Potential of the zebrafish (Danio rerio) embryo test to discriminate between chemicals of similar molecular structure-a study with valproic acid and 14 of its analogues.

Valproic acid is a frequently used antiepileptic drug and known pediatric hepatotoxic agent. In search of pharmaceuticals with increased effectiveness and reduced toxicity, analogue chemicals came into focus. So far, toxicity and teratogenicity data of drugs and metabolites have usually been collected from mammalian model systems such as mice and rats. However, in an attempt to reduce mammalian testing while maintaining the reliability of toxicity testing of new industrial chemicals and drugs, alternative test methods are being developed. To this end, the potential of the zebrafish (Danio rerio) embryo to discriminate between valproic acid and 14 analogues was investigated by exposing zebrafish embryos for 120 h post fertilization in the extended version of the fish embryo acute toxicity test (FET; OECD TG 236), and analyzing liver histology to evaluate the correlation of liver effects and the molecular structure of each compound. Although histological evaluation of zebrafish liver did not identify steatosis as the prominent adverse effect typical in human and mice, the structure-activity relationship (SAR) derived was comparable not only to human HepG2 cells, but also to available in vivo mouse and rat data. Thus, there is evidence that zebrafish embryos might serve as a tool to bridge the gap between subcellular, cell-based systems and vertebrate models.

Multistate models of developmental toxicity: Application to valproic acid-induced malformations in the zebrafish embryo.

For the determination of acute toxicity of chemicals in zebrafish (Danio rerio) embryos, the OECD test guideline 236, relative to the Fish Embryo Toxicity Test (FET), stipulates a dose-response analysis of four lethal core endpoints and a quantitative characterization of abnormalities including their time-dependency. Routinely, the data are analyzed at the different observation times separately. However, observations at a given time strongly depend on the previous effects and should be analyzed jointly with them. To solve this problem, we developed multistate models for occurrence of developmental malformations and live events in zebrafish embryos exposed to eight concentrations of valproic acid (VPA) the first five days of life. Observations were recorded daily per embryo. We statistically infer on model structure and parameters using a numerical Bayesian framework. Hatching probability rate changed with time and we compared five forms of its time-dependence; a constant rate, a piecewise constant rate with a fixed hatching time at 48 h post fertilization, a piecewise constant rate with a variable hatching time, as well as a Hill and Gaussian form. A piecewise constant function of time adequately described the hatching data. The other transition rates were conditioned on the embryo body concentration of VPA, obtained using a physiologically-based pharmacokinetic model. VPA impacted mostly the malformation probability rate in hatched and non-hatched embryos. Malformation reversion probability rates were lowered by VPA. Direct mortality was low at the concentrations tested, but increased linearly with internal concentration. The model makes full use of data and gives a finer grain analysis of the teratogenic effects of VPA in zebrafish than the OECD-prescribed approach. We discuss the use of the model for obtaining toxicological reference values suitable for inter-species extrapolation. A general result is that complex multistate models can be efficiently evaluated numerically.

Pre-validation of choriogenin H transgenic medaka eleutheroembryos as a quantitative estrogenic activity test method.

The choriogenin H - EGFP transgenic medaka (Oryzias melastigma) has been used to test estrogenic substances and quantify estrogenic activity into 17ß-estradiol (E2) equivalency (EEQ). The method uses 8 eleutheroembryos in 2 ml solution per well and 3 wells per treatment in 24-well plates at 26 ± 1 °C for 24 ± 2 h, with subsequent measurements of induced GFP signal intensity. EEQ measurements are calculated using a E2 probit regression model with a coefficient of determination (R2) > 0.90. The selectivity was confirmed evaluating 27 known estrogenic and 5 known non-estrogenic compounds. Limit of quantitation (LOQ), recovery rate and bias were calculated to be 1 ng/ml EEQ, 104% and 4% respectively. Robustness analysis revealed exposure temperature is a sensitive parameter that should be kept at 26 ± 1 °C. The repeatability of intra- and inter-laboratories achieved CV < 30% for most tested food and cosmetics samples. The lot-lot stability was confirmed by the stable EEQ qualitative control (QC, 1 ng/mL E2) and calibration curve results. The stability of standard reagents, samples and sample extracts was also investigated. These data demonstrated this method to be an accurate indicator of estrogenic activity for both chemicals and extracts.

