Extrapolation of cytotoxic masked effects in planar in vitro assays.

The masking of specific effects in in vitro assays by cytotoxicity is a commonly known phenomenon. This may result in a partial or complete loss of effect signals. For common in vitro assays, approaches for identifying and quantifying cytotoxic masking are partly available. However, a quantification of cytotoxicity-affected signals is not possible. As an alternative, planar bioassays that combine high-performance thin layer chromatography with in vitro assays, such as the planar yeast estrogen screen (p-YES), might allow for a quantification of cytotoxically affected signals. Affected signals form a typical ring structure with a supressed or completely lacking centre that results in a double peak chromatogram. This study investigates whether these double peaks can be used for fitting a peak function to extrapolate the theoretical, unaffected signals. The precision of the modelling was evaluated for four individual peak functions, using 42 ideal, undistorted peaks from estrogenic model compounds in the p-YES. Modelled ED50-values from bisphenol A (BPA) experiments with cytotoxically disturbed signals were 13 times higher than for the apparent data without compensation for cytotoxicity (320 ± 63 ng versus 24 ± 17 ng). This finding has a high relevance for the modelling of mixture effects according to concentration addition that requires unaffected, complete dose-response relationships. Finally, we applied the approach to results of a p-YES assay on leachate samples of an elastomer material used in water engineering. In summary, the fitting approach enables the quantitative evaluation of cytotoxically affected signals in planar in vitro assays and also has applications for other fields of chemical analysis like distorted chromatography signals.

Enhanced Detection of Estrogen-like Compounds by Genetically Engineered Yeast Sensor Strains.

The release of endocrine-disrupting compounds (EDCs) to the environment poses a health hazard to both humans and wildlife. EDCs can activate or inhibit endogenous endocrine functions by binding hormone receptors, leading to potentially adverse effects. Conventional analytical methods can detect EDCs at a high sensitivity and precision, but are blind to the biological activity of the detected compounds. To overcome this limitation, yeast-based bioassays have previously been developed as a pre-screening method, providing an effect-based overview of hormonal-disruptive activity within the sample prior to the application of analytical methods. These yeast biosensors express human endocrine-specific receptors, co-transfected with the relevant response element fused to the specific fluorescent protein reporter gene. We describe several molecular manipulations of the sensor/reporter circuit in a Saccharomyces cerevisiae bioreporter strain that have yielded an enhanced detection of estrogenic-like compounds. Improved responses were displayed both in liquid culture (96-well plate format) as well as in conjunction with sample separation using high-performance thin-layer chromatography (HPTLC). The latter approach allows for an assessment of the biological effect of individual sample components without the need for their chemical identification at the screening stage.

Results of an international interlaboratory study on dioxin-like activities in drinking-, river surface- and wastewater using DR CALUX bioassay.

Aquatic animals and consumers of aquatic animals are exposed to increasingly complex mixtures of known and as-yet-unknown chemicals with dioxin-like toxicities in the water cycle. Effect- and cell-based bioanalysis can cover known and yet unknown dioxin and dioxin-like compounds as well as complex mixtures thereof but need to be standardized and integrated into international guidelines for environmental testing. In an international laboratory testing (ILT) following ISO/CD 24295 as standard procedure for rat cell-based DR CALUX un-spiked and spiked extracts of drinking-, surface-, and wastewater were validated to generate precision data for the development of the full ISO-standard. We found acceptable repeatability and reproducibility ranges below 36 % by DR CALUX bioassay for the tested un-spiked and spiked water of different origins. The presence of 17 PCDD/Fs and 12 dioxin-like PCBs was also confirmed by congener-specific GC-HRMS analysis. We compared the sum of dioxin-like activity levels measured by DR CALUX bioassay (expressed in 2,3,7,8-TCDD Bioanalytical Equivalents, BEQ; ISO 23196, 2022) with the obtained GC-HRMS chemical analysis results converted to toxic equivalents (TEQ; van den Berg et al., 2013).

Planar chromatography-bioassays for the parallel and sensitive detection of androgenicity, anti-androgenicity and cytotoxicity.

