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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.

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.

Hydroxylation of polyaromatic compounds through cytochromes P450 (CYPs) is known to result in potentially estrogenic transformation products. Recently, there has been an increasing awareness of the importance of alternative pathways such as aldehyde oxidases (AOX) or N-methyltransferases (NMT) in bioactivation of small molecules, particularly N-heterocycles. Therefore, this study investigated the biotransformation and activity of methylated quinolines, a class of environmentally relevant N-heterocycles that are no native ligands of the estrogen receptor (ER), in the estrogen-responsive cell line ERα CALUX. We found that this widely used cell line overexpresses AOXs and NMTs while having low expression of CYP enzymes. Exposure of ERα CALUX cells to quinolines resulted in estrogenic effects, which could be mitigated using an inhibitor of AOX/NMTs. No such mitigation occurred after coexposure to a CYP1A inhibitor. A number of N-methylated but no hydroxylated transformation products were detected using liquid chromatography-mass spectrometry, which indicated that biotransformations to estrogenic metabolites were likely catalyzed by NMTs. Compared to the natural ER ligand 17β-estradiol, the products formed during the metabolization of quinolines were weak to moderate agonists of the human ERα. Our findings have potential implications for the risk assessment of these compounds and indicate that care must be taken when using in vitro estrogenicity assays, for example, ERα CALUX, for the characterization of N-heterocycles or environmental samples that may contain them.

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.

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.

In order to contribute to a comprehensive understanding of the regulating mechanisms of the aryl-hydrocarbon-receptor (AHR) in zebrafish embryos, we aimed to elucidate the interaction of proteins taking part in this signaling pathway during early development of the zebrafish (Danio rerio) after chemical exposure. We managed to illustrate initial transcription processes of the implemented proteins after exposure to two environmentally relevant chemicals: polychlorinated biphenyl 126 (PCB126) and β-Naphthoflavone (BNF). Using qPCR, we quantified mRNA every 4 h until 118 h post fertilization and found the expression of biotransformation enzymes (cyp1 family) and the repressor of the AHR (ahr-r) to be dependent on the duration of chemical exposure and the biodegradability of the compounds. PCB126 induced persistently increased amounts of transcripts as it is not metabolized, whereas activation by BNF was limited to the initial period of exposure. We did not find a clear relation between the amount of transcripts and activity of the induced CYP-proteins, so posttranscriptional mechanisms are likely to regulate biotransformation of BNF. With regard to zebrafish embryos and their application in risk assessment of hazardous chemicals, our examination of the AHR pathway especially supports the relevance of the time point or period of exposure that is used for bioanalytical investigations and consideration of chemical properties determining biodegradability.

Endocrine-active substances can adversely impact the aquatic ecosystems. A special emphasis is laid, among others, on the effects of estrogens and estrogen mimicking compounds. Effect-based screening methods like in vitro bioassays are suitable tools to detect and quantify endocrine activities of known and unknown mixtures. This study describes the validation of the Arxula-Yeast Estrogen Screen (A-YES®) assay, an effect-based method for the detection of the estrogenic potential of water and waste water. This reporter gene assay, provided in ready to use format, is based on the activation of the human estrogen receptor alpha. The user-friendly A-YES® enables inexperienced operators to rapidly become competent with the assay. Fourteen laboratories from four countries with different training levels analyzed 17β-estradiol equivalent concentrations (EEQ) in spiked and unspiked waste water effluent and surface water samples, in waste water influent and spiked salt water samples and in a mixture of three bisphenols. The limit of detection (LOD) for untreated samples was 1.8 ng/L 17β-estradiol (E2). Relative repeatability and reproducibility standard deviation for samples with EEQ above the LOD (mean EEQ values between 6.3 and 20.4 ng/L) ranged from 7.5 to 21.4% and 16.6 to 28.0%, respectively. Precision results are comparable to other frequently used analytical methods for estrogens. The A-YES® has been demonstrated to be an accurate, precise and robust bioassay. The results have been included in the ISO draft standard. The assay was shown to be applicable for testing of typical waste water influent, effluent and saline water. Other studies have shown that the assay can be used with enriched samples, which lower the LOD to the pg/L range. The validation of the A-YES® and the development of a corresponding international standard constitute a step further towards harmonized and reliable bioassays for the effect-based analysis of estrogens and estrogen-like compounds in water samples.

