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The hypothalamic-pituitary-thyroid (HPT) axis is known to play a crucial role in the development of teleost fish. However, knowledge of endogenous transcription profiles of thyroid-related genes in developing teleosts remains fragmented. We selected two model teleost species, the fathead minnow ( Pimephales promelas ) and the zebrafish ( Danio rerio ), to compare the gene transcription ontogeny of the HPT axis. Control organisms were sampled at several time points during embryonic and larval development until 33 days post-fertilization. Total RNA was extracted from pooled, whole fish, and thyroid-related mRNA expression was evaluated using quantitative polymerase chain reaction. Gene transcripts examined included: thyrotropin-releasing hormone receptor ( trhr ), thyroid-stimulating hormone receptor ( tshr ), sodium-iodide symporter ( nis ), thyroid peroxidase ( tpo ), thyroglobulin ( tg ), transthyretin ( ttr ), deiodinases 1, 2, 3a, and 3b ( dio1, dio2, dio3a and 3b ), and thyroid hormone receptors alpha and beta ( thrα and β ). A loess regression method was successful in identifying maxima and minima of transcriptional expression during early development of both species. Overall, we observed great similarities between the species, including maternal transfer, at least to some extent, of almost all transcripts (confirmed in unfertilized eggs), increasing expression of most transcripts during hatching and embryo-larval transition, and indications of a fully functional HPT axis in larvae. These data will aid in the development of hypotheses on the role of certain genes and pathways during development. Furthermore, this provides a background reference dataset for designing and interpreting targeted transcriptional expression studies both for fundamental research and for applications such as toxicology. [ABSTRACT FROM AUTHOR]

Environmental pollutants capable of interfering with the thyroid hormone (TH) system increasingly raise concern for both human and environmental health. Recently, resorcinol has received attention as a compound of concern due to its endocrine disrupting properties. It is a known inhibitor of thyroperoxidase (TPO), an enzyme required in TH synthesis, and therapeutic use of resorcinol exposure has led to hypothyroidism in humans. There is limited evidence concerning ecotoxicologically relevant effects of resorcinol in fish. A set of adverse outcome pathways (AOPs) has recently been developed linking thyroid hormone system disruption (THSD) to impaired swim bladder inflation and eye development in fish. In the present study, these AOPs were used to provide the background for testing potential THSD effects of resorcinol in zebrafish eleutheroembryos. We exposed zebrafish eleutheroembryos to resorcinol and assessed TH levels, swim bladder inflation and eye morphology. As a TPO inhibitor, resorcinol is expected to affect TH levels and eye morphology but not swim bladder inflation during embryonic development. Indeed, thyroxine (T4) levels were significantly decreased following resorcinol exposure. In contrast to our hypothesis, swim bladder inflation was impaired at 5 days post fertilization (dpf) and no effects on eye morphology were detected. Therefore, in vitro assays were performed to identify potential additional thyroid hormone system disruption-related mechanisms through which resorcinol may act. Two new mechanisms were identified: TH receptor (TR) antagonism and transthyretin (TTR) binding inhibition. Both of these mechanisms can plausibly be linked to impaired swim bladder inflation and could, therefore, explain the observed effect. Overall, our study contributes to the knowledge of the THSD potential of resorcinol both in vivo in the zebrafish model as well as in vitro., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

A subgroup of endocrine-disrupting chemicals have the ability to disrupt metabolism. These metabolism-disrupting chemicals (MDCs) can end up in aquatic environments and lead to adverse outcomes in fish. Although molecular and physiological effects of MDCs have been studied in adult fish, few studies have investigated the consequences of metabolic disruption in fish during the earliest life stages. To investigate the processes affected by metabolic disruption, zebrafish embryos were exposed to peroxisome proliferator-activated receptor gamma (PPARγ) agonist rosiglitazone, the PPARγ antagonist T0070907, and the well-known environmentally relevant MDC bisphenol A. Decreased apolipoprotein Ea transcript levels indicated disrupted lipid transport, which was likely related to the observed dose-dependent increases in yolk size across all compounds. Increased yolk size and decreased swimming activity indicate decreased energy usage, which could lead to adverse outcomes because the availability of energy reserves is essential for embryo survival and growth. Exposure to T0070907 resulted in a darkened yolk. This was likely related to reduced transcript levels of genes involved in lipid transport and fatty acid oxidation, a combination of responses that was specific to exposure to this compound, possibly leading to lipid accumulation and cell death in the yolk. Paraoxonase 1 (Pon1) transcript levels were increased by rosiglitazone and T0070907, but this was not reflected in PON1 enzyme activities. The present study shows how exposure to MDCs can influence biochemical and molecular processes involved in early lipid metabolism and may lead to adverse outcomes in the earliest life stages of fish. Environ Toxicol Chem 2024;43:1880-1893. © 2024 The Author(s). Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC., (© 2024 The Author(s). Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.)

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., Competing Interests: Declaration of competing interest The authors have no relevant financial or non-financial interests to disclose., (Copyright © 2024. Published by Elsevier B.V.)

