (2) Elevated nutrient levels and eutrophication: Low to moderate nutrient concentrations together with complete canopy cover at the reference sites correlated with low densities of benthic algae (biofilms). We found no more systematic relation of algal density with nutrient concentrations at the downstream sites, suggesting that limiting concentrations are exceeded already at moderate nutrient concentrations and reduced shading by riparian vegetation. (3) Elevated organic matter levels: Wastewater treatment plants (WWTP) and stormwater drainage systems were the primary sources of bioavailable dissolved organic carbon. Consequently, planktonic bacterial production and especially extracellular enzyme activity increased downstream of those effluents showing local peaks. (4) Micropollutants and toxicity-related stress: WWTPs were the predominant source of toxic stress, resulting in a rapid increase of the toxicity for invertebrates and algae with only one order of magnitude below the acute toxic levels. This toxicity correlates negatively with the contribution of invertebrate species being sensitive towards pesticides (SPEARpesticides index), probably contributing to the loss of biodiversity recorded in response to WWTP effluents. Our longitudinal approach highlights the potential of coordinated community efforts in supplementing established monitoring methods to tackle the complex phenomenon of multiple stress.

Selection of assay, organism, and approach in biomonitoring significantly affects the evaluation of genotoxic potential in aquatic environments.

In this study, few different evaluation concepts were used for the assessment of genotoxic potential at the stretch of the Danube River identified as a significant hotspot of pollution originated through the untreated wastewaters. Three sites were chosen: one site upstream of the wastewater outlet in Novi Sad (Serbia), one at the outlet of wastewaters, and one site few kilometer downstream. Ex situ approach comprised prokaryotic SOS/umuC test on Salmonella typhimurium TA1535/pSK1005 and comet assay on human hepatoma cell line (HepG2). In situ approach was based on the active monitoring (cage approach) using freshwater mussels Sinanodonta woodiana and fish Cyprinus carpio. The comet and micronucleus assays were selected for evaluation of DNA damage in mussel haemocytes and fish blood cells. Within the ex situ part of the study, our results indicated that the eukaryotic model system is more sensitive compared to the prokaryotic one. In situ bioassays are recommended for obtaining a better insight into ecosystem status and in the case of our study the complete insight of genotoxic pressure. However, the choice of animals as bioindicators also has a significant impact on the quality of the obtained information. Differential response between fish and mussels was observed at the highly polluted site suggesting possible involvement of additional protective mechanism such as valve closure in mussels.

Assessing the fate of brown trout (Salmo trutta) environmental DNA in a natural stream using a sensitive and specific dual-labelled probe.

Environmental DNA (eDNA) analysis in the aquatic environment has emerged as a promising tool for diagnosis of the ecological status in comprehensive monitoring strategies and might become useful in context of the European Water Framework Directive (WFD) and other legislations to derive stressor-specific indicators. Despite many studies having made significant progress for the future use of eDNA in terms of ecosystem composition and detection of invasive/rare species in inland waters, much remains unknown about the transport and fate of eDNA under natural environmental conditions. We designed a specific dual-labelled probe to detect brown trout (Salmo trutta, L.) eDNA and used the probe to describe the fate of eDNA released from an aquaculture facility into the low mountain range stream Wehebach, Germany. The probe was shown to be specific to brown trout, as ponds housing rainbow trout (Oncorhynchus mykiss) did not test positive. Even though we observed different strengths of eDNA signals for three ponds containing different brown trout quantities, no significant correlation was found between biomass (kg/L) and eDNA quantity. Our results indicate that the release of DNA from brown trout might be life stage and/or age-dependent. The effluents of the aquaculture facility were a source of high levels of eDNA which resulted in the greatest abundance of brown trout eDNA directly downstream of the facility. Despite the natural occurrence of brown trout in the Wehebach, as shown by ecological investigations conducted by authorities of the federal state of North Rhine-Westphalia (Germany) and personal observations, we observed a significant decrease of relative abundance of eDNA in the Wehebach within the first 1.5 km downstream of the aquaculture. Our results suggest that concentrations of eDNA in running waters rapidly decrease under natural conditions due to dilution and degradation processes, which might have important implications for the utility of eDNA in environmental research.

