An automated computational data pipeline to rapidly acquire, score, and rank toxicological data for ecological hazard assessment.

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;00:1-15. © 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.

Evaluating potential developmental toxicity of perfluoroalkyl and polyfluoroalkyl substances in Xenopus laevis embryos and larvae.

As part of the US Environmental Protection Agency's perfluoroalkyl and polyfluoroalkyl substances (PFAS) Action Plan, the agency is committed to increasing our understanding of the potential ecological effects of PFAS. The objective of these studies was to examine the developmental toxicity of PFAS using the laboratory model amphibian species Xenopus laevis. We had two primary aims: (1) to understand the developmental toxicity of a structurally diverse set of PFAS compounds in developing embryos and (2) to characterize the potential impacts of perfluorooctanesulfonic acid (PFOS), perfluorohexanesulfonic acid (PFHxS), perfluorooctanoic acid (PFOA), and hexafluoropropylene oxide-dimer acid (HFPO-DA a.k.a. GenX), on growth and thyroid hormone-controlled metamorphosis. We employed a combination of static renewal and flow-through exposure designs. Embryos were exposed to 17 structurally diverse PFAS starting at the midblastula stage through the completion of organogenesis (96 h). To investigate impacts on PFOS, PFOA, PFHxS, and HFPO-DA on development and metamorphosis, larvae were exposed from premetamorphosis (Nieuwkoop Faber stage 51 or 54) through pro metamorphosis. Of the PFAS tested in embryos, only 1H,1H,10H,10H-perfluorodecane-1,10-diol (FC10-diol) and perfluorohexanesulfonamide (FHxSA) exposure resulted in clear concentration-dependent developmental toxicity. For both of these PFAS, a significant increase in mortality was observed at 2.5 and 5 mg/L. For FC10-diol, 100% of the surviving embryos were malformed at 1.25 and 2.5 mg/L, while for FHxSA, a significant increase in malformations (100%) was observed at 2.5 and 5 mg/L. Developmental stage achieved was the most sensitive endpoint with significant effects observed at 1.25 and 0.625 mg/L for FC10-diol and FHxSA, respectively. In larval studies, we observed impacts on growth following exposure to PFHxS and PFOS at concentrations of 100 and 2.5 mg/L, respectively, while no impacts were observed in larvae when exposed to PFOA and HFPO-DA at concentration of 100 mg/L. Further, we did not observe impacts on thyroid endpoints in exposed larvae. These experiments have broadened our understanding of the impact of PFAS on anuran development.

Comparison of in silico, in vitro, and in vivo toxicity benchmarks suggests a role for ToxCast data in ecological hazard assessment.

Large repositories of in vitro bioactivity data such as US EPA's Toxicity Forecaster (ToxCast) provide a wealth of publicly accessible toxicity information for thousands of chemicals. These data can be used to calculate point-of-departure (POD) estimates via concentration-response modeling that may serve as lower bound, protective estimates of in vivo effects. However, the data are predominantly based on mammalian models and discussions to date about their utility have largely focused on potential integration into human hazard assessment, rather than application to ecological risk assessment. The goal of the present study was to compare PODs based on (1) quantitative structure-activity relationships (QSARs), (2) the 5th centile of the activity concentration at cutoff (ACC), and (3) lower-bound cytotoxic burst (LCB) from ToxCast, with the distribution of in vivo PODs compiled in the Ecotoxicology Knowledgebase (ECOTOX). While overall correlation between ToxCast ACC5 and ECOTOX PODs for 649 chemicals was weak, there were significant associations among PODs based on LCB and ECOTOX, LCB and QSARs, and ECOTOX and QSARs. Certain classes of compounds showed moderate correlation across datasets (eg, antimicrobials/disinfectants), while others, such as organophosphate insecticides, did not. Unsurprisingly, more precise classifications of the data based on ECOTOX effect and endpoint type (eg, apical vs biochemical; acute vs chronic) had a significant effect on overall relationships. Results of this research help to define appropriate roles for data from new approach methodologies in chemical prioritization and screening of ecological hazards.

A biologically based computational model for the hypothalamic-pituitary-thyroid (HPT) axis in Xenopus laevis larvae.

A biologically based computational model was developed to describe the hypothalamic-pituitary-thyroid (HPT) axis in developing Xenopus laevis larvae. The goal of this effort was to develop a tool that can be used to better understand mechanisms of thyroid hormone-mediated metamorphosis in X. laevis and predict organismal outcomes when those mechanisms are perturbed by chemical toxicants. In this report, we describe efforts to simulate the normal biology of control organisms. The structure of the model borrows from established models of HPT axis function in mammals. Additional features specific to X. laevis account for the effects of organism growth, growth of the thyroid gland, and developmental changes in regulation of thyroid stimulating hormone (TSH) by circulating thyroid hormones (THs). Calibration was achieved by simulating observed changes in stored and circulating levels of THs during a critical developmental window (Nieuwkoop and Faber stages 54-57) that encompasses widely used in vivo chemical testing protocols. The resulting model predicts that multiple homeostatic processes, operating in concert, can act to preserve circulating levels of THs despite profound impairments in TH synthesis. Represented in the model are several biochemical processes for which there are high-throughput in vitro chemical screening assays. By linking the HPT axis model to a toxicokinetic model of chemical uptake and distribution, it may be possible to use this in vitro effects information to predict chemical effects in X. laevis larvae resulting from defined chemical exposures.