Bisphenol A alternatives are manufactured as potentially less harmful substitutes of bisphenol A (BPA) that offer similar functionality. These alternatives are already in the market, entering the environment and thus raising ecological concerns. However, it can be expected that levels of BPA alternatives will dominate in the future, they are limited information on their environmental safety. The EU PARC project highlights BPA alternatives as priority chemicals and consolidates information on BPA alternatives, with a focus on environmental relevance and on the identification of the research gaps. The review highlighted aspects and future perspectives. In brief, an extension of environmental monitoring is crucial, extending it to cover BPA alternatives to track their levels and facilitate the timely implementation of mitigation measures. The biological activity has been studied for BPA alternatives, but in a non-systematic way and prioritized a limited number of chemicals. For several BPA alternatives, the data has already provided substantial evidence regarding their potential harm to the environment. We stress the importance of conducting more comprehensive assessments that go beyond the traditional reproductive studies and focus on overlooked relevant endpoints. Future research should also consider mixture effects, realistic environmental concentrations, and the long-term consequences on biota and ecosystems., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

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)., (© 2023 The Authors. Integrated Environmental Assessment and Management published by Wiley Periodicals LLC on behalf of Society of Environmental Toxicology & Chemistry (SETAC).)

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)., (© 2023 The Authors. Integrated Environmental Assessment and Management published by Wiley Periodicals LLC on behalf of Society of Environmental Toxicology & Chemistry (SETAC).)

Introduction: The Adverse Outcome Pathway (AOP) concept facilitates rapid hazard assessment for human health risks. AOPs are constantly evolving, their number is growing, and they are referenced in the AOP-Wiki database, which is supported by the OECD. Here, we present a study that aims at identifying well-defined biological areas, as well as gaps within the AOP-Wiki for future research needs. It does not intend to provide a systematic and comprehensive summary of the available literature on AOPs but summarizes and maps biological knowledge and diseases represented by the already developed AOPs (with OECD endorsed status or under validation). Methods: Knowledge from the AOP-Wiki database were extracted and prepared for analysis using a multi-step procedure. An automatic mapping of the existing information on AOPs (i.e., genes/proteins and diseases) was performed using bioinformatics tools (i.e., overrepresentation analysis using Gene Ontology and DisGeNET), allowing both the classification of AOPs and the development of AOP networks (AOPN). Results: AOPs related to diseases of the genitourinary system, neoplasms and developmental anomalies are the most frequently investigated on the AOP-Wiki. An evaluation of the three priority cases (i.e., immunotoxicity and non-genotoxic carcinogenesis, endocrine and metabolic disruption, and developmental and adult neurotoxicity) of the EU-funded PARC project (Partnership for the Risk Assessment of Chemicals) are presented. These were used to highlight under- and over-represented adverse outcomes and to identify and prioritize gaps for further research. Discussion: These results contribute to a more comprehensive understanding of the adverse effects associated with the molecular events in AOPs, and aid in refining risk assessment for stressors and mitigation strategies. Moreover, the FAIRness (i.e., data which meets principles of findability, accessibility, interoperability, and reusability (FAIR)) of the AOPs appears to be an important consideration for further development., Competing Interests: Author AB-P was employed by Joint Research Centre and Author EF was employed by DNTOX GmbH. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Jaylet, Coustillet, Smith, Viviani, Lindeman, Vergauwen, Myhre, Yarar, Gostner, Monfort-Lanzas, Jornod, Holbech, Coumoul, Sarigiannis, Antczak, Bal-Price, Fritsche, Kuchovska, Stratidakis, Barouki, Kim, Taboureau, Wojewodzic, Knapen and Audouze.)

Paraoxonase 1 (PON1) is an antioxidant enzyme linked to metabolic disorders by genome-wide association studies in humans. Exposure to metabolic disrupting chemicals (MDCs) such as bisphenol A (BPA), together with genetic and dietary factors, can increase the risk of metabolic disorders. The objective of this study was to investigate how PON1 responds to the metabolic changes and oxidative stress caused by a western diet, and whether exposure to BPA alters the metabolic and PON1 responses. Zebrafish larvae at 14 days post fertilization were fed a custom-made western diet with and without aquatic exposure to two concentrations of BPA for 5 days. A combination of western diet and 150 μg/L BPA exposure resulted in a stepwise increase in weight, length and oxidative stress, suggesting that BPA amplifies the western diet-induced metabolic shift. PON1 arylesterase activity was increased in all western diet and BPA exposure groups and PON1 lactonase activity was increased when western diet was combined with exposure to 1800 μg/L BPA. Both PON1 activities were positively correlated to oxidative stress. Based on our observations we hypothesize that a western diet caused a shift towards fatty acid-based metabolism, which was increased by BPA exposure. This shift resulted in increased oxidative stress, which in turn was associated with a PON1 activity increase as an antioxidant response. This is the first exploration of PON1 responses to metabolic challenges in zebrafish, and the first study of PON1 in the context of MDC exposure in vertebrates., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

