RESUMO
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.
Assuntos
Benzoquinonas , Hidroquinonas , Benzoquinonas/toxicidade , Bioensaio , Oxirredução , Fenóis , QuinonasRESUMO
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.
Assuntos
Modelos Biológicos , Testes de Toxicidade/métodos , Animais , Bioensaio , Linhagem Celular , Meios de Cultura , Embrião não Mamífero/efeitos dos fármacos , Interações Hidrofóbicas e Hidrofílicas , Microalgas/efeitos dos fármacos , Oncorhynchus mykiss , Reprodutibilidade dos Testes , Peixe-Zebra/embriologiaRESUMO
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.
Assuntos
Mucosa Intestinal/citologia , Mucosa Intestinal/metabolismo , Oncorhynchus mykiss/metabolismo , Animais , Técnicas de Cultura de Células , Linhagem Celular , Permeabilidade da Membrana Celular/efeitos dos fármacos , Mucosa Intestinal/efeitos dos fármacos , Modelos Animais , Prata/toxicidade , Testes de ToxicidadeRESUMO
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) in the range of 3-8 × 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, UV photolysis), quinoline (OH, ozone), methamidophos (OH), N-nitrosopyrolidine (OH), N-nitrosodi-n-propylamine (OH), 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. 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.
Assuntos
Água Potável/química , Radical Hidroxila/química , Ozônio/química , Poluentes Químicos da Água/química , Purificação da Água/métodos , Compostos de Anilina/química , Compostos de Anilina/toxicidade , Cinética , Estrutura Molecular , Mutagênicos/química , Mutagênicos/toxicidade , Nitrobenzenos/química , Nitrobenzenos/toxicidade , Nitrosaminas/química , Nitrosaminas/toxicidade , Oxirredução , Fotólise/efeitos da radiação , Quinolinas/química , Quinolinas/toxicidade , Testes de Toxicidade/métodos , Raios Ultravioleta , Estados Unidos , United States Environmental Protection Agency , Poluentes Químicos da Água/toxicidadeRESUMO
A diverse mix of micropollutants, including pesticides, biocides, and pharmaceuticals, reaches the aquatic environment through treated sewage effluents. We sampled 21 final effluents with polar organic chemical integrative samplers (POCIS) and investigated to what extent chemical analyses of six photosystem II (PS-II) inhibitors and 12 other chemicals explain the toxic burdens quantified with two bioassays. Baseline toxicity equivalent concentrations (TEQ) were determined with a bacterial bioluminescence inhibition assay using Vibrio fischeri (baseline-TEQ(bacteria)) and by assessing toxicity on algal growth using Pseudokirchneriella subcapitata (baseline-TEQ(algae)). Inhibition PS-II was also determined with algae and expressed using diuron equivalent concentrations (DEQ(bio)). Concentrations of chemicals and toxicities varied appreciably between effluents, typically spanning two orders of magnitude. Across 21 independent effluents, a DEQ calculated by concentration addition of PS-II inhibitors (DEQ(chem)) proved a very good predictor of DEQ(bio); DEQ(chem) explained 65% of DEQ(bio). However, baseline-TEQ(bacteria,bio) correlated poorly with baseline-TEQ(algae,bio), because baseline-TEQ(algae) were strongly influenced by PS-II inhibitors. Using data on the 18 quantified compounds, and their estimated toxicities in the bacterial assay, we calculated a baseline-TEQ(bacteria,chem). With one exception, a site with a high load of diclofenac, less than 1% of baseline-TEQ(bacteria,bio) was explained by the analyzed chemicals. We conclude that for the analyses of final effluents, DEQ(bio) is a robust endpoint and useful screening tool for PS-II inhibitors; in the presence of herbicides, baseline-TEQ(bacteria,bio) proves a more robust measure of baseline toxicity than baseline-TEQ(algae,bio).