The present study compares two alternative in vivo approaches for the measurement of ethoxyresorufin-O-deethylase (EROD) activity in zebrafish (Danio rerio) following exposure to acetonic model sediment extracts: (1) the live-imaging EROD assay for the direct detection of EROD induction in individual livers via epifluorescence, and (2) the fish embryo EROD assay in subcellular fractions derived from entire zebrafish embryos after in vivo exposure. For toxicity assessment, each sediment extract was tested with the standard fish embryo test (FET). Upon completion of a functioning liver after 72h, the embryos gave a distinct fluorescent signal in the liver, and a corresponding EROD activity could be detected in the fish embryo EROD assay. The exposure time in the live-imaging EROD assay was reduced to 3h, which resulted in a stronger, less variable and more sensitive EROD response. Overall, the live-imaging and the fish embryo EROD assays showed the same tendencies and gave comparable results, e.g. a concentration-dependent increase in EROD activity at concentrations one order of magnitude below concentrations producing macroscopically visible abnormalities. At higher concentrations, however, a decrease of EROD activity was observed in either test. Both tests ranked the three model sediment extracts in the same order. Results indicate that both test systems complement each other and together provide a rapid and reliable in vivo tool to investigate the presence of dioxin-like substances in environmental samples.

Numerous environmental pollutants have the potential to accumulate in sediments, and among them are endocrine-disrupting chemicals (EDCs). It is well documented that water-borne exposure concentrations of some potent EDCs, more specifically estrogenic- active compounds (ECs), can impair the reproduction of fish. In contrast, little is known about the bioavailability and effects of sediment-associated ECs on fish. Particularly, when sediments are disturbed, e.g., during flood events, chemicals may be released from the sediment and become bioavailable. The main objectives of this study were to evaluate a) whether ECs from the sediment become bioavailable to fish when the sediment is suspended, and b) whether such exposure leads to endocrine responses in fish. Juvenile rainbow trout (Oncorhynchus mykiss) were exposed over 21 days to constantly suspended sediments in the following treatments: i) a contaminated sediment from the Luppe River, representing a "hotspot" for EC accumulation, ii) a reference sediment (exhibiting only background contamination), iii) three dilutions, 2-, 4- and 8-fold of Luppe sediment diluted with the reference sediment, and iv) a water-only control. Measured estrogenic activity using in vitro bioassays as well as target analysis of nonylphenol and estrone via LC-MS/MS in sediment, water, fish plasma, as well as bile samples, confirmed that ECs became bioavailable from the sediment during suspension. ECs were dissolved in the water phase, as indicated by passive samplers, and were readily taken up by the exposed trout. An estrogenic response of fish to Luppe sediment was indicated by increased abundance of transcripts of typical estrogen responsive genes, i.e. vitelline envelope protein α in the liver and vitellogenin induction in the skin mucus. Altered gene expression profiles of trout in response to suspended sediment from the Luppe River suggest that in addition to ECs a number of other contaminants such as dioxins, polychlorinated biphenyls (PCBs) and heavy metals were remobilized during suspension. The results of the present study demonstrated that sediments not only function as a sink for ECs but can turn into a significant source of pollution when sediments are resuspended as during flood-events. This highlights the need for sediment quality criteria considering bioavailability sediment-bound contaminants in context of flood events.

