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Diamide insecticides, specifically chlorantraniliprole (CHL), have been rising in popularity over the past decade, becoming one of the most widely used insecticide classes globally. These insecticides target the ryanodine receptor (RyR), primarily for control of lepidopteran agricultural pests. Field studies have revealed that some lepidopteran species have developed mutations where a methionine in a particular position (e.g., I4790M) increases resistance to CHL. The toxicity data for CHL across species is limited, as is the case for many chemicals, which creates an opportunity to apply both traditional toxicity test methods and new approach methods (NAMs) to address data gaps. Here, the U.S. Environmental Protection Agency's Sequence Alignment to Predict Across Species Susceptibility (SeqAPASS) tool was used to query the RyR to generate susceptibility predictions for species exposed to CHL to fill those data gaps. These SeqAPASS results generated testable hypotheses that were used to guide focused acute aquatic toxicity studies using Daphnia magna, Daphnia pulex, Pimephales promelas, and Danio rerio. The fish species were not sensitive to CHL, whereas D. magna and D. pulex were found to be sensitive to CHL at environmentally relevant concentrations, despite having the methionine residue in the position of the I4790M resistance mutation. Additional SeqAPASS results showed that many other species, including beneficial pollinators and Lepidoptera, are predicted as likely susceptible to CHL. This study provided multiple lines of evidence toward the unlikelihood for the I4790M mutation to be the primary cause of resistance across species, filled knowledge gaps concerning CHL toxicity across species, and generated predictions of susceptibility for nontarget species that are not generally amenable to toxicity testing. This work presents a case example that demonstrates how NAMs can be used in combination with other types of data to direct targeted testing and build confidence in predictive approaches for their use in risk assessment., (Published by Oxford University Press on behalf of the Society of Environmental Toxicology and Chemistry 2025.)

Biological Evaluations support Endangered Species Act (ESA) consultation with the US Fish and Wildlife Service and National Marine Fisheries Service by federal action agencies, such as the USEPA, regarding impacts of federal activities on threatened or endangered species. However, they are often time-consuming and challenging to conduct. The identification of pollutant benchmarks or guidance to protect taxa for states and tribes when USEPA has not yet developed criteria recommendations is also of importance to ensure a streamlined approach to Clean Water Act program implementation. Due to substantial workloads, tight regulatory timelines, and the often-protracted length of ESA consultations, there is a need to streamline the development of biological evaluation toxicity assessments for determining the impact of chemical pollutants on ESA-listed species. Moreover, there is limited availability of species-specific toxicity data for many contaminants, further complicating the consultation process. New approach methodologies are being increasingly used in toxicology and chemical safety assessment to rapidly and cost-effectively provide data that can fill gaps in hazard and/or exposure characterization. Here, we present the development of an automated computational pipeline-RASRTox (Rapidly Acquire, Score, and Rank Toxicological data)-to rapidly extract and categorize ecological toxicity benchmark values from curated data sources (ECOTOX, ToxCast) and well-established quantitative structure-activity relationships (TEST, ECOSAR). As a proof of concept, points-of-departure (PODs) generated in RASRTox for 13 chemicals were compared against benchmark values derived using traditional methods-toxicity reference values (TRVs) and water quality criteria (WQC). The RASRTox PODs were generally within an order of magnitude of corresponding TRVs, though less concordant compared with WQC. The greatest utility of RASRTox, however, lies in its ability to quickly and systematically identify critical studies that may serve as a basis for screening value derivation by toxicologists as part of an ecological hazard assessment. As such, the strategy described in this case study can potentially be adapted for other risk assessment contexts and stakeholder needs. Integr Environ Assess Manag 2024;20:2203-2217. © 2024 Society of Environmental Toxicology & Chemistry (SETAC). This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA., (© 2024 Society of Environmental Toxicology & Chemistry (SETAC). This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA.)

Accounting for intraspecific and interspecific competition when assessing the effects of chemical and nonchemical stressors is an important uncertainty in ecological risk assessments. We developed novel projection of interspecific competition (PIC) matrices that allow for analysis of population dynamics of two or more species exposed to a given stressor(s) that compete for shared resources within a landscape. We demonstrate the application of PIC matrices to investigate the population dynamics of two hypothetical fish species that compete with one another and have differences in net reproductive rate and intrinsic rate of population increase. Population status predictions were made under scenarios that included exposure to a chemical stressor that reduced fecundity for one or both species. The results of our simulations demonstrated that measures obtained from the life table and Leslie matrix of an organism, including net reproductive rate and intrinsic rate of increase, can result in erroneous conclusions of population status and viability in the absence of a consideration of resource limitation and interspecific competition. This modeling approach can be used in conjunction with field monitoring efforts and/or laboratory testing to link effects due to stressors to possible outcomes within an ecosystem. In addition, PIC matrices could be combined with adverse outcome pathways to allow for ecosystem projection based on taxonomic conservation of molecular targets of chemicals to predict the likelihood of relative cross-species susceptibility. Overall, the present study shows how PIC matrices can integrate effects across the life cycles of multiple species, provide a linkage between endpoints observed in individual and population-level responses, and project outcomes at the community level for multiple generations for multiple species that compete for limited resources. Environ Toxicol Chem 2024;43:1406-1422. Published 2024. This article is a U.S. Government work and is in the public domain in the USA., (Published 2024. This article is a U.S. Government work and is in the public domain in the USA.)

Translation of environmental science to the practice aims to protect biodiversity and ecosystem services, and our future ability to do so relies on the development of a precision ecotoxicology approach wherein we leverage the genetics and informatics of species to better understand and manage the risks of global pollution. A little over a decade ago, a workshop focusing on the risks of pharmaceuticals and personal care products (PPCPs) in the environment identified a priority research question, "What can be learned about the evolutionary conservation of PPCP targets across species and life stages in the context of potential adverse outcomes and effects?" We review the activities in this area over the past decade, consider prospects of more recent developments, and identify future research needs to develop next-generation approaches for PPCPs and other global chemicals and waste challenges. Environ Toxicol Chem 2024;43:526-536. © 2023 SETAC. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA., (© 2023 SETAC. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA.)

There are insufficient toxicity data to assess the ecological risks of many pharmaceuticals and personal care products (PPCPs). While data limitations are not uncommon for contaminants of environmental concern, PPCPs are somewhat unique in that an a priori understanding of their biological activities in conjunction with measurements of molecular, biochemical, or histological responses could provide a foundation for understanding mode(s) of action and predicting potential adverse apical effects. Over the past decade significant progress has been made in the development of new approach methodologies (NAMs) to efficiently quantify these types of endpoints using computational models and pathway-based in vitro and in vivo assays. The availability of open-access knowledgebases to curate biological response (including NAM) data and sophisticated bioinformatics tools to help interpret the information also has significantly increased. Finally, advances in the development and implementation of the adverse outcome pathway framework provide the critical conceptual underpinnings needed to translate NAM data into predictions of the ecologically relevant outcomes required by risk assessors and managers. The evolution and convergence of these various data streams, tools, and concepts provides the basis for a fundamental change in how ecological risks of PPCPs can be pragmatically assessed. Environ Toxicol Chem 2024;43:537-548. © 2022 SETAC. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA., (© 2022 SETAC. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA.)

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.)

Several adverse outcome pathways (AOPs) have linked molecular initiating events like aromatase inhibition, androgen receptor (AR) agonism, and estrogen receptor (ER) antagonism to reproductive impairment in adult fish. Estrogen receptor agonists can also cause adverse reproductive effects, however, the early key events (KEs) in an AOP leading to this are mostly unknown. The primary aim of this study was to develop hypotheses regarding the potential mechanisms through which exposure to ER agonists might lead to reproductive impairment in female fish. Mature fathead minnows were exposed to 1 or 10 ng 17α-ethynylestradiol (EE2)/L or 10 or 100 µg bisphenol A (BPA)/L for 14 d. The response to EE2 and BPA was contrasted with the effects of 500 ng/L of 17β-trenbolone (TRB), an AR agonist, as well as TRB combined with the low and high concentrations of EE2 or BPA tested individually. Exposure to 10 ng EE2/L, 100 µg BPA/L, TRB, or the various mixtures with TRB caused significant decreases in plasma concentrations of 17β-estradiol. Exposure to TRB alone caused a significant reduction in plasma vitellogenin (VTG), but VTG was unaffected or even increased in females exposed to EE2 or BPA alone or, in most cases, in mixtures with TRB. Over the course of the 14-d exposure, the only treatments that clearly did not affect egg production were 1 ng EE2/L and 10 µg BPA/L. Based on these results and knowledge of hypothalamic-pituitary-gonadal axis function, we hypothesize an AOP whereby decreased production of maturation-inducing steroid leading to impaired oocyte maturation and ovulation, possibly due to negative feedback or direct inhibitory effects of membrane ER activation, could be responsible for causing adverse reproductive impacts in female fish exposed to ER agonists., 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., (Published by Elsevier B.V.)