Cytochrome P450-dependent biotransformation capacities in embryonic, juvenile and adult stages of zebrafish (Danio rerio)-a state-of-the-art review.

Given the strong trend to implement zebrafish (Danio rerio) embryos as translational model not only in ecotoxicological, but also toxicological testing strategies, there is an increasing need for a better understanding of their capacity for xenobiotic biotransformation. With respect to the extrapolation of toxicological data from zebrafish embryos to other life stages or even other organisms, qualitative and quantitative differences in biotransformation pathways, above all in cytochrome P450-dependent (CYP) phase I biotransformation, may lead to over- or underestimation of the hazard and risk certain xenobiotic compounds may pose to later developmental stages or other species. This review provides a comprehensive state-of-the-art overview of the scientific knowledge on the development of the CYP1-4 families and corresponding phase I biotransformation and bioactivation capacities in zebrafish. A total of 68 publications dealing with spatiotemporal CYP mRNA expression patterns, activities towards mammalian CYP-probe substrates, bioactivation and detoxification activities, as well as metabolite profiling were analyzed and included in this review. The main results allow for the following conclusions: (1) Extensive work has been done to document mRNA expression of CYP isoforms from earliest embryonic stages of zebrafish, but juvenile and adult zebrafish have been largely neglected so far. (2) There is insufficient understanding of how sex- and developmental stage-related differences in expression levels of certain CYP isoforms may impact biotransformation and bioactivation capacities in the respective sexes and in different developmental stages of zebrafish. (3) Albeit qualitatively often identical, many studies revealed quantitative differences in metabolic activities of zebrafish embryos and later developmental stages. However, the actual relevance of age-related differences on the outcome of toxicological studies still needs to be clarified. (4) With respect to current remaining gaps, there is still an urgent need for further studies systematically assessing metabolic profiles and capacities of CYP isoforms in zebrafish. Given the increasing importance of Adverse Outcome Pathway (AOP) concepts, an improved understanding of CYP capacities appears essential for the interpretation and outcome of (eco)toxicological studies.

Neurotoxic effects in zebrafish embryos by valproic acid and nine of its analogues: the fish-mouse connection?

Since teratogenicity testing in mammals is a particular challenge from an animal welfare perspective, there is a great need for the development of alternative test systems. In this context, the zebrafish (Danio rerio) embryo has received increasing attention as a non-protected embryonic vertebrate in vivo model. The predictive power of zebrafish embryos for general vertebrate teratogenicity strongly depends on the correlation between fish and mammals with respect to both overall general toxicity and more specific endpoints indicative of certain modes-of-action. The present study was designed to analyze the correlation between (1) effects of valproic acid and nine of its analogues in zebrafish embryos and (2) their known neurodevelopmental effects in mice. To this end, zebrafish embryos exposed for 120 h in an extended version of the acute fish embryo toxicity test (FET; OECD TG 236) were analyzed with respect to an extended list of sublethal endpoints. Particular care was given to endpoints putatively related to neurodevelopmental toxicity, namely jitter/tremor, deformation of sensory organs (eyes) and craniofacial deformation, which might correlate to neural tube defects caused by valproic acid in mammals. A standard evaluation of lethal (LC according to OECD TG 236) and sublethal toxicity (EC) merely indicated that four out of ten compounds tested in zebrafish correlate with positive results in mouse in vivo studies. A detailed assessment of more specific effects, however, namely, jitter/tremor, small eyes and craniofacial deformation, resulted in a correspondence of 75% with in vivo mouse data. A refinement of endpoint analysis from an integration of all observations into one LCx or ECx data (as foreseen by current ecotoxicology-driven OECD guidelines) to a differential evaluation of endpoints specific of selected modes-of-action thus increases significantly the predictive power of the zebrafish embryo model for mammalian teratogenicity. However, for some of the endpoints observed, e.g., scoliosis, lordosis, pectoral fin deformation and lack of movement, further experiments are required for the identification of underlying modes-of-action and an unambiguous interpretation of their predictive power for mammalian toxicity.

