Amelia A. Rand. Copyright by Amelia A. Rand 2013

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1 Direct and Indirect Sources of Human Exposure to Perfluorinated Carboxylates: Investigating the Significance of Perfluorinated Carboxylate Reactive Precursor Metabolites by Amelia A. Rand A thesis submitted in conformity with the requirements for the degree of Doctor of Philosophy Department of Chemistry University of Toronto Copyright by Amelia A. Rand 2013

2 Direct and Indirect Sources of Human Exposure to Perfluorinated Carboxylates: Investigating the Significance of Perfluorinated Carboxylate Reactive Precursor Metabolites Amelia A. Rand Doctor of Philosophy Degree Department of Chemistry University of Toronto 2013 ABSTRACT Perfluorinated carboxylates (PFCAs) are persistent and ubiquitous in the environment. Humans are exposed to PFCAs through direct and indirect sources, although the relative importance of each is uncertain. Direct sources of PFCAs have been attributed to two primary fluorochemical manufacturing processes: electrochemical fluorination (ECF) and telomerization. A focus of this thesis was to elucidate an additional direct source of PFCAs resulting from the direct fluorination of polyolefin materials. High density polyethylene bottles with varying levels of fluorination were observed to contain significant amounts of PFCAs, particularly those with carbon chain-lengths C6, marking an unexplored source of PFCA exposure. PFCAs are also produced indirectly from the biotransformation of fluorotelomer-based compounds, such as polyfluoroalkyl phosphate esters (PAPs) and fluorotelomer alcohols (FTOHs). During this transformation process, two predominant classes of metabolic intermediates are formed: the fluorotelomer unsaturated aldehydes (FTUALs) and the fluorotelomer unsaturated carboxylic acids (FTUCAs). Another focus of this thesis was to examine the reactivity of FTUALs and FTUCAs with endogenous nucleophiles such as glutathione (GSH), select amino acids, and model proteins. FTUALs formed adducts with all nucleophiles examined, where those having ii

3 shorter carbon chain lengths (i.e. 6:2 and 8:2 FTUAL) were more reactive than longer carbon chains (i.e. 10:2 FTUAL). By contrast, FTUCAs had comparably limited reactivity; although FTUCAs showed mild reactivity with GSH, they did not react with any other nucleophiles. In vitro and in vivo experiments were carried out to determine the extent of protein binding formed from the biotransformation of fluorotelomer-based compounds, including the 8:2 FTOH and the 6:2 PAP diester. A significant portion of these biotransformations yielded covalent protein binding at nmol/mg protein concentrations. Protein adducts were observed predominantly in rat liver and also in plasma and kidney. The formation of reactive intermediates may be toxicologically important through protein deactivation. Cellular toxicity of FTUALs was significantly higher compared to PFCAs and the acid metabolic intermediates (i.e. FTUCAs). The EC 50 values calculated from dose-response incubations were dependant on chain length and functional group. The work in this thesis examined an unexplored consequence of indirect exposure to PFCAs, potentially impacting the relative importance of PFCA exposure sources. iii

4 ACKNOWLEDGEMENTS The foundation of this thesis was built by many hands: friends, teachers, family, and colleagues. It s hard to imagine what form this work would have without the culmination of support, advice, and encouragement from many. Although words can only go so far, here are a few to show my appreciation. Scott, you have a knack for making analogies stick: The supervisor points the compass, but you must be the roadmap, and you are the driver. You summed up the purpose of a graduate degree within seconds, which has been influencing me for the last five years. Under your wing, I ve grown significantly as a scientist. Thanks for being curious about the big picture, for asking questions that lead to answers in fields that may be out of our comfort area, and for having the flexibility that problems can be solved in many ways, using the many tools and instruments available. A gracious thanks also for rounding this degree with many opportunities to teach, lecture, supervise, travel, and interact with other giants in the field. I also appreciated the occasional prompt for non-scientific exploration (i.e. swimming through underwater tunnels at the Bruce and white water rafting down the New River Gorge). Thank you to Shana Kelley and Jamie Donaldson for being my committee members. Jamie, regardless of your schedule, you have always been available to provide guidance with challenges and opportunities. I value your friendship and for always having the time to talk. Shana, I appreciate how readily you joined the committee, jumping in just before one of the busiest times for a committee member: the oral examination. Many thanks also to Derek Muir for acting as the external internal committee member for both the oral and defence examination. Thanks to Christopher Lau, for travelling from the warmer, more temperate climate of North Carolina to the tail end of winter in order to be my external thesis examiner. To all the people who have come and gone from the Mabury Lab, I ve been fortunate to be part of it through some of its generations. Choice in who we work with isn t usually available, and this group is no exception, yet many close friendships have developed regardless. Amila, Cora, Craig, and Jessica, what a set of inspiring role models! Amila, although you left shortly after I joined, your legend in the lab continued on. I ve appreciated getting to know you over the years, and am counting the days until we can take another slippery slide down the Swiss Alps iv

5 after a good week of scientific discussion. Craig, thanks for being such a great scientist to look up to and I appreciated your ability for giving critical support. Thanks also for providing me with a home during our collaboration at Duke. I look forward to more collaborations and outdoor adventures. Jess, I can remember thinking that I wanted to emulate your scientific personality right from the start. You work hard and intelligently, yet are easy going, humble, and a fantastic partner to have when travelling the globe. Cora, you have never failed to impress. You re talent at making things work for you is topnotch, yet you know how to balance and appreciate life outside the lab. For the others that have come-and-gone, thanks for contributing to the group. Angela, I look forward to seeing you go far and anticipate our climbing trip this summer. I would also like to thank Barbara, who provided much advancement in a challenging synthesis. I wonder sometimes whether I would still be synthesizing the 6:2 FTUAL without your practical knowledge of synthetic organic chemistry and NMR spectroscopy. Holly and Derek, you have been by my side since the very beginning. It has been amazing to work alongside both of you - think of all the hurdles we ve jumped! Derek, you are one smart scientist, and I don t know anyone who explains complexity with such coherent simplicity. But what I think makes you really shine is the selfless way in which you help others. I ve learned much from you, and can only hope that you ll get to spread your love of science (and astronomy, and genealogy, and aquatic and aerial transportation, ) to hundreds of future students. Holly, how fortunate it was to face this big life chapter literally side-by-side. You are a scientific powerhouse, driven to get things to where you want them to be. You have been a high integrity scientist, advice-giver, and friend, all wrapped into one and I am so thankful that we have shared many experiences, both in the lab and around the globe. To all of the current Mabury members: Anne, Keegan, Leo, and Lisa, you ll have to carry the torch for the next generation! Anne, it s wonderful that you came back for round two, and I have really appreciated your friendship in the lab and such places as the airport runway. Keegan, what a balance you brought to the group with your relatively relaxed personality. Leo, how grateful I am for your analytical knowledge and patience. You are certainly someone I will think about when I need to conjure patience when things aren t going as planned. Lisa, it has been great getting to know you. Your talent for recognizing mass spectroscopic fragmentation patterns is uncanny. v