Anti-androgens entering the aquatic environment, e.g., by effluents from wastewater treatment plants or agricultural settings are contributing to endocrine disruption in wildlife and humans. Due to the simultaneous presence of agonistic compounds, common in vitro bioassays can underestimate the risk posed by androgen antagonists. On the other hand, cytotoxic effects might lead to false positive assessments of anti-androgenic effects in conventional bioassays. In the present study, a combination of normal phase high-performance thin-layer chromatography (NP-HPTLC) with a yeast-based reporter gene assay is established for the detection of anti-androgenicity as a promising tool to reduce interferences of androgenic and anti-androgenic compounds present in the same sample. To avoid a misinterpretation of anti-androgenicity with cytotoxic effects, cell viability was assessed in parallel on the same plate using a resazurin viability assay adapted to HPTLC plates. The method was characterized by establishing dose-response curves for the model compounds flutamide and bisphenol A. Calculated effective doses at 10% (ED10) were 27.9 ± 1.3 ng zone-1 for flutamide and 20.1 ± 5.1 ng zone-1 for bisphenol A. Successful distinction between anti-androgenicity and cytotoxicity was exemplarily demonstrated with 4-nitroquinoline 1-oxide. As a proof of concept, the detection and quantification of anti-androgenicity in an extract of a landfill leachate is demonstrated. This study shows that the hyphenation of HPTLC with the yeast anti-androgen screen is a matrix-robust, cost-efficient and fast screening tool for the sensitive and simultaneous detection of anti-androgenic and cytotoxic effects in environmental samples. The method offers a wide range of possible applications in environmental monitoring and contributes to the identification of anti-androgenicity drivers in the course of an effect-directed analysis.

Prevalence and Distribution of Potentially Human Pathogenic Vibrio spp. on German North and Baltic Sea Coasts.

Global ocean warming results in an increase of infectious diseases including an elevated emergence of Vibrio spp. in Northern Europe. The European Centre for Disease Prevention and Control reported annual periods of high to very high risks of infection with Vibrio spp. during summer months along the North Sea and Baltic Sea coasts. Based on those facts, the risk of Vibrio infections associated with recreational bathing in European coastal waters increases. To obtain an overview of the seasonal and spatial distribution of potentially human pathogenic Vibrio spp. at German coasts, this study monitored V. cholerae, V. parahaemolyticus, and V. vulnificus at seven recreational bathing areas from 2017 to 2018, including the heat wave event in summer 2018. The study shows that all three Vibrio species occurred in water and sediment samples at all sampling sites. Temperature was shown to be the main driving factor of Vibrio abundance, whereas Vibrio community composition was mainly modulated by salinity. A species-specific rapid increase was observed at water temperatures above 10°C, reaching the highest detection numbers during the heat wave event with abundances of 4.5 log10 CFU+1/100 ml of seawater and 6.5 log10 CFU+1/100 g of sediment. Due to salinity, the dominant Vibrio species found in North Sea samples was V. parahaemolyticus, whereas V. vulnificus was predominantly detected in Baltic Sea samples. Most detections of V. cholerae were associated with estuarine samples from both seas. Vibrio spp. concentrations in sediments were up to three log higher compared to water samples, indicating that sediments are an important habitat for Vibrio spp. to persist in the environment. Antibiotic resistances were found against beta-lactam antibiotics (ampicillin 31%, cefazolin 36%, and oxacillin and penicillin 100%) and trimethoprim-sulfamethoxazole (45%). Moreover, isolates harboring pathogenicity-associated genes such as trh for V. parahaemolyticus as well as vcg, cap/wcv, and the 16S rRNA-type B variant for V. vulnificus were detected. All sampled V. cholerae isolates were identified as non-toxigenic non-O1/non-O139 serotypes. To sum up, increasing water temperatures at German North Sea and Baltic Sea coasts provoke elevated Vibrio numbers and encourage human recreational water activities, resulting in increased exposure rates. Owing to a moderate Baltic Sea salinity, the risk of V. vulnificus infections is of particular concern.