Anthropogenic activities introduce complex mixtures into aquatic environments, necessitating mixture toxicity evaluation during risk assessment. There are many alternative approaches that can be used to complement traditional techniques for mixture assessment. Our study aimed to demonstrate how these approaches could be employed for mixture evaluation in a target watershed. Evaluations were carried out over 2 years (2017-2018) across 8-11 study sites in the Milwaukee Estuary (WI, USA). Whole mixtures were evaluated on a site-specific basis by deploying caged fathead minnows (Pimephales promelas) alongside composite samplers for 96 h and characterizing chemical composition, in vitro bioactivity of collected water samples, and in vivo effects in whole organisms. Chemicals were grouped based on structure/mode of action, bioactivity, and pharmacological activity. Priority chemicals and mixtures were identified based on their relative contributions to estimated mixture pressure (based on cumulative toxic units) and via predictive assessments (random forest regression). Whole mixture assessments identified target sites for further evaluation including two sites targeted for industrial/urban chemical mixture effects assessment; three target sites for pharmaceutical mixture effects assessment; three target sites for further mixture characterization; and three low-priority sites. Analyses identified 14 mixtures and 16 chemicals that significantly contributed to cumulative effects, representing high or medium priority targets for further ecotoxicological evaluation, monitoring, or regulatory assessment. Overall, our study represents an important complement to single-chemical prioritizations, providing a comprehensive evaluation of the cumulative effects of mixtures detected in a target watershed. Furthermore, it demonstrates how different tools and techniques can be used to identify diverse facets of mixture risk and highlights strategies that can be considered in future complex mixture assessments. Environ Toxicol Chem 2023;42:1229-1256. © 2023 SETAC., (© 2023 SETAC.)

The U.S. Environmental Protection Agency's Endocrine Disruptor Screening Program (EDSP) is tasked with assessing chemicals for their potential to perturb endocrine pathways, including those controlled by androgen receptor (AR). To address challenges associated with traditional testing strategies, EDSP is considering in vitro high-throughput screening assays to screen and prioritize chemicals more efficiently. The ability of these assays to accurately reflect chemical interactions in nonmammalian species remains uncertain. Therefore, a goal of the EDSP is to evaluate how broadly results can be extrapolated across taxa. To assess the cross-species conservation of AR-modulated pathways, computational analyses and systematic literature review approaches were used to conduct a comprehensive analysis of existing in silico, in vitro, and in vivo data. First, molecular target conservation was assessed across 585 diverse species based on the structural similarity of ARs. These results indicate that ARs are conserved across vertebrates and are predicted to share similarly susceptibility to chemicals that interact with the human AR. Systematic analysis of over 5000 published manuscripts was used to compile in vitro and in vivo cross-species toxicity data. Assessment of in vitro data indicates conservation of responses occurs across vertebrate ARs, with potential differences in sensitivity. Similarly, in vivo data indicate strong conservation of the AR signaling pathways across vertebrate species, although sensitivity may vary. Overall, this study demonstrates a framework for utilizing bioinformatics and existing data to build weight of evidence for cross-species extrapolation and provides a technical basis for extrapolating hAR-based data to prioritize hazard in nonmammalian vertebrate species., (Published by Oxford University Press on behalf of the Society of Toxicology 2023.)

New approach methodologies (NAMs) are being developed to reduce and replace vertebrate animal testing in support of ecotoxicology and risk assessment. The US Environmental Protection Agency's Sequence Alignment to Predict Across Species Susceptibility (SeqAPASS) bioinformatic tool was used to evaluate amino acid sequence conservation of the type 3 iodothyronine deiodinase (DIO3) enzyme across species to demonstrate NAM applications for understanding effects of chemical interactions with a specific protein target. Existing literature was used to identify critical amino acids for thyroid hormone binding and interaction with a reducing cofactor. The SeqAPASS tool identifies whether known critical amino acids involved in ligand binding are exact, partial, or not matches across species compared with a template species based on molecular weight and side chain classification. This evaluation guided the design of variant proteins representing critical amino acid substitutions found in various species. Site-directed mutagenesis of the wild-type (WT) human DIO3 gene sequence was used to create six variant proteins expressed in cell culture, which were then tested in vitro for chemical inhibition. Significant differences in in vitro median inhibitory concentration results were observed among variants for potential competitive inhibitors. A molecular model representing the WT human DIO3 was constructed using Molecular Operating Environment (MOE) software and mutated in silico to create the six variants. The MOE Site Finder tool identified the proposed catalytic and cofactor sites and potential alternative binding sites. Virtual docking did not provide affinity scores with sufficient resolution to rank the potency of the chemical inhibitors. Chemical characteristics, function and location of substituted amino acids, and complexities of the protein target are important considerations in developing NAMs to evaluate chemical susceptibility across species. Environ Toxicol Chem 2023;42:1032-1048. © 2023 University of Wisconsin-Madison. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA., (© 2023 University of Wisconsin-Madison. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA.)

Multiple in vivo test guidelines focusing on the estrogen, androgen, thyroid, and steroidogenesis pathways have been developed and validated for mammals, amphibians, or fish. However, these tests are resource-intensive and often use a large number of laboratory animals. Developing alternatives for in vivo tests is consistent with the replacement, reduction, and refinement principles for animal welfare considerations, which are supported by increasing mandates to move toward an "animal-free" testing paradigm worldwide. New approach methodologies (NAMs) hold great promise to identify molecular, cellular, and tissue changes that can be used to predict effects reliably and more efficiently at the individual level (and potentially on populations) while reducing the number of animals used in (eco)toxicological testing for endocrine disruption. In a collaborative effort, experts from government, academia, and industry met in 2020 to discuss the current challenges of testing for endocrine activity assessment for fish and amphibians. Continuing this cross-sector initiative, our review focuses on the current state of the science regarding the use of NAMs to identify chemical-induced endocrine effects. The present study highlights the challenges of using NAMs for safety assessment and what work is needed to reduce their uncertainties and increase their acceptance in regulatory processes. We have reviewed the current NAMs available for endocrine activity assessment including in silico, in vitro, and eleutheroembryo models. New approach methodologies can be integrated as part of a weight-of-evidence approach for hazard or risk assessment using the adverse outcome pathway framework. The development and utilization of NAMs not only allows for replacement, reduction, and refinement of animal testing but can also provide robust and fit-for-purpose methods to identify chemicals acting via endocrine mechanisms. Environ Toxicol Chem 2023;42:757-777. © 2023 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC., (© 2023 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.)

The US Environmental Protection Agency Sequence Alignment to Predict Across Species Susceptibility (SeqAPASS) tool is a fast, freely available, online screening application that allows researchers and regulators to extrapolate toxicity information across species. For biological targets in model systems such as human cells, mice, rats, and zebrafish, toxicity data are available for a variety of chemicals. Through the evaluation of protein target conservation, this tool can be used to extrapolate data generated from such model systems to thousands of other species lacking toxicity data, yielding predictions of relative intrinsic chemical susceptibility. The latest releases of the tool (versions 2.0-6.1) have incorporated new features that allow for the rapid synthesis, interpretation, and use of the data for publication plus presentation-quality graphics. Among these features are customizable data visualizations and a comprehensive summary report designed to summarize SeqAPASS data for ease of interpretation. This paper describes the protocol to guide users through submitting jobs, navigating the various levels of protein sequence comparisons, and interpreting and displaying the resulting data. New features of SeqAPASS v2.0-6.0 are highlighted. Furthermore, two use-cases focused on transthyretin and opioid receptor protein conservation using this tool are described. Finally, SeqAPASS' strengths and limitations are discussed to define the domain of applicability for the tool and highlight different applications for cross-species extrapolation.