Norfluoxetine Is the Only Metabolite of Fluoxetine in Zebrafish (Danio rerio) Embryos That Accumulates at Environmentally Relevant Exposure Scenarios.

Fluoxetine has been recognized as one of the most toxic pharmaceuticals in the aquatic environment. Since there is growing evidence that the toxic potential of fluoxetine in surface waters is markedly influenced by its own metabolism in aquatic species, this study investigated the biotransformation of fluoxetine in the zebrafish embryo - an aquatic model organism of intermediate complexity. Zebrafish embryos were exposed to 0.1, 1.0, 10, 50, and 5000 µg/L of fluoxetine from 48 to 120 h post-fertilization (hpf), and the accumulation of fluoxetine and its metabolites was analyzed over time. Additionally, depuration of fluoxetine and its metabolites from 96 to 120 hpf was investigated, and autoinhibitory effects of fluoxetine on phase I biotransformation were analyzed. Exposure to 5000 µg/L fluoxetine resulted in elevated 7-ethoxyresorufin-O-deethylase (EROD) activity of cytochrome P450 enzymes and continuous accumulation of fluoxetine and 11 fluoxetine metabolites. Embryos exposed to 10 and 50 µg/L fluoxetine were able to reduce fluoxetine accumulation from 94 to 120 hpf. During depuration, accumulation of fluoxetine and most metabolites was clearly reduced, and biotransformation shifted in favor of norfluoxetine, the primary fluoxetine metabolite in humans. Findings demonstrated that norfluoxetine is the only metabolite of fluoxetine that accumulates in zebrafish embryos at environmentally relevant exposure scenarios.

Development of a capillary electrophoresis-mass spectrometry method for the analysis of metformin and its transformation product guanylurea in biota.

A method with capillary electrophoresis coupled to mass spectrometry was optimized to determine the uptake of metformin and its metabolite guanylurea by zebrafish (Danio rerio) embryos and brown trout (Salmo trutta f. fario) exposed under laboratory conditions. Metformin was extracted from fish tissues by sonication in methanol, resulting in an absolute recovery of almost 90%. For the extraction of guanylurea from brown trout, solid-phase extraction was implemented with a recovery of 84%. The use of a mixture of methanol and glacial acetic acid as a non-aqueous background electrolyte was vital to achieve robust analysis using a bare fused-silica capillary with an applied voltage of +30 kV. Problems with adsorption associated with an aqueous background electrolyte were eliminated using a non-aqueous background electrolyte made of methanol/acetic acid (97:3) with 25 mM ammonium acetate (for zebrafish embryos) or 100 mM ammonium acetate (for brown trouts), depending on the sample complexity and matrix influences. High resolution and high separation selectivity from matrix components were achieved by optimization of the ammonium acetate concentration in the background electrolyte. An extensive evaluation of matrix effects was conducted with regard to the complex matrices present in the fish samples. They required adapting the background electrolyte to higher concentrations. Applying this method to extracts of zebrafish embryos and brown trout tissue samples, limits of detection for both metformin and guanylurea in zebrafish embryos (12.2 µg/l and 15 µg/l) and brown trout tissues (15 ng/g and 34 ng/g) were in the low µg/l or ng/g range. Finally, metformin and guanylurea could be both quantified for the first time in biota samples from exposure experiments.

The EU-ToxRisk method documentation, data processing and chemical testing pipeline for the regulatory use of new approach methods.