Wastewater-based monitoring of the spread of the new SARS-CoV-2 virus, also referred to as wastewater-based epidemiology (WBE), has been suggested as a tool to support epidemiology. An extensive sampling campaign, including nine municipal wastewater treatment plants, has been conducted in different cities of the Federal State of North Rhine-Westphalia (Germany) on the same day in April 2020, close to the first peak of the corona crisis. Samples were processed and analysed for a set of SARS-CoV-2-specific genes, as well as pan-genotypic gene sequences also covering other coronavirus types, using reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Additionally, a comprehensive set of chemical reference parameters and bioindicators was analysed to characterize the wastewater quality and composition. Results of the RT-qPCR based gene analysis indicate the presence of SARS-CoV-2 genetic traces in different raw wastewaters. Furthermore, selected samples have been sequenced using Sanger technology to confirm the specificity of the RT-qPCR and the origin of the coronavirus. A comparison of the particle-bound and the dissolved portion of SARS-CoV-2 virus genes shows that quantifications must not neglect the solid-phase reservoir. The infectivity of the raw wastewater has also been assessed by viral outgrowth assay with a potential SARS-CoV-2 host cell line in vitro, which were not infected when exposed to the samples. This first evidence suggests that wastewater might be no major route for transmission to humans. Our findings draw attention to the need for further methodological and molecular assay validation for enveloped viruses in wastewater., Graphical abstract Unlabelled Image, Highlights • The first study that reports the detection of SARS-CoV-2 in wastewater in Germany using RT-qPCR. • The presence of SARS-CoV-2 was confirmed by sequencing, but also the risk of false-positive results has been elucidated. • In raw wastewater, 3.0 to 20 gene equivalents/mL was found in raw wastewater. • The replication potential tests were negative for wastewater samples. • Sanger sequencing was required to differentiate the genetic pattern clearly.

Fish are important sentinel organisms for the assessment of water quality and play a central role in ecotoxicological research. Of particular importance to the assessment of health and fitness of fish stocks in response to environmental conditions or pollution are morphometric (e.g. Fulton's condition index) and somatic indices (e.g. hepatosomatic, and gonadosomatic index). Standard measurements of somatic indices are invasive and require, by definition, the sacrifice of examined animals, thus prohibiting longitudinal studies and relocation of animals captured in the field. As a potential solution, in the present study, we propose the use of micro-computed tomography (μCT) as imaging modality to non-invasively tomographically image rainbow trout (Oncorhynchus mykiss) exposed to different sediment suspensions. We here demonstrate that μCT can be used as a tool to reliably measure the volumes of different organs, which could then be applied as a substitute of their weights in calculation of somatic indices. To the best of our knowledge, this study is the first to report the results of μCT analyses in the context of ecotoxicological research in rainbow trout. It has the potential to greatly increase the information value of experiments conducted with fish and also to potentially reduce the number of animals required for studying temporal effects through facilitating longitudinal studies within the same individuals.

Fish strongly rely on olfaction as a variety of essential behaviors such as foraging and predator avoidance are mediated by the olfactory system. Cadmium (Cd) is known to impair olfaction and accumulate in the olfactory epithelium (OE) and bulb (OB) of fishes. In the present study, the acute toxicity of Cd on olfaction in zebrafish (Danio rerio) was characterized on the molecular and behavioral level. To this end, quantitative real-time PCR was performed in order to analyze the expression of selected genes in both the OE and OB. Moreover, the response of zebrafish to an alarm cue was investigated. Following 24 h of exposure to Cd, the expression of genes associated with olfactory sensory neurons was reduced in the OE. Furthermore, the antioxidant genes peroxiredoxin 1 (prdx1) and heme oxygenase 1 (hmox1), as well as the metallothionein 2 gene (mt2) were upregulated in the OE, whereas hmox1 and the stress-inducible heat shock protein 70 gene (hsp70) were upregulated in the OB upon exposure to Cd. Following stimulation with a conspecific skin extract, zebrafish displayed a considerable disruption of the antipredator behavior with increasing Cd concentration. Taken together, Cd impaired olfaction in zebrafish, thereby disrupting the antipredator response, which is crucial for the survival of individuals. Cellular stress followed by disruption of olfactory sensory neurons may have contributed to the observed behavioral deficits.

