Current testing programs for pesticides adequately capture endocrine activity and adversity for protection of vertebrate wildlife.

The toxicity and ecotoxicity of pesticide active ingredients are evaluated by a number of standardized test methods using vertebrate animals. These standard test methods are required under various regulatory programs for the registration of pesticides. Over the past two decades, additional test methods have been developed with endpoints that are responsive to endocrine activity and subsequent adverse effects. This article examines the available test methods and their endpoints that are relevant to an assessment of endocrine-disrupting properties of pesticides. Furthermore, the article highlights how weight-of-evidence approaches should be applied to determine whether an adverse response in (eco)toxicity tests is caused by an endocrine mechanism of action. The large number of endpoints in the current testing paradigms for pesticides make it unlikely that endocrine activity and adversity is being overlooked. Integr Environ Assess Manag 2023;19:1089-1109. © 2023 Bayer CropScience and The Authors. Integrated Environmental Assessment and Management published by Wiley Periodicals LLC on behalf of Society of Environmental Toxicology & Chemistry (SETAC).

A field spray drift study to determine the downwind effects of isoxaflutole herbicide to nontarget plants.

Spray drift buffers are often required on herbicide labels to prevent potential drift effects to nontarget plants. Buffers are typically derived by determining the distance at which predicted exposure from spray drift equals the ecotoxicology threshold for sensitive plant species determined in greenhouse tests. Field studies performed under realistic conditions have demonstrated, however, that this approach is far more conservative than necessary. In 2016, the US Environmental Protection Agency estimated that isoxaflutole (IFT), a herbicide used to control grass and broadleaf weeds, could adversely affect downwind nontarget dicot plants at distances of ≥304 m from the edge of the treated field due to spray drift. This prediction implies that a buffer of at least 304 m is required to protect nontarget plants. To refine the predicted buffer distance for IFT, we conducted a field study in which sensitive nontarget plants (lettuce and navy bean, two to four leaf stage) were placed at various distances downwind from previously harvested soybean fields sprayed with Balance® Flexx Herbicide. The test plants were then transported to a greenhouse for grow out following the standard vegetative vigor test protocol. There were three trials. One had vegetation in the downwind deposition area (i.e., test plants placed in mowed grass; typical exposure scenario) and two had bare ground deposition areas (worst-case exposure scenario). For both plant species in bare ground deposition areas, effects on shoot height and weight were observed at 1.52 m but not at downwind distances of ≥9.14 m from the edge of the treated area. No effects were observed at any distance for plants placed in the vegetated deposition area. The field study demonstrated that a buffer of 9.14 m protects nontarget terrestrial plants exposed to IFT via spray drift even under worst-case conditions. Integr Environ Assess Manag 2022;18:757-769. © 2021 Bayer. Integrated Environmental Assessment and Management published by Wiley Periodicals LLC on behalf of Society of Environmental Toxicology & Chemistry (SETAC).

The Extended Amphibian Metamorphosis Assay: A Thyroid-Specific and Less Animal-Intensive Alternative to the Larval Amphibian Growth and Development Assay.

The amphibian metamorphosis assay (AMA; US Environmental Protection Agency [USEPA] test guideline 890.1100 and Organisation for Economic Co-Operation and Development test guideline 231) has been used for more than a decade to assess the potential thyroid-mediated endocrine activity of chemicals. In 2013, in the context of the Endocrine Disruptor Screening Program of the USEPA, a Scientific Advisory Panel reviewed the results from 18 studies and recommended changes to the AMA test guideline, including a modification to a fixed-stage design rather than a fixed-time (i.e., 21-d) design. We describe an extended test design for the AMA (or EAMA) that includes thyroid histopathology and time to metamorphosis (Nieuwkoop-Faber [NF] stage 62), to address both the issues with the fixed-time design and the specific question of thyroid-mediated adversity in a shorter assay than the larval amphibian growth and development assay (LAGDA; Organisation for Economic Co-Operation and Development test guideline 241), using fewer animals and resources. A demonstration study was conducted with the EAMA (up to NF stage 58) using sodium perchlorate. Data analyses and interpretation of the fixed-stage design of the EAMA are more straightforward than the fixed-time design because the fixed-stage design avoids confounded morphometric measurements and thyroid histopathology caused by varying developmental stages at test termination. It also results in greater statistical power to detect metamorphic delays than the fixed-time design. By preferentially extending the AMA to NF stage 62, suitable data can be produced to evaluate thyroid-mediated adversity and preclude the need to perform a LAGDA for thyroid mode of action analysis. The LAGDA remains of further interest should investigations of longer term effects related to sexual development modulated though the hypothalamus-pituitary-gonadal axis be necessary. However, reproduction assessment or life cycle testing is currently not addressed in the LAGDA study design. This is better addressed by higher tier studies in fish, which should then include specific thyroid-related endpoints. Environ Toxicol Chem 2021;40:2135-2144. © 2021 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Critical Review of Read-Across Potential in Testing for Endocrine-Related Effects in Vertebrate Ecological Receptors.

