Toxicokinetics of Polar Chemicals in Zebrafish Embryo (Danio rerio): Influence of Physicochemical Properties and of Biological Processes.

The time-resolved uptake of 17 nonionic and ionic polar compounds (logD ≤ 2) with a diversity of functional groups into zebrafish embryos (ZFE) was studied over 96 h of exposure. Among them were pharmaceuticals, pesticides and plant active ingredients. Uptake rates for the diffusion controlled passive uptake through the ZFE membrane ranged from 0.02 to 24 h(-1) for the nonionic compounds and were slower for ionic compounds (<0.008-0.08 h(-1)). The study compounds did not enrich much in the ZFE (median bioconcentration factor of 1, max. 7). Biotransformation significantly influenced the internal concentration of some of the test compounds over time (benzocaine, phenacetin, metribuzin, phenytoin, thiacloprid, valproic acid). For benzocaine, valproic acid and phenacetin several transformation products (TPs) were observed by LC-MS already at early life-stages (before 28 hpf); for benzocaine the TPs comprised >90% of the initial amount taken up into the ZFE. For six compounds internal concentrations remained very low (rel. int. conc. < 0.2). Besides biotransformation (sulfamethoxazole), poor membrane permeability (cimetidine, colchicine) and also affinity to efflux transporters (atropine and chloramphenicol) are the likely reasons for these low internal concentrations. This study outlines that the uptake of polar compounds into ZFE is influenced by their physicochemical properties. However, biological processes, biotransformation and, likely, efflux can strongly affect the internal concentrations already in early developmental stages of the ZFE. This should be considered in future toxicokinetic modeling. The evaluation of the toxicity of chemicals by ZFE requires toxicokinetic studies of the test compounds and their TPs to increase comparability to effects in fish.

A quantitative HPLC-MS/MS method for studying internal concentrations and toxicokinetics of 34 polar analytes in zebrafish (Danio rerio) embryos.

An analytical method using high-performance liquid chromatography-tandem mass spectrometry was developed to determine internal concentrations of 34 test compounds such as pharmaceuticals and pesticides in zebrafish embryos (ZFE), among them, cimetidine, 2,4-dichlorophenoxyacetic acid, metoprolol, atropine and phenytoin. For qualification and quantification, multiple reaction monitoring mode was used. The linear range extends from 0.075 ng/mL for thiacloprid and metazachlor and 7.5 ng/mL for coniine and clofibrate to 250 ng/mL for many of the test compounds. Matrix effects were strongest for nicotine, but never exceeded ±20 % for any of the developmental stages of the ZFE. Method recoveries ranged from 90 to 110 % from an analysis of nine pooled ZFE. These findings together with the simple sample preparation mean this approach is suitable for the determination of internal concentrations from only nine individual ZFE in all life stages up to 96 h post-fertilization. Exemplarily, the time course of the internal concentrations of clofibric acid, metribuzin and benzocaine in ZFE was studied over 96 h, and three different patterns were distinguished, on the basis of the speed and extent of uptake and whether or not a steady state was reached. Decreasing internal concentrations may be due to metabolism in the ZFE.

Potential of nature-based solutions to reduce antibiotics, antimicrobial resistance, and pathogens in aquatic ecosystems. a critical review.

This comprehensive scientific review evaluates the effectiveness of nature-based solutions (NBS) in reducing antibiotics (ABs), combating antimicrobial resistance (AMR), and controlling pathogens in various aquatic environments at different river catchment levels. It covers conventional and innovative treatment wetland configurations for wastewater treatment to reduce pollutant discharge into the aquatic ecosystems as well as exploring how river restoration and saltmarshes can enhance pollutant removal. Through the analysis of experimental studies and case examples, the review shows NBS's potential for providing sustainable and cost-effective solutions to improve the health of aquatic ecosystems. It also evaluates the use of diagnostic indicators to predict NBS effectiveness in removing specific pollutants such as ABs and AMR. The review concludes that NBS are feasible for addressing the new challenges stemming from human activities such as the presence of ABs, AMR and pathogens, contributing to a better understanding of NBS, highlighting success stories, addressing knowledge gaps, and providing recommendations for future research and implementation.

