AN ABSTRACT OF THE DISSERTATION OF Emerson C. Christie for the degree of Doctor of Philosophy in Toxicology presented on March 11, 2021. Title: Per- and Polyfluoroalkyl Substances (PFAS): Protein Binding and Partitioning and Sorption in Light Non-Aqueous Phase Liquids Abstract approved: ______________________________________________________ Jennifer A. Field Per- and polyfluoroalkyl substances (PFAS) are anthropogenic surfactants that have recently been identified as persistent organic pollutants. These so called “Forever Chemicals” have been detected in drinking waters, ground waters, soils, and consumer and industrial products globally; with environmental impacts stretching into the artic, far from known PFAS sources. The increase in awareness regarding PFAS distribution in the environment has generated interest into how PFAS interact with humans, what PFAS specific properties may be involved, and what additional environmental compartments may they be found in. In Chapter 2 we discuss the use of molecular dynamics (MD) modeling to screen for protein – PFAS binding affinity to inform experimental measurements of binding affinity via equilibrium dialysis (Eq D). The equilibrium dissociation constants (KD) of six perfluoroalkyl carboxylates (PFCAs) and three perfluoroalkyl sulfonates (PFSAs) to liver and intestinal fatty acid binding proteins (L- and I-FABPs) and peroxisome proliferator activated nuclear receptors (PPAR-α, - δ and - γ) were determined via liquid chromatography mass spectrometry. The MD models were found to predict relative and not absolute binding for all protein – PFAS combinations. This research was the first to identify sub micromolar binding between short chain PFAS (6 or less carbons) and PPAR-α and δ, which may have implications for the assumed safety of shorter chain PFAS due to rapid clearance. Chain length dependent binding was observed for L- FABP but not observed for PPAR proteins which means that for these proteins binding affinity cannot be inferred by PFAS chain length. Additionally, a comparison was made between KDs derived from EqD and other in-vitro approaches, using these experimental results and results from literature. It was discovered that KDs derived from EqD were lower (i.e. higher binding affinity) than other in-vitro approaches which has implications for comparisons between methodologies and raises an important question regarding which KDs should be considered most relevant in-vivo. Research discussed in Chapter 3 surrounds the development of an extraction and analytical method to quantify PFAS in environmental non-aqueous phase liquids (NAPL). As mentioned above, PFAS are used in industrial products and one common group of industrial products that have been identified as the root cause for environmental PFAS contamination at U.S. military sites are aqueous film forming foams (AFFF). AFFF are complex mixtures known to contain high concentrations of many surfactants including PFAS. At U.S. military sites it is also common to encounter NAPL in the subsurface. Co-disposal of PFAS (AFFF) with NAPL has happened historically through intentional use (e.g. firefighting) or unintentionally at waste sites. In order to quantify PFAS within NAPL, a liquid-liquid extraction method was developed that could successfully extract anionic, cationic, and zwitterionic PFAS. This research discovered the presence of PFAS in recovered NAPL at microgram per liter concentrations at non-source zone sites. Concentrations of PFAS in NAPL are likely much higher at source zone sites and could have implications for NAPL remediation. Chapter 4 discusses the partitioning and interfacial adsorption of PFAS into NAPL at environmentally relevant concentrations (i.e. nano – microgram per liter). Given the discovery of low microgram per liter concentrations of PFAS in recovered NAPL discussed in Chapter 3, it is relevant to investigate what partitioning and sorption processes are occurring at these concentrations. Current research in this area has focused on the NAPL – water interface and has done so at high concentrations, milli – gram per liter. Here we performed batch equilibrium experiments at low concentrations (2,000 – 100,000 ng/L) between jet fuel A (NAPL) and synthetic freshwater. Single point partition coefficients (Kn) were calculated for PFAS of carbon chain length 8-14 across the concentration range. Values for Kn decreased with increasing PFAS concentration indicating non-ideal partitioning, which become more evident with increasing chain length. Partitioning into jet fuel A was not observed for PFAS below eight carbons. Interfacial sorption (Knw) was estimated by mass difference and found to be orders of magnitude higher than previously reported literature values. ©Copyright by Emerson