Recent regulatory testing programs have been designed to evaluate whether a chemical has the potential to interact with the endocrine system and could cause adverse effects. Some endocrine pathways are highly conserved among vertebrates, providing a potential to extrapolate data generated for one vertebrate taxonomic group to others (i.e., biological read-across). To assess the potential for biological read-across, we reviewed tools and approaches that support species extrapolation for fish, amphibians, birds, and reptiles. For each of the estrogen, androgen, thyroid, and steroidogenesis (EATS) pathways, we considered the pathway conservation across species and the responses of endocrine-sensitive endpoints. The available data show a high degree of confidence in the conservation of the hypothalamus-pituitary-gonadal axis between fish and mammals and the hypothalamus-pituitary-thyroid axis between amphibians and mammals. Comparatively, there is less empirical evidence for the conservation of other EATS pathways between other taxonomic groups, but this may be due to limited data. Although more information on sensitive pathways and endpoints would be useful, current developments in the use of molecular target sequencing similarity tools and thoughtful application of the adverse outcome pathway concept show promise for further advancement of read-across approaches for testing EATS pathways in vertebrate ecological receptors. Environ Toxicol Chem 2020;39:739-753. © 2020 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals, Inc. on behalf of SETAC.

Scientific integrity issues in Environmental Toxicology and Chemistry: Improving research reproducibility, credibility, and transparency.

High-profile reports of detrimental scientific practices leading to retractions in the scientific literature contribute to lack of trust in scientific experts. Although the bulk of these have been in the literature of other disciplines, environmental toxicology and chemistry are not free from problems. While we believe that egregious misconduct such as fraud, fabrication of data, or plagiarism is rare, scientific integrity is much broader than the absence of misconduct. We are more concerned with more commonly encountered and nuanced issues such as poor reliability and bias. We review a range of topics including conflicts of interests, competing interests, some particularly challenging situations, reproducibility, bias, and other attributes of ecotoxicological studies that enhance or detract from scientific credibility. Our vision of scientific integrity encourages a self-correcting culture that promotes scientific rigor, relevant reproducible research, transparency in competing interests, methods and results, and education. Integr Environ Assess Manag 2019;00:000-000. © 2019 SETAC.

Distinguishing between endocrine disruption and non-specific effects on endocrine systems.

The endocrine system is responsible for growth, development, maintaining homeostasis and for the control of many physiological processes. Due to the integral nature of its signaling pathways, it can be difficult to distinguish endocrine-mediated adverse effects from transient fluctuations, adaptive/compensatory responses, or adverse effects on the endocrine system that are caused by mechanisms outside the endocrine system. This is particularly true in toxicological studies that require generation of effects through the use of Maximum Tolerated Doses (or Concentrations). Endocrine-mediated adverse effects are those that occur as a consequence of the interaction of a chemical with a specific molecular component of the endocrine system, for example, a hormone receptor. Non-endocrine-mediated adverse effects on the endocrine system are those that occur by other mechanisms. For example, systemic toxicity, which perturbs homeostasis and affects the general well-being of an organism, can affect endocrine signaling. Some organs/tissues can be affected by both endocrine and non-endocrine signals, which must be distinguished. This paper examines in vitro and in vivo endocrine endpoints that can be altered by non-endocrine processes. It recommends an evaluation of these issues in the assessment of effects for the determination of endocrine disrupting properties of chemicals. This underscores the importance of using a formal weight of evidence (WoE) process to evaluate potential endocrine activity.

