Reproducibility, reliability, and regulatory relevance of plant visual injury assessments.

The registration of herbicides in the European Union requires an assessment of risks to nontarget terrestrial plants (NTTPs). Regulatory plant studies are performed to determine risk-assessment-relevant endpoints (50% effect rate) for quantitative parameters, mostly biomass and survival. Recently, the European Food Safety Authority stated that endpoints for qualitatively assessed plant visual injuries (PVIs) such as necrosis, chlorosis, and so forth should be considered for the risk assessment as equal to endpoints derived from quantitatively determined parameters. However, the lack of guidance in the NTTP test guidelines on how to assess PVI and how to derive a statistically meaningful endpoint for PVI makes their use in risk assessments challenging. To evaluate and improve the reliability, reproducibility, and regulatory relevance of PVI assessments in NTTP studies, the PVI Working Group was formed in 2022 within the SETAC Plant Interest Group. In a first exercise, research needs, guidance gaps, and shortcomings in current methodologies were identified and are presented together with recommendations for a future, validated, and harmonized method for the assessment of PVI. Survey results revealed a high variability in how PVI are currently assessed, and that the reliability of these data is unclear. Under current conditions, the PVI data can rather be seen as supportive information instead of using the data for the statistically sound determination of a regulatory endpoint. Consequently, standardization and harmonization of procedures for the assessment of PVI are needed. An improved scoring methodology should be developed that allows for a precise, statistically sound endpoint determination. Regarding the regulatory relevance of PVI, further research is required to assess the biological meaning of PVI data and how this is connected to the regulatory requirements and protection goals. Last but not least, guidance is required on how to evaluate the historically available PVI data that are based on various assessment methodologies. Integr Environ Assess Manag 2023;00:1-9. © 2023 The Authors. Integrated Environmental Assessment and Management published by Wiley Periodicals LLC on behalf of Society of Environmental Toxicology & Chemistry (SETAC).

Effects of 17 beta-estradiol exposure on Xenopus laevis gonadal histopathology.

The natural estrogen 17 beta-estradiol (E2) is a potential environmental contaminant commonly employed as a positive control substance in bioassays involving estrogenic effects. The aquatic anuran Xenopus laevis is a frequent subject of reproductive endocrine disruptor research; however, histopathological investigations have tended to be less than comprehensive. Consequently, a study was designed to characterize gross and microscopic changes in the gonads of X. laevis as a result of E2 exposure. Additional goals of this study, which consisted of three separate experiments, included the standardization of diagnostic terminology and criteria, the validation of statistical methodology, and the establishment of a half maximal effective concentration (EC50) for E2 as defined by an approximately 50% conversion of presumptive genotypic males to phenotypic females. In the first experiment, frogs were exposed to nominal concentrations of 0, 0.2, 1.5, or 6.0 microg/L E2. From these experimental results and those of a subsequent range finding trial, the EC50 for E2 was determined to be approximately 0.2 microg/L. This E2 concentration was utilized in the other two experiments, which were performed at different facilities to confirm the reproducibility of results. Experiments were conducted according to Good Laboratory Practice guidelines, and the histopathologic evaluations were peer reviewed by an independent pathologist. Among the three trials, the histopathological findings that were strongly associated with E2-exposure (p<0.001 to 0.0001) included an increase in the proportion of phenotypic females, mixed sex, dilated testis tubules, dividing gonocytes in the testis, and dilated ovarian cavities in phenotypic ovaries. A comparison of the gross and microscopic evaluations suggested that some morphologic changes in the gonads may potentially be missed if studies rely entirely on macroscopic assessment.

Development of an in vitro diagnostic method to determine the genotypic sex of Xenopus laevis.

A genotypic sex determination assay provides accurate gender information of individuals with well-developed phenotypic characters as well as those with poorly developed or absent of phenotypic characters. Determination of genetic sex for Xenopus laevis can be used to validate the outcomes of Tier 2 amphibian assays, and is a requirement for conducting the larval amphibian growth and development assay (LAGDA), in the endocrine disruptor screening program (EDSP), test guidelines. The assay we developed uses a dual-labeled TaqMan probe-based real-time polymerase chain reaction (real-time PCR) method to determine the genotypic sex. The reliability of the assay was tested on 37 adult specimens of X. laevis collected from in-house cultures in Eurofins EAG Agroscience, Easton. The newly designed X. laevis-specific primer pair and probe targets the DM domain gene linked-chromosome W as a master female-determining gene. Accuracy of the molecular method was assessed by comparing with phenotypic sex, determined by necropsy and histological examination of gonads for all examined specimens. Genotypic sex assignments were strongly concordant with observed phenotypic sex, confirming that the 19 specimens were male and 18 were female. The results indicate that the TaqMan® assay could be practically used to determine the genetic sex of animals with poorly developed or no phenotypic sex characteristics with 100% precision. Therefore, the TaqMan® assay is confirmed as an efficient and feasible method, providing a diagnostic molecular sex determination approach to be used in the amphibian endocrine disrupting screening programs conducted by regulatory industries. The strength of an EDSP is dependent on a reliable method to determine genetic sex in order to identify reversals of phenotypic sex in animals exposed to endocrine active compounds.

Development, standardization and refinement of procedures for evaluating effects of endocrine active compounds on development and sexual differentiation of Xenopus laevis.