UV aged epoxy coatings - Ecotoxicological effects and released compounds.

Organic coatings can guarantee long-term protection of steel structures due to causing a physical barrier against water and oxygen. Because of their mechanical properties and resistances to heat and chemicals, epoxy resin-based coatings are widely used for corrosion protection. Despite of the aromatic backbone and the resulting susceptibility to UV degradation, epoxy resins are frequently used as binding agent in top layers of anti-corrosion coating systems. Consequently, these organic polymers are directly exposed to sunlight and thus UV radiation. The present study was designed to investigate if toxic effects of epoxy resin-based-coatings are changed by UV-A irradiation. For this purpose, two epoxide-based top coatings were examined with and without UV aging for their bacterial toxicity and estrogenicity. In addition, chemical analyses were performed to identify released compounds as well as photolytic degradation products and to assign toxic effects to individual substances. UV-A irradiation of epoxy resin based top coatings resulted in an overall decrease of acute and specific ecotoxicological effects but as well to the formation of toxic transformation products. Both, in leachates of untreated and UV-A irradiated coatings, 4tBP was identified as the main driver of estrogenicity and toxicity to luminescent bacteria. BPA and structural analogs contributing to estrogenic effects in leachates were formed by UV-A irradiation. The combination of HPTLC coupled bioassays and LC-MS analyses supported the identification of bioactive compounds in terms of an effect-directed analysis. The present findings indicate that epoxide-based coatings are less suitable for the application as top coatings and more UV stable coatings like aliphatic polyurethanes should be preferred.

Ingestion and Toxicity of Polystyrene Microplastics in Freshwater Bivalves.

The ubiquity of microplastics in aquatic ecosystems has raised concerns over their interaction with biota. However, microplastics research on freshwater species, especially mollusks, is still scarce. We, therefore, investigated the factors affecting microplastics ingestion in the freshwater mussel Dreissena polymorpha. Using polystyrene spheres (5, 10, 45, 90 µm), we determined the body burden of microplastics in the mussels in relation to 1) exposure and depuration time, 2) body size, 3) food abundance, and 4) microplastic concentrations. D. polymorpha rapidly ingested microplastics and excreted most particles within 12 h. A few microplastics were retained for up to 1 wk. Smaller individuals had a higher relative body burden of microplastics than larger individuals. The uptake of microplastics was concentration-dependent, whereas an additional food supply (algae) reduced it. We also compared the ingestion of microplastics by D. polymorpha with 2 other freshwater species (Anodonta anatina, Sinanodonta woodiana), highlighting that absolute and relative uptake depends on the species and the size of the mussels. In addition, we determined toxicity of polystyrene fragments (≤63 µm, 6.4-100 000 p mL-1 ) and diatomite (natural particle, 100 000 p mL-1 ) in D. polymorpha after 1, 3, 7, and 42 d of exposure, investigating clearance rate, energy reserves, and oxidative stress. Despite ingesting large quantities, exposure to polystyrene fragments only affected the clearance rate of D. polymorpha. Further, results of the microplastic and diatomite exposure did not differ significantly. Therefore, D. polymorpha is unaffected by or can compensate for polystyrene fragment toxicity even at concentrations above current environmental levels. Environ Toxicol Chem 2021;40:2247-2260. © 2021 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Coupling high-performance thin-layer chromatography with a battery of cell-based assays reveals bioactive components in wastewater and landfill leachates.

