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.

The generation gap in endocrine disruption: Can the integrated fish endocrine disruptor test (iFEDT) bridge the gap by assessing intergenerational effects of thyroid hormone system disruption?

Thyroid hormones (THs) act early in ontogenesis, even prior to the differentiation of thyrocytes. Maternal transfer of THs is therefore known to play an essential role in early development. Current OECD test guidelines for the assessment of TH system disruption (THSD) do not address inter- or transgenerational effects. The integrated fish endocrine disruptor test (iFEDT), a test combining parental and developmental exposure of filial fish, may fill this gap. We tested the ability of the iFEDT to detect intergenerational effects in zebrafish (Danio rerio): Parental fish were exposed to propylthiouracil (PTU), an inhibitor of TH synthesis, or not exposed. The offspring was submitted to a crossed experimental design to obtain four exposure scenarios: (1) no exposure at all, (2) parental exposure only, (3) embryonic exposure only, and (4) combined parental and embryonic exposure. Swim bladder inflation, visual motor response (VMR) and gene expression of the progeny were analysed. Parental, but not embryonic PTU exposure reduced the size of the swim bladder of 5 d old embryos, indicating the existence of intergenerational effects. The VMR test produced opposite responses in 4.5 d old embryos exposed to PTU vs. embryos derived from exposed parents. Embryonic exposure, but not parental exposure increased gene expression of thyroperoxidase, the target of PTU, most likely due to a compensatory mechanism. The gene expression of pde-6h (phosphodiesterase) was reduced by embryonic, but not parental exposure, suggesting downregulation of phototransduction pathways. Hence, adverse effects on swim bladder inflation appear more sensitive to parental than embryonic exposure and the iFEDT represents an improvement in the testing strategy for THSD.

Development of a Zebrafish Embryo-Based Test System for Thyroid Hormone System Disruption: 3Rs in Ecotoxicological Research.

There is increasing concern regarding pollutants disrupting the vertebrate thyroid hormone (TH) system, which is crucial for development. Thus, identification of TH system-disrupting chemicals (THSDCs) is an important requirement in the Organisation for Economic Co-operation and Development (OECD) testing framework. The current OECD approach uses different model organisms for different endocrine modalities, leading to a high number of animal tests. Alternative models compatible with the 3Rs (replacement, reduction, refinement) principle are required. Zebrafish embryos, not protected by current European Union animal welfare legislation, represent a promising model. Studies show that zebrafish swim bladder inflation and eye development are affected by THSDCs, and the respective adverse outcome pathways (AOPs) have been established. The present study compared effects of four THSDCs with distinct molecular modes of action: Propylthiouracil (PTU), potassium perchlorate, iopanoic acid, and the TH triiodothyronine (T3) were tested with a protocol based on the OECD fish embryo toxicity test (FET). Effects were analyzed according to the AOP concept from molecular over morphological to behavioral levels: Analysis of thyroid- and eye-related gene expression revealed significant effects after PTU and T3 exposure. All substances caused changes in thyroid follicle morphology of a transgenic zebrafish line expressing fluorescence in thyrocytes. Impaired eye development and swimming activity were observed in all treatments, supporting the hypothesis that THSDCs cause adverse population-relevant changes. Findings thus confirm that the FET can be amended by TH system-related endpoints into an integrated protocol comprising molecular, morphological, and behavioral endpoints for environmental risk assessment of potential endocrine disruptors, which is compatible with the 3Rs principle. Environ Toxicol Chem 2024;00:1-18. © 2024 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

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.

Development of the integrated fish endocrine disruptor test (iFEDT)-Part A: Merging of existing fish test guidelines.