Xenopus laevis has been introduced as a model to study effects of endocrine-active compounds (EAC) on development and sexual differentiation. However, variable and inconsistent data have raised questions about the reliability of the test methods applied. The current study was conducted in two laboratories to develop, refine, and standardize procedures and protocols. Larvae were exposed in flow-through systems to 17beta-estradiol (E2), at concentrations from 0.2 to 6.0 microg E2 L(-1) in Experiment 1A, and 0.015 to 2.0 microg E2 L(-1) in Experiment 1B. In both studies survival (92%, 99%) and percentage of animals that completed metamorphosis (97%, 99%) indicated reproducible biological performance. Furthermore, minor variations in husbandry led to significant differences in snout-to-vent length, weight, and gonad size. In Experiment 1A, almost complete feminization occurred in all E2 treatment groups whereas a concentration response was observed in Experiment 1B resulting in an EC(50) of 0.12 microg E2 L(-1). The final verified protocol is suitable for determining effects of EAC on development and sexual differentiation in X. laevis.

Assessment of an approach to estimating aquatic bioconcentration factors using reduced sampling.

For a broad range of circumstances, we show that reliable bioconcentration factor (BCF) estimates can be made using a study design that is based on standard regulatory guideline test procedures but that uses significantly fewer animals and resources. This minimized design involves taking tissue samples only twice during a 14-d depuration period. The utility of the minimized test design was first assessed by resampling data from a series of standard guideline tests and calculating the BCF estimates that would have been obtained if the test had been performed using the minimized design. Data from 25 bioconcentration curves giving BCF estimates ranging from approximately 0.3 to over 20,000 were used. The correlation of log BCF estimates from the guideline study with log BCF estimates from the simulated minimized tests was r=0.99, and the slope of the regression line was 0.96. The robustness of BCF estimates to random variation in measurement of chemicals in fish and water (coefficients of variation of concentrations ranging up to 25%) was evaluated using Monte Carlo simulations. For chemicals with depuration half-lives of less than the length of the depuration period, median BCF estimates from the Monte Carlo simulations of the minimized design were always within 7% of the true BCE The ratio of the 95th to the 5th percentile BCF estimates was always less than or equal to 3.7. Furthermore, the span from the 95th to the 5th percentile of BCF estimates was only 15% wider in the minimized test than in the full guideline test, even though animal use and analytical effort was markedly reduced.

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).

Recovery of duckweed from time-varying exposure to atrazine.

The purpose of the present study was to evaluate the recovery of duckweed (Lemna gibba L. G3) after being removed from multiple duration exposures to the herbicide atrazine. Consequently, L. gibba were exposed under various scenarios to atrazine at nominal concentrations ranging from 5 to 160 µg/L and durations of 1, 3, 5, 7, 9, and 14 d under static-renewal test conditions. Exposures were followed by a recovery phase in untreated media for either 7 or 14 d. The 3-, 5-, 7-, 9-, and 14-d median effective concentration (EC50) values were >137, >137, 124, >77, and >75 µg/L, respectively, based on mean growth rate. No clear effect trends were apparent between exposure duration and the magnitude of effective concentrations (EC50s or EC10s). No phytocidal effects of chlorosis or necrosis were identified for any treatment scenario. Nearly all L. gibba plants transferred from treatment groups of different exposure scenarios to media without atrazine during the recovery phase had growth rates that demonstrated immediate recovery, indicating effects were phytostatic in nature and reversible. Only the 1- and 5-d exposure scenarios had growth rates indicating marginally prolonged recovery at the higher concentrations (160 µg/L; additionally, at 40 µg/L for the 5-d exposure). Time to recovery, therefore, was found to be largely independent of exposure duration except at the highest concentrations assessed. Based on growth rate by interval, all treatments demonstrated recovery by the final assessment interval (days 5-7), indicating complete recovery in all exposure scenarios by 7 d, consistent with the mode of action of atrazine.

Does atrazine influence larval development and sexual differentiation in Xenopus laevis?

Debate and controversy exists concerning the potential for the herbicide atrazine to cause gonadal malformations in developing Xenopus laevis. Following review of the existing literature the U.S. Environmental Protection Agency required a rigorous investigation conducted under standardized procedures. X. laevis tadpoles were exposed to atrazine at concentrations of 0.01, 0.1, 1, 25, or 100 microg/l from day 8 postfertilization (dpf) until completion of metamorphosis or dpf 83, whichever came first. Nearly identical experiments were performed in two independent laboratories: experiment 1 at Wildlife International, Ltd. and experiment 2 at the Leibniz-Institute of Freshwater Ecology and Inland Fisheries (IGB). Both experiments employed optimized animal husbandry procedures and environmental conditions in validated flow-through exposure systems. The two experiments demonstrated consistent survival, growth, and development of X. laevis tadpoles, and all measured parameters were within the expected ranges and were comparable in negative control and atrazine-treated groups. Atrazine, at concentrations up to 100 microg/l, had no effect in either experiment on the percentage of males or the incidence of mixed sex as determined by histological evaluation. In contrast, exposure of larval X. laevis to 0.2 microg 17beta-estradiol/l as the positive control resulted in gonadal feminization. Instead of an even distribution of male and female phenotypes, percentages of males:females:mixed sex were 19:75:6 and 22:60:18 in experiments 1 and 2, respectively. These studies demonstrate that long-term exposure of larval X. laevis to atrazine at concentrations ranging from 0.01 to 100 microg/l does not affect growth, larval development, or sexual differentiation.