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Although frequently examined as a target organ for non-endocrine toxicity, histopathological evaluation of the liver is becoming a routine component of endocrine disruption studies that utilize various fish species as test subjects. However, the interpretation of microscopic liver findings can be challenging, especially when attempting to distinguish adverse changes associated with endocrine disrupting substances from those caused by systemic or direct hepatic toxicity. The purpose of this project was to conduct a critical assessment of the available peer-reviewed and grey literature concerning the histopathologic effects of reproductive endocrine active substances (EAS) and non-endocrine acting substances in the livers of fish models, and to determine if liver histopathology can be used to reliably distinguish endocrine from non-endocrine etiologies. The results of this review suggest that few compound-specific histopathologic liver effects have been identified, among which are estrogen agonist-induced increases in hepatocyte basophilia and proteinaceous intravascular fluid in adult male teleosts, and potentially, decreased hepatocyte basophilia in female fish exposed to substances that possess androgenic, anti-estrogenic, or aromatase inhibitory activity. This review also used published standardized methodology to assess the credibility of the histopathology data in each of the 117 articles that reported liver effects of treatment, and consequently it was determined that in only 37% of those papers were the data considered either highly credible or credible. The outcome of this work highlights the value of histopathologic liver evaluation as an investigative tool for EAS studies, and provides information that may have implications for EAS hazard assessment. [ABSTRACT FROM AUTHOR]

Highlights: [•] Solvent carriers are discussed in terms of the practical, statistical and regulatory considerations. [•] Analysis using a database of 141 responses (endpoints) from 73 experiments. [•] Conditional strategy to control choice is recommended. [•] Wherever possible a solvent should not be employed or at least the concentration minimized. [Copyright &y& Elsevier]

Abstract: Fish in vivo screening methods to detect endocrine active substances, specifically interacting with the hypothalamic–pituitary–gonadal axis, have been developed by both the Organization for Economic Co-operation and Development (OECD) and United States Environmental Protection Agency (US-EPA). In application of these methods, i.e. regulatory testing, this paper provides a proposal on the setting of test concentrations using all available acute and chronic data and also discusses the importance of avoiding the confounding effects of systemic toxicity on endocrine endpoints. This guidance is aimed at reducing the number of false positives and subsequently the number of inappropriate definitive vertebrate studies potentially triggered by effects consequent to systemic, rather than endocrine, toxicity. At the same time it provides a pragmatic approach that maximizes the probability of detecting an effect, if it exists, thus limiting the potential for false negative outcomes. [Copyright &y& Elsevier]

Secondary sexual characteristics (SSCs) are important features that have evolved in many fish species because of inter-individual competition for mates. SSCs are crucial not only for sexual selection, but also for other components of the reproductive process and parental care. Externally, they are especially clear in males (for instance, tubercles, fatpad, anal finnage, colouration) but are also externally present in the females (for instance, ovipositor). These characters are under hormonal control and as such there has been much interest in incorporating them as measures in fish test methods to assess the potential endocrine activity of chemicals. Here we describe the external SSCs in typical laboratory test species for endocrine testing - fathead minnow (Pimephales promelas), Japanese medaka (Oryzias latipes), zebrafish (Danio rerio) and the three-spined stickleback (Gasterosteus aculeatus L.). We also provide some examples and discuss the utility of SSC responses to the endocrine activity of chemicals in the field and the laboratory. This paper is not aimed to provide a comprehensive review of SSCs in fish but presents a view on the assessment of SSCs in regulatory testing. Due to the current regulatory importance of establishing an endocrine mode-of-action for chemicals, we also consider other, non-endocrine factors that may lead to SSC responses in fish. We conclude with recommendations for how the assessment of SSCs in fish could be usefully incorporated into the endocrine hazard and risk assessment of chemicals. [ABSTRACT FROM AUTHOR]