6 To everyone else in environmental chemistry, AIMS, and the NMR facility, thanks for making these past five years so enjoyable! It has been great working with you as scientists, and knowing you as friends. To my friends who have been in my life for years, thanks for your ever-continuous support. I m so glad that much of the Q.E. and Mt. A crew migrated west so that living in the big city was an easy transition, filled with some great memories. Specifically, to Ben who has really been a fantastic friend literally from the first plane ride to Toronto. I m counting the months to celebrate your defence! Also, to Erin who not only sought out many like-minded musical and outdoor adventures, but also meticulously read several of these chapters with an editing prowess. Thanks also to the people at Bloor St., especially David, Martha, and the choir, who permitted a scientist of all things to fulfill their passion for music every other Sunday. Lastly, I would like to extend a heart-felt thanks to Damien and my family. Damien, what a road it has been. I thank you everyday for being a part of my life, for celebrating the victories, providing a shoulder to lean on when challenges arose, and simply making me laugh. I can t wait for our next big adventure! To my whole family, you are the most influential people in my life. From the countless opportunities, triumphs, and losses, you have always been present. Just as we used to climb those granite rocks by the sea, you ve challenged me to live with three points on, embracing the world with heart, soul, and mind. vi

7 TABLE OF CONTENTS CHAPTER ONE - Overview of Perfluoroalkyl and Polyfluoroalkyl Substances: Production, Exposure, and Fate 1.1 Overview Synthesis of Fluorinated Chemicals: Electrochemical Fluorination, Telomerization, 4 and Direct Fluorination Electrochemical Fluorination Telomerization Direct Fluorination Sources of Perfluorinated Carboxylates and their Precursors to the Environment Direct Sources Indirect Sources Atmospheric Transformation of Fluorotelomer-Based Compounds Biotransformation of Fluorotelomer-Based Compounds Formation of PFCAs and Intermediates Mass-Balance of FTOH Biotransformation: Production of PFCAs and Acid Metabolites Formation of GSH Conjugates: Reactivity of FTUCAs and FTUALs Human Exposure to Perfluoroalkyl and Polyfluoroalkyl Substances Direct Sources of Human Exposure Indirect Sources of Human Exposure Toxicity of PFCAs and their Metabolic Precursors Mammalian Toxicity of PFOA: Influence of Peroxisome Proliferator-Activated Receptors Evaluating Toxicity of PFCAs to Humans Epidemiological Studies Effect of Chain Length on PFCA Toxicity Relative toxicity between PFCAs, FTCAs, and FTUCAs Production and Reactivity of Electrophilic Intermediates formed from Metabolic Activation of other Pollutants, Drugs, and Natural Products Metabolism of Bioactive Compounds and Mechanisms of Adduct Formation Bromobenzene α,β-unsaturated Carbonyls: Acrolein and Hydroxynonenal Acetominaphen Halothane Relationship between Covalent Binding and Toxicity Goals and Hypotheses Literature Cited 49 vii

8 CHAPTER TWO - Perfluorinated Carboxylic Acids in Directly Fluorinated High Density Polyethylene Material 2.1 Introduction Materials and Methods Chemicals Sample Collection Extraction of Perfluorinated Acids from Directly Fluorinated HDPE Bottles Instrumental Analysis ATR-IR Spectroscopic Analysis Qualitative NMR Analysis Quality Control Results and Discussion Chemical Composition of Directly Fluorinated HDPE PFCAs in Directly Fluorinated HDPE Bottles F NMR Results Implications Acknowledgements Literature Cited 89 CHAPTER THREE - Assessing the Structure-Activity Relationships of Flurotelomer Unsaturated Acids and Aldehydes with Glutathione 3.1 Introduction Materials and Methods Chemicals Synthesis of the Fluorotelomer Unsaturated Aldehydes (FTUALs) Reactivity with Glutathione Results and Discussion Range-Finding Experiments with Glutathione % Effect Concentration for α,β-unsaturated Carbonyls Conclusions Acknowledgements Literature Cited 108 CHAPTER FOUR - In Vitro Interactions of Biological Nucleophiles with Fluorotelomer Unsaturated Acids and Aldehydes: Fate and Consequences 4.1 Introduction Materials and Methods Materials Determination of 8:2 FTUAL and FTUCA Half-Lives Using 19 F NMR Spectroscopy 115 viii

9 4.2.3 In Vitro Modifications of Human Serum Albumin (HSA) and Apomyoglobin (ApoMg) by FTUCAs and FTUALs Time of Flight-Mass Spectroscopic Analysis Determination of the Strength of Adducts TOF-Combustion Ion Chromatorgraphy Analysis Results and Discussion F NMR Pseudo First-Order Kinetics of FTUCAs and FTUALs with Nucleophilic Amino Acids ToF-MS of FTUCA and FTUAL Adducts with ApoMg ToF-MS of FTUCA and FTUAL Adducts with HSA Implications Acknowledgements Literature Cited 130 CHAPTER FIVE - Covalent Binding of Flurotelomer Unsaturated Aldehydes (FTUALs) and Carboxylic Acids (FTUCAs) to Proteins 5.1 Introduction Materials and Methods Materials Covalent Binding of FTUCAs and FTUALs to Plasma and Microsomes TOF-CIC Analysis Statistical Analysis Isolating Serum Albumin Protein Adducts from Plasma ToF-MS Analysis Biotransformation of 8:2 FTUAL and 8:2 FTOH LC-MS/MS Quantitative Analysis Aldehyde Derivization and Quantification Results and Discussion Covalent Binding of FTUCAs and FTUALs to Plasma and Microsomes Isolating Serum Albumin Protein Adducts from Plasma Biotransformation of 8:2 FTOH Aldehyde Derivatization and Quantification Implications Acknowledgements Literature Cited 155 CHAPTER SIX - Protein binding associated with exposure to fluorotelomer alcohols (FTOHs) and polyfluoroalkyl phosphate esters (PAPs) in rats 6.1 Introduction Materials and Methods Materials Animal Care and Sampling Toxic End Points 165 ix

