Predicting exposure concentrations of chemicals with a wide range of volatility and hydrophobicity in different multi-well plate set-ups

Quantification of chemical toxicity in small-scale bioassays is challenging owing to small volumes used and extensive analytical resource needs. Yet, relying on nominal concentrations for effect determination maybe erroneous because loss processes can significantly reduce the actual exposure. Mechanistic models for predicting exposure concentrations based on distribution coefficients exist but require further validation with experimental data. Here we developed a complementary empirical model framework to predict chemical medium concentrations using different well-plate formats (24/48-well), plate covers (plastic lid, or additionally aluminum foil or adhesive foil), exposure volumes, and biological entities (fish, algal cells), focusing on the chemicals’ volatility and hydrophobicity as determinants. The type of plate cover and medium volume were identified as important drivers of volatile chemical loss, which could accurately be predicted by the framework. The model focusing on adhesive foil as cover was exemplary cross-validated and extrapolated to other set-ups, specifically 6-well plates with fish cells and 24-well plates with zebrafish embryos. Two case study model applications further demonstrated the utility of the empirical model framework for toxicity predictions. Thus, our approach can significantly improve the applicability of small-scale systems by providing accurate chemical concentrations in exposure media without resource- and time-intensive analytical measurements.ISSN:2045-232

A fish intestinal epithelial barrier model established from the rainbow trout (Oncorhynchus mykiss) cell line, RTgutGC

The intestine of fish is a multifunctional organ: lined by only a single layer of specialized epithelial cells, it has various physiological roles including nutrient absorption and ion regulation. It moreover comprises an important barrier for environmental toxicants, including metals. Thus far, knowledge of the fish intestine is limited largely to in vivo or ex vivo investigations. Recently, however, the first fish intestinal cell line, RTgutGC, was established, originating from a rainbow trout (Oncorhynchus mykiss). In order to exploit the opportunities arising from RTgutGC cells for exploring fish intestinal physiology and toxicology, we present here the establishment of cells on commercially available permeable membrane supports and evaluate its suitability as a model of polarized intestinal epithelia. Within 3 weeks of culture, RTgutGC cells show epithelial features by forming tight junctions and desmosomes between adjacent cells. Cells develop a transepithelial electrical resistance comparable to in vivo measured values, reflecting the leaky nature of the fish intestine. Immunocytochemistry reveals evidence of polarization, such as basolateral localization of Na+/K+-ATPase (NKA) and apical localization of the tight junction protein ZO-1. NKA mRNA abundance was induced as physiological response toward a saltwater buffer, mimicking the migration of rainbow trout from fresh to seawater. Permeation of fluorescent molecules proved the barrier function of the cells, with permeation coefficients being comparable to those reported in fish. Finally, we demonstrate that cells on permeable supports are more resistant to the toxicity elicited by silver ions than cells grown the conventional way, likely due to improved cellular silver excretion

In vitro assessment of modes of toxic action of pharmaceuticals in aquatic life

An ecotoxicological test battery based on a mode-of-action approach was designed and applied to the hazard identification and classification of modes of action of six pharmaceuticals (carbamazepine, diclofenac, ethinyl estradiol, ibuprofen, propranolol, and sulfamethoxazole). The rationale behind the design of the battery was to cover the relevant interactions that a compound may have with biological targets. It is thus not comprehensive but contains representative examples of each category of mode of toxic action including nonspecific, specific, and reactive toxicity. The test battery consists of one test system for nonspecific toxicity (baseline toxicity or narcosis), two test systems for specific effects, and two test systems for reactive toxicity. The baseline toxicity was quantified with the Kinspec test, which detects membrane leakage via measurements of membrane potential. This test system may also be used to detect the specific effects on energy transduction, although this was not relevant to any compound investigated in this study. As examples of specific receptor-mediated toxicity, we chose the yeast estrogen screen (YES) as a specific test for estrogenicity, and the inhibition of chlorophyll fluorescence in algae to assess specific effects on photosynthesis. Reactive modes of action were assessed indirectly by measuring the relevance of cellular defense systems. Differences in growth inhibition curves between a mutant of Escherichia coli that could not synthesize glutathione and its parent strain indicate the relevance of conjugation with glutathione as a defense mechanism, which is an indirect indicator of protein damage. DNA damage was assessed by comparing the growth inhibition in a strain that lacks various DNA repair systems with that in its competent parent strain. Most compounds acted merely as baseline toxicants in all test systems. As expected, ethinylestradiol was the only compound showing estrogenic activity. Propranolol was baseline-toxic in all test systems except for the photosynthesis inhibition assay, where it surprisingly showed a 100-fold excess toxicity over the predicted baseline effect. The exact mode of toxic action could not be confirmed, but additional chlorophyll fluorescence induction experiments excluded the possibility of direct interference with photosynthesis through photosystem II inhibition. Mixture experiments were performed as a diagnostic tool to analyze the mode of toxic action. Compounds with the same mode of toxic action showed the expected concentration addition. In the photosynthesis inhibition assay, agreement between experimental results and prediction was best for two-stage predictions considering the assigned modes of action. In a two-stage prediction, concentration addition was used as a model to predict the mixture effect of the baseline toxicants followed by their independent action as a single component combined with the specifically acting compound propranolol and the reference compound diuron. A comparison with acute toxicity data for algae, daphnia, and fish showed generally good agreement for the nonspecifically acting compounds but also that the proposed test battery offered better diagnostic value in the case of the specifically acting compounds