Hazard assessment, based on new approach methods (NAM), requires the use of batteries of assays, where individual tests may be contributed by different laboratories. A unified strategy for such collaborative testing is presented. It details all procedures required to allow test information to be usable for integrated hazard assessment, strategic project decisions and/or for regulatory purposes. The EU-ToxRisk project developed a strategy to provide regulatorily valid data, and exemplified this using a panel of > 20 assays (with > 50 individual endpoints), each exposed to 19 well-known test compounds (e.g. rotenone, colchicine, mercury, paracetamol, rifampicine, paraquat, taxol). Examples of strategy implementation are provided for all aspects required to ensure data validity: (i) documentation of test methods in a publicly accessible database; (ii) deposition of standard operating procedures (SOP) at the European Union DB-ALM repository; (iii) test readiness scoring accoding to defined criteria; (iv) disclosure of the pipeline for data processing; (v) link of uncertainty measures and metadata to the data; (vi) definition of test chemicals, their handling and their behavior in test media; (vii) specification of the test purpose and overall evaluation plans. Moreover, data generation was exemplified by providing results from 25 reporter assays. A complete evaluation of the entire test battery will be described elsewhere. A major learning from the retrospective analysis of this large testing project was the need for thorough definitions of the above strategy aspects, ideally in form of a study pre-registration, to allow adequate interpretation of the data and to ensure overall scientific/toxicological validity.

Microplastic particles reduce EROD-induction specifically by highly lipophilic compounds in RTL-W1 cells.

Microplastic particles (MPs) from lipophilic polymers have been shown to efficiently accumulate hydrophobic organic contaminants (HOCs) in aquatic environments. MPs have, therefore, frequently been discussed as vectors for contaminants, enhancing HOC uptake by various organisms after ingestion followed by pollutant release; however, integrative models of sorption argue against this mechanism and even predict cleansing of pollutants from biological systems under particular circumstances. In order to experimentally investigate such a depuration mechanism, RTL-W1 cells were dosed with three 7-ethoxyresorufin-O-deethylase (EROD) inducers of distinct lipophilicity via the medium before adding both native and hexane-purified polyethylene MPs (20-25 µm) to the medium surface. EROD activity was significantly reduced in the presence of MP, the extent of which correlated with the inducers' lipophilicity (KOW) and thus affinity to MP. For hexane-purged MPs and TCDD (KOW = 6.8), MPs reduce the bioavailability by up to 79%; the effect was marginally weaker with benzo[k]fluoranthene (KOW = 6.11) and almost absent with ß-Naphthoflavone (KOW = 4.68). Compared to hexane-purged MPs, native particles possessed slightly less detoxification potential. These experimental results corroborate theoretically predicted mechanisms of detoxification via MPs. Yet, it is unclear if, under corresponding conditions in the environment, MPs can compete with organismal tissues for highly lipophilic compounds and, if so, to which degree they may act as a sink reducing the amount of bioavailable pollutants in situ. However, the present results suggest that in scenarios where pollutant-free MPs interact with organisms that accumulated HOCs via other routes of uptake, qualitatively the presence of such a mechanism is likely.

In vivo fluorescence-based characterization of cytochrome P450 activity during embryonic development of zebrafish (Danio rerio).

Zebrafish (Danio rerio) early life-stages are increasingly gaining attention as an alternative model in both human and environmental toxicology. Whereas there is amble knowledge about the transcription of various cytochrome P450 isoforms, the level of information about functional implications is still limited. This study investigated the development of CYP2-dependent 7-methoxycoumarin-O-demethylase (MCOD) activity throughout the early zebrafish development from 5 to 118 h post-fertilization (hpf) via confocal laser scanning microscopy. Results demonstrate that zebrafish embryos exhibit constitutive MCOD activity from as early as 5.5 hpf. Characteristic spatiotemporal patterns were documented with MCOD activities localized in several tissues and organs, namely the cardiovascular system, the brain, the digestive system, and the urinary tract. The study thereby contributes to a better understanding of the development and functional role of CYP enzymes in zebrafish early life-stages.