Assuntos
Aliivibrio fischeri/efeitos dos fármacos , Clorófitas/efeitos dos fármacos , Compostos Orgânicos/toxicidade , Esgotos/química , Poluentes Químicos da Água/toxicidade , Compostos Orgânicos/análise , Complexo de Proteína do Fotossistema II/antagonistas & inibidores , Testes de Toxicidade , Poluentes Químicos da Água/análiseRESUMO
Tamiflu (oseltamivir ethylester) is an antiviral agent for the treatment of influenza A and B. The pro-drug Tamiflu is converted in the human body to the pharmacologically active metabolite, oseltamivir acid, with a yield of 75%. Oseltamivir acid is indirectly photodegradable and slowly biodegradable in sewage works and sediment/water systems. A previous environmental risk assessment has concluded that there is no bioaccumulation potential of either of the compounds. However, little was known about the ecotoxicity of the metabolite. Ester hydrolysis typically reduces the hydrophobicity and thus the toxicity of a compound. In this case, a zwitterionic, but overall neutral species is formed from the charged parent compound. If the speciation and predicted partitioning into biological membranes is considered, the metabolite may have a relevant contribution to the overall toxicity. These theoretical considerations triggered a study to investigate the toxicity of oseltamivir acid (OA), alone and in binary mixtures with its parent compound oseltamivir ethylester (OE). OE and OA were found to be baseline toxicants in the bioluminescence inhibition test with Vibrio fischeri. Their mixture effect lay between predictions for concentration addition and independent action for the mixture ratio excreted in urine and nine additional mixture ratios of OE and OA. In contrast, OE was an order of magnitude more toxic than OA towards algae, with a more pronounced effect when the direct inhibition of photosystem II was used as toxicity endpoint opposed to the 24h growth rate endpoint. The binary mixtures in this assay yielded experimental mixture effects that agreed with predictions for independent action. This is consistent with the finding that OE exhibits slightly enhanced toxicity, while OA acts as baseline toxicant. Therefore, with respect to mixture classification, the two compounds can be considered as acting according to different modes of toxic action, although there are indications that the difference is a toxicokinetic effect, not a true difference of mechanism of toxicity. The general mixture results illustrate the need to consider the role of metabolites in the risk assessment of pharmaceuticals. However, in the concentration ratio of parent to metabolite excreted by humans, the experimental results confirm that the active metabolite does not significantly contribute to the risk quotient of the mixture.
Assuntos
Antivirais/toxicidade , Oseltamivir/toxicidade , Poluentes Químicos da Água/toxicidade , Aliivibrio fischeri/efeitos dos fármacos , Animais , Antivirais/química , Carpas/fisiologia , Clorófitas/efeitos dos fármacos , Cyprinidae/fisiologia , Daphnia/efeitos dos fármacos , Dose Letal Mediana , Oseltamivir/análogos & derivados , Oseltamivir/química , Poluentes Químicos da Água/químicaRESUMO
Tertiary treatment of wastewater with ozone is a promising technique for removing residual micropollutants that remain after secondary biological treatment. We monitored the performance of a full-scale ozonation reactor on a sewage treatment plant in Switzerland with a screening battery of bioassays. Six toxicity endpoints were selected that covered non-specific toxicity, as well as selected receptor-mediated modes of action and reactive toxicity. Non-specific toxicity was assessed with two bioassays, the bioluminescence inhibition of the marine luminescent bacterium Vibrio Fischeri and the growth inhibition of the green algae Pseudokirchneriella subcapitata. Treatment efficiency was around 90% for the secondary treatment, but only 65% and 76% for the ozonation step in the two non-specific endpoints, respectively. This finding is consistent with this type of oxidation reaction because ozone only modifies the organic molecules but does not mineralize them fully leaving residual toxicity of the transformation products. In contrast, the specific receptor-mediated endpoints of inhibition of photosystem II in algae and estrogenicity were largely reduced by ozonation. While compounds inhibiting photosynthesis proved to be rather recalcitrant toward biological treatment with only 47% removal, an additional 86% removal by ozonation yielded an overall treatment efficiency in the entire treatment chain of 89%. The effect on estrogenicity, quantified with the yeast estrogen screen, was even more significant: A treatment efficiency of 95% in the secondary treatment, 86% during ozonation plus a small effect by biological sand filtration yielded an overall treatment efficiency of 99.5%. Insecticides that inhibit acetylcholinesterase were fairly resistant to degradation, but an overall treatment efficiency of 91% was achieved in two steps: 72% in biological treatment and 60% during ozonation. Finally, no significant genotoxicity was observed with the umuC test after ozonation, while the influent showed a genotoxic response when it was enriched by a factor of 15 to 60. Treatment efficiency increased with the ozone dose and remained virtually unchanged over ozone doses above 500 g ozone per kg dissolved organic carbon. The reduction of toxicity can be rationalized by the chemical oxidation processes likely to occur for each group of chemicals that are typical for a given mode of toxic action. For comparison, tertiary treatment with powdered activated carbon was also evaluated, which poses a viable alternative to ozonation with respect to removal of micropollutants.