A European perspective on alternatives to animal testing for environmental hazard identification and risk assessment.

Tests with vertebrates are an integral part of environmental hazard identification and risk assessment of chemicals, plant protection products, pharmaceuticals, biocides, feed additives and effluents. These tests raise ethical and economic concerns and are considered as inappropriate for assessing all of the substances and effluents that require regulatory testing. Hence, there is a strong demand for replacement, reduction and refinement strategies and methods. However, until now alternative approaches have only rarely been used in regulatory settings. This review provides an overview on current regulations of chemicals and the requirements for animal tests in environmental hazard and risk assessment. It aims to highlight the potential areas for alternative approaches in environmental hazard identification and risk assessment. Perspectives and limitations of alternative approaches to animal tests using vertebrates in environmental toxicology, i.e. mainly fish and amphibians, are discussed. Free access to existing (proprietary) animal test data, availability of validated alternative methods and a practical implementation of conceptual approaches such as the Adverse Outcome Pathways and Integrated Testing Strategies were identified as major requirements towards the successful development and implementation of alternative approaches. Although this article focusses on European regulations, its considerations and conclusions are of global relevance.

Neurotoxicity in complex environmental mixtures-a case-study at River Danube in Novi Sad (Serbia) using zebrafish embryos.

Acetylcholinesterase (AChE) inhibitors are an important class of neuroactive chemicals that are often detected in aquatic and terrestrial environments. The correct functionality of the AChE enzyme is linked to many important physiological processes such as locomotion and respiration. Consequently, it is necessary to develop new analytical strategies to identify harmful AChE inhibitors in the environment. It has been shown that mixture effects and oxidative stress may jeopardize the application of in vivo assays for the identification of AChE inhibitors in the environment. To confirm that in vivo AChE assays can be successfully applied when dealing with complex mixtures, an extract from river water impacted by non-treated wastewater was bio-tested using the acute toxicity fish embryo test (FET) and AChE inhibition assay with zebrafish. The zebrafish FET showed high sensitivity for the extract (LC10 = relative extraction factor 2.8) and we observed a significant inhibition of the AChE (40%, p < 0.01) after 4-day exposure. Furthermore, the extract was chromatographically fractionated into a total of 26 fractions to dilute the mixture effect and separate compounds according to their physico-chemical properties. As expected, non-specific acute effects (i.e., mortality) disappeared or evenly spread among the fractions, while AChE inhibition was still detected in five fractions. Chemical analysis did not detect any known AChE inhibitors in these active fractions. These results confirm that the AChE assay with Danio rerio can be applied for the detection of neuroactive effects induced in complex environmental samples, but also, they highlight the need to increase analytical and identification techniques for the detection of neurotoxic substances.

Concentration-response concept in ecotoxicoproteomics: effects of different phenanthrene concentrations to the zebrafish (Danio rerio) embryo proteome.

Concentration-response experiments, based on the testing of less replicates in favour of more exposure concentrations, represent the typical design of choice applied in toxicological and ecotoxicological effect assessment studies using traditional endpoints such as lethality. However, to our knowledge this concept has not found implementation in the increasingly applied OMICS techniques studying thousands of molecular endpoints at the same time. The present study is among the first applying the concentration-response concept for an ecotoxicoproteomics study. The effects of six different concentrations in the low effect range (

Assessing Combined Effects for Mixtures of Similar and Dissimilar Acting Neuroactive Substances on Zebrafish Embryo Movement.

