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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.

Originally designed as an alternative for the acute fish toxicity test according to, e.g., OECD TG 203, the fish embryo test (FET) with the zebrafish ( Danio rerio ) has been optimized, standardized, and validated during an OECD validation study and adopted as OECD TG 236 as a test to assess toxicity of embryonic forms of fish. Given its excellent correlation with the acute fish toxicity test and the fact that non-feeding developmental stages of fish are not categorized as protected stages according to the new European Directive 2010/63/EU on the protection of animals used for scientific purposes, the FET is ready for use not only for range-finding but also as a true alternative for the acute fish toxicity test, as required for a multitude of national and international regulations. If—for ethical reasons—not accepted as a full alternative, the FET represents at least a refinement in the sense of the 3Rs principle. Objections to the use of the FET have mainly been based on the putative lack of biotransformation capacity and the assumption that highly lipophilic and/or high molecular weight substances might not have access to the embryo due to the protective role of the chorion. With respect to bioactivation, the only substance identified so far as not being activated in the zebrafish embryo is allyl alcohol; all other biotransformation processes that have been studied in more detail so far were found to be present, albeit, in some cases, at lower levels than in adult fish. With respect to larger molecules, the extension of the test duration to 96 h (i.e., beyond hatch) has—at least for the substances tested so far—compensated for the reduced access to the embryo; however, more research is necessary to fully explore the applicability of the FET to substances with a molecular weight >3 kDa as well as substances with a neurotoxic mode of action. An extension of the endpoints to also cover sublethal endpoints makes the FET a powerful tool for the detection of teratogenicity, dioxin-like activity, genotoxicity and mutagenicity, neurotoxicity, as well as various forms of endocrine disruption.

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.

Over the last 10 years, there has been a plethora of experimental studies estimating the potential of microplastic particles (MPs) to exert toxic effects in the environment, many specifically focusing on their postulated capacity to enhance the transfer of environmental pollutants into organisms after ingestion. Obviously, there is little to no consensus on appropriate experimental design, which is mainly owing to the novelty, the interdisciplinarity of the subject, and the complexity of parameters involved. This results in fundamental discrepancies regarding the materials applied, the approach for spiking MPs with pollutants, and the exact exposure scenario. Aiming for a non-chemist audience and providing illustrative, representative, and comparative examples, this review first outlines the theoretical essentials of processes involved in sorption. Also, it discusses the implications for designing experimental approaches using MPs and interpreting the results obtained under consideration of their relevance for environmental conditions. It may help to improve the interpretation of studies on MP toxicity already published, while also calling experimenters’ attention to various aspects important to consider when designing and performing environmentally relevant experiments with MPs.

Background The fish embryo acute toxicity (FET) test with the zebrafish ( Danio rerio ) was developed to assess the acute fish toxicity of chemicals or environmental samples as a replacement for the Acute Fish Test (AFT) with juvenile fish. However, the FET is not yet established in the regulatory context. One reason is the (postulated) difference between the biotransformation capacities of embryos and juvenile fish. The present study was designed to develop a procedure for external metabolization of test substances prior to testing in the FET. The workflow allows simultaneous exposure of the embryos to the maternal substances and their potential metabolites throughout the entire exposure period. After a 2 h incubation of the samples at 37 °C with non-toxic concentrations of a rat liver S9 homogenate or an animal-free (ewoS9R) metabolization system, freshly fertilized zebrafish embryos are added and incubated up to 120 h post-fertilization at 26 °C. Five biotransformable model substances (allyl alcohol, benzo[a]pyrene (B[a]P), chlorpyrifos (CP), tris(1,3-dichloro-2-propyl) phosphate (TDCPP) and bisphenol A (BPA)) were evaluated for embryotoxicity with and without external metabolization. Results Only for allyl alcohol, external metabolization with both rat S9 and ewoS9R resulted in significantly higher embryotoxicity than under non-premetabolized conditions and, thus, in a better correlation of FET and AFT data. For B[a]P, CP, TDCPP and BPA, there was no relevant difference between data derived from the FET (with and without pre-metabolization) and literature AFT data; even though the FET results with and without pre-metabolization differed significantly for BPA (with rat S9 and ewoS9R) and TDCPP (rat S9 only). Conclusions External pre-metabolization appears a promising add-on to the FET protocol to improve the correlation with AFT data of certain biotransformable substances and might help to strengthen the FET as an alternative to the AFT and finally to reduce or replace sentient animals used for acute fish toxicity data in the regulatory context. Graphical Abstract