[Display omitted] • We propose a decision logic to identify thyroid disruption in aquatic vertebrates. • The decision logic is evidence-based and includes internationally agreed guidelines. • Thyroid-mediated effect patterns must consider consistency and directions of changes. • The weight-of-evidence considers mode-of-action and population relevance assessments. • The decision logic supports regulatory assessments while minimising animal testing. According to the World Health Organisation and European Commission definitions, substances shall be considered as having endocrine disrupting properties if they show adverse effects, have endocrine activity and the adverse effects are a consequence of the endocrine activity (using a weight-of-evidence approach based on biological plausibility), unless the adverse effects are not relevant to humans or non-target organisms at the (sub)population level. To date, there is no decision logic on how to establish endocrine disruption via the thyroid modality in non-mammalian vertebrates. This paper describes an evidence-based decision logic compliant with the integrated approach to testing and assessment (IATA) concept, to identify thyroid-mediated effect patterns in aquatic vertebrates using amphibians as relevant models for thyroid disruption assessment. The decision logic includes existing test guidelines and methods and proposes detailed considerations on how to select relevant assays and interpret the findings. If the mammalian dataset used as the starting point indicates no thyroid concern, the Xenopus Eleutheroembryonic Thyroid Assay allows checking out thyroid-mediated activity in non-mammalian vertebrates, whereas the Amphibian Metamorphosis Assay or its extended, fixed termination stage variant inform on both thyroid-mediated activity and potentially population-relevant adversity. In evaluating findings, the response patterns of all assay endpoints are considered, including the direction of changes. Thyroid-mediated effect patterns identified at the individual level in the amphibian tests are followed by mode-of-action and population relevance assessments. Finally, all data are considered in an overarching weight-of-evidence evaluation. The logic has been designed generically and can be adapted, e.g. to accommodate fish tests once available for thyroid disruption assessments. It also ensures that all scientifically relevant information is considered, and that animal testing is minimised. The proposed decision logic can be included in regulatory assessments to facilitate the conclusion on whether substances meet the endocrine disruptor definition for the thyroid modality in non-mammalian vertebrates. [ABSTRACT FROM AUTHOR]

Highlights: [•] Feasibility of the threshold approach for formulated PPPs fish acute toxicity tests. [•] Database analysis demonstrates that a reduction of approximately 38% of the fish might be achieved. [•] Proposal for the threshold approach in the development of a regulatory dataset. [Copyright &y& Elsevier]

• The Amphibian Metamorphosis Assay (AMA) and Fish Short Term Reproduction Assay (FSTRA) are standardized regulatory tests used to determine if a chemical might affect the hypothalamic-pituitary-thyroid (HPT) axis or hypothalamic-pituitary-gonadal (HPG) axis, respectively. • Histopathology historical control data were assembled and evaluated from 55 AMA and 45 fathead minnow FSTRA studies. • Historical control data were found useful for investigating the existence of outlier results and aberrant control groups, identifying potential confounding variables, providing context for rare diagnoses, differentiating endocrine from non-endocrine effects, identifying background lesions, and helping to determine if specific improvements to established test guidance may be warranted. The Amphibian Metamorphosis Assay (AMA) is used to determine if a tested chemical has potential to impact the hypothalamic-pituitary-thyroid (HPT) axis of Xenopus laevis tadpoles, while the Fish Short Term Reproduction Assay (FSTRA) assesses potential effects on the hypothalamic-pituitary-gonadal (HPG) axis of fish such as the fathead minnow (Pimephales promelas). Several global regulatory programs routinely require these internationally validated tests be performed to determine the potential endocrine activity of chemicals. As such, they are conducted in accordance with standardized protocols and test criteria, which were originally developed more than a decade ago. Sizeable numbers of AMA and FSTRA studies have since been carried out, which allows for the mining of extensive historical control data (HCD). Such data are useful for investigating the existence of outlier results and aberrant control groups, identifying potential confounding variables, providing context for rare diagnoses, discriminating target from non-target effects, and for refining current testing paradigms. The present paper provides histopathology HCD from 55 AMA studies and 45 fathead minnow FSTRA studies, so that these data may become publicly available and thus aid in the interpretation of future study outcomes. Histopathology is a key endpoint in these assays, in which it is considered to be one of the most sensitive indicators of endocrine perturbation. In the current review, granular explorations of HCD data were used to identify background lesions, to assess the utility of particular diagnostic findings for distinguishing endocrine from non-endocrine effects, and to help determine if specific improvements to established regulatory guidance may be warranted. Knowledge gleaned from this investigation, supplemented by information from other recent studies, provided further context for the interpretation of AMA and FSTRA histopathology results. We recommend HCDs for the AMA and FSTRA be maintained to support the interpretation of study results. [ABSTRACT FROM AUTHOR]