Risk assessment of chemicals is usually conducted for individual chemicals whereas mixtures of chemicals occur in the environment. Considering that neuroactive chemicals are a group of contaminants that dominate the environment, it is then imperative to understand the combined effects of mixtures. The commonly used models to predict mixture effects, namely concentration addition (CA) and independent action (IA), are thought to be suitable for mixtures of similarly or dissimilarly acting components, respectively. For mixture toxicity prediction, one important challenge is to clarify whether to group neuroactive substances based on similar mechanisms of action, e.g., same molecular target or rather similar toxicological response, e.g., hyper- or hypoactivity (effect direction). We addressed this by using the spontaneous tail coiling (STC) of zebrafish embryos, which represents the earliest observable motor activity in the developing neural network, as a model to elucidate the link between the mechanism of action and toxicological response. Our objective was to answer the following two questions: (1) Can the mixture models CA or IA be used to predict combined effects for neuroactive chemical mixtures when the components share a similar mode of action (i.e., hyper- or hypoactivity) but show different mechanism of action? (2) Will a mixture of chemicals where the components show opposing effect directions result in an antagonistic combined effect? Results indicate that mixture toxicity of chemicals such as propafenone and abamectin as well as chlorpyrifos and hexaconazole that are known to show different mechanisms of action but similar effect directions were predictable using CA and IA models. This could be interpreted with the convergence of effects on the neural level leading to either a collective activation or inhibition of synapses. We also found antagonistic effects for mixtures containing substances with opposing effect direction. Finally, we discuss how the STC may be used to amend risk assessment.

Automated measurement of the spontaneous tail coiling of zebrafish embryos as a sensitive behavior endpoint using a workflow in KNIME.

Neuroactive substances are the largest group of chemicals detected in European surface waters. Mixtures of neuroactive substances occurring at low concentrations can induce adverse neurological effects in humans and organisms in the environment. Therefore, there is a need to develop new screening tools to detect these chemicals. Measurement of behavior or motor effects in rodents and fish are usually performed to assess potential neurotoxicity for risk assessment. However, due to pain and stress inflicted on these animals, the scientific community is advocating for new alternative methods based on the 3R principle (reduce, replace and refine). As a result, the behavior measurement of early stages of zebrafish embryos such as locomotor response, photomotor response and spontaneous tail coiling are considered as a valid alternative to adult animal testing. In this study, we developed a workflow to investigate the spontaneous tail coiling (STC) of zebrafish embryos and to accurately measure the STC effect in the KNIME software. We validated the STC protocol with 3 substances (abamectin, chlorpyrifos-oxon and pyracostrobin) which have different mechanisms of action. The KNIME workflow combined with easy and cost-effective method of video acquisition makes this STC protocol a valuable method for neurotoxicity testing.•Video acquisition duration of 60 s at 25 ± 1 hpf was used•20 embryos exposed per dish and acclimatized for 30 min before video acquisition•Capability to inspect and correct errors for high accuracy.

Pesticides are the dominant stressors for vulnerable insects in lowland streams.

Despite elaborate regulation of agricultural pesticides, their occurrence in non-target areas has been linked to adverse ecological effects on insects in several field investigations. Their quantitative role in contributing to the biodiversity crisis is, however, still not known. In a large-scale study across 101 sites of small lowland streams in Central Europe, Germany we revealed that 83% of agricultural streams did not meet the pesticide-related ecological targets. For the first time we identified that agricultural nonpoint-source pesticide pollution was the major driver in reducing vulnerable insect populations in aquatic invertebrate communities, exceeding the relevance of other anthropogenic stressors such as poor hydro-morphological structure and nutrients. We identified that the current authorisation of pesticides, which aims to prevent unacceptable adverse effects, underestimates the actual ecological risk as (i) measured pesticide concentrations exceeded current regulatory acceptable concentrations in 81% of the agricultural streams investigated, (ii) for several pesticides the inertia of the authorisation process impedes the incorporation of new scientific knowledge and (iii) existing thresholds of invertebrate toxicity drivers are not protective by a factor of 5.3 to 40. To provide adequate environmental quality objectives, the authorisation process needs to include monitoring-derived information on pesticide effects at the ecosystem level. Here, we derive such thresholds that ensure a protection of the invertebrate stream community.

Biochemical, metabolic, and behavioural responses and recovery of Daphnia magna after exposure to an organophosphate.

The responses of various suborganismal and organismal endpoints of Daphnia magna to pulse exposure to sublethal levels of the organophosphate paraoxon-methyl were compared. The changes and recovery of biochemical, metabolic, and behavioural variables, as well as physiological responses, were observed. The cholinesterase (ChE), filtration, and swimming activities were all affected in a concentration-dependent manner, and these effects reached significance at concentrations of 1.0, 1.5, and 0.7 microg L(-1), respectively. The levels of these variables recovered significantly after detoxification for 24h in clean medium. ChE and swimming activities were affected significantly by lower concentrations of paraoxon-methyl than filtration activity, which had the same threshold as the physiological responses ((15)N abundance and body size). This study showed that among the parameters studied, swimming activity was the most sensitive, whereas changes in filtration activity had the most significant physiological consequences, and were therefore important in terms of effects propagation to the population level.