Toxicity of 10 organic micropollutants and their mixture: Implications for aquatic risk assessment.

Micropollutants, as a serious water pollution issue, raise considerable toxicological concerns, particularly when present as components of complex mixtures. Due to the interactions of environmental pollution components (contaminant), the micropollutant problem is increasingly complex, thus, water quality of organic chemical contamination assessed substance-by-substance might lead to underestimation in aquatic environmental risk assessment. To assess the aquatic environmental risk of micropollutants mixture, a total of 10 organic micropollutants were selected and analysed by an approach of integration of literature data, laboratory experiments and prediction techniques. The experiment results showed that all 10 micropollutants were capable of causing toxicity in zebrafish embryos, aquatic invertebrates and algae with the LC50 (50% lethal concentration) values from 1.14 mg/L to 14.37 mg/L. Triclosan, carbamazepine, diazinon and diuron were the most hazardous compounds in the Danube River and the Rhine River. The artificial mixture presented a strong antagonistic relationship, which demonstrated an independent action (IA) model of the mixture. Based on the observed toxicity data, the risk quotients (RQs) of environmental mixtures of the Danube River and the Rhine River were extrapolated. It can be concluded that the micropollutant mixture may pose a potential risk for aquatic ecosystems with the present environmentally measured concentrations in the Danube River and Rhine River. Mixture risk assessment results suggested that the toxicity of studied chemicals might be induced by dissimilar actions, which is in agreement with the mixture toxicity prediction of the IA model. The observed findings could be useful to establish an overview of the pressures, vision, measures and expectations for hazardous substances pollution, which can help in making to informed decisions to reduce the concentration and bioactive fraction of pollutants.

Integrating bioassays, chemical analysis and in silico techniques to identify genotoxicants in surface water.

Identification of hazardous compounds, as the first step of water protection and regulation, is still challenged by the difficulty to establish a linkage between toxic effects and suspected contaminants. Genotoxic compounds are one type of highly relevant toxicants in surface water, which may attack the DNA and lead to cancer in individual organism, or even damaged germ cells to be passed on to future generations. Thus, the establishment of a linkage between genotoxic effects and genotoxicant is important for environmental toxicologists and chemists. For this purpose, in the present study in silico methods were integrated with bioassays, chemical analysis and literature information to identify genotoxicants in surface water. Large volume water samples from 22 sampling sites of the Danube were collected and subjected to biological and chemical analysis. Samples from the most toxic sites (JDS32, JDS44 and JDS63) induced significant genotoxic effects in the micronucleus assay, and two of them caused mutagenicity in the Ames fluctuation assay. Chemical analysis showed that 68 chemicals were detected in these most toxic samples. Literature findings and in silico techniques using the OECD QSAR Toolbox and the ChemProp software package revealed genotoxic potentials for 29 compounds out of 68 targeted chemicals. To confirm the integrative technical data, the micronucleus assay and the Ames fluctuation assay were applied with artificial mixtures of those compounds and the raw water sample extracts. The results showed that 18 chemicals explained 48.5% of the genotoxicity in the micronucleus assay. This study highlights the capability of in silico techniques in linking adverse biological effect to suspicious hazardous compounds for the identification of toxicity drivers, and demonstrates the genotoxic potential of pollutants in the Danube.

Ecotoxicity of Nitrogen, Sulfur, or Oxygen Heterocycles and Short-Chained Alkyl Phenols Commonly Detected in Contaminated Groundwater.