There has been increasing interest in endocrine-disrupting chemicals (EDCs) among scientists and public authorities over the last 30 years, notably because of their wide use and the increasing evidence of detrimental effects on humans and the environment. However, test systems for the detection of potential EDCs as well as testing strategies still require optimization. Thus, the aim of the present project was the development of an integrated test protocol that merges the existing OECD test guidelines (TGs) 229 (fish short-term reproduction assay) and 234 (fish sexual development test) and implements thyroid-related endpoints for fish. The integrated fish endocrine disruptor test (iFEDT) represents a comprehensive approach for fish testing, which covers reproduction, early development, and sexual differentiation, and will thus allow the identification of multiple endocrine-disruptive effects in fish. Using zebrafish (Danio rerio) as a model organism, two exposure tests were performed with well-studied EDCs: 6-propyl-2-thiouracil (PTU), an inhibitor of thyroid hormone synthesis, and 17α-ethinylestradiol (EE2), an estrogen receptor agonist. In part A of this article, the effects of PTU and EE2 on established endpoints of the two existing TGs are reported, whereas part B focuses on the novel thyroid-related endpoints. Results of part A document that, as expected, both PTU and EE2 had strong effects on various endocrine-related endpoints in zebrafish and their offspring. Merging of TGs 229 and 234 proved feasible, and all established biomarkers and endpoints were responsive as expected, including reproductive and morphometric changes (PTU and EE2), vitellogenin levels, sex ratio, gonad maturation, and histopathology (only for EE2) of different life stages. A validation of the iFEDT with other well-known EDCs will allow verification of the sensitivity and usability and confirm its capacity to improve the existing testing strategy for EDCs in fish. Integr Environ Assess Manag 2024;20:817-829. © 2023 The Authors. Integrated Environmental Assessment and Management published by Wiley Periodicals LLC on behalf of Society of Environmental Toxicology & Chemistry (SETAC).

Development of the integrated fish endocrine disruptor test-Part B: Implementation of thyroid-related endpoints.

Given the vital role of thyroid hormones (THs) in vertebrate development, it is essential to identify chemicals that interfere with the TH system. Whereas, among nonmammalian laboratory animals, fish are the most frequently utilized test species in endocrine disruptor research, for example, in guidelines for the detection of effects on the sex hormone system, there is no test guideline (TG) using fish as models for thyroid-related effects; rather, amphibians are used. Therefore, the objective of the present project was to integrate thyroid-related endpoints for fish into a test protocol combining OECD TGs 229 (Fish Short-Term Reproduction Assay) and 234 (Fish Sexual Development Test). The resulting integrated Fish Endocrine Disruption Test (iFEDT) was designed as a comprehensive approach to covering sexual differentiation, early development, and reproduction and to identifying disruption not only of the sexual and/or reproductive system but also the TH system. Two 85-day exposure tests were performed using different well-studied endocrine disruptors: 6-propyl-2-thiouracil (PTU) and 17α-ethinylestradiol (EE2). Whereas the companion Part A of this study presents the findings on effects by PTU and EE2 on endpoints established in existing TGs, the present Part B discusses effects on novel thyroid-related endpoints such as TH levels, thyroid follicle histopathology, and eye development. 6-Propyl-2-thiouracil induced a massive proliferation of thyroid follicles in any life stage, and histopathological changes in the eyes proved to be highly sensitive for TH system disruption especially in younger life stages. For measurement of THs, further methodological development is required. 17-α-Ethinylestradiol demonstrated not only the well-known disruption of the hypothalamic-pituitary-gonadal axis, but also induced effects on thyroid follicles in adult zebrafish (Danio rerio) exposed to higher EE2 concentrations, suggesting crosstalk between endocrine axes. The novel iFEDT has thus proven capable of simultaneously capturing endocrine disruption of both the steroid and thyroid endocrine systems. Integr Environ Assess Manag 2024;20:830-845. © 2023 The Authors. Integrated Environmental Assessment and Management published by Wiley Periodicals LLC on behalf of Society of Environmental Toxicology & Chemistry (SETAC).

The sensitivity of the zebrafish embryo coiling assay for the detection of neurotoxicity by compounds with diverse modes of action.