Fish are warned about the presence of predators via an alarm cue released from the skin of injured conspecifics. The detection of this odor inherently initiates an antipredator response, which increases the chance of survival for the individual. In the present study, we assessed the effect of three commonly used pesticides on the antipredator response of zebrafish (Danio rerio). For this, we analyzed the behavioral response of zebrafish to a conspecific skin extract following 24 h of exposure to the respective contaminants. Results demonstrate that fish exposed to 20 μg/L of the organophosphate insecticide chlorpyrifos significantly reduced bottom-dwelling and freezing behavior, suggesting an impairment of the antipredator response. For the urea-herbicide linuron and the pyrethroid insecticide permethrin, no statistically significant effects could be detected. However, linuron-exposed fish appeared to respond in an altered manner to the skin extract; some individuals failed to perform the inherent behaviors such as erratic movements and instead merely increased their velocity. Furthermore, we determined whether zebrafish would avoid the pesticides in a choice maze. While fish avoided permethrin, they behaved indifferently to chlorpyrifos and linuron. The study demonstrates that pesticides may alter the olfactory-mediated antipredator response of zebrafish in distinct ways, revealing that particularly fish exposed to chlorpyrifos may be more prone to predation.

The project DemO3AC (demonstration of large-scale wastewater ozonation at the Aachen-Soers wastewater treatment plant, Germany) of the Eifel-Rur Waterboard contains the construction of a large-scale ozonation plant for advanced treatment of the entire 25 million m³/yr of wastewater passing through its largest wastewater treatment plant (WWTP). In dry periods, up to 70% of the receiving water consists of treated wastewater. Thus, it is expected that effects of ozonation on downstream water biocoenosis will become observable. Extensive monitoring of receiving water and the WWTP shows a severe pollution with micropollutants (already prior to WWTP inlet). (Eco-)Toxicological investigations showed increased toxicity at the inlet of the WWTP for all assays. However, endocrine-disrupting potential was also present at other sampling points at the WWTP and in the river and could not be eliminated sufficiently by the WWTP. Total cell counts at the WWTP are slightly below average. Investigations of antibiotic resistances show no increase after the WWTP outlet in the river. However, cells carrying antibiotic-resistant genes seem to be more stress resistant in general. Comparing investigations after implementation of ozonation should lead to an approximation of the correlation between micropollutants and water quality/biocoenosis and the effects that ozonation has on this matter.