The fish embryo acute toxicity (FET) test with the zebrafish ( Danio rerio ) embryo according to OECD TG 236 was originally developed as an alternative test method for acute fish toxicity testing according to, e.g., OECD TG 203. Given the versatility of the protocol, however, the FET test has found application beyond acute toxicity testing as a common tool in environmental hazard and risk assessment. Whereas the standard OECD guideline is restricted to four core endpoints (coagulation as well as lack of somite formation, heartbeat, and tail detachment) for simple, rapid assessment of acute toxicity, further endpoints can easily be integrated into the FET test protocol. This has led to the hypothesis that an extended FET test might allow for the identification of different classes of toxicants via a “fingerprint” of morphological observations. To test this hypothesis, the present study investigated a set of 18 compounds with highly diverse modes of action with respect to acute and sublethal endpoints. Especially at higher concentrations, most observations proved toxicant-unspecific. With decreasing concentrations, however, observations declined in number, but gained in specificity. Specific observations may at best be made at test concentrations ≤ EC 10 . The existence of a “fingerprint” based on morphological observations in the FET is, therefore, highly unlikely in the range of acute toxicity, but cannot be excluded for experiments at sublethal concentrations.

In order to develop a test battery based on a variety of neurological systems in fish, three sensory systems (vision, olfaction, and lateral line) as well as nerve transmission (acetylcholine esterase) were analyzed in zebrafish ( Danio rerio ) embryos with respect to their suitability as a model for the screening of neurotoxic trace substances in aquatic ecosystems. As a selection of known or putative neurotoxic compounds, amidotrizoic acid, caffeine, cypermethrin, dichlorvos, 2,4-dinitrotoluene, 2,4-dichlorophenol, 4-nonylphenol, perfluorooctanoic acid, and perfluorooctane sulfonic acid were tested in the fish embryo test (OECD test guideline 236) to determine EC 10 values, which were then used as maximum test concentration in subsequent neurotoxicity tests. Whereas inhibition of acetylcholinesterase was investigated biochemically both in vivo and in vitro (ex vivo), the sensory organs were studied in vivo by means of fluorescence microscopy and histopathology in 72- or 96-h-old zebrafish embryos, which are not regarded as protected developmental stages in Europe and thus — at least de jure — represent alternative test methods. Various steps of optimization allowed this neurotoxicity battery to identify neurotoxic potentials for five out of the nine compounds: Cypermethrin and dichlorvos could be shown to specifically modulate acetylcholinesterase activity; dichlorvos, 2,4-dichlorophenol, 4-nonylphenol, and perfluorooctane sulfonic acid led to a degeneration of neuromasts, whereas both vision and olfaction proved quite resistant to concentrations ≤ EC 10 of all of the model neurotoxicants tested. Comparison of neurotoxic effects on acetylcholinesterase activity following in vivo and in vitro (ex vivo) exposure to cypermethrin provided hints to a specific enzyme-modulating activity of pyrethroid compounds. Enhancement of the neuromast assay by applying a simultaneous double-staining procedure and implementing a 4-scale scoring system (Stengel et al. 2017 ) led to reduced variability of results and better statistical resolution and allowed to differentiate location-dependent effects in single neuromasts. Since acetylcholinesterase inhibition and neuromast degeneration can be analyzed in 72- and 96-h-old zebrafish embryos exposed to neurotoxicants according to the standard protocol of the fish embryo toxicity test (OECD TG 236), the fish embryo toxicity test can be enhanced to serve as a sensitive neurotoxicity screening test in non-protected stages of vertebrates.