Thyroid hormone system disrupting compounds are considered potential threats for human and environmental health. Multiple adverse outcome pathways (AOPs) for thyroid hormone system disruption (THSD) are being developed in different taxa. Combining these AOPs results in a cross-species AOP network for THSD which may provide an evidence-based foundation for extrapolating THSD data across vertebrate species and bridging the gap between human and environmental health. This review aimed to advance the description of the taxonomic domain of applicability (tDOA) in the network to improve its utility for cross-species extrapolation. We focused on the molecular initiating events (MIEs) and adverse outcomes (AOs) and evaluated both their plausible domain of applicability (taxa they are likely applicable to) and empirical domain of applicability (where evidence for applicability to various taxa exists) in a THSD context. The evaluation showed that all MIEs in the AOP network are applicable to mammals. With some exceptions, there was evidence of structural conservation across vertebrate taxa and especially for fish and amphibians, and to a lesser extent for birds, empirical evidence was found. Current evidence supports the applicability of impaired neurodevelopment, neurosensory development (eg, vision) and reproduction across vertebrate taxa. The results of this tDOA evaluation are summarized in a conceptual AOP network that helps prioritize (parts of) AOPs for a more detailed evaluation. In conclusion, this review advances the tDOA description of an existing THSD AOP network and serves as a catalog summarizing plausible and empirical evidence on which future cross-species AOP development and tDOA assessment could build., (© The Author(s) 2023. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The zebrafish (Danio rerio, Hamilton, 1822) is one of the most important fish model species in scientific research, with millions of fish housed in laboratory animal facilities around the world. During husbandry, it is necessary to regularly handle the fish, which could cause short- and long-term stress, possibly affecting both fish welfare and experimental outcomes. In two experiments, the authors studied effects of transferring adult zebrafish, by chasing them with a net and/or exposing them to air (netting) on different endpoints: cortisol levels, reproduction parameters and behavioural parameters. They used realistic chase and air-exposure times to mimic normal zebrafish husbandry and investigated the potential to habituate to handling stressors. Finally, the potential welfare improvements of a nutritional reward after handling were studied. All types of handling induced a stress response, but the authors did not find a correlation with the intensity of the stressor. Realistic (short) handling routines also caused stress, both after the first time and after regular handling over a long period of time. Cortisol levels peaked after 15 min, were still elevated after 30 min and dropped to resting level after 60 min. This should be taken into account by researchers when carrying out measurements or behavioural trials within an hour after handling. There is a minor potential benefit of nutritional rewards that may contribute to a faster recovery of normal behaviour. They did not find evidence of habituation to chasing and netting stress. Taking the stress response after handling into consideration will improve fish welfare and health and minimise husbandry-associated sources of variation., (© 2023 Fisheries Society of the British Isles.)

Thyroid hormone system disruption (THSD) negatively affects multiple developmental processes and organs. In fish, inhibition of deiodinases, which are enzymes crucial for (in)activating thyroid hormones (THs), leads to impaired swim bladder inflation. Until now, the underlying mechanism has remained largely unknown. Therefore, the objective of this study was to identify the process during swim bladder development that is impacted by deiodinase inhibition. Zebrafish embryos were exposed to 6 mg/L iopanoic acid (IOP), a model deiodinase inhibitor, during 8 different exposure windows (0-60, 60-120, 24-48, 48-72, 72-96, 96-120, 72-120 and 0-120 h post fertilization (hpf)). Exposure windows were chosen based on the three stages of swim bladder development: budding (24-48 hpf), pre-inflation, i.e., the formation of the swim bladder tissue layers (48-72 hpf), and inflation phase (72-120 hpf). Exposures prior to 72 hpf, during either the budding or pre-inflation phase (or both), impaired swim bladder inflation, while exposure during the inflation phase did not. Based on our results, we hypothesize that DIO inhibition before 72 hpf leads to a local decrease in T3 levels in the developing swim bladder. Gene transcript analysis showed that these TH level alterations disturb both Wnt and hedgehog signaling, known to be essential for swim bladder formation, eventually resulting in impaired development of the swim bladder tissue layers. Improper development of the swim bladder impairs swim bladder inflation, leading to reduced swimming performance. This study demonstrates that deiodinase inhibition impacts processes underlying the formation of the swim bladder and not the inflation process, suggesting that these processes primarily rely on maternal rather than endogenously synthetized THs since TH measurements showed that THs were not endogenously synthetized during the sensitive period., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

In past times, the analysis of endocrine disrupting properties of chemicals has mainly been focused on (anti-)estrogenic or (anti-)androgenic properties, as well as on aspects of steroidogenesis and the modulation of thyroid signaling. More recently, disruption of energy metabolism and related signaling pathways by exogenous substances, so-called metabolism-disrupting chemicals (MDCs) have come into focus. While general effects such as body and organ weight changes are routinely monitored in animal studies, there is a clear lack of mechanistic test systems to determine and characterize the metabolism-disrupting potential of chemicals. In order to contribute to filling this gap, one of the project within EU-funded Partnership for the Assessment of Risks of Chemicals (PARC) aims at developing novel in vitro methods for the detection of endocrine metabolic disruptors. Efforts will comprise projects related to specific signaling pathways, for example, involving mTOR or xenobiotic-sensing nuclear receptors, studies on hepatocytes, adipocytes and pancreatic beta cells covering metabolic and morphological endpoints, as well as metabolism-related zebrafish-based tests as an alternative to classic rodent bioassays. This paper provides an overview of the approaches and methods of these PARC projects and how this will contribute to the improvement of the toxicological toolbox to identify substances with endocrine disrupting properties and to decipher their mechanisms of action., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The handling editor TV declared a shared research group PARC PROJECT: European Partnership on the Assessment of Risks from Chemicals (PARC) with the authors at the time of review., (Copyright © 2023 Braeuning, Balaguer, Bourguet, Carreras-Puigvert, Feiertag, Kamstra, Knapen, Lichtenstein, Marx-Stoelting, Rietdijk, Schubert, Spjuth, Stinckens, Thedieck, van den Boom, Vergauwen, von Bergen, Wewer and Zalko.)

