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.

Comparing Transcriptomic Points of Departure to Apical Effect Concentrations For Larval Fathead Minnow Exposed to Chemicals with Four Different Modes Of Action.

It is postulated that below a transcriptomic-based point of departure, adverse effects are unlikely to occur, thereby providing a chemical concentration to use in screening level hazard assessment. The present study extends previous work describing a high-throughput fathead minnow assay that can provide full transcriptomic data after exposure to a test chemical. One-day post-hatch fathead minnows were exposed to ten concentrations of three representatives of four chemical modes of action: organophosphates, ecdysone receptor agonists, plant photosystem II inhibitors, and estrogen receptor agonists for 24 h. Concentration response modeling was performed on whole body gene expression data from each exposure, using measured chemical concentrations when available. Transcriptomic points of departure in larval fathead minnow were lower than apical effect concentrations across fish species but not always lower than toxic effect concentrations in other aquatic taxa like crustaceans and insects. The point of departure was highly dependent on measured chemical concentration which were often lower than the nominal concentration. Differentially expressed genes between chemicals within modes of action were compared and often showed statistically significant overlap. In addition, reproducibility between identical exposures using a positive control chemical (CuSO4) and variability associated with the transcriptomic point of departure using in silico sampling were considered. Results extend a transcriptomic-compatible fathead minnow high-throughput assay for possible use in ecological hazard screening.

Transcriptomics-Based Points of Departure for Daphnia magna Exposed to 18 Per- and Polyfluoroalkyl Substances.

Per- and polyfluoroalkyl substances (PFAS) represent a large group of contaminants of concern based on their widespread use, environmental persistence, and potential toxicity. Many traditional models for estimating toxicity, bioaccumulation, and other toxicological properties are not well suited for PFAS. Consequently, there is a need to generate hazard information for PFAS in an efficient and cost-effective manner. In the present study, Daphnia magna were exposed to multiple concentrations of 22 different PFAS for 24 h in a 96-well plate format. Following exposure, whole-body RNA was extracted and extracts, each representing five exposed individuals, were subjected to RNA sequencing. Following analytical measurements to verify PFAS exposure concentrations and quality control on processed cDNA libraries for sequencing, concentration-response modeling was applied to the data sets for 18 of the tested compounds, and the concentration at which a concerted molecular response occurred (transcriptomic point of departure; tPOD) was calculated. The tPODs, based on measured concentrations of PFAS, generally ranged from 0.03 to 0.58 µM (9.9-350 µg/L; interquartile range). In most cases, these concentrations were two orders of magnitude lower than similarly calculated tPODs for human cell lines exposed to PFAS. They were also lower than apical effect concentrations reported for seven PFAS for which some crustacean or invertebrate toxicity data were available, although there were a few exceptions. Despite being lower than most other available hazard benchmarks, D. magna tPODs were, on average, four orders of magnitude greater than the maximum aqueous concentrations of PFAS measured in Great Lakes tributaries. Overall, this high-throughput transcriptomics assay with D. magna holds promise as a component of a tiered hazard evaluation strategy employing new approach methodologies. Environ Toxicol Chem 2024;00:1-16. © 2024 SETAC. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA.

Derivation of Transcriptomics-Based Points of Departure for 20 Per- or Polyfluoroalkyl Substances Using a Larval Fathead Minnow (Pimephales promelas) Reduced Transcriptome Assay.