Computational screening for potentially bioactive molecules using advanced molecular modeling approaches including molecular docking and molecular dynamic simulation is mainstream in certain fields like drug discovery. Significant advances in computationally predicting protein structures from sequence information have also expanded the availability of structures for nonmodel species. Therefore, the objective of the present study was to develop an analysis pipeline to harness the power of these bioinformatics approaches for cross-species extrapolation for evaluating chemical safety. The Sequence Alignment to Predict Across Species Susceptibility (SeqAPASS) tool compares protein-sequence similarity across species for conservation of known chemical targets, providing an initial line of evidence for extrapolation of toxicity knowledge. However, with the development of structural models from tools like the Iterative Threading ASSEmbly Refinement (ITASSER), analyses of protein structural conservation can be included to add further lines of evidence and generate protein models across species. Models generated through such a pipeline could then be used for advanced molecular modeling approaches in the context of species extrapolation. Two case examples illustrating this pipeline from SeqAPASS sequences to I-TASSER-generated protein structures were created for human liver fatty acid-binding protein (LFABP) and androgen receptor (AR). Ninety-nine LFABP and 268 AR protein models representing diverse species were generated and analyzed for conservation using template modeling (TM)-align. The results from the structural comparisons were in line with the sequence-based SeqAPASS workflow, adding further evidence of LFABL and AR conservation across vertebrate species. The present study lays the foundation for expanding the capabilities of the web-based SeqAPASS tool to include structural comparisons for species extrapolation, facilitating more rapid and efficient toxicological assessments among species with limited or no existing toxicity data. Environ Toxicol Chem 2023;42:463-474. © 2022 SETAC. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA., (© 2022 SETAC. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA.)

For the majority of developed adverse outcome pathways (AOPs), the taxonomic domain of applicability (tDOA) is typically narrowly defined with a single or a handful of species. Defining the tDOA of an AOP is critical for use in regulatory decision-making, particularly when considering protection of untested species. Structural and functional conservation are two elements that can be considered when defining the tDOA. Publicly accessible bioinformatics approaches, such as the Sequence Alignment to Predict Across Species Susceptibility (SeqAPASS) tool, take advantage of existing and growing databases of protein sequence and structural information to provide lines of evidence toward structural conservation of key events (KEs) and KE relationships (KERs) of an AOP. It is anticipated that SeqAPASS results could readily be combined with data derived from empirical toxicity studies to provide evidence of both structural and functional conservation, to define the tDOA for KEs, KERs, and AOPs. Such data could be incorporated in the AOP-Wiki as lines of evidence toward biological plausibility for the tDOA. We present a case study describing the process of using bioinformatics to define the tDOA of an AOP using an AOP linking the activation of the nicotinic acetylcholine receptor to colony death/failure in Apis mellifera. Although the AOP was developed to gain a particular biological understanding relative to A. mellifera health, applicability to other Apis bees, as well as non-Apis bees, has yet to be defined. The present study demonstrates how bioinformatics can be utilized to rapidly take advantage of existing protein sequence and structural knowledge to enhance and inform the tDOA of KEs, KERs, and AOPs, focusing on providing evidence of structural conservation across species. Environ Toxicol Chem 2023;42:71-87. © 2022 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA., (© 2022 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA.)

The USEPA's 1985 guidelines for the derivation of aquatic life criteria (ALC) are robust but data-intensive. For many chemicals, the extensive in vivo data sets required for ALC derivation are not available. Thus, alternative analyses and processes that can provide provisional values to guide states, tribes, and other stakeholders while data accumulate and more rigorous criteria are derived would be beneficial. The overarching purpose of this study was to assess the feasibility of using data from new approach methodologies (NAMs) like ToxCast to derive first-pass, provisional values to guide chemical prioritization and resource management as a complement to traditional ALC derivation. To address this goal, the study objectives were to (1) estimate chemical potency using data from NAMs for nine compounds with available aquatic benchmarks, (2) evaluate the utility of using NAM data to elucidate potential mechanisms of toxicity to guide problem formulation, and (3) determine the species relevance of toxicity pathways for compounds with clearly defined mechanisms of action as a means to evaluate whether minimum data requirements could potentially be waived when deriving a more formal ALC. Points of departure were derived from ToxCast data based on the fifth percentile of the distribution of activity concentration above cutoff values falling below the cytotoxic burst. Mechanistic inferences were made based on active target hits in ToxCast and, where applicable, assessed for taxonomic conservation using SeqAPASS. ToxCast-based point-of-departure aligned relatively closely (six of nine test chemicals within a factor of 10; eight of nine within a factor of 100) with aquatic benchmarks from the USEPA and US Department of Energy (DOE). Moreover, pathways of toxicity gleaned from NAM data were reflective of in vivo-based findings from the literature. These results, while preliminary, and based on a limited number of substances, support the potential application of NAM data to complement traditional ALC derivation approaches and prioritization. Integr Environ Assess Manag 2023;19:224-238. © 2022 Society of Environmental Toxicology & Chemistry (SETAC). This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA., (© 2022 Society of Environmental Toxicology & Chemistry (SETAC). This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA.)

U.S. regulatory and research agencies use ecotoxicity test data to assess the hazards associated with substances that may be released into the environment, including but not limited to industrial chemicals, pharmaceuticals, pesticides, food additives, and color additives. These data are used to conduct hazard assessments and evaluate potential risks to aquatic life (e.g., invertebrates, fish), birds, wildlife species, or the environment. To identify opportunities for regulatory uses of non-animal replacements for ecotoxicity tests, the needs and uses for data from tests utilizing animals must first be clarified. Accordingly, the objective of this review was to identify the ecotoxicity test data relied upon by U.S. federal agencies. The standards, test guidelines, guidance documents, and/or endpoints that are used to address each of the agencies' regulatory and research needs regarding ecotoxicity testing are described in the context of their application to decision-making. Testing and information use, needs, and/or requirements relevant to the regulatory or programmatic mandates of the agencies taking part in the Interagency Coordinating Committee on the Validation of Alternative Methods Ecotoxicology Workgroup are captured. This information will be useful for coordinating efforts to develop and implement alternative test methods to reduce, refine, or replace animal use in chemical safety evaluations., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

The need for assembled existing and new toxicity data has accelerated as the amount of chemicals introduced into commerce continues to grow and regulatory mandates require safety assessments for a greater number of chemicals. To address this evolving need, the ECOTOXicology Knowledgebase (ECOTOX) was developed starting in the 1980s and is currently the world's largest compilation of curated ecotoxicity data, providing support for assessments of chemical safety and ecological research through systematic and transparent literature review procedures. The recently released version of ECOTOX (Ver 5, www.epa.gov/ecotox) provides single-chemical ecotoxicity data for over 12,000 chemicals and ecological species with over one million test results from over 50,000 references. Presented is an overview of ECOTOX, detailing the literature review and data curation processes within the context of current systematic review practices and discussing how recent updates improve the accessibility and reusability of data to support the assessment, management, and research of environmental chemicals. Relevant and acceptable toxicity results are identified from studies in the scientific literature, with pertinent methodological details and results extracted following well-established controlled vocabularies and newly extracted toxicity data added quarterly to the public website. Release of ECOTOX, Ver 5, included an entirely redesigned user interface with enhanced data queries and retrieval options, visualizations to aid in data exploration, customizable outputs for export and use in external applications, and interoperability with chemical and toxicity databases and tools. This is a reliable source of curated ecological toxicity data for chemical assessments and research and continues to evolve with accessible and transparent state-of-the-art practices in literature data curation and increased interoperability to other relevant resources. Environ Toxicol Chem 2022;41:1520-1539. © 2022 SETAC. This article has been contributed to by US Government employees and their work is in the public domain in the USA., (© 2022 SETAC. This article has been contributed to by US Government employees and their work is in the public domain in the USA.)

Exposure to certain anthropogenic chemicals can inhibit the activity to cytochrome P450 aromatase (CYP19) in fishes leading to decreased plasma 17β-estradiol (E2), plasma vitellogenin (VTG), and egg production. Reproductive dysfunction resulting from exposure to aromatase inhibitors has been extensively investigated in several laboratory model species of fish. These model species have ovaries that undergo asynchronous oocyte development, but many fishes have ovaries with group-synchronous oocyte development. Fishes with group-synchronous oocyte development have dynamic reproductive cycles which typically occur annually and are often triggered by complex environmental cues. This has resulted in a lack of test data and uncertainty regarding sensitivities to and adverse effects of aromatase inhibition. The present study used the western mosquitofish (Gambusia affinis) as a laboratory model to investigate adverse effects of chemical aromatase inhibition on group-synchronous oocyte development. Adult female western mosquitofish were exposed to either 0, 2, or 30 μg/L of the model nonsteroidal aromatase inhibiting chemical, fadrozole, for a complete reproductive cycle. Fish were sampled at four time-points representing pre-vitellogenic resting, early vitellogenesis, late vitellogenesis/early ovarian recrudescence, and late ovarian recrudescence. Temporal changes in numerous reproductive parameters were measured, including gonadosomatic index (GSI), plasma sex steroids, and expression of selected genes in the brain, liver, and gonad that are important for reproduction. In contrast to fish from the control treatment, fish exposed to 2 and 30 μg/L of fadrozole had persistent elevated expression of cyp19 in the ovary, depressed expression of vtg in the liver, and a low GSI. These responses suggest that completion of a group-synchronous reproductive cycle was unsuccessful during the assay in fish from either fadrozole treatment. These adverse effects data show that exposure to aromatase inhibitors has the potential to cause reproductive dysfunction in a wide range of fishes with both asynchronous and group-synchronous reproductive strategies., (Published by Elsevier B.V.)