Optimization of the spontaneous tail coiling test for fast assessment of neurotoxic effects in the zebrafish embryo using an automated workflow in KNIME®.

Neuroactive chemicals are frequently detected in the environment. At sufficiently high concentrations or within mixtures, they could provoke neurotoxic effects and neurological diseases to organisms and humans. Fast identification of such neuroactive compounds in the environment could help in hazard assessment and risk mitigation. Behavior change is considered as an important endpoint and might be directly or indirectly connected to a neuroactive mode of action. For a fast evaluation of environmental samples and pure substances, we optimized the measurement of a behavioral endpoint in zebrafish embryos - the spontaneous tail coiling (STC). Evaluation of results is automated via the use of a workflow established with the KNIME® software. Analysis duration and developmental stage were optimized to 1 min and 25 ± 1 hpf respectively during measurement. Exposing the embryos in a group of 10 or 20 and acclimatizing for 30 min at room temperature proved to be reliable. The optimized method was used to investigate neurotoxic effects of 18 substances with different modes of action (MoA). The STC test accurately detected the effect of 8 out of 11 neuroactive substances (chlorpyrifos, chlorpyrifos-oxon, diazinon, paraoxon-methyl, abamectin, carbamazepine, propafenone and diazepam). Aldicarb and nicotine showed subtle effects which were considered to be conditional and imidacloprid showed no effect. For substances with unknown neuroactive MoA, 3 substances did not provoke any effect on the STC (pyraclostrobin, diuron and daunorubicin-hydrochloride) while 4 other substances provoked an increased STC (hexaconazole, aniline, dimethyl-sulfoxide and 3,4-dichloroaniline). Such unexpected effects indicate possible neuroactive side effects or unknown mechanisms of action that impact on the STC. In conclusion, the optimized STC parameters and the automated analysis in KNIME® indicate opportunities for the harmonization of the STC test and further development for prospective and diagnostic testing.

Effects of Five Substances with Different Modes of Action on Cathepsin H, C and L Activities in Zebrafish Embryos.

Cathepsins have been proposed as biomarkers of chemical exposure in the zebrafish embryo model but it is unclear whether they can also be used to detect sublethal stress. The present study evaluates three cathepsin types as candidate biomarkers in zebrafish embryos. In addition to other functions, cathepsins are also involved in yolk lysosomal processes for the internal nutrition of embryos of oviparous animals until external feeding starts. The baseline enzyme activity of cathepsin types H, C and L during the embryonic development of zebrafish in the first 96 h post fertilisation was studied. Secondly, the effect of leupeptin, a known cathepsin inhibitor, and four embryotoxic xenobiotic compounds with different modes of action (phenanthrene-baseline toxicity; rotenone-an inhibitor of electron transport chain in mitochondria; DNOC (Dinitro-ortho-cresol)-an inhibitor of ATP synthesis; and tebuconazole-a sterol biosynthesis inhibitor) on in vivo cathepsin H, C and L total activities have been tested. The positive control leupeptin showed effects on cathepsin L at a 20-fold lower concentration compared to the respective LC50 (0.4 mM) of the zebrafish embryo assay (FET). The observed effects on the enzyme activity of the four other xenobiotics were not or just slightly more sensitive (factor of 1.5 to 3), but the differences did not reach statistical significance. Results of this study indicate that the analysed cathepsins are not susceptible to toxins other than the known peptide-like inhibitors. However, specific cathepsin inhibitors might be identified using the zebrafish embryo.

How to deal with lipophilic and volatile organic substances in microtiter plate assays.

