Effects of Metabolic Disruption on Lipid Metabolism and Yolk Retention in Zebrafish Embryos.

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.

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.

Exploring BPA alternatives - Environmental levels and toxicity review.

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.

Corrigendum: Development of new approach methods for the identification and characterization of endocrine metabolic disruptors-a PARC project.

[This corrects the article DOI: 10.3389/ftox.2023.1212509.].

Comprehensive mapping of the AOP-Wiki database: identifying biological and disease gaps.

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.

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).

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).

Combined western diet and bisphenol A exposure induces an oxidative stress-based paraoxonase 1 response in larval zebrafish.

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.

Cross-species applicability of an adverse outcome pathway network for thyroid hormone system disruption.

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.

Deiodinase inhibition impairs the formation of the three posterior swim bladder tissue layers during early embryonic development in zebrafish.

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.

Development of new approach methods for the identification and characterization of endocrine metabolic disruptors-a PARC project.

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.

New approach methods to improve human health risk assessment of thyroid hormone system disruption-a PARC project.

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.

Chronic handling stress in zebrafish Danio rerio husbandry.

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.

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.

Lipidomics profiling of zebrafish liver through untargeted liquid chromatography-high resolution mass spectrometry.

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 103 -107 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.

Mass Spectrometry-Based Zebrafish Toxicometabolomics: A Review of Analytical and Data Quality Challenges.

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.

A Pragmatic Approach to Adverse Outcome Pathway Development and Evaluation.

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.

Sublethal Effect Concentrations for Nonpolar Narcosis in the Zebrafish Embryo.

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.