Progress 06/01/24 to 05/31/25

Outputs
Target Audience: Nothing Reported Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals?In summer 2025, we will conduct a 7-day exposure experiment in which oysters are exposed to water spiked with EtFOSE/EtFOSA, with two water changes during the exposure period. Following exposure, oysters will be transferred to individual containers for a 10-day depuration phase, during which we will manipulate environmental conditions--such as salinity, temperature, and dissolved organic carbon (DOC)--across at least three treatment levels. The schematic below outlines the experimental design. PFAS bioaccumulation in organisms is known to be influenced by their affinity for biomolecules such as proteins and phospholipids. To support this aspect of the study, we have developed lab protocols for total protein and phospholipid assays, informed by literature review and validated through lab trials. We also plan to investigate the mechanisms of PFAS biotransformation and depuration in bivalves using bioinformatic approaches. While microbial transformation of PFAS precursors has been demonstrated in vitro and linked to specific microbes, the role of the bivalve-associated microbiome in these processes remains largely unexplored. Our initial focus will be on characterizing the microbial communities in key organs such as the digestive system, hemolymph, and gills. We are currently in the trial phase for DNA extraction from these tissues and for optimizing sequence data analysis workflows. Once we establish the microbial community profiles under conditions of high PFAS exposure, we will examine the expression of functional genes and enzymes involved in xenobiotic metabolism. We will begin with genes previously associated with EtFOSE transformation, such as pmoA. Enzymes like laccases and those involved in β-oxidation of fatty acids--implicated in the transformation of 6:2 FTOH, a product of 6:2 FTAB--will also be investigated. In addition, we will investigate the metabolic pathways in host bivalves involved in PFAS precursor transformation through differential gene expression analysis. Although in vivo biotransformation of 6:2 FTAB and N-EtFOSE has been observed in aquatic organisms, the underlying mechanisms remain poorly understood. To address this gap, we will collect hemolymph and digestive gland samples from bivalves at multiple time points during the exposure period and compare gene expression profiles to those of control samples. RNA will be extracted using Zymo Direct-zol RNA extraction kits, followed by RNA-seq analysis. Differential expression and enrichment analyses will then be used to identify active metabolic pathways involved in PFAS biotransformation in treated bivalves.

Impacts
What was accomplished under these goals? With USDA funding confirmed in April 2024, we began refining the experimental plan and preparing for project implementation. In early 2025, I recruited a graduate student to join the project and worked with our Co-PI, Dr. McIntosh at Delaware State University, to finalize the logistics and detailed procedures for our preliminary experiments. By the end of May 2025, we had completed preparations to investigate PFAS bioaccumulation, biotransformation, and depuration in two commercially important species: Eastern oyster and Atlantic blue mussel. Our biotransformation experiments focus on two PFAS precursor compounds--EtFOSE (N-ethylperfluorooctanesulfonamidoethanol or EtFOSA) and 6:2 FTAB (6:2 fluorotelomer sulfonamidoalkyl betaine)--selected for their frequent presence in PFAS-containing products and contaminated sites, their potential to degrade into highly toxic terminal PFAS, and their relatively rapid microbial biotransformation rates. For our preliminary experiments, we obtained oysters from aquaculture growers in Delaware's Inland Bays. Standard aquaria for rearing oysters and fish typically use air stones for aeration. However, given that PFAS are surfactants, excessive bubbling could enhance volatilization and loss of PFAS from water to air during exposure. To address this concern, Co-PI McIntosh's group modified the tanks and aerate water using circulation pumps instead of air stones. This setup resulted in over 99% oyster survival after one month, demonstrating its effectiveness. Given the physiological similarities between oysters and blue mussels, we expect this system will also be suitable for mussels and will validate it once we receive mussel samples from our suppliers in Maine.

Publications