Feminization of male fish and the role of endocrine-active chemicals in this phenomenon has been an area of intense study for many years. Estrone (E1), a natural steroid, is found in aquatic environments sometimes at high concentrations relative to the estrogenic steroids 17β-estradiol (E2) and 17α-ethynylestradiol. However, E1 has been less thoroughly studied than E2 or 17α-ethynylestradiol due in part to a relatively lower potency in metabolically limited estrogen receptor (ER) binding/activation assays. Recent evidence suggests that in vivo biotransformation of E1 to E2 may occur in fathead minnows (Pimephales promelas) residing in environments with high concentrations of E1, such as near wastewater treatment plants. The enzymes likely responsible for this biotransformation, 17β-hydroxysteroid dehydrogenases (17βHSDs), have been well characterized in mammals but to a lesser extent in fish species. In the present study, a novel systematic analysis of amino acid sequence data from the National Center for Biotechnology Information database demonstrated that multiple 17βHSD isoforms are conserved across different fish species. Experimentally, we showed that metabolically active hepatic cytosolic preparations from 2 commercially important salmonid species, rainbow trout and lake trout, biotransformed E1 to E2 to a degree sufficient to alter results of competitive ER binding assays. These results from in silico and in vitro analyses indicate that E1 and biotransformation may play a significant role in adverse effects on development and reproduction of a variety of fish species in contaminated aquatic environments. Environ Toxicol Chem 2020;39:2028-2040. Published 2020. This article is a US Government work and is in the public domain in the USA., (Published 2020. This article is a US Government work and is in the public domain in the USA.)

More than 1000 per- and polyfluoroalkyl substances (PFASs) have been discovered by nontarget analysis (NTA), but their prioritization for health concerns is challenging. We developed a method by incorporating size-exclusion column co-elution (SECC) and NTA, to screen PFASs binding to human liver fatty acid binding protein ( h L-FABP). Of 74 PFASs assessed, 20 were identified as h L-FABP ligands in which eight of them have high binding affinities. Increased PFAS binding affinities correlate with stronger responses in electrospray ionization (ESI - ) and longer retention times on a C18 column. This is well explained by a mechanistic model, which revealed that both polar and hydrophobic interactions are crucial for binding affinities. Encouraged by this, we then developed an SECC method to identify h L-FABP ligands, and all eight high-affinity ligands were selectively captured from 74 PFASs. The method was further applied to an aqueous film-forming foam (AFFF) product in which 31 new h L-FABP ligands were identified. Suspect and nontargeted screening revealed these ligands as analogues of perfluorosulfonic acids and homologues of alkyl ether sulfates (C 8 - and C 10 /EO n , C 8 H 17 (C 2 H 4 O) n SO 4 - , and C 10 H 21 (C 2 H 4 O) n SO 4 - ). The SECC method was then applied to AFFF-contaminated surface waters. In addition to perfluorooctanesulfonic acid and perfluorohexanesulfonic acid, eight other AFFF chemicals were discovered as novel ligands, including four C 14 - and C 15 /EO n . This study implemented a high-throughput method to prioritize PFASs and revealed the existence of many previously unknown h L-FABP ligands.

Predictive approaches to assessing the toxicity of contaminant mixtures have been largely limited to chemicals that exert effects through the same biological molecular initiating event. However, by understanding specific pathways through which chemicals exert effects, it may be possible to identify shared "downstream" nodes as the basis for forecasting interactive effects of chemicals with different molecular initiating events. Adverse outcome pathway (AOP) networks conceptually support this type of analysis. We assessed the utility of a simple AOP network for predicting the effects of mixtures of an aromatase inhibitor (fadrozole) and an androgen receptor agonist (17β-trenbolone) on aspects of reproductive endocrine function in female fathead minnows. The fish were exposed to multiple concentrations of fadrozole and 17β-trenbolone individually or in combination for 48 or 96 h. Effects on 2 shared nodes in the AOP network, plasma 17β-estradiol (E2) concentration and vitellogenin (VTG) production (measured as hepatic vtg transcripts) responded as anticipated to fadrozole alone but were minimally impacted by 17β-trenbolone alone. Overall, there were indications that 17β-trenbolone enhanced decreases in E2 and vtg in fadrozole-exposed fish, as anticipated, but the results often were not statistically significant. Failure to consistently observe hypothesized interactions between fadrozole and 17β-trenbolone could be due to several factors, including lack of impact of 17β-trenbolone, inherent biological variability in the endpoints assessed, and/or an incomplete understanding of interactions (including feedback) between different pathways within the hypothalamic-pituitary-gonadal axis. Environ Toxicol Chem 2020;39:913-922. © 2020 SETAC., (© 2020 SETAC.)

Quantitative adverse outcome pathways (qAOPs) describe quantitative response-response relationships that can predict the probability or severity of an adverse outcome for a given magnitude of chemical interaction with a molecular initiating event. However, the taxonomic domain of applicability for these predictions is largely untested. The present study began defining this applicability for a previously described qAOP for aromatase inhibition leading to decreased fecundity developed using data from fathead minnow ( Pimephales promelas ). This qAOP includes quantitative response-response relationships describing plasma 17β-estradiol (E2) as a function of plasma fadrozole, plasma vitellogenin (VTG) as a function of plasma E2, and fecundity as a function of plasma VTG. These quantitative response-response relationships simulated plasma E2, plasma VTG, and fecundity measured in female zebrafish ( Danio rerio ) exposed to fadrozole for 21 days but not these responses measured in female Japanese medaka ( Oryzias latipes ). However, Japanese medaka had different basal levels of plasma E2, plasma VTG, and fecundity. Normalizing basal levels of each measurement to equal those of female fathead minnow enabled the relationships to accurately simulate plasma E2, plasma VTG, and fecundity measured in female Japanese medaka. This suggests that these quantitative response-response relationships are conserved across these three fishes when considering relative change rather than absolute measurements. The present study represents an early step toward defining the appropriate taxonomic domain of applicability and extending the regulatory applications of this qAOP.

There is significant concern regarding potential impairment of fish reproduction associated with endocrine disrupting chemicals. Aromatase (CYP19) is a steroidogenic enzyme involved in the conversion of androgens to estrogens. Inhibition of aromatase by chemicals can result in reduced concentrations of estrogens leading to adverse reproductive effects. These effects have been extensively investigated in a small number of laboratory model fishes, such as fathead minnow (Pimephales promelas), Japanese medaka (Oryzias latipes), and zebrafish (Danio rerio). But, differences in sensitivity among species are largely unknown. Therefore, this study took a first step toward understanding potential differences in sensitivity to aromatase inhibitors among fishes. Specifically, a standard in vitro aromatase inhibition assay using subcellular fractions of whole tissue homogenates was used to evaluate the potential sensitivity of 18 phylogenetically diverse species of freshwater fish to the nonsteroidal aromatase inhibitor fadrozole. Sensitivity to fadrozole ranged by more than 52-fold among these species. Five species were further investigated for sensitivity to up to 4 additional nonsteroidal aromatase inhibitors, letrozole, imazalil, prochloraz, and propiconazole. Potencies of each of these chemicals relative to fadrozole ranged by up to 2 orders of magnitude among the 5 species. Fathead minnow, Japanese medaka, and zebrafish were among the least sensitive to all the investigated chemicals; therefore, ecological risks of aromatase inhibitors derived from these species might not be adequately protective of more sensitive native fishes. This information could guide more objective ecological risk assessments of native fishes to chemicals that inhibit aromatase., (Published by Oxford University Press on behalf of the Society of Toxicology 2019.)

High-throughput screening (HTS) and computational technologies have emerged as important tools for chemical hazard identification. The US Environmental Protection Agency (EPA) launched the Toxicity ForeCaster (ToxCast) Program, which has screened thousands of chemicals in hundreds of mammalian-based HTS assays for biological activity. The data are being used to prioritize toxicity testing on those chemicals likely to lead to adverse effects. To use HTS assays in predicting hazard to both humans and wildlife, it is necessary to understand how broadly these data may be extrapolated across species. The US EPA Sequence Alignment to Predict Across Species Susceptibility (SeqAPASS; https://seqapass.epa.gov/seqapass/ ) tool was used to assess conservation of the 484 protein targets represented in the suite of ToxCast assays and other HTS assays. To demonstrate the utility of the SeqAPASS data for guiding extrapolation, case studies were developed which focused on targets of interest to the US Endocrine Disruptor Screening Program and the Organisation for Economic Cooperation and Development. These case studies provide a line of evidence for conservation of endocrine targets across vertebrate species, with few exceptions, and demonstrate the utility of SeqAPASS for defining the taxonomic domain of applicability for HTS results and identifying organisms for suitable follow-up toxicity tests.