10 6.2.4 Extraction Procedure BCA Assay for Protein Determination LC-MS/MS Analysis Statistical Analysis TOF-CIC Analysis Results and Discussion :2 FTOH Exposure :2 dipap Exposure Detection of Phase II Metabolites Covalent Protein Binding Implications Acknowledgments Literature Cited 180 CHAPTER SEVEN - Cellular Toxicity Associated with Exposure to Perfluorinated Carboxylates (PFCAs) and their Metabolic Precursors 7.1 Introduction Materials and Methods Materials Cell Culture and Treatments Cell Viability Assays Statistical Analysis qpcr Determination of Adduct Formation with DNA TOF CIC Determination of Adduct Formation with DNA Results and Discussions Effect of PFCAs and their Metabolic Precursors on Cell Viability Comparison of EC 50 Values: PFCAs vs. Metabolic Acid Precursors vs. Metabolic Aldehyde Precursors Reactivity of FTUALs with DNA Implications Acknowledgments Literature Cited 203 CHAPTER EIGHT - Summary, Conclusions, and Future Work 8.1 Summary and Conclusions Future Directions Literature Cited 215 x

11 LIST OF TABLES Table 1.1 Table 1.2 Table 1.3 Table 3.1 Table 6.1 List of acronyms, common names, and chemical structures relevant to this thesis. Yields of isomeric primary PFOA products from the ECF manufacturing process. Calculated mass balances resulting from x:2 FTOH biotransformation in several studies. Comparative reactivity of α,β-unsaturated aldehydes, carboxylic acids, and esters. Methyl methacrylate, acrylic acid, acrolein, and 4:2 fluorotelomer unsaturated acid (FTUCA) EC 50 values include n = 3 samples, 6:2 and 10:2 fluorotelomer unsaturated aldehyde (FTUAL) values include n = 6 samples, and the 8:2 FTUAL EC 50 value includes n = 9 samples. Maximum protein binding concentrations (nmol/g) in plasma, kidney, and liver resulting from exposure to 8:2 FTOH and 6:2 dipap xi

12 LIST OF FIGURES Figure 1.1 Mechanism for electrochemical fluorination. 6 Figure 1.2 Figure 1.3 Figure 1.4 Figure 1.5 Figure 1.6 Figure 1.7 Figure 1.8 Figure 1.9 Figure 1.10 Figure 1.11 Figure 1.12 Figure 2.1 Telomerization scheme to produce fluorotelomer-based compounds for final sales products. Atmospheric transformation scheme for the production of PFCAs from fluorotelomer-based compounds. Transformation products are shown in boxes with dashed outlines, and fluorotelomer-based compounds are shown in boxes with solid outlines. Proposed biotransformation pathway for fluorotelomer-based compounds, adapted from Butt et al. Metabolic activation of bromobenzene and formation of covalently bound intermediates through the a) epoxide pathway and b) quinone pathway. Nu-H represents a nucleophilic group (i.e. amine, sulfhydryl) on a protein. Molecular orbital diagram of the 1,4-Michael addition of glutathione to acrolein. The 1,4-Michael addition of glutathione to the α,β-unsaturated aldehyde acrolein. Reaction of a free radical with a ω-6 Polyunsaturated fatty acid and subsequent rearrangement of double bonds, reaction with molecular oxygen, and formation of 4-HNE. Figure adapted from Carini et al. Formation of an enamine (Schiff-base) from a 1,2-addition reaction of a α,βunsaturated carbonyl with an nucleophilic amine. CYP enzyme-mediated metabolic activation pathway of APAP. RSH denotes a protein thiol group. Mechanism for the oxidation of halothane. Figure adapted from Srivastava et al. CYP-mediated metabolic activation pathway of AMAP. RSH denotes a protein thiol group. ATR-IR spectra of untreated and treated HDPE: (A) Comparison of untreated HDPE with Manufacturer A s five-fluorination level treatment process, (B) Comparison of two sides of a HDPE bottle fluorinated by Manufacturer B. Spectra have been shifted along the y-axis to minimize overlapping xii

13 Figure 2.2 Figure 2.3 Figure 2.4 ATR-IR spectrum of a untreated and treated HDPE bottle fluorinated using Manufacturer A s F2-level treatment process. Spectra have been shifted along the y-axis to minimize overlapping. Proposed mechanism of directly fluorinated HDPE fragmentation leading to the production of PFCAs. Comparative PFCA concentrations between fluorination levels F1 F5 obtained from manufacturer A. PFCA concentrations less than the LOD are reported as zero, and concentrations less than the LOQ are indicated with an asterisk (*) Figure 2.5 Figure 3.1 Figure 3.2 Figure 3.3 Figure F spectrum of PFCAs extracted from a level F3 bottle, acquired using a Varian 400 NMR spectrometer. An enhanced view of the region from -58 to -77 ppm is presented in the inset, and obtained using a Varian 500 NMR spectrometer. General biotransformation pathway of x:2 dipolyfluorinated alkyl phosphates (dipaps). The focus of this work was the fluorotelomer unsaturated acid and aldehyde metabolites (FTUCA and FTUALs) shown in the black box, where x = 6,8,10. The proposed mechanism of GSH reactivity at physiological ph is also highlighted. Arithmetic mean and standard error of glutathione (GSH) response to varying concentrations of 10:2 fluorotelomer unsaturated aldehyde (10:2 FTUAL), used to determine the 50% effect concentration value (EC 50 ) for the 10:2 FTUAL. Data points reflect the arithmetic mean value of n=6 samples, and error for the EC 50 value is presented as the 95% confidence interval. Percent-free glutathione versus length of exposure to (A) 6:2 fluorotelomer unsaturated aldehyde (FTUAL) and (B) 8:2 fluorotelomer unsaturated aldehyde (FTUAL), at varying concentrations. All time-points include n = 3 samples. 19 F NMR spectra of the 4:2 FTUCA reaction with cysteine at a concentration ratio of 27:1 (cysteine:4:2 FTUCA). (A) 19 F NMR spectrum of the unreacted 4:2 FTUCA control, showing the CF 3 group signal (approx. -81 ppm). (B) 19 F NMR spectrum of 4:2 FTUCA after 4 minutes, showing a new peak corresponding to the fluoride product ion (approx ppm). (C) 19 F NMR spectrum of 4:2 FTUCA reaction with cysteine after 36 minutes. The inset in the black box shows the pre-acquisition delay 19 F NMR experiment with loss of the CF 3 peak corresponding to the 4:2 FTUCA reacting with cysteine. Peak height was normalized to the height of the internal standard, 4-TFMeAc (approx. -58 ppm), to determine the pseudo-first order rate constant, k obs xiii