River sediments may contain a huge variety of environmental contaminants and play a key role in the ecological status of aquatic ecosystems. Contaminants adsorbed to sediments and suspended solids may contribute directly or after remobilization to an adverse ecological and chemical status of surface water. In this subproject of the joint research project DanTox, acetonic Soxhlet extracts from three German river sediments from the River Rhine (Altrip and Ehrenbreitstein with moderate contamination) and River Elbe (Veringkanal Hamburg heavily contaminated) were prepared and redissolved in dimethyl sulfoxide (DMSO). These extracts were analyzed with a standard bioassay battery with organisms from different trophic levels (bacteria, algae, Daphnia, fish) as well as in the Ames test and the umuC test for bacterial mutagenicity and genotoxicity according to the respective OECD and ISO guidelines. In total, 0.01% (standard) up to 0.25% (only fish embryo test) of the DMSO sediment extract was dosed to the test systems resulting in maximum sediment equivalent concentrations (SEQ) of 2 up to 50 g l(-1). The sediment of Veringkanal near Hamburg harbor was significantly more toxic in most tests compared to the sediment extracts from Altrip and Ehrenbreitstein from the River Rhine. The most toxic effect found for Veringkanal was in the algae test with an ErC50 (72 h) of 0.00226 g l(-1) SEQ. Ehrenbreitstein and Altrip samples were about factor 1,000 less toxic. In the Daphnia, Lemna, and acute fish toxicity tests, no toxicity at all was found at 2 g l(-1) SEQ. corresponding to 0.01% DMSO. Only when increasing the DMSO concentration the fish embryo test showed a 22-fold higher toxicity for Veringkanal than for Ehrenbreitstein and Altrip samples, while the toxicity difference was less evident for the Daphnia test due to the overlaying solvent toxicity above 0.05% dimethyl sulfoxide (DMSO). The higher toxicities observed with the Veringkanal sample are supported by the PAH and PCB concentrations analyzed in the sediments. The sediment extracts of Altrip and Veringkanal were mutagenic in the Ames tester strain TA98 with metabolic activation (S9-mix). The findings allow a better ecotoxicological characterization of the sediments extensively analyzed in all subprojects of the DanTox project (e.g., Garcia-Kaeufer et al. Environ Sci Pollut Res. doi: 10.1007/s11356-014-3894-4 , 2014; Schiwy et al. Environ Sci Pollut Res. doi: 10.1007/s11356-014-3185-0 , 2014; Hollert and Keiter 2015). In the absence of agreed limit values for sediment extracts in standard tests, further data with unpolluted reference sediments are required for a quantitative risk assessment of the investigated polluted sediments.

The embryotoxic potential of three model sediment samples with a distinct and well-characterized pollutant burden from the main German river basins Rhine and Elbe was investigated. The Fish Embryo Contact Test (FECT) in zebrafish (Danio rerio) was applied and submitted to further development to allow for a comprehensive risk assessment of such complex environmental samples. As particulate pollutants are constructive constituents of sediments, they underlay episodic source-sink dynamics, becoming available to benthic organisms. As bioavailability of xenobiotics is a crucial factor for ecotoxicological hazard, we focused on the direct particle-exposure pathway, evaluating throughput-capable endpoints and considering toxicokinetics. Fish embryo and larvae were exposed toward reconstituted (freeze-dried) sediment samples on a microcosm-scale experimental approach. A range of different developmental embryonic stages were considered to gain knowledge of potential correlations with metabolic competence during the early embryogenesis. Morphological, physiological, and molecular endpoints were investigated to elucidate induced adverse effects, placing particular emphasis on genomic instability, assessed by the in vivo comet assay. Flow cytometry was used to investigate the extent of induced cell death, since cytotoxicity can lead to confounding effects. The implementation of relative toxicity indices further provides inter-comparability between samples and related studies. All of the investigated sediments represent a significant ecotoxicological hazard by disrupting embryogenesis in zebrafish. Beside the induction of acute toxicity, morphological and physiological embryotoxic effects could be identified in a concentration-response manner. Increased DNA strand break frequency was detected after sediment contact in characteristic non-monotonic dose-response behavior due to overlapping cytotoxic effects. The embryonic zebrafish toxicity model along with the in vivo comet assay and molecular biomarker analysis should prospectively be considered to assess the ecotoxicological potential of sediments allowing for a comprehensive hazard ranking. In order to elucidate mode of action, novel techniques such as flow cytometry have been adopted and proved to be valuable tools for advanced risk assessment and management.