Chemical hazard assessment requires extrapolation of information from model organisms to all species of concern. The Sequence Alignment to Predict Across Species Susceptibility (SeqAPASS) tool was developed as a rapid, cost-effective method to aid cross-species extrapolation of susceptibility to chemicals acting on specific protein targets through evaluation of protein structural similarities and differences. The greatest resolution for extrapolation of chemical susceptibility across species involves comparisons of individual amino acid residues at key positions involved in protein-chemical interactions. However, a lack of understanding of whether specific amino acid substitutions among species at key positions in proteins affect interaction with chemicals made manual interpretation of alignments time consuming and potentially inconsistent. Therefore, this study used in silico site-directed mutagenesis coupled with docking simulations of computational models for acetylcholinesterase (AChE) and ecdysone receptor (EcR) to investigate how specific amino acid substitutions impact protein-chemical interaction. This study found that computationally derived substitutions in identities of key amino acids caused no change in protein-chemical interaction if residues share the same side chain functional properties and have comparable molecular dimensions, while differences in these characteristics can change protein-chemical interaction. These findings were considered in the development of capabilities for automatically generated species-specific predictions of chemical susceptibility in SeqAPASS. These predictions for AChE and EcR were shown to agree with SeqAPASS predictions comparing the primary sequence and functional domain sequence of proteins for more than 90% of the investigated species, but also identified dramatic species-specific differences in chemical susceptibility that align with results from standard toxicity tests. These results provide a compelling line of evidence for use of SeqAPASS in deriving screening level, species-specific, susceptibility predictions across broad taxonomic groups for application to human and ecological hazard assessment.

Adverse outcome pathways (AOPs) describe toxicant effects as a sequential chain of causally linked events beginning with a molecular perturbation and culminating in an adverse outcome at an individual or population level. Strategies for developing AOPs are still evolving and depend largely on the intended use or motivation for development and data availability. The present review describes 4 ecotoxicological AOP case studies, developed for different purposes. In each situation, creation of the AOP began in a manner determined by the initial motivation for its creation and expanded either to include additional components of the pathway or to address the domains of applicability in terms of chemical initiators, susceptible species, life stages, and so forth. Some general strategies can be gleaned from these case studies, which a developer may find to be useful for supporting an existing AOP or creating a new one. Several web-based tools that can aid in AOP assembly and evaluation of weight of evidence for scientific robustness of AOP components are highlighted. Environ Toxicol Chem 2017;36:1429-1449. © 2017 SETAC., (© 2017 SETAC.)

Our ability to conduct whole-organism toxicity tests to understand chemical safety has been outpaced by the synthesis of new chemicals for a wide variety of commercial applications. As a result, scientists and risk assessors are turning to mechanistically based studies to increase efficiencies in chemical risk assessment and making greater use of in vitro and in silico methods to evaluate potential environmental and human health hazards. In this context, the adverse outcome pathway (AOP) framework has gained traction in regulatory science because it offers an efficient and effective means for capturing available knowledge describing the linkage between mechanistic data and the apical toxicity end points required for regulatory assessments. A number of international activities have focused on AOP development and various applications to regulatory decision-making. These initiatives have prompted dialogue between research scientists and regulatory communities to consider how best to use the AOP framework. Although expert-facilitated discussions and AOP development have been critical in moving the science of AOPs forward, it was recognized that a survey of the broader scientific and regulatory communities would aid in identifying current limitations while guiding future initiatives for the AOP framework. To that end, a global horizon scanning exercise was conducted to solicit questions concerning the challenges or limitations that must be addressed to realize the full potential of the AOP framework in research and regulatory decision-making. The questions received fell into several broad topical areas: AOP networks, quantitative AOPs, collaboration on and communication of AOP knowledge, AOP discovery and development, chemical and cross-species extrapolation, exposure/toxicokinetics considerations, and AOP applications. Expert ranking was then used to prioritize questions for each category, where 4 broad themes emerged that could help inform and guide future AOP research and regulatory initiatives. In addition, frequently asked questions were identified and addressed by experts in the field. Answers to frequently asked questions will aid in addressing common misperceptions and will allow for clarification of AOP topics. The need for this type of clarification was highlighted with surprising frequency by our question submitters, indicating that improvements are needed in communicating the AOP framework among the scientific and regulatory communities. Overall, horizon scanning engaged the global scientific community to help identify key questions surrounding the AOP framework and guide the direction of future initiatives. Environ Toxicol Chem 2017;36:1411-1421. © 2017 SETAC., (© 2017 SETAC.)

Ongoing honey bee (Apis mellifera) colony losses are of significant international concern because of the essential role these insects play in pollinating crops. Both chemical and non-chemical stressors have been implicated as possible contributors to colony failure; however, the potential role(s) of commonly-used neonicotinoid insecticides has emerged as particularly concerning. Neonicotinoids act on the nicotinic acetylcholine receptors (nAChRs) in the central nervous system to eliminate pest insects. However, mounting evidence indicates that neonicotinoids also may adversely affect beneficial pollinators, such as the honey bee, via impairments on learning and memory, and ultimately foraging success. The specific mechanisms linking activation of the nAChR to adverse effects on learning and memory are uncertain. Additionally, clear connections between observed impacts on individual bees and colony level effects are lacking. The objective of this review was to develop adverse outcome pathways (AOPs) as a means to evaluate the biological plausibility and empirical evidence supporting (or refuting) the linkage between activation of the physiological target site, the nAChR, and colony level consequences. Potential for exposure was not a consideration in AOP development and therefore this effort should not be considered a risk assessment. Nonetheless, development of the AOPs described herein has led to the identification of research gaps which, for example, may be of high priority in understanding how perturbation of pathways involved in neurotransmission can adversely affect normal colony functions, causing colony instability and subsequent bee population failure. A putative AOP network was developed, laying the foundation for further insights as to the role of combined chemical and non-chemical stressors in impacting bee populations. Insights gained from the AOP network assembly, which more realistically represents multi-stressor impacts on honey bee colonies, are promising toward understanding common sensitive nodes in key biological pathways and identifying where mitigation strategies may be focused to reduce colony losses., (Published by Elsevier B.V.)

Cyclooxygenase (COX) inhibitors are ubiquitous in aquatic systems and have been detected in fish tissues. The exposure of fish to these pharmaceuticals is concerning because COX inhibitors disrupt the synthesis of prostaglandins (PGs), which modulate a variety of essential biological functions, including reproduction. In this study, we investigated the effects of well-characterized mammalian COX inhibitors on female fathead minnow reproductive health. Fish (n = 8) were exposed for 96 h to water containing indomethacin (IN; 100 µg/l), ibuprofen (IB; 200 µg/l) or celecoxib (CX; 20 µg/l), and evaluated for effects on liver metabolome and ovarian gene expression. Metabolomic profiles of IN, IB and CX were not significantly different from control or one another. Exposure to IB and CX resulted in differential expression of comparable numbers of genes (IB = 433, CX = 545). In contrast, 2558 genes were differentially expressed in IN-treated fish. Functional analyses (canonical pathway and gene set enrichment) indicated extensive effects of IN on PG synthesis pathway, oocyte meiosis, and several other processes consistent with physiological roles of PGs. Transcriptomic data were congruent with PG data; IN-reduced plasma PG F2α concentration, whereas IB and CX did not. Five putative AOPs were developed linking the assumed molecular initiating event of COX inhibition, with PG reduction and the adverse outcome of reproductive failure via reduction of: (1) ovulation, (2) reproductive behaviors mediated by exogenous or endogenous PGs, and (3) oocyte maturation in fish. These pathways were developed using, in part, empirical data from the present study and other publicly available data., (© The Author 2017. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

One objective in developing adverse outcome pathways (AOPs) is to connect biological changes that are relevant to risk assessors (i.e., fecundity) to molecular and cellular-level alterations that might be detectable at earlier stages of a chemical exposure. Here, we examined biochemical responses of fathead minnows (Pimephales promelas) to inform an AOP relevant to spironolactone's activation of the androgen receptor, as well as explore other biological impacts possibly unrelated to this receptor. Liquid chromatography with high resolution mass spectrometry (LC-MS) was used to measure changes in endogenous polar metabolites in livers of male and female fish that were exposed to five water concentrations of spironolactone (0, 0.05, 0.5, 5, or 50μgL -1 ) for 21days. Metabolite profiles were affected at the two highest concentrations (5 and 50μgL -1 ), but not in the lower-level exposures, which agreed with earlier reported results of reduced female fecundity and plasma vitellogenin (VTG) levels. We then applied partial least squares regression to assess whether metabolite alterations covaried with changes in fecundity, VTG gene expression and protein concentrations, and plasma 17β-estradiol and testosterone concentrations. Metabolite profiles significantly covaried with all measured endpoints in females, but only with plasma testosterone in males. Fecundity reductions occurred in parallel with changes in metabolites important in osmoregulation (e.g., betaine), membrane transport (e.g., l-carnitine), and biosynthesis of carnitine (e.g., methionine) and VTG (e.g., glutamate). Based on a network analysis program (i.e., mummichog), spironolactone also affected amino acid, tryptophan, and fatty acid metabolism. Thus, by identifying possible key events related to changes in biochemical pathways, this approach built upon an established AOP describing spironolactone's androgenic properties and highlighted broader implications potentially unrelated to androgen receptor activation, which could form a basis for the development of an AOP network., (Published by Elsevier B.V.)