Current test strategies to identify thyroid hormone (TH) system disruptors are inadequate for conducting robust chemical risk assessment required for regulation. The tests rely heavily on histopathological changes in rodent thyroid glands or measuring changes in systemic TH levels, but they lack specific new approach methodologies (NAMs) that can adequately detect TH-mediated effects. Such alternative test methods are needed to infer a causal relationship between molecular initiating events and adverse outcomes such as perturbed brain development. Although some NAMs that are relevant for TH system disruption are available-and are currently in the process of regulatory validation-there is still a need to develop more extensive alternative test batteries to cover the range of potential key events along the causal pathway between initial chemical disruption and adverse outcomes in humans. This project, funded under the Partnership for the Assessment of Risk from Chemicals (PARC) initiative, aims to facilitate the development of NAMs that are specific for TH system disruption by characterizing in vivo mechanisms of action that can be targeted by in embryo/in vitro/in silico/in chemico testing strategies. We will develop and improve human-relevant in vitro test systems to capture effects on important areas of the TH system. Furthermore, we will elaborate on important species differences in TH system disruption by incorporating non-mammalian vertebrate test species alongside classical laboratory rat species and human-derived in vitro assays., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Ramhøj, Axelstad, Baert, Cañas-Portilla, Chalmel, Dahmen, De La Vieja, Evrard, Haigis, Hamers, Heikamp, Holbech, Iglesias-Hernandez, Knapen, Marchandise, Morthorst, Nikolov, Nissen, Oelgeschlaeger, Renko, Rogiers, Schüürmann, Stinckens, Stub, Torres-Ruiz, Van Duursen, Vanhaecke, Vergauwen, Wedebye and Svingen.)

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., (© 2022 SETAC.)

Lipidomics analysis of zebrafish tissues has shown promising results to understand disease-related outcomes of exposure to toxic substances at a molecular level. However, knowledge about their lipidome is limited, as most untargeted studies only identify the lipids that are statistically significant in their setup. In this work, liquid chromatography-high resolution mass spectrometry was used to study different aspects of the analytical workflow, that is, extraction solvents (methanol/chloroform/water (3/2/2, v/v/v), methanol/dichloromethane/water (2/3/2, v/v/v) and methanol/methyl-tert-butyl ether/water (3/10/2.5, v/v/v), instrumental response, and strategies used for lipid annotation. The number of high-quality features (relative standard deviation of the intensity values ≤ 10% in the range 10 3 -10 7 counts) was affected by the dilution of lipid extracts, indicating that it is an important parameter for developing untargeted methods. The workflows used allowed the selection of a dilution factor to annotate 712 lipid species (507 bulk lipids) in zebrafish liver using four software (LipidMatch, LipidHunter, MS-DIAL, and Lipostar). Retention time mapping was a valuable tool to filter lipid annotations obtained from automatic software annotations. The lipid profiling of zebrafish livers will help in a better understanding of the true constitution of their lipidome at the species level, as well as in the use of zebrafish in toxicological studies., (© 2022 Wiley-VCH GmbH.)

Competing Interests: DK is a member of the Handbook, Guidance, and Gardening (HGG) subgroup of the OECD’s Extended Advisory Group on Molecular Screening and Toxicogenomics (EAGMST). The consideration of systematic approaches for AOP development is one of the tasks of HGG. The views expressed in this paper are those of the authors and do not necessarily reflect the views or policies of the OECD. OVM is one of the representatives of the European Parliament on the management board of the European Chemical Agency. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The handling editor AB declared a past co-authorship with one of the authors OVM.

The adverse outcome pathway (AOP) framework provides a practical means for organizing scientific knowledge that can be used to infer cause-effect relationships between stressor events and toxicity outcomes in intact organisms. It has reached wide acceptance as a tool to aid chemical safety assessment and regulatory toxicology by supporting a systematic way of predicting adverse health outcomes based on accumulated mechanistic knowledge. A major challenge for broader application of the AOP concept in regulatory toxicology, however, has been developing robust AOPs to a level where they are peer reviewed and accepted. This is because the amount of work required to substantiate the modular units of a complete AOP is considerable, to the point where it can take years from start to finish. To help alleviate this bottleneck, we propose a more pragmatic approach to AOP development whereby the focus becomes on smaller blocks. First, we argue that the key event relationship (KER) should be formally recognized as the core building block of knowledge assembly within the AOP knowledge base (AOP-KB), albeit framing them within full AOPs to ensure regulatory utility. Second, we argue that KERs should be developed using systematic review approaches, but only in cases where the underlying concept does not build on what is considered canonical knowledge. In cases where knowledge is considered canonical, rigorous systematic review approaches should not be required. It is our hope that these approaches will contribute to increasing the pace at which the AOP-KB is populated with AOPs with utility for chemical safety assessors and regulators., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Society of Toxicology.)