Plastic particles have been proven to be abundant in the aquatic environment, raising concerns about their potential toxic effects. In the present study, we determined the bioaccumulation potential of bisphenol A (BPA) in adult zebrafish (Danio rerio) in the absence and presence of nano-sized plastic particles (nanoplastics, NPPs). Results show that BPA can accumulate in the viscera, gill, head and muscle of zebrafish with 85, 43, 20, and 3 μg/g ww after 1 d exposure. NPPs were also found to accumulate in different tissues of the fish. Relative equilibrium was reached after 1 d exposure in different tissues with 39 to 636 mg/kg ww. Co-exposure of NPPs and BPA led to a 2.2 and 2.6-fold significant increment of BPA uptake in the head and viscera, if compared with BPA alone treatment after 3 d exposure. As such, we further investigated several neurotoxic biomarker alterations in the fish head. It was found that either BPA or NPPs can cause myelin basic protein (MBP)/gene up-regulation in the central nervous system (CNS); meanwhile, both contaminants exhibited significant inhibition of acetylcholinesterase (AChE) activity, which is a well-known representative biomarker for neurotoxicity. Moreover, for the co-exposure treatment, biomarkers of myeline and tubulin protein/gene expressions, dopamine content, and the mRNA expression of mesencephalic astrocyte derived neurotrophic factor (MANF) were all significantly up-regulated, suggesting that an enhanced neurotoxic effects in both CNS and dopaminergic system occurred. However, AChE activity was no more inhibited in the co-exposure treatment, which implies that solely AChE measurement may not be sufficient to identify neurotoxic effects in the cholinergic system. Overall, the present study demonstrates that the presence of NPPs can increase BPA bioavailability and cause neurotoxicity in adult zebrafish.

Wastewater discharge is one of the main sources of micropollutants within the aquatic environment. To reduce the risks for the aquatic environment, the reduction of the chemical load of wastewater treatment plant effluent is critical. Based on this need, additional treatment methods, such as ozonation, are currently being tested in several wastewater treatment plants (WWTPs). In the present study, effects were investigated using in situ feeding experiments with Gammarus pulex and body burden analyses of frequently detected micropollutants which used a Quick Easy Cheap Effective Rugged and Safe (QuEChERS) multi-residue method to quantify internal concentrations in collected gammarids. Information obtained from these experiments complemented data from the chemical analysis of water samples and bioassays, which predominantly cover hydrophilic substances. When comparing up- and downstream feeding rates of Gammarus pulex for seven days, relative to the WWTPs, no significant acute effects were detected, although a slight trend of increased feeding rate downstream of the WWTP Aachen-Soers was observed. The chemical load released by the WWTP or at other points, or by diffuse sources, might be too low to lead to clear acute effects on G. pulex. However, some compounds found in wastewater are able to alter the microbial community on its leaves, leading to an increase in the feeding rate of G. pulex. Chemical analysis of internal concentrations of pollutants in the tissues of collected gammarids suggests a potential risk for chronic effects with the chemicals imidacloprid, thiacloprid, carbendazim, and 1H-benzotriazole when exceeding the critical toxic unit value of &minus, 3. This study has demonstrated that a combination of acute testing and measurement of the internal concentration of micropollutants that might lead to chronic effects is an efficient tool for investigating river systems, assuming all relevant factors (e.g., species or season) are taken into account.

Zebrafish embryos are being increasingly used as model organisms for the assessment of single substances and complex environmental samples for regulatory purposes. Thus, it is essential to fully understand the xenobiotic metabolism during the different life-stages of early development. The aim of the present study was to determine arylhydrocarbon receptor (AhR)-mediated activity during selected times of early development using qPCR, enzymatic activity through measurement of 7-ethoxyresorufin-O-deethylase (EROD) activity, and protein expression analysis. In the present study, gene expression of cyp1a, cyp1b1, cyp1c1, cyp1c2, and ahr2 as well as EROD activity were investigated up to 120 h postfertilization (hpf) after exposure to either β-naphthoflavone (BNF) or a polycyclic aromatic hydrocarbons (PAH)-contaminated sediment extract from Vering Kanal in Hamburg (VK). Protein expression was measured at 72 hpf after exposure to 20 μg/L BNF. Altered proteins were identified by matrix assisted laser desorption ionization time-of-flight (MALDI-TOF) peptide mass fingerprinting. Distinct patterns of basal messenger RNA (mRNA) expression were found for each of the cyp1 genes, suggesting specific roles during embryonic development. All transcripts were induced by BNF and VK. ahr2 mRNA expression was significantly upregulated after exposure to VK. All cyp1 genes investigated showed a temporal decline in expression at 72 hpf. The significant decline of Hsp 90β protein at 72 hpf after exposure to BNF may suggest an explanation for the decline of cyp1 genes at this time point as Hsp 90β is of major importance for the functioning of the Ah-receptor. EROD activity measured in embryos was significantly induced after 96 hpf of exposure to BNF or VK. Together, these results demonstrate distinct temporal patterns of cyp1 genes and protein activities in zebrafish embryos as well as show a need to investigate further the xenobiotic biotransformation system during early development of zebrafish.