Monitoring of the ecotoxicological hazard potential by polar organic micropollutants in sewage treatment plants and surface waters using a mode-of-action based test battery

We propose and evaluate a mode-of-action based test battery of low-complexity and in-vitro bioassays that can be used as a routine monitoring tool for sewage treatment efficiency and water quality assessment. The test battery comprises five bioassays covering five different modes of toxic action. The bioluminescence inhibition test with Vibrio fischeri and a growth rate inhibition test with the green algae Pseudokirchneriella subcapitata are measures of non-specific integrative effects. A second endpoint in the algae test, the specific inhibition of the efficiency of photosynthesis, gives an account of the presence of herbicides. An enzymatic assay covers an important aspect of insecticidal activity, the inhibition of the acetylcholine esterase activity. Estrogenic effects are assessed with the yeast estrogen screen (YES) and genotoxicity with the umuC test. Three field studies, each lasting six to seven consecutive days, were undertaken at a sewage treatment plant (STP) in Switzerland. Samples were collected in summer and late autumn, under dry and rainy conditions. None of the bioassays gave positive results with raw water in whole effluent toxicity testing. Therefore, water samples from various sites during wastewater treatment and from surface water were enriched with solid-phase extraction. The focus was on non-volatile compounds of average to moderate hydrophobicity, a range that includes most pesticides, biocides and pharmaceuticals. Various polar solid phases were evaluated for their extraction efficiency, disturbance by matrix components and overall performance. We finally selected a mixture of a polymeric sorbent and a C18-sorbent, Lichrolut EN and RP-18 or, alternatively, Empore SDB-RPS disks. All bioassays gave clear and robust responses with the SPE extracts. With the bioassay data the treatment efficiency of the STP can be assessed with respect to different modes of toxic action and accordingly different groups of micropollutants. Furthermore, the data allowed for a comparison between the effluent and the receiving river. In all bioassays the primary effluent had a strong effect and this effect was reduced after passing the STP. Treatment efficiency was high (typically over 90%) but varied from bioassay to bioassay, which is expected because each bioassay detects different types of micropollutants and therefore we cannot expect a common answer

Screening test battery for pharmaceuticals in urine and wastewater

A test battery for identifying ecotoxicological hazards was applied to six pharmaceuticals (carbamazepine, diclofenac, ethinylestradiol, ibuprofen, propranolol, and sulfamethoxazole), to their mixtures, and to urine spiked with pharmaceuticals to test the suitability of biotests for screening urine and wastewater and for monitoring the efficiency of wastewater treatment. The test battery comprised the bioluminescence inhibition test with Vibrio fischeri, the yeast estrogen screen, and a photosynthesis inhibition assay in algae based on chlorophyll fluorescence measurements. Mixture and additional experiments with a cocktail of pharmaceuticals added to urine confirmed the applicability of the test systems as an integrated measure of the overall micropollutant burden. Because the concentration of pharmaceuticals in wastewater is low and the nutrients and salts may have a negative impact on the bioassays, urine and wastewater samples were cleaned and concentrated by solid-phase extraction (SPE). The compounds of interest ranged from polar to nonpolar and from positively charged to neutral and negatively charged. Consequently, the SPE method was optimized for universality rather than for specificity. Results of preliminary experiments with raw and treated urine and wastewater indicate the suitability of the proposed test battery for screening urine and wastewater

Membrane-water partitioning, membrane permeability, and baseline toxicity of the parasiticides ivermectin, albendazole, and morantel

