Eberhard Küster

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

Background, aim, and scopeThe 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 featuresBeyond 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 discussionsThe 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.ConclusionsThe 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 perspectivesChallenges 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.

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.

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.

Identification of toxic products of anthracene photomodification in simulated sunlight

Currently, the evidence of a rapid photomodification of anthracene under sunlight resulting in enhanced toxicity exists; however, the chemical causes of toxicity are still unknown. The present study aimed at filling this gap by irradiation of an anthracene suspension with simulated sunlight and subsequent effect-directed fractionation and analysis of toxic products with respect to the inhibition of bacterial energy metabolism of Vibrio fischeri, reproduction of the green algae Scenedesmus vacuolatus, and genotoxicity in the umuC test. Algal toxicity of anthracene was hardly modified by irradiation prior to testing and distributed over all fractions with emphasis on the fractions containing anthracene-9,10-dione and a photometabolite suggested to be 10-hydroxyanthrone. Bacterial toxicity and genotoxicity in contrast emerged only when anthracene was irradiated. Anthracene-1,4-dione, a so-far-unknown trace photometabolite, was identified as a very potent toxicant dominating the toxicity of photomodified anthracene to V. fischeri. In genotoxic fractions, 1-hydroxyanthracene-9,10-dione and 1,4-dihydroxyanthracene-9,10-dione were identified and confirmed as genotoxicants. The results stress the potential of effect-directed analysis approaches in contrast to mere chemical analysis in studies aiming at toxicologically relevant photomodified substances.

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.

The internal concentration of organic substances in fish embryos—A toxicokinetic approach

In ecotoxicity assessment, the ambient exposure concentration is typically applied to quantify the toxic potential of xenobiotic substances. However, exposure and organism-related differences in bioconcentration often cause a considerable variability of toxicity data. This can be minimized by using the internal organism concentration, because toxicokinetic modifying factors are considered implicitly. In the present study, the relationship between ambient and internal concentration-time profiles was investigated for zebrafish (Danio rerio) embryos. The aim was to gain a better understanding and interpretation of exposure-based methods using this model organism. For this purpose, a simple and effective approach to determine the internal concentration was developed. Embryos were exposed to a series of 4 neutral organic substances (naphthalene, fluorene, fluoranthene, benz[a]anthracene) of different hydrophobicity for 72 h. The internal and ambient concentrations were measured at 8 to 9 time points. Kinetics of uptake and elimination were modeled using a first-order 1-compartment model. Biotransformation processes appeared to influence the internal concentrations of fluoranthene and benz[a]anthracene after 48 h. The bioconcentration factors (BCFs) obtained are in excellent agreement with those determined in previous studies using radiolabeled substances. The method demonstrated in the present study is a further step toward a refined ecotoxicity assessment using fish embryos, which links toxicity to the chemical concentration within the organism. This system may also be considered as an alternative to animal testing for BCF determination.

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 (<LC₂₀) of the PAH phenanthrene to the proteome of the ecotoxicological vertebrate model zebrafish (Danio rerio) embryo were investigated (two replicates per concentration) after 5 days exposure. Proteomics analyses were performed on organism extracts using 2-DE DIGE. Protein abundance profiles of around 713 protein spots were studied. About one-third of the protein signals could be detected to show robust reactions correlating with stressor concentration. Within this group, 65 protein signals showed significant changes compared to controls already at 1% lethal concentration (LC₀₁). Interestingly, 28 proteins significantly reacted at very low concentrations (<LC₀₁) and showed an exposure concentration dependent regulation status. Characteristic protein spots were identified by mass spectrometry. With the results of the present study the utility and several benefits using a concentration-response approach in proteomics studies could be shown. These included (i) knowledge about and the ability to model concentration dependent dynamics of molecular endpoints, (ii) to gain information about sensitivity of the molecular response in comparison to traditional endpoints and (iii) to help selecting the most promising protein spots for further investigations such as protein identification and biomarker studies. Using this experimental design based on testing of several exposure concentrations and less replicates might provide a step forward in getting increased output from toxicoproteomics studies.