The US Environmental Protection Agency has responsibility for assessing endocrine activity of more than 10 000 chemicals, a task that cannot reasonably be achieved solely through use of available mammalian and nonmammalian in vivo screening assays. Hence, it has been proposed that chemicals be prioritized for in vivo testing using data from in vitro high-throughput assays for specific endocrine system targets. Recent efforts focused on potential estrogenic chemicals-specifically those that activate estrogen receptor-alpha (ERα)-have broadly demonstrated feasibility of the approach. However, a major uncertainty is whether prioritization based on mammalian (primarily human) high-throughput assays accurately reflects potential chemical-ERα interactions in nonmammalian species. The authors conducted a comprehensive analysis of cross-species comparability of chemical-ERα interactions based on information concerning structural attributes of estrogen receptors, in vitro binding and transactivation data for ERα, and the effects of a range of chemicals on estrogen-signaling pathways in vivo. Overall, this integrated analysis suggests that chemicals with moderate to high estrogenic potency in mammalian systems also should be priority chemicals in nonmammalian vertebrates. However, the degree to which the prioritization approach might be applicable to invertebrates is uncertain because of a lack of knowledge of the biological role(s) of possible ERα orthologs found in phyla such as annelids. Further, comparative analysis of in vitro data for fish and reptiles suggests that mammalian-based assays may not effectively capture ERα interactions for low-affinity chemicals in all vertebrate classes. Environ Toxicol Chem 2016;35:2806-2816. Published 2016 Wiley Periodicals Inc. on behalf of SETAC. This article is a US Government work and, as such, is in the public domain in the United States of America., (Published 2016 Wiley Periodicals Inc. on behalf of SETAC. This article is a US Government work and, as such, is in the public domain in the United States of America.)

Conservation of a molecular target across species can be used as a line-of-evidence to predict the likelihood of chemical susceptibility. The web-based Sequence Alignment to Predict Across Species Susceptibility (SeqAPASS; https://seqapass.epa.gov/seqapass/) application was developed to simplify, streamline, and quantitatively assess protein sequence/structural similarity across taxonomic groups as a means to predict relative intrinsic susceptibility. The intent of the tool is to allow for evaluation of any potential protein target while remaining amenable to variable degrees of protein characterization, in the context of available information about the chemical/protein interaction and the molecular target itself. To accommodate this flexibility in the analysis, 3 levels of evaluation were developed. The first level of the SeqAPASS analysis compares primary amino acid sequences to a query sequence, calculating a metric for sequence similarity (including detection of orthologs); the second level evaluates sequence similarity within selected functional domains (eg, ligand-binding domain); and the third level of analysis compares individual amino acid residue positions of importance for protein conformation and/or interaction with the chemical upon binding. Each level of the SeqAPASS analysis provides additional evidence to apply toward rapid, screening-level assessments of probable cross species susceptibility. Such analyses can support prioritization of chemicals for further evaluation, selection of appropriate species for testing, extrapolation of empirical toxicity data, and/or assessment of the cross-species relevance of adverse outcome pathways. Three case studies are described herein to demonstrate application of the SeqAPASS tool: the first 2 focused on predictions of pollinator susceptibility to molt-accelerating compounds and neonicotinoid insecticides, and the third on evaluation of cross-species susceptibility to strobilurin fungicides. These analyses illustrate challenges in species extrapolation and demonstrate the broad utility of SeqAPASS for risk-based decision making and research., (Published by Oxford University Press on behalf of the Society of Toxicology 2016. This work is written by US Government employees and is in the public domain in the US.)

The potential for pharmaceuticals in the environment to cause adverse ecological effects is of increasing concern. Given the thousands of active pharmaceutical ingredients (APIs) that can enter the aquatic environment through human and/or animal (e.g., livestock) waste, a current challenge in aquatic toxicology is identifying those that pose the greatest risk. Because empirical toxicity information for aquatic species is generally lacking for pharmaceuticals, an important data source for prioritization is that generated during the mammalian drug development process. Applying concepts of species read-across, mammalian pharmacokinetic data were used to systematically prioritize APIs by estimating their potential to cause adverse biological consequences to aquatic organisms, using fish as an example. Mammalian absorption, distribution, metabolism, and excretion (ADME) data (e.g., peak plasma concentration, apparent volume of distribution, clearance rate, and half-life) were collected and curated, creating the Mammalian Pharmacokinetic Prioritization For Aquatic Species Targeting (MaPPFAST) database representing 1070 APIs. From these data, a probabilistic model and scoring system were developed and evaluated. Individual APIs and therapeutic classes were ranked based on clearly defined read-across assumptions for translating mammalian-derived ADME parameters to estimate potential hazard in fish (i.e., greatest predicted hazard associated with lowest mammalian peak plasma concentrations, total clearance and highest volume of distribution, half-life). It is anticipated that the MaPPFAST database and the associated API prioritization approach will help guide research and/or inform ecological risk assessment., (Published 2015 Wiley Periodicals Inc. on behalf of SETAC. This article is a US Government work and, as such, is in the public domain in the United States of America.)

Wastewater treatment plant (WWTP) effluents are known contributors of chemical mixtures into the environment. Of particular concern are endocrine-disrupting compounds, such as estrogens, which can affect the hypothalamic-pituitary-gonadal axis function in exposed organisms. The present study examined reproductive effects in fathead minnows exposed for 21 d to a historically estrogenic WWTP effluent. Fathead minnow breeding pairs were held in control water or 1 of 3 effluent concentrations (5%, 20%, and 100%) in a novel onsite, flow-through system providing real-time exposure. The authors examined molecular and biochemical endpoints representing key events along adverse outcome pathways linking estrogen receptor activation and other molecular initiating events to reproductive impairment. In addition, the authors used chemical analysis of the effluent to construct a chemical-gene interaction network to aid in targeted gene expression analyses and identifying potentially impacted biological pathways. Cumulative fecundity was significantly reduced in fish exposed to 100% effluent but increased in those exposed to 20% effluent, the approximate dilution factor in the receiving waters. Plasma vitellogenin concentrations in males increased in a dose-dependent manner with effluent concentration; however, male fertility was not impacted. Although in vitro analyses, analytical chemistry, and biomarker responses confirmed the effluent was estrogenic, estrogen receptor agonists were unlikely the primary driver of impaired reproduction. The results provide insights into the significance of pathway-based effects with regard to predicting adverse reproductive outcomes., (Published 2015 by Wiley Periodicals Inc. on behalf of SETAC. This article is a US Government work, and as such, is in the public domain in the United States of America.)

Early-life stage fish can be more sensitive to toxicants than adults, so delineating mechanisms of perturbation of biological pathways by chemicals during this life stage is crucial. Whole-mount in situ hybridization (WISH) paired with quantitative real-time polymerase chain reaction (QPCR) assays can enhance pathway-based analyses through determination of specific tissues where changes in gene expression are occurring. While WISH has frequently been used in zebrafish (Danio rerio), this technology has not previously been applied to fathead minnows (Pimephales promelas), another well-established small fish model species. The objective of the present study was to adapt WISH to fathead minnow embryos and larvae, and use the approach to evaluate the effects of estrone, an environmentally-relevant estrogen receptor (ER) agonist. Embryos were exposed via the water to 0, 18 or 1800 ng estrone/L (0, 0.067 and 6.7nM) for 3 or 6 days in a solvent-free, flow-through test system. Relative transcript abundance of three estrogen-responsive genes, estrogen receptor-α (esr1), cytochrome P450-aromatase B (cyp19b), and vitellogenin (vtg) was examined in pooled whole embryos using QPCR, and the spatial distribution of up-regulated gene transcripts was examined in individual fish using WISH. After 3 days of exposure to 1800 ng estrone/L, esr1 and cyp19b were significantly up-regulated, while vtg mRNA expression was not affected. After 6 days of exposure to 1800 ng estrone/L, transcripts for all three genes were significantly up-regulated. Corresponding WISH assays revealed spatial distribution of esr1 and vtg in the liver region, an observation consistent with activation of the hepatic ER. This study clearly demonstrates the potential utility of WISH, in conjunction with QPCR, to examine the mechanistic basis of the effects of toxicants on early-life stage fathead minnows., (Copyright © 2015 Elsevier B.V. All rights reserved.)