14 Figure 4.2 ESI-ToF mass spectra of apomyoglobin and stoichiometry of 8:2 FTUAL adduct formation. Apomyoglobin was incubated for 2 h at 37 C in the absence (control) and presence of 8:2 FTUAL at the following apomyoglobin:8:2 FTUAL molar ratios: 1:0.5 and 1:1. The arrows in the 1:0.5 spectrum indicate the presence of adducts, which increase in intensity with increasing molar ratio. 124 Figure 4.3 ESI-ToF mass spectrum of HSA-8:2 FTUAL adduct formation at the 1:0.25 (HSA:8:2 FTUAL) molar ratio compared to the native control spectrum. Panel (A) shows unadducted HSA. The inset in panel (A) is the spectrum of native HSA between m/z values of , showing m/z signals representing the mass of the native HSA ( ± 0.46 Da) and the cysteinylated adduct ( ± 0.87 Da). Panel (B) depicts the stoichiometry of 8:2 FTUAL adducts to HSA. The arrows in the inset of panel (B) indicate adducts formed from reaction of 8:2 FTUAL with HSA, with masses of ± 2.12 Da and ± 0.76 Da compared to native and cysteinylated HSA, respectively. 127 Figure 4.4 TOF-CIC analysis showing an increase in the total fluoride concentration (ppm) as a result of combusting the protein adduct samples at varying HSA:8:2 FTUAL molar ratios (1:0.75 1:5). Error bars are shown for n=3 samples. 128 Figure 5.1 Figure 5.1. Binding of 6:2, 8:2, and 10:2 FTUAL at concentrations of 1, 5, 10, and 15 µm to the microsome and plasma protein fractions (sample size, n =3). All values have been corrected for protein recovery. 145 Figure 5.2 Figure 5.3 ESI-ToF mass spectra of the native control spectrum and the BSA-8:2 FTUAL adducts formed after the addition of100 µm 8:2 FTUAL. Panel (A) shows unadducted BSA. Panel (B) depicts the stoichiometry of 8:2 FTUAL adducts to BSA. The arrows in panel (B) indicate observed adducts formed from reaction of 8:2 FTUAL with BSA, with masses of ±3.8 Da, ± 7.9 Da, and ± 1.0 Da compared to the natural adducts in native BSA, respectively. The % fluoride in the three fractions (XAD cartridge, supernatant, and protein pellet) taken from the 8:2 FTOH biotransformation experiments. The volatile 8:2 FTOH in the headspace was trapped by an XAD cartridge, the unreacted 8:2 FTOH, biotransformation intermediates and PFCAs were detected in the supernatant, and the covalently bound intermediates (i.e. 8:2 FTUAL) were extracted in the protein pellet. NADPH was added to initiate the biotransformation of 8:2 FTOH ((+) NADPH, n = 3). T 0 samples (T 0 (+) NADPH control) and samples without NADPH ((-) NADPH control) were used as experimental controls (n = 2) xiv

15 Figure 6.1 Mean liver, plasma, and liver concentrations (ng/g w/w) of 7:3 FTCA, 8:2 FTCA, 8:2 FTUCA, 7:3 FTUCA, PFOA, PFNA, and PFHpA produced from exposure to 8:2 FTOH. Error bars represent standard error for n = 3 sample replicates. Figure 6.2 Mean liver, plasma, and liver concentrations (ng/g w/w) of 5:3 FTCA, 6:2 FTCA, 6:2 FTUCA, 6:3 FTUCA, PFHxA, PFHpA, and PFPeA produced from exposure to 6:2 dipap. Error bars represent standard error for n = 3 sample replicates. Figure 6.3 Normalized concentrations (%) of protein binding of corresponding to 6:2 dipap and 8:2 FTOH exposure in liver, plasma, kidney. Concentration values (ng/g) were normalized to the highest binding concentration observed in all tissues collected over all time points. Error bars represent standard error for n =3 sample replicates Figure 7.1 Figure 7.2 Figure 7.3 Figure 7.4 General biotransformation pathway of fluorotelomer-based compounds (i.e. x:2 dipap). In this work, toxicity of FTALs (x = 6, 8), FTUALs (x = 6, 8, 10), FTCAs (x = 4, 6, 8, 10), FTUCAs (x = 4, 6, 8, 10), and PFCAs (x = 3 9) was examined. Cellular dose-response curves for FTUALs, FTALs, FTUCAs, FTCAs, and PFCAs. Error bars represent standard error for n = 3 sample replicates. 50% Effect Concentrations (EC 50 ) calculated from the dose-response curves for PFCAs, FTUCAs, FTCAs, FTUALs, and FTALs. Error bars represent standard error for n = 3 sample replicates. Relative EC 50 concentrations of PFCAs with their corresponding acid (FTUCA and FTCA) and aldehyde (FTUAL and FTAL) precursors. Error bars represent standard error for n = 3 sample replicates xv

16 LIST OF APPENDICES Appendix A Supporting Information for Chapter Two 218 Appendix B Supporting Information for Chapter Three 225 Appendix C Supporting Information for Chapter Four 234 Appendix D Supporting Information for Chapter Five 253 Appendix E Supporting Information for Chapter Six 273 Appendix F Supporting Information for Chapter Seven 287 xvi