Life-cycle studies with 2 marine species and bisphenol A: The mysid shrimp (Americamysis bahia) and sheepshead minnow (Cyprinodon variegatus).

Bisphenol A (BPA) is a high production volume compound primarily used to produce epoxy resins and polycarbonate plastic. Exposure to low concentrations of BPA occurs in freshwater and marine systems, primarily from wastewater treatment plant discharges. The dataset for chronic toxicity of BPA to freshwater organisms includes studies on fish, amphibians, invertebrates, algae, and aquatic plants. To broaden the dataset, a 1.5-generation test with sheepshead minnow (Cyprinodon variegatus) and a full life-cycle test with mysid shrimp (Americamysis bahia) were conducted. Testing focused on apical endpoints of survival, growth and development, and reproduction. The respective no-observed-effect concentration (NOEC) and lowest-observed-effect concentration (LOEC) values of 170 and 370 µg/L for mysid and 66 and 130 µg/L for sheepshead were based on reduced fecundity. The hazardous concentrations for 5% of the species (HC5) values of 18 µg/L were calculated from species sensitivity distributions (SSDs) with freshwater-only data and combined freshwater and marine data. Inclusion of marine data resulted in no apparent difference in SSD shape, R2 values for the distributions, or HC5 values. Upper-bound 95th percentile concentrations of BPA measured in marine waters of North America and Europe (0.024 and 0.15 µg/L, respectively) are below the HC5 value of 18 µg/L. These results suggest that marine and freshwater species are of generally similar sensitivity and that chronic studies using a diverse set of species can be combined to assess the aquatic toxicity of BPA. Environ Toxicol Chem 2018;37:398-410. © 2017 The Authors. Environmental Toxicology and Chemistry Published by Wiley Periodicals, Inc. on behalf of SETAC.

Recommended approaches to the scientific evaluation of ecotoxicological hazards and risks of endocrine-active substances.

A SETAC Pellston Workshop® "Environmental Hazard and Risk Assessment Approaches for Endocrine-Active Substances (EHRA)" was held in February 2016 in Pensacola, Florida, USA. The primary objective of the workshop was to provide advice, based on current scientific understanding, to regulators and policy makers; the aim being to make considered, informed decisions on whether to select an ecotoxicological hazard- or a risk-based approach for regulating a given endocrine-disrupting substance (EDS) under review. The workshop additionally considered recent developments in the identification of EDS. Case studies were undertaken on 6 endocrine-active substances (EAS-not necessarily proven EDS, but substances known to interact directly with the endocrine system) that are representative of a range of perturbations of the endocrine system and considered to be data rich in relevant information at multiple biological levels of organization for 1 or more ecologically relevant taxa. The substances selected were 17α-ethinylestradiol, perchlorate, propiconazole, 17ß-trenbolone, tributyltin, and vinclozolin. The 6 case studies were not comprehensive safety evaluations but provided foundations for clarifying key issues and procedures that should be considered when assessing the ecotoxicological hazards and risks of EAS and EDS. The workshop also highlighted areas of scientific uncertainty, and made specific recommendations for research and methods-development to resolve some of the identified issues. The present paper provides broad guidance for scientists in regulatory authorities, industry, and academia on issues likely to arise during the ecotoxicological hazard and risk assessment of EAS and EDS. The primary conclusion of this paper, and of the SETAC Pellston Workshop on which it is based, is that if data on environmental exposure, effects on sensitive species and life-stages, delayed effects, and effects at low concentrations are robust, initiating environmental risk assessment of EDS is scientifically sound and sufficiently reliable and protective of the environment. In the absence of such data, assessment on the basis of hazard is scientifically justified until such time as relevant new information is available. Integr Environ Assess Manag 2017;13:267-279. © 2017 The Authors. Integrated Environmental Assessment and Management published by Wiley Periodicals, Inc. on behalf of Society of Environmental Toxicology & Chemistry (SETAC).

Challenges in assigning endocrine-specific modes of action: Recommendations for researchers and regulators.