Over the last two decades, effect-directed analysis (EDA) gained importance as a seminal screening tool for tracking biological effects of environmental organic micro-pollutants (MPs). As EDA using high-performance liquid chromatography and bioassays is costly and time consuming, recent implementations of this approach have combined high-performance thin-layer chromatography (HPTLC) with effect-based methods (EBMs) using cell-based bioassays, enabling the detection of estrogenic, androgenic, genotoxic, photosystem II (PSII)- inhibiting, and dioxin-like sample components on a HPTLC plate. In the present study, the developed methodologies were applied as a HPTLC-based bioassay battery, to investigate toxicant elimination efficiency of wastewater treatment plants (WWTPs), and to characterize the toxic potential of landfill leachates. Activity levels detected in untreated landfill leachates, expressed as reference compound equivalence (EQ) concentration, were up to 16.8 µg ß-naphthoflavone-EQ L-1 (indicating the degree of dioxin-like activity), 1.9 µg estradiol-EQ L-1 (estrogenicity) and 8.3 µg diuron-EQ L­1 (PSII-inhibition), dropping to maximal concentrations of 47 ng ß-naphthoflavone-EQ L-1, 0.7 µg estradiol-EQ L-1 and 53.1 ng diuron-EQ L-1 following treatment. Bisphenol A (BPA) is suggested to be the main contributor to estrogenic activity, with concentrations determined by the planar yeast estrogen screen corresponding well to results from chemical analysis. In the investigated WWTP samples, a decrease of estrogenic activity of 6-100% was observed following treatment for most of the active fractions, except of a 20% increase in one fraction (Rf = 0.568). In contrast, androgenicity with concentrations up to 640 ng dihydrotestosterone-EQ L-1 was completely removed by treatment. Interestingly, genotoxic activity increased over the WWTP processes, releasing genotoxic fractions into receiving waters. We propose this combined HPTLC and EBM battery to contribute to an efficient, cheap, fast and robust screening of environmental samples; such an assay panel would allow to gain an estimate of potential biological effects for prioritization prior to substance identification, and its routine application will support an inexpensive identification of the toxicity drivers as a first tier in an EDA strategy.

Combined sediment desorption and bioconcentration model to predict levels of dioxin-like chemicals in fish.

Flooding and other sediment disturbances can lead to increases in sediment resuspension. In this context, it is of central importance to understand the kinetics of release from these sediments and the uptake of pollutants, such as polychlorinated biphenyls (PCBs) and polychlorinated dioxins and furans (PCDD/Fs), into aquatic organisms. In the present study, we parameterized a sediment desorption model based on experimentally determined rapidly-desorbing fractions of dioxin-like chemicals (DLCs). We coupled this desorption model with a physiologically-based toxicokinetic model for rainbow trout. This combined model was used to predict DLC concentrations in the muscle of exposed fish. The performance of this model was evaluated using a previously published dataset on DLC uptake from sediment suspensions during simulated re-suspension events. Predictions generally differed less than 10-fold from measured values, and the model showed a good global coefficient of determination (R2) of 0.95. The root mean squared error (RMSE) for PCBs was 0.31 log units and 0.53 log units for PCDD/Fs. The results of our study demonstrate that the prediction of bioconcentration and related risk to fish resulting from sediment resuspension can be accurately predicted using coupled desorption and toxicokinetic models.

Tyre and road wear particles - A calculation of generation, transport and release to water and soil with special regard to German roads.

Tyre and road wear is one of the main emission sources of particulate plastics (microplastics). In this study, the emissions of tyre wear particles (TWP) which are annually generated on the German road network were calculated. Emissions are calculated by applying two different data sets of emission factors and the annual mileage for distinct vehicles and road types (urban roads, rural roads, highways). Environmental entry paths of tyre and road wear particles (TRWP) were considered including releases to ambient air, soils and surface waters. Road runoff treatment was taken into account differentiated into sewage systems in urban areas and at non-urban roads. This study identifies the soundest data available concerning emission factors, data on traffic and road systems as well as comprehensive information on road runoff treatment in Germany. Applying this data and assuming that 5% of the total emissions are 'fine' air-borne particles, the emissions of coarse 'non-airborne' particles are 75,200-98,400 t/a; . the transport to road banks and soils near roads is 57,300-65,400 t/a (66-76%) including runoff and drift; 8700-19,800 t/a (12-20%) are released to surface waters. Due to lack of data, degradation in soils and surface waters was not considered. Besides soils, urban wastewater treatment plants are expected to be an important sink of TRWP assuming that most of the particles are incorporated in the sewage sludge. Due to the application of sewage sludge as a fertilizer, 1400-2800 t/a TWP are currently deposited on agricultural areas. No reliable data was available to estimate the masses of TWP which are transported from freshwaters into the marine environment. Existing mitigation measures should be improved according to the principle of precaution by installation of road runoff treatment systems and retrofitting of existing plants regarding optimum fine particle retention.