In the aim to determine neurotoxicity, new methods are being validated, including tests and test batteries comprising in vitro and in vivo approaches. Alternative test models such as the zebrafish (Danio rerio) embryo have received increasing attention, with minor modifications of the fish embryo toxicity test (FET; OECD TG 236) as a tool to assess behavioral endpoints related to neurotoxicity during early developmental stages. The spontaneous tail movement assay, also known as coiling assay, assesses the development of random movement into complex behavioral patterns and has proven sensitive to acetylcholine esterase inhibitors at sublethal concentrations. The present study explored the sensitivity of the assay to neurotoxicants with other modes of action (MoAs). Here, five compounds with diverse MoAs were tested at sublethal concentrations: acrylamide, carbaryl, hexachlorophene, ibuprofen, and rotenone. While carbaryl, hexachlorophene, and rotenone consistently induced severe behavioral alterations by ~ 30 h post fertilization (hpf), acrylamide and ibuprofen expressed time- and/or concentration-dependent effects. At 37-38 hpf, additional observations revealed behavioral changes during dark phases with a strict concentration-dependency. The study documented the applicability of the coiling assay to MoA-dependent behavioral alterations at sublethal concentrations, underlining its potential as a component of a neurotoxicity test battery.

A novel automated method for the simultaneous detection of breathing frequency and amplitude in zebrafish (Danio rerio) embryos and larvae.

Stress responses of fish to disruption of oxygen homeostasis include adjusted oxygen consumption rate (MO2) as well as the hyperventilation consisting of changes in breathing frequency (fv) and amplitude (fampl). However, studying the HVR in very small organisms such as zebrafish (Danio rerio) embryos and larvae is challenging, and breathing movements (i.e., fv) are usually manually counted, which is time- and human resource-intense, error-prone and does not provide information on the amplitude of breathing movements of the response, the breathing amplitude (fampl). Hence, in the present study, a new automated method was developed to simultaneously measure fv and fampl in small zebrafish embryos and larvae with the computer software DanioScope™. To compare HVR strategies at different life-stages of zebrafish and the physiologically linked MO2, hatched 4 d old embryos and early gill-breathing 12 d old larvae were treated with the HVR-inducing neurotoxic compound lindane (γ-hexachlorocyclohexane; γ-HCH) as a model substance. Comparison of manually counted fv with fv data measured by DanioScope™ at both life-stages showed high to moderate agreement between the two methods with respect to fv in control fish and in fish treated with lower lindane concentrations (3 - 18% deviation at 25 µg/L γ-HCH). With increasing lindane concentrations (100 and 400 µg/L γ-HCH), however, manual counts showed an average underestimation of fv by up to 30%, mainly due to very fast, rapidly successive, and indistinct movements of the fish, which cannot be properly detected by manual counts. Automated measurement thus proved significantly more sensitive, although several pre- and post-processing steps are needed. The improved automated detection of fv and the first reliable estimation of fampl in small fish embryos and larvae, as well as the inclusion of MO2, may provide new insights into different respiratory strategies and may, thus, represent a tool to lower the detection limit for reactions of different life-stages of fish to environmental stressors. In the present study, this became evident, as early gill-breathing 12 d old zebrafish larvae showed symptoms of respiratory failure (i.e., increase in fv, fampl and MO2, followed by subsequent lethargy) after exposure to lindane, whereas skin-breathing in 4 d old embryos proved mainly insensitive to the paralytic effects of lindane.

Reversibility of Thyroid Hormone System-Disrupting Effects on Eye and Thyroid Follicle Development in Zebrafish (Danio rerio) Embryos.

Early vertebrate development is partially regulated by thyroid hormones (THs). Environmental pollutants that interact with the TH system (TH system-disrupting chemicals [THSDCs]) can have massively disrupting effects on this essential phase. Eye development of fish is directly regulated by THs and can, therefore, be used as a thyroid-related endpoint in endocrine disruptor testing. To evaluate the effects of THSDC-induced eye malformations during early development, zebrafish (Danio rerio) embryos were exposed for 5 days postfertilization (dpf) to either propylthiouracil, a TH synthesis inhibitor, or tetrabromobisphenol A, which interacts with TH receptors. Subsequently, one half of the embryos were exposed further to the THSDCs until 8 dpf, while the other half of the embryos were raised in clean water for 3 days to check for reversibility of effects. Continued THSDC exposure altered eye size and pigmentation and induced changes in the cellular structure of the retina. This correlated with morphological alterations of thyroid follicles as revealed by use of a transgenic zebrafish line. Interestingly, effects were partly reversible after a recovery period as short as 3 days. Results are consistent with changes in TH levels measured in different tissues of the embryos, for example, in the eyes. The results show that eye development in zebrafish embryos is very sensitive to THSDC treatment but able to recover quickly from early exposure by effective repair mechanisms. Environ Toxicol Chem 2023;42:1276-1292. © 2023 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Developmental exposure to triclosan and benzophenone-2 causes morphological alterations in zebrafish (Danio rerio) thyroid follicles and eyes.