An adverse outcome pathway (AOP) is a conceptual framework that organizes existing knowledge concerning biologically plausible, and empirically supported, links between molecular-level perturbation of a biological system and an adverse outcome at a level of biological organization of regulatory relevance. Systematic organization of information into AOP frameworks has potential to improve regulatory decision-making through greater integration and more meaningful use of mechanistic data. However, for the scientific community to collectively develop a useful AOP knowledgebase that encompasses toxicological contexts of concern to human health and ecological risk assessment, it is critical that AOPs be developed in accordance with a consistent set of core principles. Based on the experiences and scientific discourse among a group of AOP practitioners, we propose a set of five fundamental principles that guide AOP development: (1) AOPs are not chemical specific; (2) AOPs are modular and composed of reusable components-notably key events (KEs) and key event relationships (KERs); (3) an individual AOP, composed of a single sequence of KEs and KERs, is a pragmatic unit of AOP development and evaluation; (4) networks composed of multiple AOPs that share common KEs and KERs are likely to be the functional unit of prediction for most real-world scenarios; and (5) AOPs are living documents that will evolve over time as new knowledge is generated. The goal of the present article was to introduce some strategies for AOP development and detail the rationale behind these 5 key principles. Consideration of these principles addresses many of the current uncertainties regarding the AOP framework and its application and is intended to foster greater consistency in AOP development., (Published by Oxford University Press on behalf of the Society of Toxicological 2014. This work is written by US Government employees and is in the public domain in the US.)

Organization of existing and emerging toxicological knowledge into adverse outcome pathway (AOP) descriptions can facilitate greater application of mechanistic data, including those derived through high-throughput in vitro, high content omics and imaging, and biomarker approaches, in risk-based decision making. The previously ad hoc process of AOP development is being formalized through development of internationally harmonized guidance and principles. The goal of this article was to outline the information content desired for formal AOP description and some rules of thumb and best practices intended to facilitate reuse and connectivity of elements of an AOP description in a knowledgebase and network context. For example, key events (KEs) are measurements of change in biological state that are indicative of progression of a perturbation toward a specified adverse outcome. Best practices for KE description suggest that each KE should be defined as an independent measurement made at a particular level of biological organization. The concept of "functional equivalence" can help guide both decisions about how many KEs to include in an AOP and the specificity with which they are defined. Likewise, in describing both KEs and evidence that supports a causal linkage or statistical association between them (ie, a key event relationship; KER), best practice is to build from and contribute to existing KE or KER descriptions in the AOP knowledgebase rather than creating redundant descriptions. The best practices proposed address many of the challenges and uncertainties related to AOP development and help promote a consistent and reliable, yet flexible approach., (Published by Oxford University Press on behalf of the Society of Toxicology 2014. This work is written by a US Government employees and is in the public domain in the US.)

Medicinal innovation has led to the discovery and use of thousands of human and veterinary drugs. With this comes the potential for unintended effects on non-target organisms exposed to pharmaceuticals inevitably entering the environment. The impracticality of generating whole-organism chronic toxicity data representative of all species in the environment has necessitated prioritization of drugs for focused empirical testing as well as field monitoring. Current prioritization strategies typically emphasize likelihood for exposure (i.e. predicted/measured environmental concentrations), while incorporating only rather limited consideration of potential effects of the drug to non-target organisms. However, substantial mammalian pharmacokinetic and mechanism/mode of action (MOA) data are produced during drug development to understand drug target specificity and efficacy for intended consumers. An integrated prioritization strategy for assessing risks of human and veterinary drugs would leverage available pharmacokinetic and toxicokinetic data for evaluation of the potential for adverse effects to non-target organisms. In this reiview, we demonstrate the utility of read-across approaches to leverage mammalian absorption, distribution, metabolism and elimination data; analyse cross-species molecular target conservation and translate therapeutic MOA to an adverse outcome pathway(s) relevant to aquatic organisms as a means to inform prioritization of drugs for focused toxicity testing and environmental monitoring., (© 2014 The Author(s) Published by the Royal Society. All rights reserved.)

Adverse outcome pathways (AOPs) are designed to describe linkages of key events within a biological pathway that result in an adverse outcome associated with chemical perturbation of a well-defined molecular initiating event. Risk assessors have traditionally relied on data from apical endpoints (e.g., mortality, growth, reproduction) to derive benchmark values for use in determining the potential adverse impacts of chemicals. One goal in building reliable and well-characterized AOPs is to identify relevant in vitro assays and/or in vivo biomarkers that could be used in screening the potential hazard of substances, thereby reducing costs and increasing the number of chemicals that can be evaluated in a timely fashion. The purpose of this review article is to build an AOP for substances with a molecular initiating event of acetylcholinesterase inhibition leading to acute mortality following guidance developed by the Organisation for Economic Cooperation and Development. In contrast to most other AOPs developed to date, in which coverage is for a relatively limited taxonomic group or life stage, this AOP is applicable to a wide range of species at multiple life stages. Furthermore, while development of most AOPs has relied on data for a few model chemicals, the AOP described in the present review captures information from a large number of studies with a diversity of organophosphate and carbamate insecticides., (Published 2014 Wiley Periodicals Inc. on behalf of SETAC. This article is a US government work and, as such, is in the public domain in the United States of America.)

Animal waste from livestock farming operations can contain varying levels of natural and synthetic androgens and/or estrogens, which can contaminate surrounding waterways. In the present study, surface stream water was collected from 6 basins containing livestock farming operations. Aqueous concentrations of 12 hormones were determined via chemical analyses. Relative androgenic and estrogenic activity was measured using in vitro cell assays (MDA-kb2 and T47D-Kbluc assays, respectively). In parallel, 48-h static-renewal in vivo exposures were conducted to examine potential endocrine-disrupting effects in fathead minnows. Mature fish were exposed to surface water dilutions (0%, 25%, 50%, and 100%) and 10-ng/L of 17α-ethynylestradiol or 50-ng/L of 17β-trenbolone as positive controls. Hepatic expression of vitellogenin and estrogen receptor α mRNA, gonadal ex vivo testosterone and 17β-estradiol production, and plasma vitellogenin concentrations were examined. Potentially estrogenic and androgenic steroids were detected at low nanogram per liter concentrations. In vitro estrogenic activity was detected in all samples, whereas androgenic activity was detected in only 1 sample. In vivo exposures to the surface water had no significant dose-dependent effect on any of the biological endpoints, with the exception of increased male testosterone production in 1 exposure. The present study, which combines analytical chemistry measurements, in vitro bioassays, and in vivo fish exposures, highlights the integrated value and future use of a combination of techniques to obtain a comprehensive characterization of an environmental chemical mixture., (Published 2014 Wiley Periodicals, Inc. This article is a US Government work and, as such, is in the public domain in the United States of America.)

Assessment of potential risks of complex contaminant mixtures in the environment requires integrated chemical and biological approaches. In support of the US Great Lakes Restoration Initiative, the US Environmental Protection Agency lab in Duluth, MN, is developing these types of methods for assessing possible risks of aquatic contaminants in near-shore Great Lakes (USA) sites. One component involves an exposure system for caged fathead minnow (Pimephales promelas) adults suitable for the wide range of habitat and deployment situations encountered in and around the Great Lakes. To complement the fish exposure system, the authors developed an automated device for collection of composite water samples that could be simultaneously deployed with the cages and reflect a temporally integrated exposure of the animals. The present study describes methodological details of the design, construction, and deployment of a flexible yet comparatively inexpensive (<600 USD) caged-fish/autosampler system. The utility and performance of the system were demonstrated with data collected from deployments at several Great Lakes sites. For example, over 3 field seasons, only 2 of 130 deployed cages were lost, and approximately 99% of successfully deployed adult fish were recovered after exposures of 4 d or longer. A number of molecular, biochemical, and apical endpoints were successfully measured in recovered animals, changes in which reflected known characteristics of the study sites (e.g., upregulation of hepatic genes involved in xenobiotic metabolism in fish held in the vicinity of wastewater treatment plants). The automated composite samplers proved robust with regard to successful water collection (>95% of deployed units in the latest field season), and low within- and among-unit variations were found relative to programmed collection volumes. Overall, the test system has excellent potential for integrated chemical-biological monitoring of contaminants in a variety of field settings., (© 2014 SETAC.)