17 PREFACE This thesis is comprised of a series of manuscripts that have been published or are in preparation for submission to be published in peer-reviewed scientific journals. Consequently, repetition of introductory and experimental details was inevitable. All manuscripts were written by Amelia Rand with critical comments provided by Scott Mabury. The contributions of coauthors are detailed below. Chapter One Overview of Perfluoroalkyl and Polyfluoroalkyl Substances: Production, Exposure, and Fate Contributions Amelia Rand prepared this chapter with editorial comments by Scott Mabury Chapter Two - Perfluorinated Carboxylic Acids in Directly Fluorinated High Density Polyethylene Material Published in Environ. Sci. Technol. 2011, 45, Contributions Amelia Rand conceived and performed all method development, LC-MS/MS analysis, and data interpretation. Amelia Rand prepared this manuscript under the guidance of Scott Mabury. Chapter Three Assessing the Structure-Activity Relationships of Fluorotelomer Unsaturated Acids and Aldehydes with Glutathione Published in Cell Biol. Toxicol. 2012, 28, Contributions The FTUAL and FTUCA sysnthesis, dose-response method, analysis, and data interpretation were performed by Amelia Rand. All research and manuscript preparation was conducted under the guidance of Scott Mabury. Chapter Four In Vitro Interactions of Biological Nucleophiles with Fluorotelomer Unsaturated Acids and Aldehydes: Fate and Consequences xvii

18 Published in Environ. Sci. Technol. 2012, 46, Contributions Amelia Rand conceived and performed all kinetic NMR and ToF-MS experiments, method development, analysis, and data interpretation. The research and manuscript preparation was performed under the guidance of Scott Mabury. Chapter Five Covalent Binding of Fluorotelomer Unsaturated Aldehydes (FTUALs) and Carboxylic Acids (FTUCAs) to Proteins Published in Environ. Sci. Technol. 2013, 47, Contributions Amelia Rand conceived and performed all protein binding and 8:2 FTOH biotransformation experiments, 8:2 FTUAL LC-MS/MS method development, LC-MS/MS and GC-MS analysis, and data interpretation. The research and manuscript preparation was conducted under the guidance of Scott Mabury. Chapter Six Protein Binding Associated with Exposure to Fluorotelomer Alcohols (FTOHs) and Polyfluoroalkyl Phosphate Esters (PAPs) in Rats In preparation for submission to Environmental Science and Technology Contributions Amelia Rand conceived and performed all biotransformation experiments, extraction method development, LC-MS/MS analysis, and data interpretation. Research and manuscript preparation was done under the guidance of Scott Mabury. Chapter Seven Cellular Toxicity Associated with Exposure to Perfluorinated Carboxylates (PFCAs) and their Metabolic Precursors Author List Amelia A. Rand, John P. Rooney, Craig M. Butt, Joel N. Meyer, and Scott A. Mabury In preparation for submission to Chemico-Biological Interactions Contributions Synthesis of the FTUALs, FTALs, and FTUCAs was done by Amelia Rand. All cellular and DNA toxicity studies were done at Duke University, in Joel Meyer s research lab. This collaboration was initiated by Craig Butt. Amelia Rand performed all experiments with significant assistance by John Rooney. Amelia Rand performed the analysis, with input from xviii

19 John Rooney and Joel Meyer. Results were interpreted by Amelia Rand with minor contributions from Craig Butt. Amelia Rand prepared this manuscript under the guidance of Scott Mabury. Chapter Eight Summary, Conclusions, and Future Work Contributions Amelia Rand prepared this chapter with editorial comments provided by Scott Mabury. xix

20 Other Publications during PhD: Lee, H.L.; Rand, A.R.; D eon, J. High Performance Liquid Chromatography-Tandem Mass Spectrometry (HPLC-MS/MS) Analysis of Food Packaging Material as a Potential Source of Human Exposure to Fluorochemicals: An Undergraduate Experiment. 2013, to be submitted to J. Chem. Ed.

21 1 CHAPTER ONE Overview of Perfluoroalkyl and Polyfluoroalkyl Substances: Production, Exposure, and Fate Amelia A. Rand and Scott A. Mabury Contributions: Amelia Rand prepared this chapter with editorial comments by Scott Mabury

22 1.1 Overview 2 Perfluoroalkyl and polyfluoroalkyl substances (PFAS) are compounds that are defined by two structural components: a fluorinated carbon tail (F(CF 2 ) x ) and a polar headgroup (R). Their unique chemical structures renders them both hydrophobic and oleophobic (1). As such, they are used for industrial and commercial applications due to their simultaneous oil- and water-repellent properties. These properties have been heavily employed by industry for the past several decades. PFAS have been used in non-stick, grease-proofing, and surface treatment applications. High molecular weight (MW) polymers and surfactants are two classes of PFAS, both of which dominate the commercial fluorochemical production (2, 3). Additionally, a class of low MW PFAS exist: the perfluoroalkyl carboxylates (PFCAs). PFCAs have received much attention focused on elucidating their environmental fate. Depending on the fluorocarbon chain length, PFCAs are persistent and have been detected in environmental and biological matrices throughout the world. The sources of exposure to PFCAs are complex. Studies have shown that some high MW PFAS, as well as the compounds used industrially to produce high MW PFAS can transform to produce PFCAs (4 13). This pathway, thought to be a significant source of exposure to PFCAs, also is responsible for producing several intermediate compounds with unknown reactivity. The potential for reactivity to biological nucleophiles highlights a relatively unrecognized pathway in terms of the consequences of PFAS exposure. The goal of this thesis is to investigate the exposure to PFCAs. It is organized into the following two categories: direct exposure to PFCAs and indirect exposure to PFCAs, and the relative importance of each. Table 1.1 lists the names, structures, and abbreviations of various PFASs that are of interest to this work.