Transformation Products of Fluoxetine Formed by Photodegradation in Water and Biodegradation in Zebrafish Embryos ( Danio rerio).

The present study investigates the transformation of the antidepressant fluoxetine (FLX) by photo- and biodegradation and shows similarities and differences in transformation products (TPs). TPs were identified using LC-high-resolution mass spectrometry with positive and negative electrospray ionization. In a sunlight simulator, photodegradation was carried out using ultrapure water (pH 6, 8, and 10) and surface water (pH 8) to study the effect of direct and indirect photolysis, respectively. The well-known metabolite norfluoxetine (NFLX) proved to be a minor TP in photolysis (≤2% of degraded FLX). In addition, 26 TPs were detected, which were formed by cleavage of the phenolether bond ( O-dealkylation) which primarily formed 3-(methylamino)-1-phenyl-1-propanol (TP 166) and 4-(trifluoromethyl)phenol, by hydroxylation of the benzyl moiety, by CF3 substitution to benzoic aldehyde/acid, and by adduct formation at the amine group ( N-acylation with aldehydes and carboxylic acids). Higher pH favors the neutral species of FLX and the neutral/anionic species of primary TPs and, therefore, photodegradation. In zebrafish embryos, the bioconcentration factor of FLX was found to be 110, and about 1% of FLX taken up by the embryos was transformed to NFLX. Seven metabolites known from photodegradation and formed by hydrolysis, hydroxylation, and N-acylation as well as three new metabolites formed by N-hydroxylation, N-methylation, and attachment of an amine group were identified in zebrafish embryos. The study highlights the importance of considering a broad range of TPs of FLX in fresh water systems and in ecotoxicity tests and to include TP formation in both environmental processes and metabolism in organisms.

Analysis of tail coiling activity of zebrafish (Danio rerio) embryos allows for the differentiation of neurotoxicants with different modes of action.

In (eco)toxicology, there is a critical need for efficient methods to evaluate the neurotoxic potential of environmental chemicals. Recent studies proposed analysis of early coiling activity in zebrafish embryos as a powerful tool for the identification of neurotoxic compounds. In order to demonstrate that the analysis of early tail movements of zebrafish embryos allows for the discrimination of neurotoxicants acting via different mechanisms, the present study investigated the effects of four different neurotoxicants on the embryogenesis (fish embryo toxicity test) and early tail coiling movements of zebrafish embryos. Cadmium predominantly increased the frequency of tail coiling at the late pharyngula stage. Dichlorvos delayed embryonic development and caused convulsive tail movements resulting in prolonged duration of tail coils. Embryos exposed to teratogenic concentrations of fluoxetine and citalopram displayed absence of spontaneous tail movements at 24 h post-fertilization. In contrast, a non-teratogenic test concentration of citalopram decreased coiling frequency at multiple time points. Results demonstrated that the analysis of tail coiling movements of zebrafish embryos has the potential to discriminate neurotoxic compounds with different primary modes of action. In addition, chemical-induced effects on coiling activity were shown to potentially overlap with effects on embryogenesis. Further studies are needed to clarify the interplay of unspecific developmental toxicity of neurotoxic chemicals and effects resulting from specific neurotoxic mechanisms.

Intrafollicular thyroid hormone staining in whole-mount zebrafish (Danio rerio) embryos for the detection of thyroid hormone synthesis disruption.