Thyroid hormones (THs) regulate a multitude of developmental and metabolic processes, which are responsible for vertebrate development, growth, and maintenance of homeostasis. THs also play a key role in neurogenesis of vertebrates and thus affect eye development, which is vital for foraging efficiency and for effective escape from predation. Currently, there are no validated test guidelines for the assessment of TH system-disrupting chemicals (THSDCs) in fish. Consequently, the present study was designed to demonstrate the suitability of novel thyroid-related endpoints in early life-stages of fish. Embryos of a transgenic zebrafish (Danio rerio) line expressing the reporter gene tg:mCherry in their thyrocytes were used to investigate the effects of the environmental THSDCs triclosan (TCS, antibacterial agent) and benzophenone-2 (BP-2, UV filter) on thyroid follicle and eye development. Both BP-2 and TCS caused thyroid follicle hyperplasia in transgenic zebrafish, thus confirming their role as THSDCs. The effect intensity on follicle size and fluorescence was comparable with a 1.7-fold increase for BP-2 and 1.6-fold for TCS. Alterations of the cellular structures of the retina indicate an impact of both substances on eye development, with a stronger impact of TCS. With respect to guideline development, results provide further evidence for the suitability of morphological changes in thyroid follicles and the eyes as novel endpoints for the sensitive assessment of THSD-related effects in fish.

Adapting classic paradigms to analyze alterations of shoal-wide behavior in early-life stages of zebrafish (Danio rerio) - A case study with fluoxetine.

Given the strong increase in prescription of neuroactive pharmaceuticals, neurotoxicity has received growing concern in science and the public. Regulatory requirements stimulated the development of new methods to evaluate the risk of neurotoxic substances for humans and the environment, and, with respect to potential damage to aquatic ecosystems, a variety of behavior-based assays have been proposed for neurotoxicity testing, most of which, however, are restricted to changes in the behavior of individual fish. Since many fish species form shoals under natural conditions, this may cause important aspects of behavior to be overlooked and there is a need for behavior assays integrating individual behavior with behavior of the entire swarm. In order to combine more environmentally realistic sub-chronic exposure scenarios with undistorted social behavior and animal welfare considerations, two behavioral assays are proposed that might be integrated into early-life stage toxicity studies according to OECD TG 210, which are commonly run for a multitude of regulations: To this end, protocols for a novel tank test and a predator response assay were adapted to also record the behavior of free-swimming zebrafish (Danio rerio) juveniles within shoals. Comparisons of the diving response (novel tank) or the shoal's coherence and position relative to the stimulus (predator) with control groups allow conclusions about the anxiety state of the fish, which might well have an impact on survival chances in the wild. As a model substance, the antidepressant fluoxetine ((RS)-N-Methyl-3-phenyl-3-(4-trifluoromethylphenoxy)propylamine) produced adverse effects down to concentrations three orders of magnitude below the EC10 from acute fish embryo toxicity tests according to OECD TG 236. With the integration of such behavior tests into OECD TG 210, important population-relevant information on potential neurotoxicity can be collected without increasing the number of experimental animals.

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.

AOP Report: Thyroperoxidase Inhibition Leading to Altered Visual Function in Fish Via Altered Retinal Layer Structure.