Highlights: [•] We developed an individual-based zebrafish population model. [•] We investigated the population-level impact of two endocrine disrupting chemicals. [•] Acute exposure had no effects at the population-level. [•] Chronic exposure had significant effects at the population-level. [•] Ecological models can link individual-level effects to population-level impacts. [Copyright &y& Elsevier]

Abstract: Fish bioconcentration tests are time consuming, expensive, and use many animals. Alternative methods that replace, reduce or refine the use of fish for BCF testing would therefore be of value. Test guidelines generally require that bioconcentration factors (BCFs) are determined at two exposure concentrations. However, recent revisions to the OECD Test Guideline for BCF testing (TG 305) provide the option to use only one exposure concentration, when justification is provided, although two concentrations may still be required for some regulatory purposes. Analysis of 55 studies on plant protection products demonstrates that BCF values do not significantly differ between the two exposure concentrations. This analysis therefore provides evidence to support the revision of OECD TG 305, and in particular provides justification for using the one test concentration approach for plant protection product active substances. [Copyright &y& Elsevier]

Population regulation is fundamental to the long-term persistence of populations and their responses to harvesting, habitat modification, and exposure to toxic chemicals. In fish and other organisms with complex life histories, regulation may involve density dependence in different life-stages and vital rates. We studied density dependence in body growth and mortality through the life-cycle of laboratory populations of zebrafish Danio rerio. When feed input was held constant at population-level (leading to resource limitation), body growth was strongly density-dependent in the late juvenile and adult phases of the life-cycle. Density dependence in mortality was strong during the early juvenile phase but declined thereafter and virtually ceased prior to maturation. Provision of feed in proportion to individual requirements (easing resource limitation) removed density dependence in growth and substantially reduced density dependence in mortality, thus indicating that 'bottom-up' effects act on growth as well as mortality, but most strongly on growth. Both growth and mortality played an important role in population regulation, with density-dependent growth having the greater impact on population biomass while mortality had the greatest impact on numbers. We demonstrate a clear ontogenic pattern of change in density-dependent processes within populations of a very small (maximum length 5 mm) fish, maintained in constant homogeneous laboratory conditions. The patterns are consistent with those distilled from studies on wild fish populations, indicating the presence of broad ontogenic patterns in density-dependent processes that are invariant to maximum body size and hold in homogeneous laboratory, as well as complex natural environments. [ABSTRACT FROM AUTHOR]

Abstract: Exogenous treatment of fish with natural sex hormones and their mimics has been shown to influence gonadal differentiation resulting in biased phenotypic sex-ratios. This has lead to the development of the Fish Sexual Development Test (FSDT) as a method for the detection of endocrine active chemicals. Proposed test organisms include the medaka, zebrafish (ZF) and stickleback, although the guideline also allows for inclusion of species such as the fathead minnow (FHM), provided the test duration allows for sufficient sexual differentiation. However, although the processes underlying sexual differentiation are known to differ for each of these species, it is not known how, or if, these differences would influence the results of the FSDT. In the experiments reported here, responses of the ZF and FHM to prochloraz, a sterol biosynthesis inhibitor and androgen antagonist, were characterized and compared. Exposure to 320μg/L of prochloraz, from embryo until 60 (ZF) or 95–125 (FHM) days post hatch inhibited somatic growth of both species, but while a negative impact on ZF larval survival was observed (LOEC 32μg/L) there was no evidence for an effect on FHM larval survival. Prochloraz influenced sexual differentiation in both species by decreasing the proportion of females (LOEC 100μg/L (ZF), 320μg/L (FHM)) and delaying completion of sexual differentiation; manifest as an increased incidence of ovotestis in the ZF (LOEC 100μg/L) and as an increased number of fish with undifferentiated gonads in the FHM (LOEC 320μg/L). However, while exposure to 320μg/L prochloraz delayed maturation of the differentiated FHM testis, there was no such effect in the ZF. These results demonstrate that the different strategy of sexual differentiation in the ZF and FHM influences the profile of responses of their gonads to the masculinising effects of prochloraz, but does not affect their overall sensitivity. [Copyright &y& Elsevier]