Endocrine-disrupting chemicals are known to impact multiple hormonal axes of vertebrates, among which the thyroid system is crucial for multiple developmental and physiological processes. Thus, the present study focused on the semi-quantitative visualization of intrafollicular triiodothyronine (T3) and thyroxin (T4) in zebrafish embryos as a potential test system for the detection of disrupted thyroid hormone synthesis. To this end, an antibody-based fluorescence double-staining protocol for whole-mount zebrafish embryos and larvae was adapted to simultaneously detect intrafollicular T3 and T4. During normal development until 10 days post-fertilization (dpf), the number of thyroid follicles increased along the ventral aorta. Concentrations of T4 and T3, measured by fluorescence intensity, increased until 6 dpf, but decreased thereafter. Exposure of zebrafish embryos to propylthiouracil (PTU), a known inhibitor of TH synthesis, resulted in a significant decrease in the number of follicles that stained for T3, whereas a trend for increase in follicles that stained for T4 was observed. In contrast, fluorescence intensity for both thyroid hormones decreased significantly after exposure to PTU. Overall, the zebrafish embryo appears to be suitable for the simultaneous visualization and detection of changing intrafollicular TH contents during normal development and after PTU treatment.

Ultrastructural Alterations in Thyrocytes of Zebrafish ( Danio rerio) after Exposure to Propylthiouracil and Perchlorate.

Histopathology is a widely used approach to evaluate effects of endocrine-active chemicals in the thyroid. However, effects at an ultrastructural level have hardly been examined in fish thyroids. In the present study, zebrafish was exposed to sublethal concentrations of propylthiouracil (PTU; 0-50 mg/L) and perchlorate (PER; 0-5,000 µg/L) for 5 weeks in a modified early life-stage test. None of the treatments caused significant mortality (no observed effect concentrations for survival ≥50 mg/L [PTU] and ≥5,000 µg/L [PER]). PTU induced dose-dependent alterations in the rough endoplasmic reticulum (rER) in all exposure groups, whereas only the 2 highest PER exposure groups (500 and 5,000 µg/L) resulted in alterations of the rER. Both substances caused an increase in the numbers of lysosomes and mitochondria, with mitochondria displaying distorted cristae. Increased mitochondrial diameters were only observed in the PTU treatment. PER-exposed samples displayed an increase in apical microvilli. The highest PTU concentration (50 mg/L) showed first signs of cellular degeneration. Ultrastructural changes in zebrafish thyrocytes thus appear specific for different chemicals, most likely depending on their specific modes of action. Additional knowledge of subcellular changes in thyrocytes can help to better understand and interpret existing histological data in the future.

Adverse outcome pathways: opportunities, limitations and open questions.

Adverse outcome pathways (AOPs) are a recent toxicological construct that connects, in a formalized, transparent and quality-controlled way, mechanistic information to apical endpoints for regulatory purposes. AOP links a molecular initiating event (MIE) to the adverse outcome (AO) via key events (KE), in a way specified by key event relationships (KER). Although this approach to formalize mechanistic toxicological information only started in 2010, over 200 AOPs have already been established. At this stage, new requirements arise, such as the need for harmonization and re-assessment, for continuous updating, as well as for alerting about pitfalls, misuses and limits of applicability. In this review, the history of the AOP concept and its most prominent strengths are discussed, including the advantages of a formalized approach, the systematic collection of weight of evidence, the linkage of mechanisms to apical end points, the examination of the plausibility of epidemiological data, the identification of critical knowledge gaps and the design of mechanistic test methods. To prepare the ground for a broadened and appropriate use of AOPs, some widespread misconceptions are explained. Moreover, potential weaknesses and shortcomings of the current AOP rule set are addressed (1) to facilitate the discussion on its further evolution and (2) to better define appropriate vs. less suitable application areas. Exemplary toxicological studies are presented to discuss the linearity assumptions of AOP, the management of event modifiers and compensatory mechanisms, and whether a separation of toxicodynamics from toxicokinetics including metabolism is possible in the framework of pathway plasticity. Suggestions on how to compromise between different needs of AOP stakeholders have been added. A clear definition of open questions and limitations is provided to encourage further progress in the field.

Assessment of cytotoxicity, genotoxicity and 7-ethoxyresorufin-O-deethylase (EROD) induction in sediment extracts from New Zealand urban estuaries.

