The objective of this project is to develop and demonstrate the utility of ecological high throughput transcriptomics assays as a new methodology for determining potential toxicity of per- and polyfluoroalkyl substances (PFAS) sites impacted by aqueous film-forming foam (AFFF). This includes the following:

1. Efficient generation of hazard estimates for individual PFAS for use in risk-based screening.
2. Determining the integrated hazard of defined PFAS mixtures and environmental samples.
3. Documenting changes in integrated hazard as a function of site remediation or treatment activities.
4. Grouping PFAS based on the similarity in elicited biological response profiles and evaluating structural correlates to those groupings.
5. Using biological response profiles to infer whether PFAS versus other chemicals are a key driver of potential toxicity at a site.
6. Using biological response profiles to infer which individual PFAS, or categories of PFAS, at a site are likely responsible for biological effects observed, based on statistical similarity of response profiles.

Application of Ecological High Throughput Transcriptomics as a New Approach Methodology for Understanding the Ecological Toxicity of PFAS at AFFF Impacted Sites

The project is structured around three tasks defined by the complexity of samples to be tested.

• In Task 1, up to 25 commercially obtainable AFFF-relevant PFAS (available in ≥ 1 mg amounts) will be tested in concentration response (minimum seven concentrations). Larval Pimephales promelas (fathead minnow; vertebrate, secondary consumer), Daphnia magna (invertebrate, primary consumer), and Raphidocelis subcapitata (algae, primary producer) will be exposed for 24 hours in 96-well plate format. Following exposure, whole body, whole transcriptome, gene expression will be measured. The concentration at which gene expression is significantly altered (a transcriptomic point of departure) will be used to characterize the relative biological potency of each tested PFAS. The profile of genes whose expression was impacted will be compared to a library of other such profiles to discern similarity in biological response among chemicals/stressors.
• In Task 2, the same assays will be applied to test defined mixtures and evaluate the hypothesis that mixtures of PFAS with similar gene expression response profiles would conform to a concentration addition model (i.e., act through the same mode of action), while mixtures of PFAS with disparate gene expression response profiles would deviate from concentration addition.
• In Task 3, the same bioassays will be employed to test AFFF formulations and environmental samples before and after treatment. Assay results will be used to determine whether applied treatments reduced the overall potency of the treated sample and significantly altered its biological response profile, indicating a change in bioactive chemical composition.

Testing of individual AFFF-relevant PFAS in Task 1 will provide novel hazard data for data poor PFAS that can be used to support screening-level risk assessment. Understanding gained in Task 2 will allow for more effective prediction of biological hazards of PFAS mixtures based on analytical monitoring data alone. Task 3 results will demonstrate how high throughput ecological assays can be used to assess remedy effectiveness of PFAS treatment or clean up methods, for example supporting assessments under the Comprehensive Environmental Response, Compensation, and Liability Act by providing quantitative measures of a reduction in biological potency. Likewise, if response profiling approaches are successful, results may provide a means to determine whether AFFF-relevant PFAS or other chemicals are key contributors to biological potency and to differentiate adaptive, general stress response, from indications of adverse response. Successful completion of this effort will ultimately lead to more cost effective PFAS management, directly benefiting the warfighter and installation communities. (Anticipated Project Completion - 2029)