It is not feasible to conduct toxicity tests with all species that may be impacted by chemical exposures. Therefore, cross-species extrapolation is fundamental to environmental risk assessment. Recognition of the impracticality of generating empirical, whole organism, toxicity data for the extensive universe of chemicals in commerce has been an impetus driving the field of predictive toxicology. We describe a strategy that leverages expanding databases of molecular sequence information together with identification of specific molecular chemical targets whose perturbation can lead to adverse outcomes to support predictive species extrapolation. This approach can be used to predict which species may be more (or less) susceptible to effects following exposure to chemicals with known modes of action (e.g., pharmaceuticals, pesticides). Primary amino acid sequence alignments are combined with more detailed analyses of conserved functional domains to derive the predictions. This methodology employs bioinformatic approaches to automate, collate, and calculate quantitative metrics associated with cross-species sequence similarity of key molecular initiating events (MIEs). Case examples focused on the actions of (a) 17α-ethinyl estradiol on the human (Homo sapiens) estrogen receptor; (b) permethrin on the mosquito (Aedes aegypti) voltage-gated para-like sodium channel; and (c) 17β-trenbolone on the bovine (Bos taurus) androgen receptor are presented to demonstrate the potential predictive utility of this species extrapolation strategy. The examples compare empirical toxicity data to cross-species predictions of intrinsic susceptibility based on analyses of sequence similarity relevant to the MIEs of defined adverse outcome pathways. Through further refinement, and definition of appropriate domains of applicability, we envision practical and routine utility for the molecular target similarity-based predictive method in chemical risk assessment, particularly where testing resources are limited., (Published by Elsevier B.V.)

Spironolactone is a pharmaceutical that in humans is used to treat conditions like hirsutism, various dermatologic afflictions, and female-pattern hair loss through antagonism of the androgen receptor. Although not routinely monitored in the environment, spironolactone has been detected downstream of a pharmaceutical manufacturer, indicating a potential for exposure of aquatic species. Furthermore, spironolactone has been reported to cause masculinization of female western mosquitofish, a response indicative of androgen receptor activation. Predictive methods to identify homologous proteins to the human and western mosquitofish androgen receptor suggest that vertebrates would be more susceptible to adverse effects mediated by chemicals like spironolactone that target the androgen receptor compared with invertebrate species that lack a relevant homolog. In addition, an adverse outcome pathway previously developed for activation of the androgen receptor suggests that androgen mimics can lead to reproductive toxicity in fish. To assess this, 21-d reproduction studies were conducted with 2 fish species, fathead minnow and Japanese medaka, and the invertebrate Daphnia magna. Spironolactone significantly reduced the fecundity of medaka and fathead minnows at 50 μg/L, whereas daphnia reproduction was not affected by concentrations as large as 500 μg/L. Phenotypic masculinization of females of both fish species was observed at 5 μg/L as evidenced by formation of tubercles in fathead minnows and papillary processes in Japanese medaka. Effects in fish occurred at concentrations below those reported in the environment. These results demonstrate how a priori knowledge of an adverse outcome pathway and the conservation of a key molecular target across vertebrates can be utilized to identify potential chemicals of concern in terms of monitoring and highlight potentially sensitive species and endpoints for testing., (© 2013 SETAC.)

Background: Traditional animal toxicity tests can be time and resource intensive, thereby limiting the number of chemicals that can be comprehensively tested for potential hazards to humans and/or to the environment., Objective: We compared several types of data to demonstrate how alternative models can be used to inform both human and ecological risk assessment., Methods: We reviewed and compared data derived from high throughput in vitro assays to fish reproductive tests for seven chemicals. We investigated whether human-focused assays can be predictive of chemical hazards in the environment. We examined how conserved pathways enable the use of nonmammalian models, such as fathead minnow, zebrafish, and Xenopus laevis, to understand modes of action and to screen for chemical risks to humans., Results: We examined how dose-dependent responses of zebrafish embryos exposed to flusilazole can be extrapolated, using pathway point of departure data and reverse toxicokinetics, to obtain human oral dose hazard values that are similar to published mammalian chronic toxicity values for the chemical. We also examined how development/safety data for human health can be used to help assess potential risks of pharmaceuticals to nontarget species in the environment., Discussion: Using several examples, we demonstrate that pathway-based analysis of chemical effects provides new opportunities to use alternative models (nonmammalian species, in vitro tests) to support decision making while reducing animal use and associated costs., Conclusions: These analyses and examples demonstrate how alternative models can be used to reduce cost and animal use while being protective of both human and ecological health.

Oligonucleotide microarrays and other 'omics' approaches are powerful tools for unsupervised analysis of chemical impacts on biological systems. However, the lack of well annotated biological pathways for many aquatic organisms, including fish, and the limited power of microarray-based analyses to detect low level differential expression of individual genes can hinder the ability to infer and understand chemical effects based on transcriptomic data. Here we report on the supervised assembly of a series of tissue-specific functional gene sets intended to aid transcriptomic analysis of chemical impacts on the female teleost reproductive axis. Gene sets were defined based on an updated graphical systems model of the teleost brain-pituitary-gonadal-hepatic axis. Features depicted in the model were organized into gene sets and mapped to specific probes on three zebrafish (Danio rerio) and two fathead minnow (Pimephales promelas) microarray platforms. Coverage of target genes on the microarrays ranged from 48% for the fathead minnow arrays to 88% for the most current zebrafish platform. Additionally, extended fathead minnow gene sets, incorporating first degree neighbors identified from a Spearman correlation network derived from a large compendium of fathead minnow microarray data, were constructed. Overall, only 14% of the 78 genes queried were connected in the network. Among those, over half had less than five neighbors, while two genes, cyclin b1 and zona pellucida glycoprotein 3, had over 100 first degree neighbors, and were neighbors to one another. Gene set enrichment analyses were conducted using microarray data from a zebrafish hypoxia experiment and fathead minnow time-course experiments conducted with three different endocrine-active chemicals. Results of these analyses demonstrate the utility of the approach for supporting biological inference from ecotoxicogenomic data and comparisons across multiple toxicogenomic experiments. The graphical model, gene mapping, and gene sets described are now available to the scientific community as tools to support ecotoxicogenomic research., (Published by Elsevier B.V.)

Diethylstilbestrol (DES) is a synthetic estrogen that has been banned for use in humans, but still is employed in livestock and aquaculture operations in some parts of the world. Detectable concentrations of DES in effluent and surface waters have been reported to range from slightly below 1 to greater than 10 ng/L. Little is known, however, concerning the toxicological potency of DES in fish. In this study, sexually mature fathead minnows (Pimephales promelas) of both sexes were exposed to 1, 10, or 100 ng of DES/L of water in a flow-through system. Tissue concentrations of DES and changes in a number of estrogen-responsive end points were measured in the fish at the end of a 4 d exposure and after a 4 d depuration/recovery period in clean water. Accumulation of DES was sex-dependent, with females exhibiting higher tissue residues than males after the 4 d exposure. The observed bioconcentration of DES in the fish was about 1 order of magnitude lower than that predicted on the basis of the octanol-water partition coefficient of the chemical, suggesting relatively efficient metabolic clearance by the fish. Exposure to 1, 10, or 100 ng of DES/L caused decreased testis weight and morphological demasculinization of males (regression of dorsal nuptial tubercles). Diethylstilbesterol induced plasma vitellogenin (VTG) in both sexes at water concentrations ≥10 ng/L; this response (especially in males) persisted through the end of the 4 d recovery period. Hepatic transcripts of VTG and estrogen receptor-α also were affected at DES concentrations ≥10 ng/L. Evaluation of transcript profiles in the liver of females using a 15K-gene fathead minnow microarray revealed a concentration-dependent change in gene expression, with mostly up-regulated transcripts after the exposure and substantial numbers of down-regulated gene products after depuration. Genes previously identified as vitellogenesis-related and regulated by 17β-estradiol were significantly enriched among those differentially expressed following exposure to DES. Overall, our studies show that DES causes a range of responses in fish at water concentrations comparable to those reported in the environment and that in vivo potency of the estrogen is on par with that of the better-studied estrogenic contaminant 17α-ethinylestradiol.