Modern breeding has formed a multitude of cattle breeds ranging from undemanding, low-productive breeds to high-productive, specialized dairy, or beef cattle. The choice of breed has important implications for farm management, but its impact on pasture vegetation is underestimated. We hypothesized (i) that anatomy, movement, and foraging behavior of cattle are allometrically related on the individual level, (ii) that differences among cattle are not explained by individual variation alone but also by breed, and (iii) that anatomy, movement, and foraging behavior of a cattle breed is related to its productivity. In order to test these hypotheses, we conducted a controlled grazing experiment in which three cattle breeds simultaneously grazed three types of heterogenous, alpine pastures: low-productive Highland cattle (average weight: 358 kg); local, dual-purpose Original Braunvieh (582 kg); and high-productive Angus × Holstein crossbreed (679 kg). We measured body weight and claw base of nine cows per breed after 10 weeks of grazing alpine pastures. Over a period of 9 days, we recorded the step frequency and lying time by pedometer and space use by GPS. Moreover, we visually observed foraging behavior on three occasions per cow. Forage selectivity and quality were calculated for every cow's diet. Allometric relationships were analyzed on the individual level by fitting standardized major axes. For most parameters measured, we detected strong allometric relationships and clear differences among breeds that depended on the level of productivity. The claws of Highland cattle were relatively large compared to their body weight and thus they exerted less static pressure than other breeds. Moreover, the more productive a breed was, the higher its selectivity and step frequency were. For example, Highland cattle foraged shrubs and thistles more frequently than high-productive Angus × Holstein. The latter walked longer distances to select higher-quality forage, while Highland cattle used the space more evenly, visited steeper slopes, and moved further away from water points. Irrespective of breed, vegetation composition influenced cattle behavior: On pastures of low forage quality, animals walked more, foraged more selectively, and used space less evenly. In conclusion, the observed breed-specific differences can be used to improve pasture management and grassland conservation.

As wide-spread pollutants in the marine environment, microplastics (MPs) have raised public concern about potential toxic effects in aquatic organisms, and, among others, MPs were suspected to act as a vector for organic pollutants to biota. The purpose of the present study was to investigate effects by three model pollutants, oxybenzone (BP3), benzo[a]pyrene (BaP) and perfluorooctane sulfonate (PFOS) adsorbed to polyethylene MPs on the basis of a standard assay, the acute fish embryo toxicity test (FET; OECD TG 236) with zebrafish (Danio rerio) supplemented by additional endpoints such as induction of ethoxyresorufin-O-deethylase (EROD) activity, modification of cyp1a gene transcription and changes in larval swimming behavior. FET assays were performed in three laboratories using slightly different husbandry and exposure conditions, which, however, were all fully compatible with the limits defined by OECD TG 236. This allowed for testing of potential changes in the FET assay due to protocol variations. The standard endpoints of the FET (acute embryotoxicity) did not reveal any acute toxicity for both virgin MPs and MPs spiked with BP3, BaP and PFOS. With respect to sublethal endpoints, EROD activity was increased after exposure to MPs spiked with BP3 (3 h pulse) and MPs spiked with BaP (96 h continuous exposure). Cyp1a transcription was increased upon exposure to MPs spiked with BP3 or BaP. For the selected combination of MPs particles and contaminants, the basic FET proved not sensitive enough to reveal effects of (virgin and spiked) MPs. However, given that the FET can easily be supplemented by a broad variety of more subtle and sensitive endpoints, an enhanced FET protocol may provide a relevant approach with developmental stages of a vertebrate animal model, which is not protected by current EU animal welfare legislation (Directive EU 2010/63).

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.