Greater flowering and response to flooding in Lythrum virgatum than L. salicaria (purple loosestrife).

Newly introduced trait diversity can spur rapid evolution and facilitate local adaptation in the introduced plant Lythrum salicaria. The horticultural plant L. virgatum might further introduce meaningful trait variation by escaping into established L. salicaria populations or by hybridizing with L. salicaria. Although many experiments have focused on L. salicaria genotypes, relatively little is known about L. virgatum ecology. We used a greenhouse common garden to compare traits and flood response of L. salicaria and L. virgatum collected from two sources each in their native range. We tested the hypotheses that these two wetland taxa have comparable responses to flooding (inundation), and that flood tolerance correlated to higher fitness. Flooding produced stronger stress responses in L. virgatum. Compared to L. salicaria, L. virgatum shifted more aboveground allocation away from reproduction, decreased inflorescence biomass by 40% more, and produced 7% more stem aerenchymatous phellum, a specialized tissue that maintains aeration. Despite these more pronounced responses to flooding stress, L. virgatum had higher fitness (inflorescence biomass and reproductive allocation) than L. salicaria. Overall, L. virgatum differed from L. salicaria in functionally important ways. Lythrum virgatum persisted under flooding and produced more reproductive biomass than L. salicaria under both flooded and non-flooded conditions. However, inundation stressed L. virgatum more than L. salicaria. Lythrum virgatum is likely able to establish into the wetland habitats in which L. salicaria prevails but may possess broader habitat tolerances.

Combining In Vitro and In Silico New Approach Methods to Investigate Type 3 Iodothyronine Deiodinase Chemical Inhibition Across Species.

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.

Autopolyploids of Arabidopsis thaliana are more phenotypically plastic than their diploid progenitors.

BACKGROUND AND AIMS: Polyploids are often hypothesized to have increased phenotypic plasticity compared with their diploid progenitors, but recent work suggests that the relationship between whole-genome duplication (WGD) and plasticity is not so straightforward. Impacts of WGD on plasticity are moderated by other evolutionary processes in nature, which has impeded generalizations regarding the effects of WGD alone. We assessed shifts in phenotypic plasticity and mean trait values accompanying WGD, as well as the adaptive consequences of these shifts. METHODS: To isolate WGD effects, we compared two diploid lineages of Arabidopsis thaliana wiht corresponding autotetraploids grown across different salt and nutrient conditions in a growth chamber. KEY RESULTS: For the few cases in which diploids and polyploids differed in plasticity, polyploids were more plastic, consistent with hypotheses that WGD increases plasticity. Under stress, increased plasticity was often adaptive (associated with higher total seed mass), but in other cases plasticity was unrelated to fitness. Mean trait values and plasticity were equally likely to be affected by WGD, but the adaptive consequences of these shifts were often context dependent or lineage specific. For example, polyploids had extended life spans, a shift that was adaptive in one polyploid lineage under amenable conditions but was maladaptive in the other lineage under stress. CONCLUSIONS: Our work shows that increased phenotypic plasticity can result from WGD alone, independent of other evolutionary processes. We find that the effects of WGD can differ depending on the genotype of the progenitor and the environmental context. Though our experiment was limited to two genotypes of a single species, these findings support the idea that WGD can indeed increase plasticity.

From Protein Sequence to Structure: The Next Frontier in Cross-Species Extrapolation for Chemical Safety Evaluations.

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.

Potential local adaptation in populations of invasive reed canary grass (Phalaris arundinacea) across an urbanization gradient.

Urban stressors represent strong selective gradients that can elicit evolutionary change, especially in non-native species that may harbor substantial within-population variability. To test whether urban stressors drive phenotypic differentiation and influence local adaptation, we compared stress responses of populations of a ubiquitous invader, reed canary grass (Phalaris arundinacea). Specifically, we quantified responses to salt, copper, and zinc additions by reed canary grass collected from four populations spanning an urbanization gradient (natural, rural, moderate urban, and intense urban). We measured ten phenotypic traits and trait plasticities, because reed canary grass is known to be highly plastic and because plasticity may enhance invasion success. We tested the following hypotheses: (a) Source populations vary systematically in their stress response, with the intense urban population least sensitive and the natural population most sensitive, and (b) plastic responses are adaptive under stressful conditions. We found clear trait variation among populations, with the greatest divergence in traits and trait plasticities between the natural and intense urban populations. The intense urban population showed stress tolerator characteristics for resource acquisition traits including leaf dry matter content and specific root length. Trait plasticity varied among populations for over half the traits measured, highlighting that plasticity differences were as common as trait differences. Plasticity in root mass ratio and specific root length were adaptive in some contexts, suggesting that natural selection by anthropogenic stressors may have contributed to root trait differences. Reed canary grass populations in highly urbanized wetlands may therefore be evolving enhanced tolerance to urban stressors, suggesting a mechanism by which invasive species may proliferate across urban wetland systems generally.