As regulatory programs evaluate substances for their endocrine-disrupting properties, careful study design and data interpretation are needed to distinguish between responses that are truly endocrine specific and those that are not. This is particularly important in regulatory environments where criteria are under development to identify endocrine-disrupting properties to enable hazard-based regulation. Irrespective of these processes, most jurisdictions use the World Health Organization/International Programme on Chemical Safety definition of an endocrine disruptor, requiring that a substance is demonstrated to cause a change in endocrine function that consequently leads to an adverse effect in an intact organism. Such a definition is broad, and at its most cautious might capture many general mechanisms that would not specifically denote an endocrine disruptor. In addition, endocrine responses may be adaptive in nature, designed to maintain homeostasis rather than induce an irreversible adverse effect. The likelihood of indirect effects is increased in (eco)toxicological studies that require the use of maximum tolerated concentrations or doses, which must produce some adverse effect. The misidentification of indirect effects as truly endocrine mediated has serious consequences for prompting animal- and resource-intensive testing and regulatory consequences. To minimize the risk for misidentification, an objective and transparent weight-of-evidence procedure based on biological plausibility, essentiality, and empirical evidence of key events in an adverse outcome pathway is recommended to describe the modes of action that may be involved in toxic responses in nontarget organisms. Confounding factors such as systemic toxicity, general stress, and infection can add complexity to such an evaluation and should be considered in the weight of evidence. A recommended set of questions is proffered to help guide researchers and regulators in discerning endocrine and nonendocrine responses. Although many examples provided in this study are based on ecotoxicology, the majority of the concepts and processes are applicable to both environmental and human health assessments. Integr Environ Assess Manag 2017;13:280-292. © 2016 SETAC.

Characterizing the effects of bisphenol A on sediment-dwelling benthic organisms.

Bisphenol A (BPA) is a high production volume chemical intermediate used primarily in the production of polycarbonate plastics and epoxy resins. It primarily enters surface water and sediment via effluent discharges during its manufacture and use. The physical properties of BPA suggest that sediment is a potential sink and may result in exposure to benthic organisms. Currently there are no studies measuring the chronic toxicity of BPA to benthic organisms via direct sediment exposure. The present study examined the chronic toxicity of BPA to 3 commonly used test organisms that are generally representative of invertebrates occupying the base of the benthic food web and for which standardized testing protocols are available: the oligochaete Lumbriculus variegatus (mean numbers and biomass), the midge Chironomus riparius (emergence and development rate), and the estuarine amphipod Leptocheirus plumulosus (survival, growth, and reproduction). No-observed-effect concentrations (NOECs) for the 3 species ranged from 12 mg/kg to 54 mg/kg dry weight. All NOEC values were higher than all measured concentrations of BPA in freshwater and marine sediments reported in reliable, fully reported studies from North America and Europe from the 1990s to the present. For the first time, there are studies with BPA measuring the chronic toxicity to 3 taxa of sediment dwelling invertebrates, which are suitable to support region-specific risk assessments.

Relevance weighting of tier 1 endocrine screening endpoints by rank order.

Weight of evidence (WoE) approaches are recommended for interpreting various toxicological data, but few systematic and transparent procedures exist. A hypothesis-based WoE framework was recently published focusing on the U.S. EPA's Tier 1 Endocrine Screening Battery (ESB) as an example. The framework recommends weighting each experimental endpoint according to its relevance for deciding eight hypotheses addressed by the ESB. Here we present detailed rationale for weighting the ESB endpoints according to three rank ordered categories and an interpretive process for using the rankings to reach WoE determinations. Rank 1 was assigned to in vivo endpoints that characterize the fundamental physiological actions for androgen, estrogen, and thyroid activities. Rank 1 endpoints are specific and sensitive for the hypothesis, interpretable without ancillary data, and rarely confounded by artifacts or nonspecific activity. Rank 2 endpoints are specific and interpretable for the hypothesis but less informative than Rank 1, often due to oversensitivity, inclusion of narrowly context-dependent components of the hormonal system (e.g., in vitro endpoints), or confounding by nonspecific activity. Rank 3 endpoints are relevant for the hypothesis but only corroborative of Ranks 1 and 2 endpoints. Rank 3 includes many apical in vivo endpoints that can be affected by systemic toxicity and nonhormonal activity. Although these relevance weight rankings (WREL ) necessarily involve professional judgment, their a priori derivation enhances transparency and renders WoE determinations amenable to methodological scrutiny according to basic scientific premises, characteristics that cannot be assured by processes in which the rationale for decisions is provided post hoc.