Traditional toxicity testing has been unable to keep pace with the introduction of new chemicals into commerce. Consequently, there are limited or no toxicity data for many chemicals to which fish and wildlife may be exposed. Per- and polyfluoroalkyl substances (PFAS) are emblematic of this issue in that ecological hazards of most PFAS remain uncharacterized. The present study employed a high-throughput assay to identify the concentration at which 20 PFAS, with diverse properties, elicited a concerted gene expression response (termed a transcriptomics-based point of departure [tPOD]) in larval fathead minnows (Pimephales promelas; 5-6 days postfertilization) exposed for 24 h. Based on a reduced transcriptome approach that measured whole-body expression of 1832 genes, the median tPOD for the 20 PFAS tested was 10 µM. Longer-chain carboxylic acids (12-13 C-F); an eight-C-F dialcohol, N-alkyl sulfonamide; and telomer sulfonic acid were among the most potent PFAS, eliciting gene expression responses at concentrations <1 µM. With a few exceptions, larval fathead minnow tPODs were concordant with those based on whole-transcriptome response in human cell lines. However, larval fathead minnow tPODs were often greater than those for Daphnia magna exposed to the same PFAS. The tPODs overlapped concentrations at which other sublethal effects have been reported in fish (available for 10 PFAS). Nonetheless, fathead minnow tPODs were orders of magnitude higher than aqueous PFAS concentrations detected in tributaries of the North American Great Lakes, suggesting a substantial margin of safety. Overall, results broadly support the use of a fathead minnow larval transcriptomics assay to derive screening-level potency estimates for use in ecological risk-based prioritization. Environ Toxicol Chem 2024;00:1-16. © 2024 SETAC. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA.

Short-Term Transcriptomic Points of Departure Are Consistent with Chronic Points of Departure for Three Organophosphate Pesticides across Mouse and Fathead Minnow.

New approach methods (NAMs) can reduce the need for chronic animal studies. Here, we apply benchmark dose (concentration) (BMD(C))-response modeling to transcriptomic changes in the liver of mice and in fathead minnow larvae after short-term exposures (7 days and 1 day, respectively) to several dose/concentrations of three organophosphate pesticides (OPPs): fenthion, methidathion, and parathion. The mouse liver transcriptional points of departure (TPODs) for fenthion, methidathion, and parathion were 0.009, 0.093, and 0.046 mg/Kg-bw/day, while the fathead minnow larva TPODs were 0.007, 0.115, and 0.046 mg/L, respectively. The TPODs were consistent across both species and reflected the relative potencies from traditional chronic toxicity studies with fenthion identified as the most potent. Moreover, the mouse liver TPODs were more sensitive than or within a 10-fold difference from the chronic apical points of departure (APODs) for mammals, while the fathead minnow larva TPODs were within an 18-fold difference from the chronic APODs for fish species. Short-term exposure to OPPs significantly impacted acetylcholinesterase mRNA abundance (FDR p-value <0.05, |fold change| ≥2) and canonical pathways (IPA, p-value <0.05) associated with organism death and neurological/immune dysfunctions, indicating the conservation of key events related to OPP toxicity. Together, these results build confidence in using short-term, molecular-based assays for the characterization of chemical toxicity and risk, thereby reducing reliance on chronic animal studies.

Demonstration of the Sequence Alignment to Predict Across Species Susceptibility Tool for Rapid Assessment of Protein Conservation.

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.

Pilot testing and optimization of a larval fathead minnow high throughput transcriptomics assay.

Concentrations at which global gene expression profiles in cells or animals exposed to a test substance start to differ significantly from those of controls have been proposed as an alternative point of departure for use in screening level hazard assessment. The present study describes pilot testing of a high throughput compatible transcriptomics assay with larval fathead minnows. One day post hatch fathead minnows were exposed to eleven different concentrations of three metals, three selective serotonin reuptake inhibitors, and four neonicotinoid-like compounds for 24 h and concentration response modeling was applied to whole body gene expression data. Transcriptomics-based points of departure (tPODs) were consistently lower than effect concentrations reported in apical endpoint studies in fish. However, larval fathead minnow-based tPODs were not always lower than concentrations reported to elicit apical toxicity in other aquatic organisms like crustaceans or insects. Random in silico subsampling of data from the pilot assays was used to evaluate various assay design and acceptance considerations such as transcriptome coverage, number of replicate individuals to sequence per treatment, and minimum number of differentially expressed genes to produce a reliable tPOD estimate. Results showed a strong association between the total number of genes for which a concentration response relationship could be derived and the overall variability in the resulting tPOD estimates. We conclude that, for our current assay design and analysis pipeline, tPODs based on fewer than 15 differentially expressed genes are likely to be unreliable for screening and that interindividual variability in gene expression profiles appears to be a more significant driver of tPOD variability than sample size alone. Results represent initial steps toward developing high throughput transcriptomics assays for use in ecological hazard screening.