Thyroid hormones (THs) are involved in the regulation of many important physiological and developmental processes, including vertebrate eye development. Thyroid hormone system-disrupting chemicals (THSDCs) may have severe consequences, because proper functioning of the visual system is a key factor for survival in wildlife. However, the sequence of events leading from TH system disruption (THSD) to altered eye development in fish has not yet been fully described. The development of this adverse outcome pathway (AOP) was based on an intensive literature review of studies that focused on THSD and impacts on eye development, mainly in fish. In total, approximately 120 studies (up to the end of 2021) were used in the development of this AOP linking inhibition of the key enzyme for TH synthesis, thyroperoxidase (TPO), to effects on retinal layer structure and visual function in fish (AOP-Wiki, AOP 363). In a weight-of-evidence evaluation, the confidence levels were overall moderate, with ample studies showing the link between reduced TH levels and altered retinal layer structure. However, some uncertainties about the underlying mechanism(s) remain. Although the current weight-of-evidence evaluation is based on fish, the AOP is plausibly applicable to other vertebrate classes. Through the re-use of several building blocks, this AOP is connected to the AOPs leading from TPO and deiodinase inhibition to impaired swim bladder inflation in fish (AOPs 155-159), together forming an AOP network describing THSD in fish. This AOP network addresses the lack of thyroid-related endpoints in existing fish test guidelines for the evaluation of THSDCs. Environ Toxicol Chem 2022;41:2632-2648. © 2022 SETAC.

The onset of active gill respiration in post-embryonic zebrafish (Danio rerio) larvae triggers an increased sensitivity to neurotoxic compounds.

Originally designed as a general alternative to acute fish toxicity testing (AFT), the fish embryo toxicity test (FET) has become subject to concerns with respect to neurotoxic substances. Whereas oxygen uptake in the fish embryo primarily occurs via diffusion across the skin, juvenile and adult fish rely on active ventilation of the gills. As a consequence, substances including, e.g., neurotoxicants which prevent appropriate ventilation of gills ("respiratory failure syndrome") might lead to suffocation in juvenile and adult fish, but not in skin-breathing embryos. To investigate if this respiratory failure syndrome might play a role for the higher sensitivity of juvenile and adult fish to neurotoxicants, a modified acute toxicity test using post-embryonic, early gill-breathing life-stages of zebrafish was developed with chlorpyrifos, permethrin, lindane, aldicarb, ziram and aniline as test substances. Additionally, a comparative study into bioaccumulation of lipophilic substances with logKow > 3.5 and swimbladder deflation as potential side effects of the respiratory failure syndrome was performed with 4 d old skin-breathing and 12 d old gill-breathing zebrafish. With respect to acute toxicity, post-embryonic 12 d larvae proved to be more sensitive than both embryos (FET) and adult zebrafish (AFT) to all test substances except for permethrin. Accumulation of chlorpyrifos, lindane and permethrin was 1.3- to 5-fold higher in 4 d old than in 12 d old zebrafish, suggesting that (intermediate) storage of substances in the yolk might reduce bioavailability and prevent metabolization, which could be a further reason for lower toxicity in 4 d than in 12 d old zebrafish. Whereas ziram and aniline showed no significant effect on the swimbladder, zebrafish exposed to chlorpyrifos, lindane and permethrin showed significantly deflated swimbladders in 12 d old larvae; in the case of aldicarb, there was a significant hyperinflation in 4 d old larvae. Swimbladder deflation in post-embryonic 12 d zebrafish larvae might be hypothesized as a reason for a lack of internal oxygen supplies during the respiratory failure syndrome, whereas in 4 d old embryos cholinergic hyperinflation of the swimbladder dominates over other effects. Regarding acute lethality, the study provides further evidence that the switch from transcutaneous to branchial respiration in post-embryonic zebrafish life-stages might be the reason for the higher sensitivity of juvenile and adult fish to neurotoxic substances.

Beyond the behavioural phenotype: Uncovering mechanistic foundations in aquatic eco-neurotoxicology.

During the last decade, there has been an increase in awareness of how anthropogenic pollution can alter behavioural traits of diverse aquatic organisms. Apart from understanding profound ecological implications, alterations in neuro-behavioural indices have emerged as sensitive and physiologically integrative endpoints in chemical risk assessment. Accordingly, behavioural ecotoxicology and broader eco-neurotoxicology are becoming increasingly popular fields of research that span a plethora of fundamental laboratory experimentations as well as applied field-based studies. Despite mounting interest in aquatic behavioural ecotoxicology studies, there is, however, a considerable paucity in deciphering the mechanistic foundations underlying behavioural alterations upon exposure to pollutants. The behavioural phenotype is indeed the highest-level integrative neurobiological phenomenon, but at its core lie myriads of intertwined biochemical, cellular, and physiological processes. Therefore, the mechanisms that underlie changes in behavioural phenotypes can stem among others from dysregulation of neurotransmitter pathways, electrical signalling, and cell death of discrete cell populations in the central and peripheral nervous systems. They can, however, also be a result of toxicity to sensory organs and even metabolic dysfunctions. In this critical review, we outline why behavioural phenotyping should be the starting point that leads to actual discovery of fundamental mechanisms underlying actions of neurotoxic and neuromodulating contaminants. We highlight potential applications of the currently existing and emerging neurobiology and neurophysiology analytical strategies that should be embraced and more broadly adopted in behavioural ecotoxicology. Such strategies can provide new mechanistic discoveries instead of only observing the end sum phenotypic effects.