A comparative hazard assessment of the antiparasitics ivermectin, albendazole, and morantel was performed, with a particular focus on bioavailability and uptake into biological membranes. The experimentally determined liposome-waterdistribution ratio at pH 7 (D^sub lipw^ (pH 7)) of the positively charged morantel was 100 L/kg lipid. The D^sub lipw^ (pH 7) of albendazole was 3,000 L/kg lipid. The membrane permeability determined with the parallel artificial membrane permeability assay was consistent with predictions from a quantitative structure-activity relationship (QSAR) for morantel but 14-fold lower than predicted for albendazole which can be rationalized because neutral albendazole is, in fact, zwitterionic and the large dipole moment hinders permeation through hydrophobic membranes. An unusually large molecule, ivermectin was suspected to show decreased bioaccumulation because of its bulkiness, but experimental determination of solubility showed that it was 40-fold less soluble than expected from a QSAR between solubility and the octanol-water partition coefficient. In contrast, its membrane permeability appeared to be typical for a compound of the given hydrophobicity, but it was not possible to determine the membrane-water partition coefficient because of its low solubility and high affinity to the dialysis membrane of the experimental device. The D^sub lipw^ (pH 7) for ivermectin of 2,700 L/kg lipid was calculated with a QSAR model. Morantel and albendazole were baseline toxicants in the bioluminescence inhibition test with Vibrio fischeri and a test for inhibition of photosynthesis in green algae. Only ivermectin exhibited a specific effect toward algae, but the excess toxicity was not very pronounced and might be biased by the uncertainty of the estimated hydrophobicity descriptor. Overall, we did not find any unexpected effect on nontarget endpoint

Toxic effects of substituted p-benzoquinones and hydroquinones in in vitro bioassays are altered by reactions with the cell assay medium

Substituted para-benzoquinones and hydroquinones are ubiquitous transformation products that arise during oxidative water treatment of phenolic precursors, for example through ozonation or chlorination. The benzoquinone structural motive is associated with mutagenicity and carcinogenicity, and also with induction of the oxidative stress response through the Nrf2 pathway. For either endpoint, toxicological data for differently substituted compounds are scarce. In this study, oxidative stress response, as indicated by the AREc32 in vitro bioassay, was induced by differently substituted para-benzoquinones, but also by the corresponding hydroquinones. Bioassays that indicate defense against genotoxicity (p53RE-bla) and DNA repair activity (UmuC) were not activated by these compounds. Stability tests conducted under incubation conditions, but in the absence of cell lines, showed that tested para-benzoquinones reacted rapidly with constituents of the incubation medium. Compounds were abated already in phosphate buffer, but even faster in biological media, with reactions attributed to amino- and thiol-groups of peptides, proteins, and free amino acids. The products of these reactions were often the corresponding substituted hydroquinones. Conversely, differently substituted hydroquinones were quantitatively oxidized to p-benzoquinones over the course of the incubation. The observed induction of the oxidative stress response was attributed to hydroquinones that are presumably oxidized to benzoquinones inside the cells. Despite the instability of the tested compounds in the incubation medium, the AREc32 in vitro bioassay could be used as an unspecific sum parameter to detect para-benzoquinones and hydroquinones in oxidatively treated waters.ISSN:0043-1354ISSN:1879-244

Transformation of Contaminant Candidate List (CCL3) compounds during ozonation and advanced oxidation processes in drinking water: Assessment of biological effects

The removal of emerging contaminants during water treatment is a current issue and various technologies are being explored. These include UV- and ozone-based advanced oxidation processes (AOPs). In this study, AOPs were explored for their degradation capabilities of 25 chemical contaminants on the US Environmental Protection Agency's Contaminant Candidate List 3 (CCL3) in drinking water. Twenty-three of these were found to be amenable to hydroxyl radical-based treatment, with second-order rate constants for their reactions with hydroxyl radicals ((OH)-O-center dot) in the range of 3-8 x 10(9) M-1 s(-1). The development of biological activity of the contaminants, focusing on mutagenicity and estrogenicity, was followed in parallel with their degradation using the Ames and YES bioassays to detect potential changes in biological effects during oxidative treatment. The majority of treatment cases resulted in a loss of biological activity upon oxidation of the parent compounds without generation of any form of estrogenicity or mutagenicity. However, an increase in mutagenic activity was detected by oxidative transformation of the following CCL3 parent compounds: nitrobenzene ((OH)-O-center dot, UV photolysis), quinoline ((OH)-O-center dot, ozone), methamidophos ((OH)-O-center dot), N-nitrosopyrolidine ((OH)-O-center dot), N-nitrosodi-n-propylamine ((OH)-O-center dot), aniline (UV photolysis), and N-nitrosodiphenylamine (UV photolysis). Only one case of formation of estrogenic activity was observed, namely, for the oxidation of quinoline by (OH)-O-center dot. Overall, this study provides fundamental and practical information on AOP-based treatment of specific compounds of concern and represents a framework for evaluating the performance of transformation-based treatment processes. (C) 2016 Elsevier Ltd. All rights reserved