Sediments represent a major sink for contaminants resulting from industrial and agricultural activities - especially lipophilic substances. This study exclusively used in vitro methodologies to characterize specific toxicity effects of contaminants in sediment extracts from two urban New Zealand estuaries. Sediment extracts were prepared and tested for a range of biological endpoints. The micronucleus and comet assays in V79 cells were used to assess genotoxicity. Induction of 7-ethoxyresorufin-O-deethylase in piscine RTL-W1 cells was determined to estimate dioxin-like toxicity. Cytotoxic potentials were analyzed by neutral red uptake and MTT reduction. There was evidence of strong dioxin-like toxicity and moderate cytotoxicity. Genotoxicity was distinct in the micronucleus assay, but low in the comet assay. The results indicate the presence of chemicals in the sediments with the potential to pose a risk through multiple mechanisms of toxicity, the identities and amounts of which will be disclosed in a parallel study alongside with in vivo toxicity data.

Reversibility of endocrine disruption in zebrafish (Danio rerio) after discontinued exposure to the estrogen 17α-ethinylestradiol.

The aim of the present study was to investigate the persistence of the feminizing effects of discontinued 17α-ethinylestradiol (EE2) exposure on zebrafish (Danio rerio). An exposure scenario covering the sensitive phase of sexual differentiation, as well as final gonad maturation was chosen to examine the estrogenic effects on sexual development of zebrafish. Two exposure scenarios were compared: continuous exposure to environmentally relevant concentrations (0.1-10 ng/L EE2) up to 100 days post-hatch (dph) and developmental exposure up to 60 dph, followed by 40 days of depuration in clean water. The persistence of effects was investigated at different biological organization levels from mRNA to population-relevant endpoints to cover a broad range of important parameters. EE2 had a strong feminizing and inhibiting effect on the sexual development of zebrafish. Brain aromatase (cyp19b) mRNA expression showed no clear response, but vitellogenin levels were significantly elevated, gonad maturation and body growth were inhibited in both genders, and sex ratios were skewed towards females and undifferentiated individuals. To a large extent, all of these effects were reversed after 40 days of recovery, leading to the conclusion that exposure to the estrogen EE2 results in very strong, but reversible underdevelopment and feminization of zebrafish. The present study is the first to show this reversibility at different levels of organization, which gives better insight into the mechanistic basis of estrogenic effects in zebrafish.

OECD validation study to assess intra- and inter-laboratory reproducibility of the zebrafish embryo toxicity test for acute aquatic toxicity testing.

The OECD validation study of the zebrafish embryo acute toxicity test (ZFET) for acute aquatic toxicity testing evaluated the ZFET reproducibility by testing 20 chemicals at 5 different concentrations in 3 independent runs in at least 3 laboratories. Stock solutions and test concentrations were analytically confirmed for 11 chemicals. Newly fertilised zebrafish eggs (20/concentration and control) were exposed for 96h to chemicals. Four apical endpoints were recorded daily as indicators of acute lethality: coagulation of the embryo, lack of somite formation, non-detachment of the tail bud from the yolk sac and lack of heartbeat. Results (LC50 values for 48/96h exposure) show that the ZFET is a robust method with a good intra- and inter-laboratory reproducibility (CV<30%) for most chemicals and laboratories. The reproducibility was lower (CV>30%) for some very toxic or volatile chemicals, and chemicals tested close to their limit of solubility. The ZFET is now available as OECD Test Guideline 236. Considering the high predictive capacity of the ZFET demonstrated by Belanger et al. (2013) in their retrospective analysis of acute fish toxicity and fish embryo acute toxicity data, the ZFET is ready to be considered for acute fish toxicity for regulatory purposes.

Neurotoxic pesticides change respiratory parameters in early gill-breathing, but not in skin-breathing life-stages of zebrafish (Danio rerio).