23 Table 1.1 List of acronyms, common names, and chemical structures relevant to this thesis. 3 Acronym Name Structure PFCAs perfluorinated carboxylates CF 3 (CF 2 ) x C(O)O - TFA trifluoroacetate CF 3 COO - PFPrA perfluoropropanoate CF 3 CF 2 COO - PFBA perfluorobutanoate CF 3 (CF 2 ) 2 COO - PFPeA perfluoropropanoate CF 3 (CF 2 ) 3 COO - PFHxA perfluorohexanoate CF 3 (CF 2 ) 4 COO - PFHpA perfluoroheptanoate CF 3 (CF 2 ) 5 COO - PFOA perfluorooctanoate CF 3 (CF 2 ) 6 COO - PFNA perfluorononanoate CF 3 (CF 2 ) 7 COO - PFDA perfluorodecanoate CF 3 (CF 2 ) 8 COO - PFUnA perfluoroundecanoate CF 3 (CF 2 ) 9 COO - PFDoA perfluorododecanoate CF 3 (CF 2 ) 10 COO - Fluorotelomer-based Precursor Compounds x:2 monopap Polyfluoroalkyl phosphate monoester - CF 3 (CF 2 ) x CH 2 CH 2 OP(O)O 2 x:2 dipap Polyfluoroalkyl phosphate diester [CF 3 (CF 2 ) x CH 2 CH 2 O] 2 P(O)O - x:2 FTI Fluorotelomer iodide CF 3 (CF 2 ) x CH 2 CH 2 I x:2 FTAc Fluorotelomer acrylate CF 3 (CF 2 ) x CH 2 CH 2 OC(O)CH=CH 2 x:2 FTOH Fluorotelomer alcohol CF 3 (CF 2 ) x CH 2 CH 2 OH x:2 FTO Fluorotleomer olefin CF 3 (CF 2 ) x CH=CH 2 Fluorotelomer-based Intermediates x:2 FTAL Fluorotelomer aldehyde CF 3 (CF 2 ) x CH 2 C(O)H x:2 FTUAL Fluorotelomer unsaturated aldehyde CF 3 (CF 2 ) x-1 CFCHC(O)H x:2 PFAL Perfluorinated aldehyde CF 3 (CF 2 ) x C(O)H x:2 FTCA Fluorotelomer carboxylic acid CF 3 (CF 2 ) x CH 2 C(O)OH x:2 FTUCA Fluorotelomer unsaturated carboxylic acid x = 3, 5, 7, 9, CF 3 (CF 2 ) x-1 CFCHC(O)OH

24 1.2 Synthesis of Fluorinated Chemicals: Electrochemical Fluorination, Telomerizaton, and Direct Fluorination 4 Two primary methods of synthesizing fluorinated chemicals for commercial use have been electrochemical fluorination and telomerization. A less prominent method, developed in the 1950s, is a process known as direct fluorination. The following is a description of each method, the reagents used, and the mechanisms involved in the formation of the fluorinated chemicals mentioned within the frame of this thesis Electrochemical Fluorination Electrochemical fluorination (ECF) was used to produce fluorinated chemicals since the late 1940s (14), although it is currently no longer being used to produce perfluorooctanesulfonyl fluoride-based materials in North America. The reaction relied on the use of hydrofluoric acid, produced from the reaction of calcium fluoride with sulphuric acid (15, 16): CaF 2 + H 2 SO 4 2HF + CaSO 4 The organic substance to be fluorinated is subsequently dispersed in a solution of anhydrous hydrogen fluoride. An electric current (5 7 ev) is then applied to the solution. Hydrogen is evolved at the nickel or iron cathode, and fluorination of the organic substance occurs at the nickel anode (1): Cathode: 2H + + 2e - H 2 (1) Anode: 2F - 2F + 2e - (2) At the anode, the fluorine evolved is retained by adsorbing onto the anode to form a nickel fluoride layer, NiF 3. The organic molecules adsorb to this layer, followed by their oxidation to carbonium ions, which react with fluoride ions as part of a free-radical mechanism, as shown in Figure 1.1 (15). Over the course of this process, all hydrogen atoms in the organic compound are replaced by fluorine.

25 Figure 1.1. Mechanism for electrochemical fluorination (15). 5 The primary fluorinated organics produced using ECF were PFOA, PFOS, and PFSAms, but based on the contents of this thesis, the production of PFOA will be the focus. To produce PFOA, the organic substance was carboxylic acid chloride or fluoride, which reacted with HF to yield a perfluoroalkanoic acid fluoride (1), as shown by the following reaction: C 7 H 17 COCl + 18HF C 7 F 17 COF + HCl + by-products (3) These acid fluorides are unstable, and are rapidly hydrolyzed to carboxylic acids. Since ECF was a relatively aggressive process, the yield of the primary product was low, ranging from 12-79% (17). As a result, the by-products tended to be isomers formed from internal radical migration in the carbocation or through biradical intermediates (18). The relative isomeric abundance in ECFderived PFOA was determined by Stevenson (19), and are shown in Table 1.2.

26 Table 1.2. Yields of isomeric primary PFOA products from the ECF manufacturing process (19). 6 Isomers of PFOA Relative Abundance (%) n-isomer 78.0 Internal methyl branched 12.5 Isopropyl 9.0 t-butyl branched 0.2 Alpha branched 0.1 Internal geminal dimethyl 0.13 The production of PFOA by ECF for commercial purposed began in 1947 and continued until 2002 with 3M as the principal worldwide manufacturer (20, 21). Until 2002, production volumes of PFOA were as high as lbs/year (22). The predominant use of PFOA was as an aid in fluoropolymer processing (22). In 2002, based on evidence that fluorinated compounds with fluorinated carbon chain lengths C8 were bioaccumulative, 3M voluntarily ceased production of all perfluorooctanesulfonyl fluoride-based products for commercial sale and are currently producing shorter, perfluorobutyl chain length materials (23, 24). Due to this shift in manufacturing policy, detection of isomeric PFOA in environmental and biological samples has declined and is regarded as a historical source of exposure, unlike telomerization, which currently stands as the dominant fluorochemical manufacturing method Telomerization The telomerization process, first developed by the Du Pont Company, has been producing commercial fluorochemicals since the early 1970s and, since the phase out of the ECF process, is currently the predominant process of fluorochemical manufacturing. Telomerization involves the reaction between two substrates known as a telogen and a taxogen (1). The telogen is usually a perfluoroalkyl iodide (i.e. pentafluoroethyl iodide), which reacts with a perfluoroalkene (i.e. tetrafluoroethylene) as the taxogen. To initiate telomerization, taxogen must first react with iodine pentafluoride and iodine to form the telogen (1):