Nonpolar narcosis, also known as baseline toxicity, has been described as the minimal toxicity that an organic chemical may elicit based on its lipophilicity. Although lethal effects of narcosis-inducing chemicals (NICs) have been thoroughly investigated, knowledge of sublethal effects is still very limited. We investigated the effects of 3 well-known NICs (phenanthrene, 1,3,5-trichlorobenzene, and pentachlorobenzene) on a variety of organismal endpoints (malformations, swim bladder inflation, respiration, heart rate, swimming activity, and turning angles), which can be plausibly linked to narcosis in zebrafish embryos. Baseline toxicity recorded as mortality is typically observed in similar exposure ranges in a wide variety of species including fish, corresponding to a chemical activity range between 0.01 and 0.1. In the present study, we found that sublethal effects occurred at concentrations approximately 5 times below lethal concentrations. Altered swimming activity and impaired swim bladder inflation were the most sensitive endpoints occurring at exposure levels below the generally accepted threshold for baseline toxicity for 2 out of 3 compounds. Overall, most effective exposure levels across the sublethal endpoints and compounds did fall within the range typically associated with baseline toxicity, and deviations were generally limited to a factor 10. Although there could be benefit in adding sublethal endpoints to toxicity tests, such as the fish embryo acute toxicity (FET) test, based on the present sublethal endpoints and available evidence from our and other studies, the underestimation of toxicity as a result of the sole assessment of mortality as an endpoint in an FET test may be limited for narcosis. Environ Toxicol Chem 2021;40:2802-2812. © 2021 SETAC., (© 2021 SETAC.)

Metabolomics has achieved great progress over the last 20 years, and it is currently considered a mature research field. As a result, the number of applications in toxicology, biomarker, and drug discovery has also increased. Toxicometabolomics has emerged as a powerful strategy to provide complementary information to study molecular-level toxic effects, which can be combined with a wide range of toxicological assessments and models. The zebrafish model has gained importance in recent decades as a bridging tool between in vitro assays and mammalian in vivo studies in the field of toxicology. Furthermore, as this vertebrate model is a low-cost system and features highly conserved metabolic pathways found in humans and mammalian models, it is a promising tool for toxicometabolomics. This short review aims to introduce zebrafish researchers interested in understanding the effects of chemical exposure using metabolomics to the challenges and possibilities of the field, with a special focus on toxicometabolomics-based mass spectrometry. The overall goal is to provide insights into analytical strategies to generate and identify high-quality metabolomic experiments focusing on quality management systems (QMS) and the importance of data reporting and sharing.

Mammalian liver microsomes are occasionally used as a metabolic activation system (MAS) to compensate for the low CYP-mediated bioactivation of drugs in zebrafish embryos, in the so-called mDarT. However, this MAS is embryotoxic and consequently zebrafish embryos are only exposed during a very limited developmental window. The main aim of this study was to try to reduce the embryotoxic properties of MAS in order to extend the exposure window in the mDarT. Removing the microsomes from the incubation medium prior to exposure of the zebrafish embryos did not reduce embryotoxicity. Free radicals (ROS) in the incubation medium were successfully reduced by antioxidants, but the medium remained embryotoxic. Single dosing of NADPH or omitting toxic components from the MAS preparation did also not reduce embryotoxicity. In conclusion, the exposure window in the mDarT could not be extended by reducing ROS levels, single dosing of NADPH or modifications of the MAS preparation., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Because of the ever increasing complexity of environmental contamination profiles, there are limitations to the use of analytical pollutant measurements for monitoring and prioritization of watercourses. The potential of biomarkers has been debated for many years, especially in laboratory settings, but there is a need for studies evaluating these approaches in the field. We evaluated the usefulness of a selection of biomarkers, mostly indicators of general physiological status and common stress responses such as oxidative stress, to discriminate among environmental pollution profiles, with the aim of prioritizing contaminated watercourses for targeted remediation efforts. To this end, juvenile common carp (Cyprinus carpio Lin.) were exposed in cages in the field to Flemish watercourses with varying pollution profiles. After six weeks of exposure, the bioaccumulation of key pollutants was measured, and a set of organismal, biochemical and transcriptional endpoints was determined in several tissue types. After data integration a discrete set of 14 parameters was identified, that could successfully distinguish all watercourses from each other. We show that an integrated biomarker approach, mainly targeting common stress responses, can offer the resolving power to discriminate among environmentally relevant exposure scenarios, and a means to prioritize watercourses for targeted remediation., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

