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).

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

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

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.

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.

Estrogenicity of chemical mixtures revealed by a panel of bioassays.

Estrogenic compounds are widely released to surface waters and may cause adverse effects to sensitive aquatic species. Three hormones, estrone, 17ß-estradiol and 17α-ethinylestradiol, are of particular concern as they are bioactive at very low concentrations. Current analytical methods are not all sensitive enough for monitoring these substances in water and do not cover mixture effects. Bioassays could complement chemical analysis since they detect the overall effect of complex mixtures. Here, four chemical mixtures and two hormone mixtures were prepared and tested as reference materials together with two environmental water samples by eight laboratories employing nine in vitro and in vivo bioassays covering different steps involved in the estrogenic response. The reference materials included priority substances under the European Water Framework Directive, hormones and other emerging pollutants. Each substance in the mixture was present at its proposed safety limit concentration (EQS) in the European legislation. The in vitro bioassays detected the estrogenic effect of chemical mixtures even when 17ß-estradiol was not present but differences in responsiveness were observed. LiBERA was the most responsive, followed by LYES. The additive effect of the hormones was captured by ERα-CALUX, MELN, LYES and LiBERA. Particularly, all in vitro bioassays detected the estrogenic effects in environmental water samples (EEQ values in the range of 0.75-304 × EQS), although the concentrations of hormones were below the limit of quantification in analytical measurements. The present study confirms the applicability of reference materials for estrogenic effects' detection through bioassays and indicates possible methodological drawbacks of some of them that may lead to false negative/positive outcomes. The observed difference in responsiveness among bioassays - based on mixture composition - is probably due to biological differences between them, suggesting that panels of bioassays with different characteristics should be applied according to specific environmental pollution conditions.

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.

Bioavailability and impacts of estrogenic compounds from suspended sediment on rainbow trout (Oncorhynchus mykiss).

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.

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.

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.

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.

Detection and Quantification of Photosystem II Inhibitors Using the Freshwater Alga Desmodesmus subspicatus in Combination with High-Performance Thin-Layer Chromatography.

We present a novel tool for detecting and monitoring photosystem II (PSII) inhibitors, using the freshwater alga Desmodesmus subspicatus, in environmental samples fractionated by high-performance thin-layer chromatography (HPTLC). After chromatographic separation of a sample on a HPTLC plate, the algal suspension is sprayed homogeneously on the plate, and PSII-inhibition by specific sample components is detected based on changes in fluorescence yield, viewed by a maxi Imaging-Pulse-Amplitude-Modulation fluorometer. Dose-dependent responses to the PSII-inhibitor herbicides atrazine and diuron, frequently detected in water bodies, are demonstrated without and with chromatographic separation. The limits of quantification for atrazine and diuron with chromatographic separation were 1.94 ng and 99 pg, respectively, allowing the detection of environmentally relevant concentrations of these herbicides. The developed method was also employed to analyze sample extracts collected during a passive sampling campaign in surface waters. The obtained data correlated well with results from LC-MS/MS chemical analysis but also revealed unknown PSII-inhibiting activities. The proposed methodology represents a rapid and sensitive screening tool for the simultaneous effect-based detection of PSII-inhibitors in environmental samples.

Combination of yeast-based in vitro screens with high-performance thin-layer chromatography as a novel tool for the detection of hormonal and dioxin-like compounds.