27 5CF 2 = CF 2 + IF 5 + 2I 2 5CF 3 CF 2 I (+ catalyst, SbF 3 ) (4) 7 taxogen telogen The telogen then reacts with the taxogen, and undergoes radical chain propagation, where tetrafluoroethylene units are repetitively added to produce a perfluoroalkyl radical (1): CF 3 CF 2 I + ncf 2 =CF 2 C 2 F 5 (C 2 F 4 ) n (5) telogen taxogen perfluoroalkyl radical The radical is transferred via reaction with trifluoromethyl iodide, and terminated by reacting with another radical species (1): C 2 F 5 (C 2 F 4 ) n + CF 3 I F(CF 2 CF 2 ) x CF 2 CF 2 I + CF 3 (6) perfluoroalkyl trifluoromethyl radical iodide 2CF 3 CF 3 CF 3 (7) From here, the perfluoroalkyl iodide can react with a number of reagents to produce either fluorotelomer compounds used in the manufacture of fluorinated polymers or surfactants, or PFCAs. The general scheme for this is shown in Figure 1.2. Since the 1970s, fluorotelomer production grew to rival production by ECF and subsequently became the predominant manufacturing method, largely filling the market gap during the ECF phase out period. Between 2000 and 2002, tonnes of perfluoroalkyl iodide were produced annually (25). Currently, fluorotelomer alcohols (FTOHs) are used to make fluorotelomer acrylate monomers (FTACs) (Figure 1.2), which are the fundamental building blocks for polymeric products. Polymeric products represent >80% of the fluorotelomer-based materials manufactured and used globally, and are applied to surfaces of fabrics, textiles, and carpets (25). The reaction to produce the acrylate monomer leaves % (wt/wt) of unreacted residual FTOH, which is present in the sales products unless removed (22). The remaining 20% of the manufacturing process is used to manufacture fluorotelomer-based surfactants as grease-proofing agents in food packaging, and as surface levelling and wetting agents in other household products (25). These surfactants vary by head group to produce a variety of different surfactants (3) for various commercial purposes, such as the polyfluoroalkyl

28 phosphate mono-, di-, and tri-esters (mono-, di-, and tripaps, collectively termed PAPs ) (1, 26, 27) used to grease- and water-proof food-contact materials. 8 Figure 1.2. Telomerization scheme to produce fluorotelomer-based compounds for final sales products (1, 22).

29 9 Polymeric materials and surfactants are primarily perfluorooctyl- and perfluorohexylbased, respectively (3, 25), but other chain lengths (C 4 -C 20 ) are present in the final commercial products as impurities due to the aggressive nature of the radical telomerization process. The Du Pont Company has recently transitioned from the production of perfluorooctyl-based materials to the perfluorohexyl chain length and has a new line of perfluorohexyl-based repellents and surfactants (28). Similar to the application of ECF-based PFCAs ( ), both PFOA and PFNA were used as an aid for fluoropolymer processing, to enhance the solubility of fluoromonomers in aqueous polymerization mediums; PFOA was primarily used for the manufacture of PFTE and PFNA was used specifically for producing the polyvinylidene difluoride (PVDF) polymer, however PFOA was produced for its application much more than PFNA. PFOA was found as a residual in the polymer production at levels ranging from < µg/g, and for PFNA, < µg/g (22). It is unclear the extent of PFOA production and use from the oxidation of perfluorooctyl iodide (CF 3 (CF 2 ) 7 I, Figure 1.2) from 2002-present (29). Several fluorotelomer companies claim not to use PFOA in their manufacturing processes (30). Currently, PFNA, produced from carbonylation of CF 3 (CF 2 ) 7 I or oxidation of 8:2 fluorotelomer olefin (8:2 FTO, Figure 1.2) (31), is produced only for its purpose to manufacture fluoropolymers (22) Direct Fluorination Direct fluorination is a surface treatment process use to create fluorinated polyolefins, where all hydrogens on the polymeric backbone are replaced with fluorine. This process utilizes elemental fluorine which dissociates to react with R-H groups to form carbon radicals and R-F groups. Although direct fluorination has been regarded as unpractical due to the highly exothermic nature of reacting elemental fluorine with C-H, industrial practices have developed that control the reaction in order to dissipate the heat generated (1). The mechanism follows a chain-radical propagation, shown below, and creates an overall ΔG 298 K equal to kcalmol -1. Initiation: F 2 F + F (8) F 2 + R-H R + H-F + F (9)

30 Propagation: R-H + F R + H-F (10) 10 R + F 2 R-F + F (11) Termination: R + F R-F (12) R + R R-R (13) Direct fluorination is controlled through the use of an inert gas such as nitrogen or helium to dilute the concentration of fluorine gas (32). In doing so, the extent of carbon-carbon fragmentation is minimized by decreasing the probability of simultaneous fluorine collisions on carbon atoms adjacent to the reaction site. Diluting with oxygen is another means to control the rate of fluorination, since oxygen can terminate the chain-radical process by reacting with the F radicals formed (33, 34). Although oxygen can be beneficial to direct fluorination, it also leads to polymer fragmentation (17, 20) and oxidation of fluorinated surfaces (21). Oxidation of these surfaces leads to the formation of acid fluoride functional groups, which upon hydrolysis could lead to the formation of carboxylic acids (33). Direct fluorination might therefore be a source of PFCA exposure to the environment, which in part may depend on the applications of the direct fluorination process. Currently, two industries in North America dominate the direct fluorination market: FluoroSeal and AirProducts. According to information on the FluoroSeal website, over 300 million containers are fluorinated annually (34). Direct fluorination has several advantages as it is known to greatly improve the polymer barrier properties, adhesion, chemical resistance, and reduces permeability of gases. These combined properties increase product preservation and make directly fluorinated polymers suitable for storage of organic liquids, medical and dental parts, pipes, tubing, food containers, polypropylene toys, polyolefin aquaculture netting, and cosmetic containers (34). 1.3 Sources of Perfluorinated Carboxylates and their Precursors to the Environment PFCAs are widely distributed in the environment and have been found at part-per-billion (µg/l) concentrations in human blood. Despite their presence in environmental and biological samples across the globe, the sources of PFCA contamination are not well understood. Two