Quantitative and qualitative evaluation of plastic particles in surface waters of the Western Black Sea.

Microplastic abundances have been studied intensively in the last years in marine and freshwater environments worldwide. Though several articles have been published about the Mediterranean Sea, only few studies about the Black Sea exist. The Black Sea drains into the Mediterranean Sea and may therefore significantly contribute to the Mediterranean marine pollution. So far, only very few articles have been published about micro-, meso- and macroplastic abundances in the Western Black Sea. In order to fill this knowledge gap and to decipher the number of plastics on the water surface, 12 samples were collected from surface waters with a neustonic net (mesh size 200 µm) in the Black Sea close to the Danube Delta and the Romanian shore. Organic matter was digested and plastic particles were isolated by density separation. The results of visual inspection, pyrolysis GC-MS (for microplastics) and ATR-FTIR (for mesoplastics >5 mm) revealed an average concentration of 7 plastic particles/m³, dominated by fibers (∼76%), followed by foils (∼13%) and fragments (∼11%). Only very few spherules were detected. The polymers polypropylene (PP) and polyethylene (PE) dominated which is in line with other studies analyzing surface waters from rivers in Western Europe as well as in China. Statistical analyses show that the plastic concentration close to the mouth of the Danube River was significantly higher than at four nearshore regions along the Romanian and Bulgarian coastline. This could be explained by plastic inputs from the Danube River into the western part of the Black Sea.

Yeast-Based Fluorescent Sensors for the Simultaneous Detection of Estrogenic and Androgenic Compounds, Coupled with High-Performance Thin Layer Chromatography.

The persistence of endocrine disrupting compounds (EDCs) throughout wastewater treatment processes poses a significant health threat to humans and to the environment. The analysis of EDCs in wastewater remains a challenge for several reasons, including (a) the multitude of bioactive but partially unknown compounds, (b) the complexity of the wastewater matrix, and (c) the required analytical sensitivity. By coupling biological assays with high-performance thin-layer chromatography (HPTLC), different samples can be screened simultaneously, highlighting their active components; these may then be identified by chemical analysis. To allow the multiparallel detection of diverse endocrine disruption activities, we have constructed Saccharomyces cerevisiae-based bioreporter strains, responding to compounds with either estrogenic or androgenic activity, by the expression of green (EGFP), red (mRuby), or blue (mTagBFP2) fluorescent proteins. We demonstrate the analytical potential inherent in combining chromatographic compound separation with a direct fluorescent signal detection of EDC activities. The applicability of the system is further demonstrated by separating influent samples of wastewater treatment plants, and simultaneously quantifying estrogenic and androgenic activities of their components. The combination of a chemical separation technique with an optical yeast-based bioassay presents a potentially valuable addition to our arsenal of environmental pollution monitoring tools.

Comparative assessment of microplastics in water and sediment of a large European river.

Aquatic ecosystems are globally contaminated with microplastics (MP). However, comparative data on MP levels in freshwater systems is still scarce. Therefore, the aim of this study is to quantify MP abundance in water and sediment of the German river Elbe using visual, spectroscopic (Fourier-transform infrared spectroscopy) and thermo analytical (pyrolysis gas chromatography mass spectrometry) methods. Samples from eleven German sites along the German part of the Elbe were collected, both in the water and sediment phase, in order to better understand MP sinks and transport mechanisms. MP concentrations differed between the water and sediment phase. Sediment concentrations (mean: 3,350,000 particles m-3, 125-5000 µm MP) were in average 600,000-fold higher than water concentrations (mean: 5.57 particles m-3, 150-5000 µm MP). The abundance varied between the sampling sites: In sediments, the abundance decreased in the course of the river while in water samples no such clear trend was observed. This may be explained by a barrage retaining sediments and limiting tidal influence in the upstream parts of the river. Particle shape differed site-specifically with one site having exceptionally high quantities of spheres, most probably due to industrial emissions of PS-DVB resin beads. Suspended MP consisted predominantly of polyethylene and polypropylene whereas sediments contained a higher diversity of polymer types. Determined MP concentrations correspond well to previous results from other European rivers. In a global context, MP levels in the Elbe relate to the lower (water) to middle section (sediment) of the global range of MP concentrations determined for rivers worldwide. This highlights that elevated MP levels are not only found in single countries or continents, but that MP pollution is an issue of global concern.