C. Christie March 11, 2021 All Rights Reserved Per- and Polyfluoroalkyl Substances (PFAS): Protein Binding and Partitioning and Sorption in Light Non-Aqueous Phase Liquids by Emerson C. Christie A DISSERTATION submitted to Oregon State University in partial fulfillment of the requirements for the degree of Doctor of Philosophy Presented March 11, 2021 Commencement June 2021 Doctor of Philosophy dissertation of Emerson C. Christie presented on March 11, 2021 APPROVED: Major Professor, representing Toxicology Head of the Department of Environmental and Molecular Toxicology Dean of the Graduate School I understand that my dissertation will become part of the permanent collection of Oregon State University libraries. My signature below authorizes release of my dissertation to any reader upon request. Emerson C. Christie, Author ACKNOWLEDGEMENTS I would like to thank my wife, Brittany Poirson, for her support during the intense ride that was finishing graduate school while parenting, buying a house, moving, and a double career change, all during a pandemic. My son, Obadiah, for mandating work life balance and providing an unconditional bright smile during acutely stressful times. My soon to be born daughter, Marigold, guiding me towards the world ahead. My parents, Kevin and Libby Christie, for continuous support throughout my life. My sister, Kara Trella, for teaching me creative vision. My brother, Jack Anson, for teaching me the strength of spirit. Thank you to Dr. Alix Robel, Dr. Justin Rewerts, and Dr. Serhan Mermer for teaching me LC MS/MS and enough about lab and instrument health to keep things moving forward. Thank you to my Field Lab members who helped me develop ideas, accomplish objectives, and keep instruments running. Thank you to the many collaborators who worked very hard on the research here-in. Thank you to my committee Dr. Markus Kleber, Dr. Jeffrey Jenkins, Dr. Charles Schaefer, and Dr. Ramesh Sagili; for all their advice in making this research possible. And a big thank you to my adviser Dr. Jennifer Field for obviously seeing something in me, sharing her expertise, and guiding me on this quest. CONTRIBUTION OF AUTHORS In Chapter 2, Manoochehr Khazaee performed equilibrium dialysis experiments and contributed to manuscript write-up and edits. Weixiao Cheng performed molecular modeling and contributed to manuscript write-up and edits. Dr. Mandy Michalsen, Dr. Jennifer Field, and Dr. Carla Ng provided expertise in experimental design and manuscript edits. In Chapter 3, Dr. Bill Diguiseppi provided expertise in light non-aqueous phase liquids and manuscript edits. Dr. Konstantinos Kosterelos facilitated the collection of field samples and provided manuscript edits, and Dr. Jennifer A. Field provided experimental design expertise and manuscript edits. In Chapter 4, Dr. Charles Schaefer provided PFAS interfacial modeling expertise and experimental design expertise. Dr. Konstantinos Kostarelos LNAPL and partitioning expertise. Dr. Jennifer A. Field provided experimental design expertise and manuscript edits. TABLE OF CONTENTS Page Chapter 1- Introduction.....…………………………………………………………… 1 1.1 Per and Polyfluoroalkyl Substances ………...…………………………………… 1 1.2 PFAS – Protein Interactions……………………………………………………… 2 1.3 PFAS – NAPL Connection.....…………………………………………………… 3 1.4 Methods to Analyze PFAS in NAPL......………………………………………… 4 1.5 PFAS – NAPL Partitioning..…...………………………………………………… 4 1.6 PFAS – NAPL Interfacial Sorption..…………..………………………………… 5 1.7 Summary of Research Performed…...…………………………………………… 6 1.8 References……….......…………………………………………………………… 7 Chapter 2 - Perfluoroalkyl acid binding with peroxisome proliferator-activated receptors , , and and fatty acid binding proteins by equilibrium dialysis with a comparison of methods.....…………………………………………………………...13 2.1 Abstract ….………………………………………………………………………14 2.2 Introduction ………...……………………………………………………………15 2.3 Materials and Methods ..…………………………………………………………17 2.4 Results and Discussion .…………………………………………………………12 2.5 Conclusion ………………………………………………………………………28 . 2.6 Acknowledgements …...…………………………………………………………29 2.7 References ……………………………………………………………………….30 Chapter 3 - Per and Polyfluorinated Alkyl Substances in Nonaqueous Phase Liquids……………………………………………………………………………….48 3.1 Abstract ….………………………………………………………………………49 3.2 Introduction ………...……………………………………………………………50 TABLE OF CONTENTS (Continued) Page 3.3 Experimental ………….…………………………………………………………52 3.4 Results and Discussion…………………………………………………………..57 3.5 Implications ……..……………………………………………………………….59 . 3.6 Acknowledgements …...…………………………………………………………60 3.7 References …………………………………………………………………….…60 Chapter 4 - Interfacial uptake and partitioning of per and polyfluorinated alkyl substances in Jet Fuel A at environmental concentrations …..………………………65