Nitrogen, sulfur, or oxygen heterocyclic aromatic hydrocarbons (NSO-HETs) and short-chained alkyl phenols (SCAPs) are commonly detected in groundwater at contaminated sites and in the surrounding environment. It is now scientific consensus that these chemicals pose a risk to human and ecosystem health. However, toxicity data are comparably fragmentary, and only few studies have addressed the ecotoxicity of NSO-HETs and SCAPs in a systematic and comparative fashion. To overcome this shortcoming, we tested 18 SCAPs, 16 NSO-HETs, as well as the homocyclic hydrocarbons indane and indene in the Microtox® assay with Aliivibrio fischeri, the growth inhibition test with Desmodesmus subspicatus, the acute immobilization assay with Daphnia magna, as well as the fish embryo toxicity test with embryos of the zebrafish (Danio rerio). Because of the physicochemical properties of the tested chemicals (limited water solubility, volatility, and sorption to test vessels), actual exposure concentrations in test media and their dissipation over time were analytically quantified by means of gas chromatography with mass spectrometry. Analytically corrected effect levels (median effect and lethal concentrations) ranged from 0.017 to 180 mg L-1 , underlining the environmental relevance of some NSO-HETs and SCAPs. Para-substituted phenols showed the overall greatest toxicities in all 4 toxicity tests. We provide, for the first time, a complete high-quality data set in support of better environmental risk assessments of these chemicals. Environ Toxicol Chem 2019;38:1343-1355. © 2019 SETAC.

Integrated zebrafish-based tests as an investigation strategy for water quality assessment.

Water pollution risks to human health and the environment are emerging as serious concerns in the European Union and worldwide. With the aim to achieve good ecological and chemical status of all European water bodies, the "European Water Framework Directive" (WFD) was enacted. With the framework, bioanalytical techniques have been recognized as an important aspect. However, there are limitations to the application of bioassays directly for water quality assessment. Such approaches often fail to identify pollutants of concern, since the defined priority and monitored pollutants often fail to explain the observed toxicity. In this study, we integrated an effect-based risk assessment with a zebrafish-based investigation strategy to evaluate water sample extracts and fractions collected from the Danube. Four tiered bioassays were implemented, namely RNA-level gene expression assay, protein-level ethoxyresorufin-O-deethylase (EROD) assay, cell-level micronucleus assay and organism-level fish embryo test (FET). The results show that teratogenicity and lethality during embryonic development might be induced by molecular or cellular damages mediated by the aryl hydrocarbon receptor (AhR) -mediated activity, estrogenic activity and genotoxic activity. With the combination of high-throughput fractionation, this effect-based strategy elucidated the major responsible mixtures of each specific toxic response. In particularly, the most toxic mixture in faction F4, covering a log Kow range from 2.83 to 3.42, was composed by 12 chemicals, which were then evaluated as a designed mixture. Our study applied tiered bioassays with zebrafish to avoid interspecies differences and highlights effect-based approaches to address toxic mixtures in water samples. This strategy can be applied for large throughput screenings to support the main toxic compounds identification in water quality assessment.

Longitudinal profile of the genotoxic potential of the River Danube on erythrocytes of wild common bleak (Alburnus alburnus) assessed using the comet and micronucleus assay.

The Joint Danube Survey 3 (JDS3; the biggest river expedition in 2013) had offered the unique opportunity for a large-scale monitoring approach for biomarker response in feral fish collected along a Danube stretch from Kehlheim (DE) to Sulina (RO). The advantage of genotoxicity as a marker for pollution exposure in fish is the early detection of possible long-term effects such as cancer. Therefore, genotoxicity was in the focus of the biomarker investigations in fish during the expedition. Blood samples of common bleak (Alburnus alburnus) for the investigation of the micronucleus frequency and comet tail intensity of fragmented DNA material in erythrocytes were collected at 18 and 12 sampling sites, respectively. For 9 sampling sites same samples were used to compare the in-situ data for the comparable genotoxic endpoint in the micronucleus (MN) and comet assay (CM). The data of both in-situ assays showed a significant correlation, indicating the strength and comparability of the data sets. Significant variation in DNA damage in fish along the longitudinal profile of the Danube was demonstrated for both assays compared to reference sites. The results suggest that DNA damage in erythrocytes of fish was mainly affected by wastewater of highly populated regions. No linkage between the results and the general health/dietary status of the fish were revealed, whereas correlation with some genotoxicity drivers in the water phase, suspended particulate matter and sediments could be demonstrated.