The biological effects and interactions of different radiation types in plants are still far from understood. Among different radiation types, external gamma radiation treatments have been mostly studied to assess the biological impact of radiation toxicity in organisms. Upon exposure of plants to gamma radiation, ionisation events can cause, either directly or indirectly, severe biological damage to DNA and other biomolecules. However, the biological responses and oxidative stress related mechanisms under chronic radiation conditions are poorly understood in plant systems. In the following study, it was questioned if the Lemna minor growth inhibition test is a suitable approach to also assess the radiotoxicity of this freshwater plant. Therefore, L. minor plants were continuously exposed for seven days to 12 different dose rate levels covering almost six orders of magnitude starting from 80 μGy h(-1) up to 1.5 Gy h(-1). Subsequently, growth, antioxidative defence system and genomic responses of L. minor plants were evaluated. Although L. minor plants could survive the exposure treatment at environmental relevant exposure conditions, higher dose rate levels induced dose dependent growth inhibitions starting from approximately 27 mGy h(-1). A ten-percentage growth inhibition of frond area Effective Dose Rate (EDR10) was estimated at 95 ± 7 mGy h(-1), followed by 153 ± 13 mGy h(-1) and 169 ± 12 mGy h(-1) on fresh weight and frond number, respectively. Up to a dose rate of approximately 5 mGy h(-1), antioxidative enzymes and metabolites remained unaffected in plants. A significant change in catalase enzyme activity was found at 27 mGy h(-1) which was accompanied with significant increases of other antioxidative enzyme activities and shifts in ascorbate and glutathione content at higher dose rate levels, indicating an increase in oxidative stress in plants. Recent plant research hypothesized that environmental genotoxic stress conditions can induce endoreduplication events. Here an increase in ploidy level was observed at the highest tested dose rate. In conclusion, the results revealed that in plants several mechanisms and pathways interplay to cope with radiation induced stress., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Drought is the most important crop yield-limiting factor, and detailed knowledge of its impact on plant growth regulation is crucial. The maize (Zea mays) leaf growth zone offers unique possibilities for studying the spatiotemporal regulation of developmental processes by transcriptional analyses and methods that require more material, such as metabolite and enzyme activity measurements. By means of a kinematic analysis, we show that drought inhibits maize leaf growth by inhibiting cell division in the meristem and cell expansion in the elongation zone. Through a microarray study, we observed the down-regulation of 32 of the 54 cell cycle genes, providing a basis for the inhibited cell division. We also found evidence for an up-regulation of the photosynthetic machinery and the antioxidant and redox systems. This was confirmed by increased chlorophyll content in mature cells and increased activity of antioxidant enzymes and metabolite levels across the growth zone, respectively. We demonstrate the functional significance of the identified transcriptional reprogramming by showing that increasing the antioxidant capacity in the proliferation zone, by overexpression of the Arabidopsis (Arabidopsis thaliana) iron-superoxide dismutase gene, increases leaf growth rate by stimulating cell division. We also show that the increased photosynthetic capacity leads to enhanced photosynthesis upon rewatering, facilitating the often-observed growth compensation., (© 2015 American Society of Plant Biologists. All Rights Reserved.)

Historically, the prediction of reproductive and developmental toxicity has largely relied on the use of animals. The adverse outcome pathway (AOP) framework forms a basis for the development of new non-animal test methods. It also provides biological context for mechanistic information from existing assays. However, a single AOP may not capture all events that contribute to any relevant toxic effect, even in single chemical exposure scenarios. AOP networks, defined as sets of AOPs sharing at least one common element, are capable of more realistically representing potential chemical effects. They provide information on interactions between AOPs and have the potential to reveal previously unknown links between biological pathways. Analysis of these AOP networks can aid the prioritization of assay development, whether the goal is to develop a single assay with predictive utility of multiple outcomes, or development of assays that are highly specific for a particular mode of action. This paper provides a brief overview of the AOPs related to reproductive and developmental toxicity currently available in the AOP Wiki (http://aopwiki.org), and gives an example of an AOP network based on five reproductive and developmental toxicity-related AOPs for fish to illustrate how AOP networks can be used for assay development and refinement., (Copyright © 2015 Elsevier Inc. All rights reserved.)

There is still a lot of contradiction on whether metal ions are solely responsible for the observed toxicity of ZnO and CuO nanoparticles to aquatic species. While most experiments have studied nanoparticle effects at organismal levels (e.g. mortality, reproduction), effects at lower levels of biological organization may clarify the role of metal ions, nanoparticles and nanoparticle aggregates. In this study, the effect of ZnO and CuO nanoparticles was tested at two lower levels: energy reserves and gene transcription and compared with zinc and copper salts. Daphnia magna was exposed during 96h to 10% immobilization concentrations of all chemicals, after which daphnids were sampled for determination of glycogen, lipid and protein concentration and for a differential gene transcription analysis using microarray. The dissolved, nanoparticle and aggregated fraction in the medium was characterized. The results showed that ZnO nanoparticles had largely dissolved directly after addition to the test medium. The CuO nanoparticles mostly formed aggregates, while only a small fraction dissolved. The exposure to zinc (both nano and metal salt) had no effect on the available energy reserves. However, in the copper exposure, the glycogen, lipid and protein concentration in the exposed daphnids was lower than in the unexposed ones. When comparing the nanoparticle (ZnO or CuO) exposed daphnids to the metal salt (zinc or copper salt) exposed daphnids, the microarray results showed no significantly differentially transcribed gene fragments. The results indicate that under the current exposure conditions the toxicity of ZnO and CuO nanoparticles to D. magna is solely caused by toxic metal ions., (Copyright © 2015 Elsevier Inc. All rights reserved.)