Lessons learned, challenges, and opportunities: the U.S. Endocrine Disruptor Screening Program.

In 1996, the U.S. Congress passed the Food Quality Protection Act and amended the Safe Drinking Water Act (SDWA) requiring the U.S. Environmental Protection Agency (EPA) to implement a screening program to investigate the potential of pesticide chemicals and drinking water contaminants to adversely affect endocrine pathways. Consequently, the EPA launched the Endocrine Disruptor Screening Program (EDSP) to develop and validate estrogen, androgen, and thyroid (EAT) pathway screening assays and to produce standardized and harmonized test guidelines for regulatory application. In 2009, the EPA issued the first set of test orders for EDSP screening and a total of 50 pesticide actives and 2 inert ingredients have been evaluated using the battery of EDSP Tier 1 screening assays (i.e., five in vitro assays and six in vivo assays). To provide a framework for retrospective analysis of the data generated and to collect the insight of multiple stakeholders involved in the testing, more than 240 scientists from government, industry, academia, and non-profit organizations recently participated in a workshop titled "Lessons Learned, Challenges, and Opportunities: The U.S. Endocrine Disruptor Screening Program." The workshop focused on the science and experience to date and was organized into three focal sessions: (a) Performance of the EDSP Tier 1 Screening Assays for Estrogen, Androgen, and Thyroid Pathways; (b) Practical Applications of Tier 1 Data; and (c) Indications and Opportunities for Future Endocrine Testing. A number of key learnings and recommendations related to future EDSP evaluations emanated from the collective sessions.

Adult fathead minnow, Pimephales promelas, partial life-cycle reproductive and gonadal histopathology study with bisphenol A.

Bisphenol A (BPA) is an intermediate used to produce epoxy resins and polycarbonate plastics. Although BPA degrades rapidly in the environment with aquatic half-lives from 0.5 to 6 d, it can be found in aquatic systems because of widespread use. To evaluate potential effects from chronic exposure, fathead minnows were exposed for 164 d to nominal concentrations of 1, 16, 64, 160, and 640 µg/L BPA. Population-level endpoints of survival, growth, and reproduction were assessed with supplemental endpoints (e.g., vitellogenin, gonad histology), including gonad cell type assessment and quantification. No statistically significant changes in growth, gonad weight, gonadosomatic index, or reproduction variables (e.g., number of eggs and spawns, hatchability) were observed; however, there was a significant impact on male survival at 640 µg/L. Vitellogenin increased in both sexes at 64 µg/L or higher. Gonad cell type frequencies were significantly different from controls at 160 µg/L or higher in males with a slight decrease in spermatocytes compared with less mature cell types, and at 640 µg/L in females with a slight decrease in early vitellogenic cells compared with less mature cells. The decrease in spermatocytes did not correspond to a decrease in the most mature sex cell type (spermatozoa) and did not impair male fertility, as hatchability was not impacted. Overall, marginal shifts in gametogenic cell maturation were not associated with any statistically significant effects on population-relevant reproductive endpoints (growth, fecundity, and hatchability) at any concentration tested.

Hypothesis-driven weight of evidence framework for evaluating data within the US EPA's Endocrine Disruptor Screening Program.

"Weight of Evidence" (WoE) approaches are often used to critically examine, prioritize, and integrate results from different types of studies to reach general conclusions. For assessing hormonally active agents, WoE evaluations are necessary to assess screening assays that identify potential interactions with components of the endocrine system, long-term reproductive and developmental toxicity tests that define adverse effects, mode of action studies aimed at identifying toxicological pathways underlying adverse effects, and toxicity, exposure and pharmacokinetic data to characterize potential risks. We describe a hypothesis-driven WoE approach for hormonally active agents and illustrate the approach by constructing hypotheses for testing the premise that a substance interacts as an agonist or antagonist with components of estrogen, androgen, or thyroid pathways or with components of the aromatase or steroidogenic enzyme systems for evaluating data within the US EPA's Endocrine Disruptor Screening Program. Published recommendations are used to evaluate data validity for testing each hypothesis and quantitative weightings are proposed to reflect two data parameters. Relevance weightings should be derived for each endpoint to reflect the degree to which it probes each specific hypothesis. Response weightings should be derived based on assay results from the test substance compared to the range of responses produced in the assay by the appropriate prototype hormone and positive and negative controls. Overall WoE scores should be derived based on response and relevance weightings and a WoE narrative developed to clearly describe the final determinations.