Validation of the micro-EROD assay with H4IIE cells for assessing sediment contamination with dioxin-like chemicals.

In vitro bioassays have been used as a bioanalytical means of detecting dioxin-like compounds (DLCs) in environmental matrices and have been suggested as a tool for quantifying DLCs in sediments. The present study evaluated the relationship between bioanalytical results from the micro-7-ethoxyresorufin-O-deethylase (EROD) bioassay and chemical analytical results in 25 sediment samples collected from rivers across Germany. Sediments were collected, polychlorinated dibenzo-p-dioxins and dibenzofurans (PCDD/Fs) and dioxin-like polychlorinated biphenyls (DL-PCBs) were extracted from the sediments, biological toxicity equivalent quotients (BEQs) were determined by micro-EROD assay and toxicity equivalent quotients (TEQs) were calculated from chemical analysis. Correlations between BEQs and TEQs were evaluated, and linear regression modeling was performed, excluding 6 samples as validation data, to derive equations for predicting TEQs from BEQs. Validation data was tested to evaluate predictive capabilities of the models. Correlations were observed between BEQ and TEQ for PCDD/Fs (r=0.987), PCBs (r=0.623), measured sum of PCDD/F and PCBs (r = 0.975) and calculated sum of PCDD/F and PCBs (r = 0.971). The modeling equations provided low variances as evaluated by mean absolute error (MAE) (≤10.3 pg/g) and root mean square error (RMSE) (≤15.8 pg/g) indicating that expected TEQs could be reasonably well calculated from BEQs. Predicted TEQs from validation data fell within the 95% probability intervals of the test data and had low variances (MAE≤6.5 pg/g) and (RMSE≤10.7 pg/g). Our results indicate that the micro-EROD bioassay can be used as a screening tool for DLCs in sediment and has the capability to be used as an alternate method to chemical analysis for quantifying dioxin-like potential of sediments.

Heavy rainfall provokes anticoagulant rodenticides' release from baited sewer systems and outdoor surfaces into receiving streams.

Prevalent findings of anticoagulant rodenticide (AR) residues in liver tissue of freshwater fish recently emphasized the existence of aquatic exposure pathways. Thus, a comprehensive wastewater treatment plant and surface water monitoring campaign was conducted at two urban catchments in Germany in 2018 and 2019 to investigate potential emission sources of ARs into the aquatic environment. Over several months, the occurrence and fate of all eight ARs authorized in the European Union as well as two pharmaceutical anticoagulants was monitored in a variety of aqueous, solid, and biological environmental matrices during and after widespread sewer baiting with AR-containing bait. As a result, sewer baiting in combined sewer systems, besides outdoor rodent control at the surface, was identified as a substantial contributor of these biocidal active ingredients in the aquatic environment. In conjunction with heavy or prolonged precipitation during bait application in combined sewer systems, a direct link between sewer baiting and AR residues in wastewater treatment plant influent, effluent, and the liver of freshwater fish was established. Moreover, study results confirmed insufficient removal of anticoagulants during conventional wastewater treatment and thus indirect exposure of aquatic organisms in receiving streams via tertiary treated effluents and combined sewer overflows. Nevertheless, further research is required to determine the ecological implications and risks for aquatic organisms as well as fish-eating predators from chronic AR exposure at environmentally relevant concentrations.

Tyre and road wear particles (TRWP) - A review of generation, properties, emissions, human health risk, ecotoxicity, and fate in the environment.