In this study, the effects of CuO nanoparticles and CuCl2·2H2O were tested on Daphnia magna under chronic exposure scenarios. During a 21-day exposure to the nanoparticles and salt, the reproduction was followed by a daily count of the number of offspring. After the exposure, the adult Daphnia length and uptake of copper was measured. The dissolved, nanoparticle and aggregated fractions were distinguished in the exposure medium. The results showed that only a small fraction of the nanoparticles dissolved, while the majority of the particles formed large aggregates (>450 nm). The dissolved fraction of the nanoparticles corresponded with the dissolved fraction of the copper salt. The effects of the nanoparticles (reproduction EC10: 0.546 mg Cu/l, EC20: 0.693 mg Cu/l, EC50: 1.041 mg Cu/l) on reproduction and length were much lower than the effects of the copper salts (reproduction EC10: 0.017 mg Cu/l, EC20: 0.019 mg Cu/l, EC50: 0.022 mg Cu/l). Based upon total body analysis, the Daphnia copper concentration appeared much higher when exposed to the nanoparticles than when exposed to the salt. These combined results indicate that the toxicity of CuO nanoparticles to D. magna is caused by copper ions formed during dissolution of the nanoparticles in the exposure medium., (Copyright © 2014 Elsevier B.V. All rights reserved.)

In this study, the uptake and elimination of ZnO and CuO nanoparticles in Daphnia magna was tested. Daphnids were exposed during 10 days to sublethal concentrations of ZnO and CuO nanoparticles and corresponding metal salts (ZnCl₂ and CuCl₂.2H₂O), after which they were transferred to unexposed medium for another 10 days. At different times during the exposure and none-exposure, the total and internal zinc or copper concentration of the daphnids was determined and the nanoparticles were localized in the organism using electron microscopy. The exposure concentrations were characterized by measuring the dissolved, nanoparticle and aggregated fraction in the medium. The results showed that the ZnO nanoparticles quickly dissolved after addition to the medium. Contrarily, only a small fraction (corresponding to the dissolved metal salt) of the CuO nanoparticles dissolved, while most of these nanoparticles formed large aggregates. Despite an initial increase in zinc and copper concentration during the first 48 h to 5 day exposure, the body concentration reached a plateau level that was comparable for the ZnO nanoparticles and ZnCl₂, but much higher for the CuO nanoparticles (with visible aggregates accumulating in the gut) than CuCl₂.2H₂O. During the remaining exposure and subsequent none-exposure phase, the zinc and copper concentration decreased fast to concentrations comparable with the unexposed daphnids. The results indicate that D. magna can regulate its internal zinc and copper concentration after exposure to ZnO and CuO nanoparticles, similar as after exposure to metal salts. The combined dissolution, accumulation and toxicity results confirm that the toxicity of ZnO and CuO nanoparticles is caused by the dissolved fraction.

Climate changes increasingly threaten plant growth and productivity. Such changes are complex and involve multiple environmental factors, including rising CO2 levels and climate extreme events. As the molecular and physiological mechanisms underlying plant responses to realistic future climate extreme conditions are still poorly understood, a multiple organizational level analysis (i.e. eco-physiological, biochemical, and transcriptional) was performed, using Arabidopsis exposed to incremental heat wave and water deficit under ambient and elevated CO2 . The climate extreme resulted in biomass reduction, photosynthesis inhibition, and considerable increases in stress parameters. Photosynthesis was a major target as demonstrated at the physiological and transcriptional levels. In contrast, the climate extreme treatment induced a protective effect on oxidative membrane damage, most likely as a result of strongly increased lipophilic antioxidants and membrane-protecting enzymes. Elevated CO2 significantly mitigated the negative impact of a combined heat and drought, as apparent in biomass reduction, photosynthesis inhibition, chlorophyll fluorescence decline, H2 O2 production, and protein oxidation. Analysis of enzymatic and molecular antioxidants revealed that the stress-mitigating CO2 effect operates through up-regulation of antioxidant defense metabolism, as well as by reduced photorespiration resulting in lowered oxidative pressure. Therefore, exposure to future climate extreme episodes will negatively impact plant growth and production, but elevated CO2 is likely to mitigate this effect., (© 2014 John Wiley & Sons Ltd.)