Zebrafish embryo neonicotinoid developmental neurotoxicity in the FET test and behavioral assays.

The need for reliable, sensitive (developmental) neurotoxicity testing of chemicals has steadily increased. Given the limited capacities for routine testing according to accepted regulatory guidelines, there is potential risk to human health and the environment. Most toxicity studies are based on mammalian test systems, which have been questioned for low sensitivity, limited relevance for humans, and animal welfare considerations. This increased the need for alternative models, one of which is the zebrafish (Danio rerio) embryo. This study assessed selected neonicotinoids at sub-lethal concentrations for their effects on embryonic development and behavior. The fish embryo acute toxicity test (OECD TG 236) determined the lowest observable effective concentrations, which were used as the highest test concentrations in subsequent behavioral assays. In the FET test, no severe compound-induced sublethal effects were seen at < 100 µM. In the coiling assay, exposure to ≥ 1.25 µM nicotine (positive control) affected both the burst duration and burst count per minute, whereas ≥ 50 µM thiacloprid affected the mean burst duration. Exposure to ≥ 50 µM acetamiprid and imidacloprid induced significant alterations in both mean burst duration and burst count per minute. In the swimming assay, 100 µM acetamiprid induced alterations in the frequency and extent of movements, whilst nicotine exposure only induced non-significant changes. All behavioral changes could be correlated to findings in mammalian studies. Given the quest for alternative test methods of (developmental) neurotoxicity, zebrafish embryo behavior testing could be integrated into a future tiered testing scheme.

Biomarker responses in zebrafish (Danio rerio) following long-term exposure to microplastic-associated chlorpyrifos and benzo(k)fluoranthene.

Continuously increasing plastic production causes a constant accumulation of microplastic particles (MPs) in the aquatic environment, especially in industrialized and urbanized areas with elevated wastewater discharges. This coincides with the release of persistent organic pollutants (polycyclic aromatic hydrocarbons (PAHs), pesticides) entering limnic ecosystems. Although the assessment of potential effects of environmental pollutants sorbed to MPs under chronic exposure scenarios seems vital, data on potential hazards and risk by combined exposure to pollutants and microplastics for aquatic vertebrates is still limited. Therefore, zebrafish (Danio rerio) were exposed over 21 days to the organophosphate insecticide chlorpyrifos (CPF; 10 and 100 ng/L) and the PAH benzo(k)fluoranthene (BkF; 0.78 and 50 µg/L) either dissolved directly in water or sorbed to different MPs (irregular polystyrene, spherical polymethyl methacrylate; ≤ 100 µm), where CPF was sorbed to polystyrene MPs and BkF was sorbed to polymethyl methacrylate MPs. Contaminant sorption to MPs and leaching were documented using GC-EI-MS; potential accumulation was studied in cryosections of the gastrointestinal tract. Enzymatic biomarkers and biotransformation were measured in liver and brain. Overall, exposure to non-contaminated MPs did not induce any adverse effects. Results of fluorescence tracking, CYP1A modulation by BkF as well as changes in acetylcholinesterase activity (AChE) by CPF were less pronounced when contaminants were sorbed to MPs, indicating reduced bioavailability of pollutants. Overall, following exposure to waterborne BkF, only minor amounts of parent BkF and biotransformation products were detected in zebrafish liver. Even high loads of MPs and sorbed contaminants did not induce adverse effects in zebrafish; thus, the potential threat of MPs as vectors for contaminant transfer in limnic ecosystems can be considered limited.