Heterocyclic aromatic hydrocarbons (hetero-PAHs) are increasingly studied at contaminated sites; especially at former industrial facilities where coal tar-oil was handled, e.g., wood treatment plants, high concentrations of hetero-PAHs are frequently detected in groundwater plumes. In previous studies, fractions of groundwater with high estrogenic activity contained hetero-PAHs and their hydroxylated metabolites. To evaluate this preliminary evidence, selected hetero-PAHs were screened for their estrogenic activity in lyticase yeast estrogen screen (LYES) and ER CALUX. All tested substances were inactive in the LYES. Hetero-PAHs such as acridine, xanthene, indole, 2-methylbenzofuran, 2,3-dimethylbenzofuran, dibenzofuran, dibenzothiophene, quinoline, and 6-methylquinoline were positive in the ER CALUX, with estradiol equivalence factors (EEFs) from 2.85 × 10(-7) to 3.18 × 10(-5). The EEF values of these substances were comparable to those of other xenoestrogens (e.g., alkylphenols or bisphenol A) that are sometimes found in surface water. Chemical analyses revealed that T47Dluc cells could metabolize most of the substances. Among the metabolites (tentatively) identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS) were hydroxides and their keto tautomers, sulfates, sulfoxides, and N-oxides. Because of their high concentrations measured in groundwater, we conclude that hetero-PAHs and metabolites may be a potential risk and should be the subject of further research.

The European Water Framework Directive aims to achieve a good ecological and chemical status in surface waters until 2015. Sediment toxicology plays a major role in this intention as sediments can act as a secondary source of pollution. In order to fulfill this legal obligation, there is an urgent need to develop whole-sediment exposure protocols, since sediment contact assays represent the most realistic scenario to simulate in situ exposure conditions. Therefore, in the present study, a vertebrate sediment contact assay to determine aryl hydrocarbon receptor (AhR)-mediated activity of particle-bound pollutants was developed. Furthermore, the activity and the expression of the CYP1 family in early life stages of zebrafish after exposure to freeze-dried sediment samples were investigated. In order to validate the developed protocol, effects of β-naphthoflavone and three selected sediment on zebrafish embryos were investigated. Results documented clearly AhR-mediated toxicity after exposure to β-naphthoflavone (β-NF) and to the sediment from the Vering canal. Upregulation of mRNA levels was observed for all investigated sediment samples. The highest levels of all investigated cyp genes (cyp1a, cyp1b1, cyp1c1, and cyp1c2) were recorded after exposure to the sediment sample of the Vering canal. In conclusion, the newly developed sediment contact assay can be recommended for the investigation of dioxin-like activities of single substances and the bioavailable fraction of complex environmental samples. Moreover, the exposure of whole zebrafish embryos to native (freeze-dried) sediment samples represents a highly realistic and ecologically relevant exposure scenario.

PLoS one 9(1), e85692 (2014). doi:10.1371/journal.pone.0085692, Published by PLoS [u.a.], Lawrence, Kan.

Environmental sciences Europe : ESEU 28, 20 (2016). doi:10.1186/s12302-016-0088-3, Published by Springer, Berlin ; Heidelberg