The toxic equivalency concept is a widely applied method to express the toxicity of complex mixtures of compounds that act via receptor-mediated mechanisms such as induction of the arylhydrocarbon or estrogen receptors. Here we propose to extend this concept to baseline toxicity, using the bioluminescence inhibition test with Vibrio fischeri, and an integrative ecotoxicity endpoint, algal growth rate inhibition. Both bioassays were validated by comparison with literature data and quantitative structure-activity relationships (QSARs) for baseline toxicity were developed for all endpoints. The novel combined algae test, with Pseudokirchneriella subcapitata, allows for the simultaneous evaluation of specific inhibition of photosynthesis and growth rate. The contributions of specific inhibition of photosynthesis and non-specific toxicity could be differentiated by comparing the time and endpoint pattern. Photosynthesis efficiency, measured with the saturation pulse method after 2 h of incubation, served as indicator of specific inhibition of photosynthesis by photosystem II inhibitors. Diuron equivalents were defined as toxicity equivalents for this effect. The endpoint of growth rate over 24 h served to derive baseline toxicity equivalent concentrations (baseline-TEQ). By performing binary mixture experiments with reference compounds and complex environmental samples from a sewage treatment plant and a river, the TEQ concept was validated. The proposed method allows for easier interpretation and communication of effect-based water quality monitoring data and provides a basis for comparative analysis with chemical analytical monitoring.

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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 exceptforthe 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 ...

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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.

We analyzed nontarget effects of the beta-blockers propranolol, metoprolol, and atenolol with a screening test battery encompassing nonspecific, receptor-mediated, and reactive modes of toxic action. All beta-blockers were baseline toxicants and showed no specific effects on energy transduction nor endocrine activity in the yeast estrogen and androgen screen, and no reactive toxicity toward proteins and DNA. However, in a phytotoxicity assay based on the inhibition of the photosynthesis efficiency in green algae, all beta-blockers were 10 times more toxic than their modeled baseline toxicity. Baseline- and phytotoxicity effects increased with hydrophobicity. The beta-blockers showed concentration addition in mixture experiments, indicating a mutual specific nontarget effect on algae. Using literature data and quantitative structure-activity relationships (QSAR), we modeled the total toxic potential of mixtures of the beta-blockers and their associated human metabolites for the phytotoxicity endpoint with two scenarios. The realistic scenario (I) assumes that the metabolites lose their specific activity and act as baseline toxicants. In the worst-case scenario (II) the metabolites exhibitthe same specific mode of action as their parent drug. For scenario (II), metabolism hardly affected the overall toxicity of atenolol and metoprolol, whereas propranolol's hazard potential decreased significantly. In scenario (I), metabolism reduced the apparent EC50 of the mixture of parent drug and metabolite even further. The proposed method is a simple approach to initial hazard assessment of pharmaceuticals and can guide higher tier testing. It can be applied to other classes of pollutants, e.g., biocides, as well as to environmental transformation products of pollutants.

Chicken egg white lysozyme exhibits antimicrobial activity against both Gram‐positive and Gram‐negative bacteria. Fractionation of clostripain‐digested lysozyme yielded a pentadecapeptide with antimicrobial activity but without muramidase activity. The peptide was isolated and its sequence found to be I‐V‐S‐D‐G‐N‐G‐M‐N‐A‐W‐V‐A‐W‐R (amino acids 98–112 of chicken egg white lysozyme). A synthesized peptide of identical sequence had the same bactericidal activity as the natural peptide. Replacement of Trp 108 with tyrosine significantly reduced the antibacterial capacity of the peptide. By replacement of Trp 111 with tyrosine the antibacterial activity was lost. Replacement of Asn 106 with the positively charged arginine strongly increased the antibacterial capacity of I‐V‐S‐D‐G‐N‐G‐M‐N‐A‐W‐V‐A‐W‐R. The peptide I‐V‐S‐D‐G‐N‐G‐M consisting of the eight amino acids of the N‐terminal side had no bactericidal properties, whereas the peptide N‐A‐W‐V‐A‐W‐R of the C‐terminal side retained some bactericidal activity. Replacement of asparagine 106 by arginine (R‐A‐W‐V‐A‐W‐R) increased the bactericidal activity considerably. The D enantiomer of R‐A‐W‐V‐A‐W‐R was as active as the L form against five of the tested bacteria, but substantially less active against Serratia marcescens, Micrococcus luteus,Staphylococcus aureus, Staphylococcus epidermidis and Staphylococcus lentus. For these bacterial species some stereospecific complementarity between receptor structures of the bacteria and the peptide can be assumed.

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.