Endocrine disruption assessment in aquatic vertebrates - Identification of substance-induced thyroid-mediated effect patterns.

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

Mechanistic modelling of amphibian body burdens after dermal uptake of pesticides from soil.

Amphibians are currently considered to be covered by pesticide Environmental Risk Assessment schemes by surrogacy assumptions of exposure and susceptibility based on typical laboratory test species such as fish, mammals, and birds. While multiple reviews have shown for this approach to be adequate in the case of aquatic stages, the same cannot be definitively stated for terrestrial stages. Concerns have risen that exposure of amphibians is likely to be highly influenced by dermal absorption, primarily due to the high permeability of their skin and the lack of a protective layer, such as fur or feathers. It is thus hypothesized that dermal uptake could be a significant route of exposure. Consequently, it is necessary to determine the relative importance of different exposure routes that might affect the integrated toxicity outcome for terrestrial amphibian life-stages. Here, a one-compartment Toxicokinetic model was derived and tested using a publicly available dataset containing relevant exposure and uptake information for juvenile anurans exposed to 13 different pesticides. Modelled body burdens were then compared to measured burdens for a total of 815 individuals. Overall, a good concordance between modelled and measured values was observed, with the predicted and measured body burdens differing by a factor of 2 on average (overall R2 of 0.80 and correlation coefficient of 0.89), suggesting good predictivity of the model. Accordingly, the model predicts realistic body burdens for a variety of frog and toad species, and overall, for anurans. As the model includes rehydration (implicit in the evaluated studies) but currently does not account for metabolism, it can be seen as a worst-case assessment. We suggest toxicokinetic models, such as the one here presented, could be used to characterize dermal exposure in amphibians, screen for pesticides of concern, and prioritize risk assessment efforts, whilst reducing the need for de novo vertebrate testing.

Historical control histopathology data from amphibian metamorphosis assays and fathead minnow fish short term reproductive assays: A tool for data interpretation.

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

An Amphibian Metamorphosis Assay Dietary Restriction Study: Lessons for Data Interpretation.

The amphibian metamorphosis assay (AMA) is a key in vivo endocrine screen to investigate chemicals with potential thyroid activity. The test guidelines and associated guidance consider that treatment-related effects on thyroid gland histomorphology automatically result in the assay being considered positive for thyroid activity, independent of the direction of change or conflicting results in the other biological endpoints. An AMA study was conducted with five different feeding rations equivalent to 50%, 30%, 20%, 10%, and 5% of the recommended feeding rate. Biological endpoints relating to growth and development, including thyroid gland histopathology, were evaluated, and the specificity of these endpoints for the determination of thyroid activity was assessed. There was no effect on survival or clinical signs of toxicity. Effects related to feed reduction generally occurred in a feeding ration-response manner and included reduced development stage; reduced body weight and body length metrics; decreased prevalence of thyroid follicular cell hyperplasia and hypertrophy, and the occurrence of thyroid atrophy; reduced liver vacuolation; and the occurrence of liver atrophy. The results indicate that treatment-related histopathological changes in the AMA can be induced by Non-chemical factors; therefore histopathological results are not necessarily diagnostically specific for chemically induced thyroid endocrine activity. Consequently, the interpretation of data from AMA studies should be adjusted accordingly. We recommend that the decision logic presented in the test guidelines and associated guidance be changed to reflect a requirement for directional agreement between the thyroid histopathology findings and the growth and developmental endpoints before it is concluded that a test substance has thyroid endocrine activity. Environ Toxicol Chem 2023;42:1061-1074. © 2023 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Validating buccal swabbing as a minimal-invasive method to detect pesticide exposure in squamate reptiles.

The use of enzymatic biomarkers constitutes a widely used approach in ecotoxicology. However, standard sampling procedures are invasive, requiring tissue, organ or blood extraction. This leads to concerns regarding conservation practice, animal welfare and restrictions in study design. New techniques are needed to avoid these problems, but still generate reliable data. Here, we aimed at validating the use of buccal swabs as a reliable method to detect pesticide exposure in reptiles. Common wall lizards (Podarcis muralis) were divided into control, dermal and oral treatment groups and exposed to different pesticide formulations. Subsequently, buccal swabs were taken and enzymatic activity was analyzed. We were able to confirm the suitability of the method to detect effects of pesticide exposure on the enzymatic level. While exposure to the formulation Roundup Ultramax® didn't match when compared to effects previously observed in situ when compared to other glyphosate based formulations, effects could still be detected. This can be seen as a strong indicator that the active ingredient of a formulation may not always be the mian driver for ecotoxicological effectsat the enzymatic level. At the same time, exposure towards the single formulation Vivando® didn't result in any effects. However, individuals residing in agricultural landscapes will mostly be exposed to pesticide mixes containing different formulations. Our results strongly advocate that buccal swabbing is a reliable minimal invasive method to generate samples for detecting effects of pesticide exposure in reptiles. Due to its easy handling, we believe it will provide new opportunities concerning study designs.

European common frog Rana temporaria (Anura: Ranidae) larvae show subcellular responses under field-relevant Bacillus thuringiensis var. israelensis (Bti) exposure levels.