Rethinking the relevance of microplastics as vector for anthropogenic contaminants: Adsorption of toxicants to microplastics during exposure in a highly polluted stream - Analytical quantification and assessment of toxic effects in zebrafish (Danio rerio).

Given the increasing amounts of plastic debris entering marine and freshwater ecosystems, there is a growing demand for environmentally relevant exposure scenarios to improve the risk assessment of microplastic particles (MPs) in aquatic environments. So far, data on adverse effects in aquatic organisms induced by naturally exposed MPs are scarce and controversially discussed. As a consequence, we investigated the potential role of MPs regarding the sorption and transfer of environmental contaminants under natural conditions. For this end, a mixture of four common polymer types (polyethylene, polypropylene, polystyrene, polyvinyl chloride) was exposed to natural surface water in a polluted stream for three weeks. Samples of water, MP mixture, sediment, and suspended matter were target-screened for the presence of pollutants using GC/LC-MS, resulting in up to 94 different compounds. Possible adverse effects were investigated using several biomarkers in early developmental stages of zebrafish (Danio rerio). Exposure to natural stream water samples significantly inhibited acetylcholinesterase activity, altered CYP450 induction and modified behavioral patterns of zebrafish. In contrast, effects by samples of both non-exposed MPs and exposed MPs in zebrafish were less prominent than effects by water samples. In fact, the analytical target screening documented only few compounds sorbed to natural particles and MPs. Regarding acute toxic effects, no clear differentiation between different MPs and natural particles could be made, suggesting that - upon exposure in natural water bodies - MPs seem to approximate the sorption behavior of natural particles, presumably to a large extent due to biofilm formation. Thus, if compared to natural inorganic particles, MPs most likely do not transfer elevated amounts of environmental pollutants to biota and, therefore, do not pose a specific additional threat to aquatic organisms.

Specificity of time- and dose-dependent morphological endpoints in the fish embryo acute toxicity (FET) test for substances with diverse modes of action: the search for a "fingerprint".

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 ≤ EC10. 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.

Integrate mechanistic evidence from new approach methodologies (NAMs) into a read-across assessment to characterise trends in shared mode of action.

Read-across approaches often remain inconclusive as they do not provide sufficient evidence on a common mode of action across the category members. This read-across case study on thirteen, structurally similar, branched aliphatic carboxylic acids investigates the concept of using human-based new approach methods, such as in vitro and in silico models, to demonstrate biological similarity. Five out of the thirteen analogues have preclinical in vivo studies. Three out of them induced lipid accumulation or hypertrophy in preclinical studies with repeated exposure, which leads to the read-across hypothesis that the analogues can potentially induce hepatic steatosis. To confirm the selection of analogues, the expression patterns of the induced differentially expressed genes (DEGs) were analysed in a human liver model. With increasing dose, the expression pattern within the tested analogues got more similar, which serves as a first indication of a common mode of action and suggests differences in the potency of the analogues. Hepatic steatosis is a well-known adverse outcome, for which over 55 adverse outcome pathways have been identified. The resulting adverse outcome pathway (AOP) network, comprised a total 43 MIEs/KEs and enabled the design of an in vitro testing battery. From the AOP network, ten MIEs, early and late KEs were tested to systematically investigate a common mode of action among the grouped compounds. The targeted testing of AOP specific MIE/KEs shows that biological activity in the category decreases with side chain length. A similar trend was evident in measuring liver alterations in zebra fish embryos. However, activation of single MIEs or early KEs at in vivo relevant doses did not necessarily progress to the late KE "lipid accumulation". KEs not related to the read-across hypothesis, testing for example general mitochondrial stress responses in liver cells, showed no trend or biological similarity. Testing scope is a key issue in the design of in vitro test batteries. The Dempster-Shafer decision theory predicted those analogues with in vivo reference data correctly using one human liver model or the CALUX reporter assays. The case study shows that the read-across hypothesis is the key element to designing the testing strategy. In the case of a good mechanistic understanding, an AOP facilitates the selection of reliable human in vitro models to demonstrate a common mode of action. Testing DEGs, MIEs and early KEs served to show biological similarity, whereas the late KEs become important for confirmation, as progression from MIEs to AO is not always guaranteed.