Estimating potential risks to terrestrial invertebrates and plants exposed to bisphenol A in soil amended with activated sludge biosolids.

Bisphenol A (BPA) is a high production volume substance primarily used to produce polycarbonate plastic and epoxy resins. During manufacture and use, BPA may enter wastewater treatment plants. During treatment, BPA may become adsorbed to activated sludge biosolids, which may expose soil organisms to BPA if added to soil as an amendment. To evaluate potential risks to organisms that make up the base of the terrestrial food web (i.e., invertebrates and plants) in accordance with international regulatory practice, toxicity tests were conducted with potworms (Enchytraeids) and springtails (Collembolans) in artificial soil, and six plant types using natural soil. No-observed-effect concentrations (NOEC) for potworms and springtails were equal to or greater than 100 and equal to or greater than 500 mg/kg (dry wt), respectively. The lowest organic matter-normalized NOEC among all tests (dry shoot weight of tomatoes) was 37 mg/kg-dry weight. Dividing by an assessment factor of 10, a predicted-no-effect concentration in soil (PNEC(soil)) of 3.7 mg/kg-dry weight was calculated. Following international regulatory guidance, BPA concentrations in soil hypothetically amended with biosolids were calculated using published BPA concentrations in biosolids. The upper 95th percentile BPA biosolids concentration in North America is 14.2 mg/kg-dry weight, and in Europe is 95 mg/kg-dry weight. Based on recommended biosolids application rates, predicted BPA concentrations in soil (PEC(soil)) would be 0.021 mg/kg-dry weight for North America and 0.14 mg/kg-dry weight for Europe. Hazard quotients (ratio of PEC(soil) and PNEC(soil)) for BPA were all equal to or less than 0.04. This indicates that risks to representative invertebrates and plants at the base of the terrestrial food web are low if exposed to BPA in soil amended with activated sludge biosolids.

Acute and chronic toxicity testing of bisphenol A with aquatic invertebrates and plants.

Bisphenol A (BPA, 4,4'-isopropylidine diphenol) is a commercially important chemical used primarily as an intermediate in the production of polycarbonate plastic and epoxy resins. Extensive effect data are currently available, including long-term studies with BPA on fish, amphibians, crustaceans, and mollusks. The aim of this study was to perform additional tests with a number of aquatic invertebrates and an aquatic plant. These studies include acute tests with the midge (Chironomus tentans) and the snail (Marisa cornuarietis), and chronic studies with rotifers (Brachionus calyciflorus), amphipods (Hyalella azteca), and plants (Lemna gibba). The effect data on different aquatic invertebrate and plant species presented in this paper correspond well with the effect and no-effect concentrations (NOECs) available from invertebrate studies in the published literature and are within the range found for other aquatic species tested with BPA.

Sources of variability in plant toxicity testing.

Published literature is investigated regarding the response of plants to various substances to determine the sensitivity of agricultural plants versus other species, the similarity of effects seen at different taxonomic levels, sensitivity of plants growing outdoors versus in a greenhouse, and the sensitivity of different measurement endpoints. We find that agricultural species are not consistently more or less sensitive to the herbicides tested than non-crop species. Genus and family taxonomic groupings may show similar responses among species, but this similarity quickly decreases as the comparison progress between orders and classes. Results from field and greenhouse studies are less in agreement between studies than data from the other topics. Shoot length will be affected at concentrations lower than for other vegetative endpoints for most species tested for inorganic substances, but for organic substances root and shoot mass were more sensitive. Overall, there is no one species or endpoint that is consistently the most sensitive for all species or all chemicals in all soils, and differences in bioavailability among compounds may confound comparison of test results. Therefore, species sensitivity distributions, adjusted for bioavailability when possible, should be considered in order to better evaluate effects to non-target terrestrial plants.