Neurotoxic compounds can interfere with active gill ventilation in fish, which might lead to premature death in adult fish, but not in skin-breathing embryos of zebrafish, since these exclusively rely on passive diffusion across the skin. Regarding lethality, this respiratory failure syndrome (RFS) has been discussed as one of the main reasons for the higher sensitivity of adult fish in the acute fish toxicity test (AFT), if compared to embryos in the fish embryo toxicity test (FET). To further elucidate the relationship between the onset of gill respiration and death by a neurotoxic mode of action, a comparative study into oxygen consumption (MO2), breathing frequency (fv) and amplitude (fampl) was performed with 4 d old skin-breathing and 12 d old early gill-breathing zebrafish. Neurotoxic model substances with an LC50 FET/AFT ratio of > 10 were used: chlorpyrifos, permethrin, aldicarb, ziram, and fluoxetine. Exposure to hypoxia served as a positive control, whereas aniline was tested as an example of a narcotic substance interfering non-specifically with gill membranes. In 12 d old larvae, all substances caused an increase in MO2, fv and partly fampl, whereas effects were minor in 4 d old embryos. An increase of fv in 4 d old embryos following exposure to chlorpyrifos, aldicarb and hypoxia could not be correlated with an increased MO2 and might be attributed either to (1) to the successfully postponed decrease of arterial partial pressure of oxygen (PO2) through support of skin respiration by increased fv, (2) to an unspecific stimulation of the sphincter muscles at the base of the gill filaments, or (3) to the establishment of oxygen sensing for later stages. In gill-breathing 12 d old zebrafish, a concentration-dependent increase of fv was detected for aniline and chlorpyrifos, whereas for aldicarb, fluoxetine and permethrin, a decline of fv at higher substance concentrations was measured, most likely due to the onset of paralysis and/or fatigue of the gill filament sphincter muscles. Since alterations of fv serve to postpone the decrease in arterial PO2 and MO2 increased with decreasing fv, the respiratory failure syndrome could clearly be demonstrated in 12 d old zebrafish larvae. Passive respiration across the skin in zebrafish embryos could thus be confirmed as a probable reason for the lower sensitivity of early life-stages to neurotoxicants. Integration of respiratory markers into existing testing protocols with non-protected developmental stages such as embryos might help to not underestimate the toxicity of early life-stages of fish.

Rearing conditions (isolated versus group rearing) affect rotenone-induced changes in the behavior of zebrafish (Danio rerio) embryos in the coiling assay.

Under regulations such as REACH, testing of novel and established compounds for their (neuro)toxic potential is a legal requirement in many countries. These are largely based on animal-, cost-, and time-intensive in vivo models, not in line with the 3 Rs' principle of animal experimentation. Thus, the development of alternative test methods has also received increasing attention in neurotoxicology. Such methods focus either on physiological alterations in brain development and neuronal pathways or on behavioral changes. An example of a behavioral developmental neurotoxicity (DNT) assay is the zebrafish (Danio rerio) embryo coiling assay, which quantifies effects of compounds on the development of spontaneous movement of zebrafish embryos. While the importance of embryo-to-embryo contact prior to hatching in response to environmental contaminants or natural threats has been documented for many other clutch-laying fish species, little is known about the relevance of intra-clutch contacts for zebrafish. Here, the model neurotoxin rotenone was used to assess the effect of grouped versus separate rearing of the embryos on the expression of the coiling behavior. Some group-reared embryos reacted with hyperactivity to the exposure, to an extent that could not be recorded effectively with the utilized software. Separately reared embryos showed reduced activity, compared with group-reared individuals when assessing. However, even the control group embryos of the separately reared cohort showed reduced activity, compared with group-reared controls. Rotenone could thus be confirmed to induce neurotoxic effects in zebrafish embryos, yet modifying one parameter in an otherwise well-established neurotoxicity assay such as the coiling assay may lead to changes in behavior influenced by the proximity between individual embryos. This indicates a complex dependence of the outcome of behavior assays on a multitude of environmental parameters.