Assuntos
Ozônio/química , Esgotos/química , Eliminação de Resíduos Líquidos/métodos , Poluentes Químicos da Água/isolamento & purificação , Aliivibrio fischeri/efeitos dos fármacos , Aliivibrio fischeri/crescimento & desenvolvimento , Bioensaio , Clorófitas/efeitos dos fármacos , Clorófitas/crescimento & desenvolvimento , Monitoramento Ambiental , Estrogênios/isolamento & purificação , Inseticidas/isolamento & purificação , Suíça , Testes de Toxicidade , Poluentes Químicos da Água/toxicidadeRESUMO
Passive sampling is a tool to monitor the presence and concentrations of micropollutants in the aquatic environment. We investigated the duration of integrative sampling and the effects of flow rate on the performance of three configurations of the Chemcatcher - a sampler for polar organic compounds. Chemcatchers were fitted with Empore styrenedivinylbenzene (SDB) XC disks (XC), SDB-RPS disks (RPS) or SDB-RPS disks covered with a polyethersulfone membrane (RPS-PES). Samplers were either exposed to treated sewage effluent for 5 days at various flow rates, or at a single flow rate with overlapping exposures of 3-24 days. Chemical analysis focused on a set of pharmaceuticals and biocides and ecotoxicological analysis measured inhibition of photosystem II in algae. For compounds with logK(OW)>2, both XC and RPS disks respond dynamically to higher flow rates; uptake increased up to five-fold when flow increased from 0.03 to 0.37ms(-1). At a flow rate of 0.13ms(-1) the integrative window of SDB disks approached 6 days for more hydrophobic compounds (logK(OW)>3.5). The RPS-PES configuration was less affected by flow and also showed an extended integrative window (up to 24 days). The membrane causes a lag phase of up to 2.3 days which thwarts a sound interpretation of data from sampling periods of less than 10 days.
Assuntos
Desinfetantes/análise , Herbicidas/análise , Estudos de Amostragem , Esgotos/análise , Toxicologia/métodos , Desinfetantes/química , Ecossistema , Monitoramento Ambiental/métodos , Eucariotos/fisiologia , Herbicidas/química , Membranas Artificiais , Polímeros , SulfonasRESUMO
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.
Assuntos
Aliivibrio fischeri/efeitos dos fármacos , Clorófitas/efeitos dos fármacos , Testes de Toxicidade , Poluentes da Água/toxicidade , Diurona/toxicidade , Complexo de Proteína do Fotossistema II/antagonistas & inibidores , Relação Quantitativa Estrutura-AtividadeRESUMO
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.
Assuntos
Aliivibrio fischeri/efeitos dos fármacos , Clorófitas/efeitos dos fármacos , Testes de Toxicidade , Poluentes da Água/toxicidade , Inibidores da Colinesterase/análise , Estrogênios/análise , Testes de Mutagenicidade , Fotossíntese/efeitos dos fármacos , Esgotos/química , Poluentes da Água/químicaRESUMO
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-water distribution ratio at pH 7 (D(lipw) (pH 7)) of the positively charged morantel was 100 L/kg lipid. The D(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(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 endpoints.
Assuntos
Albendazol/toxicidade , Antiparasitários/toxicidade , Permeabilidade da Membrana Celular , Ivermectina/toxicidade , Morantel/toxicidade , Albendazol/química , Albendazol/farmacocinética , Animais , Antiparasitários/química , Antiparasitários/farmacocinética , Relação Dose-Resposta a Droga , Estabilidade de Medicamentos , Peixes/metabolismo , Ivermectina/química , Ivermectina/farmacocinética , Lipossomos/química , Morantel/química , Morantel/farmacocinética , Relação Quantitativa Estrutura-Atividade , SolubilidadeRESUMO
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.
Assuntos
Antagonistas Adrenérgicos beta/toxicidade , Clorófitas/efeitos dos fármacos , Fotossíntese/efeitos dos fármacos , Testes de Toxicidade/métodos , Poluentes Químicos da Água/toxicidade , Antagonistas Adrenérgicos beta/metabolismo , Atenolol/toxicidade , Determinação de Ponto Final , Humanos , Lipossomos/metabolismo , Metoprolol/toxicidade , Propranolol/toxicidade , Relação Quantitativa Estrutura-Atividade , Medição de RiscoRESUMO
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 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.
Assuntos
Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos , Cadeia Alimentar , Modelos Teóricos , Poluentes Químicos da Água/toxicidade , Animais , Daphnia , Eucariotos , Peixes , Medição de Risco , Testes de ToxicidadeRESUMO
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.