In this study, the effect of ZnO nanoparticles and ZnCl2 on growth, reproduction and accumulation of zinc in Daphnia magna was determined in a 21-day chronic toxicity test. A variety of techniques were used to distinguish the free zinc ion, dissolved, nanoparticle and aggregated zinc fraction in the Daphnia test medium. The results showed similar chronic effects on growth, reproduction and accumulation for the ZnO nanoparticles (EC10, 20, 50 reproduction: 0.030, 0.049, 0.112 mg Zn/l) and the ZnCl2 (EC10, 20, 50 reproduction: 0.014, 0.027, 0.082 mg Zn/l). A large fraction of the nanoparticles rapidly dissolved after introduction in the exposure medium. Aggregation of nanoparticles was also observed but within 48 h of exposure most of these ZnO aggregates were dissolved. Based on the combined dissolution kinetics and toxicity results, it can be concluded that the toxicological effects of ZnO nanoparticles at the chronic level can be largely attributed to the dissolved fraction rather than the nanoparticles or initially formed aggregates.

Microarrays are an ideal tool to screen for differences in gene expression of thousands of genes simultaneously. However, often commercial arrays are not available. In this study, we performed microarray analyses to evaluate patterns of gene transcription following exposure to two natural and one anthropogenic stressor. cDNA microarrays compiled of three life stage specific and three stressor-specific EST libraries, yielding 1734 different EST sequences, were used. We exposed juveniles of the water flea Daphnia magna for 48, 96 and 144 h to three stressors known to exert strong selection in natural populations of this species i.e. a sublethal concentration of the pesticide carbaryl, infective spores of the endoparasite Pasteuria ramosa, and fish predation risk mimicked by exposure to fish kairomones. A total of 148 gene fragments were differentially expressed compared to the control. Based on a PCA, the exposure treatments were separated into two main groups based on the extent of the transcriptional response: a low and a high (144 h of fish or carbaryl exposure and 96 h of parasite exposure) stress group. Firstly, we observed a general stress-related transcriptional expression profile independent of the treatment characterized by repression of transcripts involved in transcription, translation, signal transduction and energy metabolism. Secondly, we observed treatment-specific responses including signs of migration to deeper water layers in response to fish predation, structural challenge of the cuticle in response to carbaryl exposure, and disturbance of the ATP production in parasite exposure. A third important conclusion is that transcription expression patterns exhibit stress-specific changes over time. Parasite exposure shows the most differentially expressed gene fragments after 96 h. The peak of differentially expressed transcripts came only after 144 h of fish exposure, while carbaryl exposure induced a more stable number of differently expressed gene fragments over time.

The zebrafish has become a valuable vertebrate model organism in a wide range of scientific disciplines, but current information concerning the physiological temperature response of adult zebrafish is rather scarce. In this study, zebrafish were experimentally acclimated for 28 days to 18, 26 or 34 °C and a suite of non-invasive and invasive methods was applied to determine the thermal dependence of zebrafish physiological condition. With decreasing temperature, the metabolic rate of zebrafish decreased, as shown by the decreasing oxygen uptake and ammonia excretion rates, limiting the critical swimming speed, probably due to a decreased muscle fibre power output. In response to exercise, fuel stores were mobilized to the liver as shown by the increased hepatosomatic index, liver total absolute energetic value and liver carbohydrate concentration but due to the low metabolic rate they could not be adequately addressed to power swimming activity at 18 °C. Conversely, the increased metabolic performance at high temperature came with an increased metabolic cost resulting in decreased energy status reflected particularly well by the non-invasive condition factor and invasive measures of carcass protein concentration, carcass total absolute energetic value and liver carbohydrate concentration. We showed that the combined measurement of the relative condition factor and critical swimming speed is a powerful non-invasive tool for long-term follow-up studies. Invasive methods were redundant for measuring general energy status but they provided detailed information concerning metabolic reorganization. With this study we proved that the usefulness of the zebrafish as a model organism can easily be expanded to include physiological studies and we provided a reference dataset for the selection of measures of physiological responses for future studies using the zebrafish.

This study was designed to evaluate if the rat H4IIE hepatoma cell line is a physiologically relevant model to study hepatic insulin responses to hint at its prospective application in pollutant-related insulin resistance research. DNA microarray analysis, real-time PCR and flow cytometric cell cycle analysis were used to assess the relevance of the insulin response in H4IIE cells. Insulin dose dependently stimulated H4IIE growth and time dependently altered the expression of the known insulin responsive genes: Fasn, Pck1 and Irs2. Microarray analysis performed on cells exposed to insulin (100nM) for 6h and 24h showed that genes related to carbohydrate and lipid metabolism were most profoundly afflicted, in accordance with in vivo hepatic insulin action. Since changes in carbohydrate and lipid metabolism are pivotal in the pathogenesis of insulin resistance, the presence of a physiological relevant insulin response in H4IIE cells pleads for further testing of its potential use in research on pollutant-driven insulin resistance., (Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.)

Two-colour microarrays are a popular platform of choice in gene expression studies. Because two different samples are hybridized on a single microarray, and several microarrays are usually needed in a given experiment, there are many possible ways to combine samples on different microarrays. The actual combination employed is commonly referred to as the 'hybridization design'. Different types of hybridization designs have been developed, all aimed at optimizing the experimental setup for the detection of differentially expressed genes while coping with technical noise. Here, we first provide an overview of the different classes of hybridization designs, discussing their advantages and limitations, and then we illustrate the current trends in the use of different hybridization design types in contemporary research.