A novel in vitro system for the determination of bioconcentration factors and the internal dose in zebrafish (Danio rerio) eggs

In this study a novel in vitro approach for the determination of bioconcentration factors (BCF) and rate constants of lipophilic substances utilizing zebrafish (Danio rerio) eggs is presented. Zebrafish eggs were exposed in a static exposure regime towards a phenanthrene solution and concentration-time profiles of the exposure solutions were analyzed over time. The rate constants and the BCF were obtained from the concentration-time profile with the use of a least-square fit to a non-linear model. The determined BCF at steady-state (after 72h of exposure) for phenanthrene was estimated to be only about 1.5 times lower, than the respective BCF value reported in the literature. For uptake of solutes in zebrafish embryos, different transport processes are assumed as substances have to pass the chorion first and subsequently the membranes of the embryo. To investigate this, the period to steady-state concentration between zebrafish eggs and the ambient medium for phenanthrene under an agitated and non-agitated static exposure regime were compared. It was found, that this equilibrium was reached within a shorter time frame under agitation, resulting in higher rate constants. In addition to the determination of bioconcentration parameters, the internal phenanthrene dose in zebrafish eggs was determined by utilizing a biomimetic extraction method with water as transfer medium. Approximately 55% of the expected accumulated phenanthrene amount in zebrafish eggs could be re-extracted with a silicone rod extraction method. These results agree very well to what has been observed in abiotic systems. The scope of the proposed in vitro protocol to serve as an alternative for BCF determinations using established in vivo animal testing protocols with adult fish is discussed.

Application of preparative capillary gas chromatography (pcGC), automated structure generation and mutagenicity prediction to improve effect-directed analysis of genotoxicants in a contaminated groundwater

Background, aim and scopeThe importance of groundwater for human life cannot be overemphasised. Besides fulfilling essential ecological functions, it is a major source of drinking water. However, in the industrial area of Bitterfeld, it is contaminated with a multitude of harmful chemicals, including genotoxicants. Therefore, recently developed methodologies including preparative capillary gas chromatography (pcGC), MOLGEN-MS structure generation and mutagenicity prediction were applied within effect-directed analysis (EDA) to reduce sample complexity and to identify candidate mutagens in the samples. A major focus was put on the added value of these tools compared to conventional EDA combining reversed-phase liquid chromatography (RP-LC) followed by GC/MS analysis and MS library search.Materials and methodsWe combined genotoxicity testing with umuC and RP-LC with pcGC fractionation to isolate genotoxic compounds from a contaminated groundwater sample. Spectral library information from the NIST05 database was combined with a computer-based structure generation tool called MOLGEN-MS for structure elucidation of unknowns. Finally, we applied a computer model for mutagenicity prediction (ChemProp) to identify candidate mutagens and genotoxicants.Results and discussionA total of 62 components were tentatively identified in genotoxic fractions. Ten of these components were predicted to be potentially mutagenic, whilst 2,4,6-trichlorophenol, 2,4-dichloro-6-methylphenol and 4-chlorobenzoic acid were confirmed as genotoxicants.Conclusions and perspectivesThe results suggest pcGC as a high-resolution fractionation tool and MOLGEN-MS to improve structure elucidation, whilst mutagenicity prediction failed in our study to predict identified genotoxicants. Genotoxicity, mutagenicity and carcinogenicity caused by chemicals are complex processes, and prediction from chemical structure still appears to be quite difficult. Progress in this field would significantly support EDA and risk assessment of environmental mixtures.

Comparison of cholin- and carboxylesterase enzyme inhibition and visible effects in the zebra fish embryo bioassay under short-term paraoxon-methyl exposure

Abstract The acute zebra fish embryo test (Danio rerio Hamilton–Buchanan, 1822) is an accepted bioassay to assess the toxicity of waste water that may be used for the replacement of testing with adult fish. It is also suggested for chemical hazard characterization and assessment, although only a few groups of substances have yet been studied. Specifically acting substances such as neurotoxic insecticides pose a potentially hazard for non-target fish. To establish whether the proposed zebra fish embryo test protocol and the inhibition of cholinesterases (acetylcholinesterase EC 3.1.1.7, propionylcholinesterase EC 3.1.1.8) and carboxylesterase (EC 3.1.1.1) enzymes can be used in a similar fashion for hazard characterization and risk assessment of chemicals and environmental samples, two types of experiments were conducted. Visual effects of exposure to the organophosphate metabolite paraoxon-methyl after 24 and 48 h in the zebra fish embryo test system were analysed with the use of an inverse microscope (rate of mortality, developmental disturbances, heart rate and others). The inhibition to cholinesterases and carboxylesterase was also measured. Enzyme inhibition as a biomarker of exposure was about 70 times more sensitive than the effects in the zebra fish embryo test with an IC50 below 1.2 µmol compared with an EC50 of 91 µmol. The dose–response relationships showed different curve characteristics with a linear increase of enzyme inhibition compared with a sigmoidal curve for the overt effects. Significant overt effects could only be seen at concentrations at which already 80% of the activities of the different esterases were inhibited.