Microtiter plate-based assays are a promising technique for toxicity assessment of substances. Chemicals with physicochemical properties such as high volatility and/or high lipophilicity, however, can be lost from the exposure solution during an experiment, so that exposure concentrations are not consistent. The aim of the present study was to determine and reduce the proportion of the reference compounds phenanthrene and phenanthridine lost during exposure in the zebra fish (Danio rerio) embryo test regime. It could be shown that under the standard exposure regime (48 h), the concentration of phenanthrene decreased strongly, by more than 99%, whereas that of phenanthridine decreased by 17% during a 48-h experiment. After modifications to the microtiter plate exposure regime, the phenanthrene concentration showed a decrease of only 40%, while the phenanthridine concentration remained unchanged. The major processes of substance loss could be assigned to accumulations of these substances into the glue of commercially available adhesive foils and the polystyrene walls of the microtiter plates. Furthermore, by investigating the sorption capacity of different plastics, it was found that the phenanthrene concentration decreased less when using a plexiglass specimen (28%) compared with the same-sized polystyrene specimen (94%). Moreover, it was found, for a constant exposure regime, that concentration profiles of different phenanthrene concentrations in the microtiter plate assay during an experiment were similar. A mathematical method is proposed to predict concentration profiles in an exposure solution by scaling a determined profile.

Oxygen decline in biotesting of environmental samples--is there a need for consideration in the acute zebrafish embryo assay?

Environmental samples such as groundwater, sediment pore water, native or freeze dried sediments may be difficult to analyze for toxic effects with organismic aquatic bioassays. These samples might evoke low oxygen concentration or oxygen depletion during the test. The toxicity assessment could thus be confounded by low oxygen concentrations. The acute zebrafish embryo assay was used to analyze the influence of oxygen deficit on the embryonic development in the first 48 h post fertilization. Embryos were exposed to varying oxygen concentrations ranging from <30 to >80% oxygen saturation of water. A clear concentration dependent retardation of fish embryo development was observed. Because of a retarded development toxic thresholds of environmental samples which might include substances slowing down the development will be altered. For the purpose of identification of critical contaminants in complex environmental samples, it is proposed to actively aerate environmental samples which are likely to be oxygen depleted during the duration of the zebrafish embryo bioassay.

The zebrafish embryo model in environmental risk assessment--applications beyond acute toxicity testing.

BACKGROUND, AIM, AND SCOPE: The use of fish embryos is not regulated by current legislations on animal welfare and is therefore considered as a refinement, if not replacement of animal experiments. Fish embryos represent an attractive model for environmental risk assessment of chemicals since they offer the possibility to perform small-scale, high-throughput analyses. MAIN FEATURES: Beyond their application for determining the acute toxicity, fish embryos are also excellent models for studies aimed at the understanding of toxic mechanisms and the indication of possible adverse and long-term effects. Therefore, we have reviewed the scientific literature in order to indicate alternative applications of the fish embryo model with focus on embryos of the zebrafish. RESULTS AND DISCUSSIONS: The analysis of the mode of action is important for the risk assessment of environmental chemicals and can assist in indicating adverse and long-term effects. Toxicogenomics present a promising approach to unravel the potential mechanisms. Therefore, we present examples of the use of zebrafish embryos to study the effect of chemicals on gene and protein patterns, and the potential implications of differential expression for toxicity. The possible application of other methods, such as kinase arrays or metabolomic profiling, is also highlighted. Furthermore, we show examples of toxicokinetic studies (bioconcentration, ABC transporters) and discuss limitations that might be caused by the potential barrier function of the chorion. Finally, we demonstrate that biomarkers of endocrine disruption, immune modulation, genotoxicity or chronic toxicity could be used as indicators or predictors of sub-acute and long-term effects. CONCLUSIONS: The zebrafish embryo represents a model with an impressive range of possible applications in environmental sciences. Particularly, the adaptation of molecular, system-wide approaches from biomedical research is likely to extend its use in ecotoxicology. RECOMMENDATIONS AND PERSPECTIVES: Challenges for future research are (1) the identification of further suitable molecular markers as indicators of the mode of action, (2) the establishment of strong links between (molecular) effects in short-term assays in embryos and long-term (toxic) effects on individuals, (3) the definition of limitations of the model and (4) the development of tests that can be used for regulatory purposes.

Suborganismic and organismic effects of aldicarb and its metabolite aldicarb-sulfoxide to the zebrafish embryo (Danio rerio).