In this paper, the current knowledge on tyre and road wear particles (TRWP) is compiled regarding all environmental and health aspects. TRWP generated on roads during driving processes contribute to airborne non-exhaust emissions and are discussed in connection with the microplastic pollution. The major amount of TRWP consists of coarser heterogenous particles released to road surface, soils and aquatic compartments. The extensive compilation of annual emissions of tyre wear for numerous countries shows per-capita-masses ranging from 0.2 to 5.5 kg/(cap*a). Ecotoxicological studies revealed effects on aquatic organisms, but test concentrations and materials do not reflect environmental conditions. Contribution of tyre wear to PM10 accounts for up to approx. 11 mass %. A recent thorough risk assessment indicates the risk for human health via inhalation to be low, but no information is available on the risk caused by intake via the food chain. Data on degradation is scarce and most studies do not use realistic materials and conditions. The only published degradation study performed under environmental conditions implies a half-life of tyre rubber particles in soils of 16 months. For truck tyres, which mainly contain natural rubber, shorter periods were observed under optimum conditions in laboratory tests. Concentrations of tyre wear compiled from environmental monitoring studies show highly variable concentrations in road runoff, road dust, roadside soils, river sediments and river water, with a general decrease following the transport paths. However, the behaviour of TRWP in freshwater referring to transport, degradation, and sedimentation is still unclarified. Environmental monitoring of TRWP is still hampered by challenges for analytics. Thus, data on environmental concentrations is rare and has mainly exemplary character. Further research is needed with regard to emission factors, development of analytical methods for environmental matrices, long-period monitoring, fate in surface waters and soils, (eco)toxicological impacts and degradation under realistic conditions.

Prevalence and seasonal dynamics of blaCTX-M antibiotic resistance genes and fecal indicator organisms in the lower Lahn River, Germany.

Antibiotic-resistant bacteria represent an emerging global health problem and are frequently detected in riverine environments. Analyzing the occurrence of corresponding antibiotic-resistant genes in rivers is of public interest as it contributes towards understanding the origin and dissemination of these emerging microbial contaminants via surface water. This is critical for devising strategies to mitigate the spread of resistances in the environment. Concentrations of blaCTX-M antibiotic resistance genes were quantified weekly over a 12-month period in Lahn River surface water at two sampling sites using quantitative real-time PCR. Gene abundances were statistically assessed with regard to previously determined concentrations of fecal indicator organisms Escherichia coli, intestinal enterococci and somatic coliphages, as well as influential environmental factors. Similar seasonal patterns and strong positive correlations between fecal indicators and blaCTX-M genes indicated identical sources. Accordingly, linear regression analyses showed that blaCTX-M concentrations could largely be explained by fecal pollution. E. coli provided the best estimates (75% explained variance) at the upstream site, where proportions of blaCTX-M genes in relation to fecal indicator organisms were highest. At this site, rainfall proved to be more influential, hinting at surface runoff as an emission source. The level of agricultural impact increased from downstream to upstream, linking increasing blaCTX-M concentrations after rainfall events to the degree of agricultural land use. Exposure assessment revealed that even participants in non-swimming recreational activities were at risk of incidentally ingesting blaCTX-M genes and thus potentially antibiotic resistant bacteria. Considering that blaCTX-M genes are ubiquitous in Lahn River and participants in bathing and non-bathing water sports are at risk of exposure, results highlight the importance of microbial water quality monitoring with an emphasis on antibiotic resistance not only in designated bathing waters. Moreover, E. coli might serve as a suitable estimate for the presence of respective antibiotic resistant strains.

Ecotoxicological characterization of emissions from steel coatings in contact with water.