The combination of classic in vitro bioassays with high-performance thin-layer chromatography (HPTLC) is a promising technique to directly link chemical analysis of contaminants to their potential adverse biological effects. With respect to endocrine disruption, much work is focused on estrogenicity. While a direct combination of HPTLC and the yeast estrogen screen is already developed, it is well accepted that further endocrine effects are relevant for monitoring environmental wellbeing. Here we show that non-estrogenic specific biological endpoints, (partly) related to the endocrine system, can also be addressed by combining respective yeast reporter gene assays with HPTLC to support effect-directed analysis (EDA). These are: androgenicity (YAS), thyroidogenicity (YTS), dioxin-like effects (YDS), effects on the vitamin D (YVS) and the retinoic acid receptor (YRaS). A proof of principle is demonstrated within this study by the characterization of dose-dependent responses to different model compounds for the respective receptors with and without chromatographic development of the HPTLC-plate. Limits of quantification (LOQ) for several model compounds were determined, e.g. 37 pg for testosterone (p-YAS), 0.476 ng for ß-naphthoflavone (p-YDS) and 1.02 ng for calcipotriol hydrate (p-YVS) with chromatographic development. The LOQ for p-YTS and p-YRaS were 10.16 pg for 3,3',5-triiodothyroacetic acid (p-YTS) and 0.41 pg for tamibarotene (p-YRaS), without chromatographic separation. Furthermore, we challenged the developed methodology using environmental samples, demonstrating an elimination efficiency of androgenic activity from municipal wastewater by a wastewater treatment plant between 99.4 and 100%. We anticipate our methodology to substantially broaden the spectrum of specific endpoints combined with HPTLC for an efficient and robust screening of environmental samples to guide a subsequent in-depth EDA.

Bioavailability of estrogenic compounds from sediment in the context of flood events evaluated by passive sampling.

Studies worldwide have demonstrated through in vitro bioassays and chemical analysis that endocrine-disrupting chemicals (EDCs) can accumulate in river sediments. However, remobilization of sediment-bound EDCs due to bioturbation or re-suspension during flood events remains poorly understood. The aim of this study was to evaluate the bioavailability of EDCs, more specifically estrogenic compounds (EC), from sediment under turbulent conditions using a passive sampling approach. Sediment was sampled along the Luppe River, Germany, previously described as a "hotspot" for ECs. The concentration of target ECs and estrogenic activity were investigated using chemical analysis (LC MS/MS) in addition to a novel screening tool (planar Yeast Estrogen Screen; p-YES) that utilizes high performance thin-layer chromatography plates in combination with an in vitro bioassay (YES). Estrone (50%, E1) and nonylphenol (35%, NP) accounted for the majority of estrogenic activity reported of up to 20 ±â€¯2.4 µg E2 equivalents per kg dry weight in the Luppe sediments. Two types of passive samplers (polar organic chemical integrative sampler (POCIS) and Chemcatcher) were used to investigate the bioavailability of ECs from suspended sediment under laboratory conditions. NP, E1, E2 and ethynylestradiol (EE2) were remobilized from Luppe sediment when subjected to turbulent conditions, such as in a flood event, and were readily bioavailable at ecotoxicologically relevant concentrations (NP 18 µg/L, E1 14 ng/L, E2 0.2 ng/L, EE2 0.5 ng/L).

Monitoring estrogenic activities of waste and surface waters using a novel in vivo zebrafish embryonic (EASZY) assay: Comparison with in vitro cell-based assays and determination of effect-based trigger values.

This study reports the use of the recently developed EASZY assay that uses transgenic cyp19a1b-GFP zebrafish (Danio rerio) embryos to assess in vivo estrogenic activity of 33 surface (SW) and waste water (WW) samples collected across Europe that were previously well-characterized for estrogen hormones and in vitro estrogenic activity. We showed that 18 out of the 33 SW and WW samples induced estrogenic responses in the EASZY assay leading to a significant and concentration-dependent up-regulation of the ER-regulated cyp19a1b gene expression in the developing brain. The in vivo 17ß-estradiol-equivalents (EEQs) were highly correlated with, both, the chemical analytical risk quotient (RQ) based on steroidal estrogen concentrations and EEQs reported from five different in vitro reporter gene assays. Regression analyses between the vitro and in vivo effect concentrations allowed us to determine an optimal cut-off value for each in vitro assay, above which in vivo responses were observed. These in vitro assay-specific effect-based trigger values (EBTs), ranging from 0.28 to 0.58 ng EEQ/L define the sensitivity and specificity of the individual in vitro assays for predicting a risk associated with substances acting through the same mode of action in water samples. Altogether, this study demonstrates the toxicological relevance of in vitro-based assessment of estrogenic activity and recommends the use of such in vitro/in vivo comparative approach to refine and validate EBTs for mechanism-based bioassays.

Coupling High-Performance Thin-Layer Chromatography with Bacterial Genotoxicity Bioreporters.