The new European chemical regulation (REACH) requires a short-term fish test for chemicals where the level of production exceeds 10tons per year. For ethical reasons (3R-concept), an alternative to the acute fish test should be introduced to decrease the number of animal testing with fish. The zebrafish embryo (Danio rerio) test became a valuable tool in ecotoxicology and already replaces the acute fish test for the evaluation of wastewater in Germany. Recent efforts are targeted to use this and other fish embryo tests for the effect assessment of chemicals. The toxic effects of the carbamate insecticide aldicarb and its metabolite aldicarb-sulfoxide to zebrafish embryos were analysed using two approaches with different endpoints. Organismic tests were conducted with zebrafish embryos exposed to the pesticides for 48h. In addition, suborganismic effects were examined analysing the enzyme inhibition of cholinesterases and carboxylesterases. On the organismic level, the only sublethal effect seen was the increase of heart rate at low and decrease at higher concentration with the use of aldicarb-sulfoxide but not with aldicarb (concentration range 0.2-300microM). In contrast, analysis of enzyme inhibitions showed high to very high effects caused by the two carbamates. The enzyme inhibition analysis of whole homogenates of exposed embryos may be advantageous for toxicant screening (biomarker of exposure) and might be used to bridge the gap of sensitivity of the (48h old) zebrafish embryos to adult fish when exposed to anti-cholinesterase substances (biomarker of prospective effect).

Biotransformation in the zebrafish embryo -temporal gene transcription changes of cytochrome P450 enzymes and internal exposure dynamics of the AhR binding xenobiotic benz[a]anthracene.

Not much is known about the biotransformation capability of zebrafish (Danio rerio) embryos. For understanding possible toxicity differences to adult fish, it might be crucial to understand the biotransformation of chemicals in zebrafish embryos i.e. as part of toxicokinetics. The biotransformation capabilities were analysed for two different stages of zebrafish embryos in conjunction with the internal concentrations of a xenobiotic. Zebrafish embryos of the late cleavage/early blastula period (2-26 hpf) and the early pharyngula period (26-50 hpf) were exposed for 24 h to the AhR binding compound benz[a]anthracene (BaA). Time dependent changes in cyp transcription (cyp1a, cyp1b1, cyp1c1 and cyp1c2) as well as concentration & time-dependent courses of BaA in the fish embryo and the exposure medium were analysed. Additionally, the CYP mediated formation of biotransformation products was investigated. We found correlations between transcriptional responses and the internal concentration for both exposure types. These correlations were depending on the start of the exposure i.e. the age of the exposed embryo. While no significant induction of the examined gene transcripts was observed in the first 12 h of exposure beginning in the blastula period a correlation was apparent when exposure started later i.e. in the pharyngula period. A significant induction of cyp1a was detected already after 1.5 h of BaA exposure. Gene transcripts for cyp1b1, cyp1c1 and cyp1c2 showed expressions distinctly different from cyp1a and were, in general, less inducible by BaA in both exposure windows. The toxicokinetic analysis showed that the biotransformation capability was fivefold higher in the older fish embryos. Biotransformation products of phase I reactions were found between 32 hpf and 50 hpf and were tentatively identified as benz[a]anthracene-phenol and benz[a]anthracene-dihydrodiol-epoxide. In conclusion, not only duration but also onset of exposure in relation to the developmental stage of zebrafish embryos is important in the analysis and interpretation of effects due to different biotransformation capabilities.

Effect-based assessment of toxicity removal during wastewater treatment.