31 general sources, direct and indirect, are attributed to most of the ubiquitous contamination observed. The relative importance of each is unclear, and has recently been reviewed by D eon and Mabury (35). In this section, some of the primary direct and indirect sources are described and compared, with emphasis on the biotransformation of fluorotelomer-based compounds to yield PFCAs as the ultimate transformation product Direct Sources When PFCAs are released to the environment as a result of their presence in the production, use, and disposal or fluorochemicals, it is referred to a direct source. The magnitude of direct sources of telomer-based compounds has been described by Prevedouros et al. (22); the following represents a brief summary of the primary direct sources of PFCAs, specifically PFOA and PFNA. As previously mentioned, historical production lasted from and in that period, a total of tonnes of ammonium perfluorooctanooate (APFO) was emitted globally from APFO manufacturers (22). In 2000, before the phase-out of perfluorooctyl-based manufacturing, the largest ECF production plant was emitting ~ 20 tonnes (5-10% of the total annual production) of PFOA in 2000, where 95% was being discharged to water, and 5% to air (36). Although 3M, the principal manufacturer of ECF-based materials, discontinued production in 2002, small manufacturers in Europe and Asia are currently still producing fluorinated chemicals via the ECF process (22). As a result, the global APFO emissions have decreased dramatically, from about 45 tonnes in 1999 to about 15 tonnes in 2000 (22). In the United States, telomerization has dominated the fluoro-chemical market share since 2002, reducing the amount of APFO emissions from tonnes to kilograms. The historical global production of ammonium perfluorononanoate (APFN) was estimated to be between 70 and 200 tonnes from , where emissions of APFN to air and water from production processes were about 10% of the amount produced. While an effort to reduce PFOA emissions has been in place, in 2006 there was no effort to reduce emissions from APFN manufacture (22). Another important direct environmental source of PFCAs is due to their use in the production of fluoropolymers. Between 1951 and 2003, the estimated historical global PFCA emissions (as APFO and APFN) from fluoropolymer manufacture ranged from

32 tonnes with 23%, 65%, and 12% distributed to air, water, and land, respectively (prev ref. 20, 31). This represented the single largest known source of PFCA emissions. Fluoropolymer production occurred world-wide in 33 fluoropolymer manufacturing sites located in North America (8), Japan (7), China (7), Europe (7), Russia (2), and India (1) (22, 37). Emission reductions are now in place, where fluoropolymer manufacturers are capturing and recycling APFO (38). In 2006, APFO and APFN emissions have been reduced by > 90% (> 50 tonnes) and >67%, respectively (22). 12 Trace levels of PFCAs were present as residuals in fluorotelomer-based products as byproducts of the manufacturing process (<1-100 ppm) (39). When fluorotelomer-based products were industrially applied to textiles, trace levels of PFOA or FTOHs present within the product were released to the air (22). Global emissions of PFCAs between 1974 and 2004 to air and water from these fluorotelomer-based products were between 0.3 and 30 tonnes (22). Based on data from a fluorotelomer-based products manufacturing facility, < 1 kg of PFOA was released to air and < 100 kg of PFOA was released to water per year (39). PFCA residuals and their precursors that may be present as byproducts in the final sales products are currently being eliminated in North America and Europe. In Canada, this strategic elimination falls under the direction of Environment Canada and Health Canada in conjunction with various Canadian fluorochemical companies (i.e. Arkema Canada Inc., Asahi Glass Company, Ltd., Ciba Canada Ltd., Clariant Canada Inc., and E.I. du Pont Canada Company). By the end of 2010, it was projected that 95% of these residuals would be removed, with total elimination by the end of 2015 (40). A similar program is present in the United States, established by the Environmental Protection Agency (EPA) with several U.S. fluorochemical companies (i.e. Arkema, Asahi, BASF Corporation, Clariant, Daikin, 3M/Dyneon, Du Pont, and Solvay Solexis). All have committed to reduce PFCAs ( 7 CFs) and the corresponding PFCA precursor residual emissions and product content levels by 95% by 2010 and eliminate them by 2015 (41) Indirect Sources When PFCAs are produced through the degradation or transformation of precursor compounds ( precursors ), it is referred to as an indirect source. The major pathways that produce indirect sources of PFCAs are atmospheric reactions and biological transformations;

33 each pathway will be discussed in the following sections. Both pathways have significance in terms of environmental and human exposure Atmospheric Transformation of Fluorotelomer-Based Compounds Production of PFCAs from the atmospheric transformation of fluorotelomer-based compounds has been extensively reviewed (42). A brief examination of the pathways forming PFCAs will be described here, with focus on atmospheric transformation of the high-production chemicals, including the fluorotelomer alocohls (FTOHs), fluorotelomer iodides (FTIs), fluorotelomer olefins (FTOs), and fluorotelomer acrylates (FTAcs). Due to their high production volumes, volatile fluorotelomer-based compounds such as the FTOHs, FTIs, FTOs, and FTAcs are present in the atmosphere at variable pg/m 3 concentrations that depend on proximity to point sources. These are all synthetic precursors of fluorotelomer-based materials, and have been measured as residual impurities in commercial fluorotelomer polymers (43). All of them have been shown in smog chamber studies to atmospherically transform via reaction with either chlorine atoms or hydroxyl radicals to produce PFCAs, as shown in Figure 1.3. This pathway contributes to the accumulation of PFCAs in the environment. The transformation of FTOHs, FTIs, FTOs, and FTACs begins differently until the formation of a common intermediate, the PFAL (CF 3 (CF 2 ) x C(O)H), after which the mechanism proceeds identically toward PFCA formation. FTOHs, FTIs, and FTACs have a similar mechanism prior to the formation of the PFAL, through the formation of the x+1:2 FTAL (CF 3 (CF 2 ) x CH 2 C(O)H, FTAL), which subsequently yields the PFAL through reaction with atmospheric oxidants or light (42). The fate of PFAL is controlled by three pathways: hydrogen abstraction from a hydroxyl radical, reaction with water to form stable hydrates (not shown) (44), or through photolysis (45 47). Of these three pathways, the primary determinant of PFAL fate is through photolysis, where the product formed from this reaction is the perfluoroalkyl radical (CF 3 (CF 2 ) x ). The PFAL product produced as a result of reaction with atmospheric oxidants is a perfluoroacyl radical (CF 3 (CF 2 ) x C(O) ), which can react with oxygen to yield the perfluoroalkyl radical (CF 3 (CF 2 )x ) after loss of carbon monoxide. Despite these varying intermediate fates through reaction with oxidants, water, or light, the ultimate products of all pathways from the PFAL are PFCAs of all chain-lengths. By forming the perfluoroalkyl radical from the PFAL, the process of unzipping