We present an innovative technological platform for monitoring the direct genotoxicity of individual components in complex environmental samples, based on bioluminescent Escherichia coli genotoxicity bioreporters, sprayed onto the surface of a high-performance thin-layer chromatography (HPTLC) plate. These sensor strains harbor plasmid-borne fusions of selected gene promoters of the E. coli SOS DNA repair system to the Photorhabdus luminescens luxABCDE gene cassette, and mark by increased luminescence the presence of potentially DNA-damaging sample components separated on the plate. We demonstrate an "on plate" quantifiable dose-dependent response to several model genotoxicants (without metabolic activation). We further demonstrate the applicability of the system by identifying as genotoxic specific components of HPTLC-separated influent and effluent samples of wastewater treatment plants, thereby alleviating the need for a comprehensive chemical analysis of the sample.

Effect-based trigger values for in vitro and in vivo bioassays performed on surface water extracts supporting the environmental quality standards (EQS) of the European Water Framework Directive.

Effect-based methods including cell-based bioassays, reporter gene assays and whole-organism assays have been applied for decades in water quality monitoring and testing of enriched solid-phase extracts. There is no common EU-wide agreement on what level of bioassay response in water extracts is acceptable. At present, bioassay results are only benchmarked against each other but not against a consented measure of chemical water quality. The EU environmental quality standards (EQS) differentiate between acceptable and unacceptable surface water concentrations for individual chemicals but cannot capture the thousands of chemicals in water and their biological action as mixtures. We developed a method that reads across from existing EQS and includes additional mixture considerations with the goal that the derived effect-based trigger values (EBT) indicate acceptable risk for complex mixtures as they occur in surface water. Advantages and limitations of various approaches to read across from EQS are discussed and distilled to an algorithm that translates EQS into their corresponding bioanalytical equivalent concentrations (BEQ). The proposed EBT derivation method was applied to 48 in vitro bioassays with 32 of them having sufficient information to yield preliminary EBTs. To assess the practicability and robustness of the proposed approach, we compared the tentative EBTs with observed environmental effects. The proposed method only gives guidance on how to derive EBTs but does not propose final EBTs for implementation. The EBTs for some bioassays such as those for estrogenicity are already mature and could be implemented into regulation in the near future, while for others it will still take a few iterations until we can be confident of the power of the proposed EBTs to differentiate good from poor water quality with respect to chemical contamination.

In vitro tools for the toxicological evaluation of sediments and dredged materials: intra- and inter-laboratory comparisons of chemical and bioanalytical methods.

The implementation of in vitro bioassays for the screening of dioxin-like compounds (DLCs) into management guidelines of dredged material is of increasing interest to regulators and risk assessors. This study reports on an intra- and inter-laboratory comparison study between four independent laboratories. A bioassay battery consisting of RTL-W1 (7-ethoxy-resorufin-O-deethylase; EROD), H4IIE (micro-EROD), and H4IIE-luc cells was used to assess aryl hydrocarbon receptor-mediated effects of sediments from two major European rivers, differently contaminated with DLCs. Each assay was validated by characterization of its limit of detection (LOD) and quantification (LOQ), z-factor, reproducibility, and repeatability. DLC concentrations were measured using high-resolution gas chromatography/high-resolution mass spectrometry (HRGC/HRMS) and compared to bioassay-specific responses via toxicity equivalents (TEQs) on intra- and inter-laboratory levels. The micro-EROD assay exhibited the best overall performance among the bioassays. It was ranked excellent (z-factor = 0.54), reached a repeatability > 75%, was highly comparable (r 2 = 0.87) and reproducible (83%) between two laboratories, and was well correlated (r 2 = 0.803) with TEQs. Its LOD and LOQ of 0.5 and 0.7 pM 2,3,7,8-TCDD, respectively, approached LOQs of HRGC/HRMS measurements. In contrast, cell lines RTL-W1 and H4IIE-luc produced LODs > 0.7 pM 2,3,7,8-TCDD, LOQs > 1.7 pM 2,3,7,8-TCDD, and repeatability < 70%. Based on the data obtained, the micro-EROD assay is the most favorable bioanalytical tool, and via a micro-EROD-based limit value, it would allow for the assessment of sediment DLC concentrations; thus, it could be considered for the implementation into testing and management guidelines for dredged materials.