Wastewaters contain complex mixtures of chemicals, which can cause adverse toxic effects in the receiving environment. In the present study, the toxicity removal during wastewater treatment at seven municipal wastewater treatment plants (WWTPs) was investigated using an effect-based approach. A battery of eight bioassays was applied comprising of cytotoxicity, genotoxicity, endocrine disruption and fish embryo toxicity assays. Human cell-based CALUX assays, transgenic larval models and the fish embryo toxicity test were particularly sensitive to WWTP effluents. The results indicate that most effects were significantly reduced or completely removed during wastewater treatment (76-100%), while embryo toxicity, estrogenic activity and thyroid disruption were still detectable in the effluents suggesting that some harmful substances remain after treatment. The responsiveness of the bioassays was compared and the human cell-based CALUX assays showed highest responsiveness in the samples. Additionally, the fish embryo toxicity test and the transgenic larval models for endocrine disrupting effects showed high responsiveness at low sample concentrations in nearly all of the effluent samples. The results showed a similar effect pattern among all WWTPs investigated, indicating that the wastewater composition could be rather similar at different locations. There were no considerable differences in the toxicity removal efficiencies of the treatment plants and no correlation was observed with WWTP characteristics, such as process configuration or sludge age. This study demonstrated that a biotest battery comprising of multiple endpoints can serve as a powerful tool when assessing water quality or water treatment efficiency in a holistic manner. Rather than analyzing the concentrations of a few selected chemicals, bioassays can be used to complement traditional methods of monitoring in the future by assessing sum-parameter based effects, such as mixture effects, and tackling chemicals that are present at concentrations below chemical analytical detection limits.

Mixture toxicity of water contaminants-effect analysis using the zebrafish embryo assay (Danio rerio).

Three water contaminants were selected to be tested in the zebrafish embryo toxicity test (DarT) in order to investigate the sensitivity of the zebrafish embryo toxicity test with respect to mixture effect detection. The concentration-response curves for the observed effects lethality and hypo-pigmentation were calculated after an exposure of the embryos for 96 h with a fungicide (carbendazim), a plasticizer or propellent precursor (2,4-DNT: 2,4- dinitrotoluene) and an aromatic compound (AαC: 2-amino-9H-pyrido[2,3-b]indol), respectively. Follow-up mixture tests were based on the calculated LC50 or EC50 of the single compounds and combined effects were predicted according to the mixture concepts of concentration addition (CA) and independent action (IA). The order of toxicity for the single substances was carbendazim (LC50 = 1.25 µM) < AαC (LC50 = 8.16 µM) < 2,4-DNT (LC50 = 177.05 µM). For AαC and 2,4 DNT hypo-pigmentation was observed in addition (AαC EC50 = 1.81 µM; 2,4-DNT EC50 = 8.81 µM). Two binary and one ternary mixture were studied on lethality and one on hypo-pigmentation: 2,4-DNT/AαC (LC50 = 119.21 µM, EC50 = 5.37 µM), carbendazim/AαC (LC50 = 4.49 µM) and AαC/Carbendazim/2,4 DNT (LC50 = 108.62 µM). Results showed that the effects were in agreement with the CA model when substances were tested in mixtures. Therefore, in a reasonable worst case scenario substance combination effects in fish embryos were at maximum only prone to overestimation when using CA as the mixture concept.

Metabolism of clofibric acid in zebrafish embryos (Danio rerio) as determined by liquid chromatography-high resolution-mass spectrometry.

The zebrafish embryo (ZFE) is increasingly used in ecotoxicology research but detailed knowledge of its metabolic potential is still limited. This study focuses on the xenobiotic metabolism of ZFE at different life-stages using the pharmaceutical compound clofibric acid as study compound. Liquid chromatography with quadrupole-time-of-flight mass spectrometry (LC-QToF-MS) is used to detect and to identify the transformation products (TPs). In screening experiments, a total of 18 TPs was detected and structure proposals were elaborated for 17 TPs, formed by phase I and phase II metabolism. Biotransformation of clofibric acid by the ZFE involves conjugation with sulfate or glucuronic acid, and, reported here for the first time, with carnitine, taurine, and aminomethanesulfonic acid. Further yet unknown cyclization products were identified using non-target screening that may represent a new detoxification pathway. Sulfate containing TPs occurred already after 3h of exposure (7hpf), and from 48h of exposure (52hpf) onwards, all TPs were detected. The detection of these TPs indicates the activity of phase I and phase II enzymes already at early life-stages. Additionally, the excretion of one TP into the exposure medium was observed. The results of this study outline the high metabolic potential of the ZFE with respect to the transformation of xenobiotics. Similarities but also differences to other test systems were observed. Biotransformation of test chemicals in toxicity testing with ZFE may therefore need further consideration.