Bacillus thuringiensis var. israelensis (Bti) is presumed to be an environmental friendly agent for the use in either health-related mosquito control or the reduction of nuisance associated with mosquitoes from seasonal wetlands. Amphibians inhabiting these valuable wetlands may be exposed to Bti products several times during their breeding season. Up until now, information regarding effects on the non-targeted group of amphibians has to be considered rather inconsistent. On this account, we evaluated how three repeated exposures to frequently used Bti formulations (VectoBac®12AS, VectoBac®WG) in field-relevant rates affect European common frog (Rana temporaria) larvae. In a laboratory approach, we assessed potential effects with regard to enzymatic biomarkers (glutathione-S-transferase (GST), glutathione reductase (GR), acetylcholine esterase (AChE)), development, body condition and survival until the end of metamorphosis. Although survival and time to metamorphosis were not significantly affected, larval development tended to be shortened in the Bti treated water phase. Furthermore, exposure to Bti induced significant increases of GST (37-550%), GR (5-140%) and AChE (38-137%) irrespectively of the applied formulation, indicating detoxification, antioxidant responses as well as an alteration of neuronal activity. GST activity increased twice as much after two repeatedly executed Bti applications within a time period of 6 days. The examination of several biochemical markers is needed to fully evaluate the ecotoxicological risk of Bti for amphibian populations, especially in the context of worldwide amphibian declines. Nevertheless, following the precautionary principle, it may be advisable to implement certain thresholds for application numbers and intervals in order to ensure environmentally friendly mosquito control programs, especially in areas designated for nature conservation.

The impact of land use intensity and associated pesticide applications on fitness and enzymatic activity in reptiles-A field study.

Environmental pollution and habitat loss are described as underlying causes for population declines in reptiles and especially affect species in agricultural landscapes. Studies dealing with effects of pesticide exposure on reptiles are limited, mainly addressing the orders Testudines and Crocodylia, but largely neglecting the most diverse reptile order Squamata (lizards and snakes). As a consequence, information regarding effects on their organisms, as well as exposure probability and pesticide uptake in the Reptilia has to be considered rather uncharted. We here ask how pesticide applications affect a widely distributed, synanthropic squamate species in Europe. We studied the common wall lizard (Podarcis muralis) with regard to enzymatic biomarkers of pesticide exposure (Glutathione-S-Transferase, Glutathione Reductase, Acetylcholinesterase) and body condition. Lizards were sampled from wild populations, along an exposure gradient (three exposed sites with differing land use intensity and one reference site). Our results suggest both dermal and oral uptake of pesticide formulations, with the former being especially relevant during the first two days after a pesticide application. Enzymatic activity slightly differed between exposure gradients, while showing overall similar patterns. Body condition of lizards decreased with increasing pesticide exposure. Furthermore, gender distribution was particularly skewed in favor to males within exposed sample sites. Although reptiles are not target organisms of pesticide applications, many species do come into contact with them, and most probably suffer from dermal and oral uptake. Thus, we believe it is indispensable for reptiles to be integrated in risk assessments in order to improve conservation practice.

The use of buccal swabs as a minimal-invasive method for detecting effects of pesticide exposure on enzymatic activity in common wall lizards.

Habitat loss and environmental pollution are among the main causes responsible for worldwide biodiversity loss. The resulting species and population declines affect all vertebrates including reptiles. Especially in industrialized countries, pollution by agrochemicals is of remarkable importance. Here, habitat loss has historically been associated with expansion of agriculture. Species persisting in such environments do not only need to cope with habitat loss, but more recently, also with chemical intensification, namely pesticide exposure. In this study, we examined effects of different fungicide and herbicide applications on the common wall lizard (Podarcis muralis) in grape-growing areas. We used three enzymatic biomarkers (GST, GR, AChE) and for the first time saliva from buccal swabs as a minimal-invasive sampling method for detection. Our results demonstrate absorption of substances by lizards and effects of pesticide exposure on enzymatic activities. Our findings are in accordance with those of previous laboratory studies, although samples were retrieved from natural habitats. We conclude that buccal swabs could become a useful tool for the detection of pesticide exposure in reptiles and have the potential to replace more invasive methods, such as organ extraction or cardiac puncture. This is an important finding, as reptiles are non-target organisms of pesticide applications, and there is a strong need to integrate them into pesticide risk assessments.

Risk of pesticide exposure for reptile species in the European Union.

Environmental pollution has an especially high impact on wildlife. This is especially the case in industrialized countries. Although, many species within the European Union benefit from protection by the Habitats Directive, no special consideration is given to possible detrimental effects of pesticides. This is in particular remarkable as negative effects, which may lead to a regional diversity loss, have already been identified in laboratory and mesocosm studies. We conducted a pesticide exposure risk evaluation for all European reptile species with sufficient literature data on the considered biological and ecological aspects and occurrence data within agricultural areas with regular pesticide applications (102 out of 141). By using three evaluation factors - (i) pesticide exposure, (ii) physiology and (iii) life history - a taxon-specific pesticide exposure risk factor (ERF) was created. The results suggest that about half of all evaluated species, and thus at least 1/3 of all European species exhibited a high exposure risk. At the same time, two of them (Mauremys leprosa and Testudo graeca) are globally classified as threatened with extinction in the IUCN Red List of Threatened Species. Variation regarding species occurrence in exposed landscapes between pesticide admission zones within the EU is rather large. This variation is mainly caused by differing land use and species abundances between zones. At the taxonomic level, significant differences in exposure risk can be observed between threatened and non-threatened species, which can be explained by the formers remote distribution areas. Lizards display the highest sensitivity toward pesticides, although no differences in overall ERFs can be observed between taxonomic groups. By identifying species at above-average risk to pesticide exposure, species-based risk evaluations can improve conservation actions for reptiles from cultivated landscapes.