In order to prevent corrosion damage, steel structures need to be protected. Coating systems achieve this by the isolation of the steel from its environment. Common binding agents are epoxide and polyurethane resins which harden by polyaddition reactions. In contact with water, various organic substances might be leached out and released into the aquatic environment potentially causing adverse effects. So far, no legal requirements are mandatory for the environmental sustainability of coating systems. To characterize emissions from steel coatings, recommendations for the ecotoxicological assessment of construction products were utilized. Seven different coating systems based on epoxide or polyurethane resins were leached in 8 steps (6 h-64 d), followed by the testing of acute toxic effects on bacteria and algae as well as estrogen-like and mutagenic effects. In addition, chemical analysis by GC-MS was performed to identify potentially toxic compounds released from the coating systems. Two systems tested did not show any significant effects in the bioassays. One coating system caused significant algal toxicity, none was found to cause mutagenic effects. The other coating systems mainly showed estrogenic effects and bacterial toxicity. The effects increased with increasing leaching time. 4-tert-butylphenol, which is used in epoxy resins as a hardener, was identified as the main contributor to acute and estrogenic effects in two coatings. The release mechanism of 4-tert-butylphenol was characterized by two different modelling approaches. It was found that the release from the most toxic coating is not explainable by an elevated content of 4-tert-butylphenol but more likely by the release mechanism that - in contrast to the less toxic coating - is controlled not only by diffusion. This finding might indicate a sub-optimal formulation of this coating system resulting in a less stable layer and thus an increased release of toxic compounds.

Microplastics and their possible sources: The example of Ofanto river in southeast Italy.

Monitoring studies have quantified microscopic plastic debris, so-called microplastics, in freshwater systems, including banks, surface waters and sediments. However, there is a lack of knowledge of freshwater and terrestrial environments. When microplastics are released in freshwater environments, they will be transported and will not remain stationary. Moreover, their transport from sink to source (land-based to river systems) may depend on several factors such as weather conditions and river hydrology. The present study aims to investigate the abundance and composition of microplastics in the most important river of Apulia Region (Southeast Italy) evaluating the main drivers and possible input sources of microplastic debris. The following work is the first study showing an Italian river context. For this research five sampling campaigns have been conducted west of the Ofanto river mouth. Microplastics were collected by three surface plankton nets fixed in the middle of the river in order to reduce the spatial and temporal variability. For each campaign, a total of six replicates were sampled during two time slots. Microplastic concentrations ranged from 0.9 ±â€¯0.4 p/m3 to 13 ±â€¯5 p/m3 showing comparable values to or greater than those ones reported in other studies. A statistically significant difference in the average microplastic concentrations in different campaigns of this study has been observed, suggesting thus a temporal variation in plastic abundances. These significant differences could be explained by the hydrology of the river that influences the particle concentration with its physical forces such as flow velocity, water level and seasonal variability. Microplastics were found at higher concentrations during wet periods indicating a land-based origin probably connected to waste produced by the surroundings agricultural areas. In fact, Spearman's correlation results show a strong positive statistically significant correlation between the concentration of microplastics and the water level (R = 0.8475, p < 0.0001).

Toxicity of microplastics and natural particles in the freshwater dipteran Chironomus riparius: Same same but different?

Microplastics (MP) are contaminants of emerging concern in aquatic ecosystems. While the number of studies is rapidly increasing, a comparison of the toxicity of MP and natural particulate matter is largely missing. In addition, research focusses on the impacts of hydrophobic chemicals sorbed to plastics. However, the interactive effects of MP and hydrophilic, dissolved chemicals remain largely unknown. Therefore, we conducted chronic toxicity studies with larvae of the freshwater dipteran Chironomus riparius exposed to unplasticised polyvinyl chloride MP (PVC-MP) as well as kaolin and diatomite as reference materials for 28 days. In addition, we investigated the effects of particles in combination with the neonicotinoid imidacloprid in a multiple-stressor experiment. High concentrations of kaolin positively affected the chironomids. In contrast, exposure to diatomite and PVC-MP reduced the emergence and mass of C. riparius. Likewise, the toxicity of imidacloprid was enhanced in the presence of PVC-MP and slightly decreased in the co-exposure with kaolin. Overall, parallel experiments and chemical analysis indicate that the toxicity of PVC-MP was not caused by leached or sorbed chemicals. Our study demonstrates that PVC-MP induce more severe effects than both natural particulate materials. However, the latter are not benign per se, as the case of diatomite highlights. Considering the high, environmentally irrelevant concentrations needed to induce adverse effects, C. riparius is insensitive to exposures to PVC-MP.