Fluorotelomer-Based Acrylate Polymers as an Indirect Source of Perfluoroalkyl Carboxylates

Transcription

1 Fluorotelomer-Based Acrylate Polymers as an Indirect Source of Perfluoroalkyl Carboxylates by Keegan Rankin A thesis submitted in conformity with the requirements for the degree of Doctor of Philosophy Chemistry University of Toronto Copyright by Keegan Rankin 2015

2 Fluorotelomer-Based Acrylate Polymers as an Indirect Source of Perfluoroalkyl Carboxylates Keegan Rankin Doctor of Philosophy Department of Chemistry University of Toronto 2015 Abstract Fluorotelomer-based acrylate polymers (FTACPs) are a class of side-chain fluorinated polymers that have been widely used as surface protectants in textile, upholstery and paper industries. Despite FTACPs constituting the largest fraction of commercial fluorotelomer products, little is known about their environmental fate. This dissertation investigates the development and application of matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry to directly analyze the environmental fate of model FTACPs, which was hypothesized to be a significant indirect source of perfluoroalkyl carboxylates (PFCAs). MALDI-TOF methods were developed using a homologous series of FTACPs with fluorotelomer and hydrocarbon acrylates. The relative degree of FTACPs fluorination was demonstrated to influence the sample preparation protocol for MALDI-TOF analysis. Application of the MALDI-TOF method was used to directly determine the biodegradation of the model FTACP, poly(8:2 FTAC). Alterations to the characteristic repeat pattern of poly(8:2 FTAC) suggested biodegradation, which was consistent with the indirect analysis of PFCAs by liquid chromatography tandem mass spectrometry (LC-MS/MS). The biodegradation half-life of ii

3 poly(8:2 FTAC) ranged from 8 to 111 years. Further MALDI-TOF method development demonstrated that FTACPs could be directly quantified using the matrix signal as an internal standard. Direct MALDI-TOF quantification measured the model FTACP, poly(8:2 FTAC-co- HDA), to within 25% of the theoretical concentration in aqueous media. Off gassing of residual fluorotelomer alcohols (FTOHs), and as the first degradation product of FTACPs supports atmospheric long-range transport (LRT) of volatile precursors as a principal mode for the global distribution of PFCAs. A collection of surface soils obtained from areas with little human impact showed PFCAs, as well as perfluoroalkanesulfonates (PFSAs), were distributed consistent with the atmospheric oxidation of volatile precursors. The principle goal of this dissertation was to directly analyze FTACP degradation. The qualitative MALDI-TOF results strongly suggest that FTACPs will degrade under environmental conditions, and likely represent a significant indirect source of PFCAs. iii

4 Acknowledgments The completion of my Ph.D. would not have been possible without the help and support of many individuals that I have been fortunate to meet along this journey. I am deeply grateful for having the opportunity to study in a department with so many talented and intelligent individuals. I would first like to thank my supervisor Scott Mabury for his mentorship. I gained a deeper appreciation and understanding of analytical and environmental chemistry. You pushed me beyond my comfort level, and I learned a tremendous amount about myself in the process. Thank you Scott for all your advice and wisdom. Thank you to my advisory committee Rebecca Jockusch and Derek Muir for offering your time and advice, and to Christopher Higgins and Eric Reiner for serving on my examination committee. I am very grateful to have had the opportunity to work on several projects with John Washington. I learned a great deal from our work together, and I am fortunate that you were so willing to discuss any challenge I encountered along the way. To all the past and present Mabury members, I couldn t have asked for a better group of talented and intelligent individuals to work with at the University of Toronto. I would particularly like to thank Amy Rand, Anne Myers, Derek Jackson, Holly Lee, Leo Yeung, Lisa D Agostino and Rob Di Lorenzo for the numerous discussions, words of encouragements and helpful advice. I will miss our daily coffee runs, group outings and conference adventures. Thank you to Lennart Trouborst and Shira Joudan for the encouragement and support during my last few months, and making our time in Vancouver a memorable one. I would also like to thank iv

5 Amila DeSilva, Angela Hong, Barbara Weiner, Craig Butt, Cora Young, Jessica D eon and Shona Robinson. Although our time in the group didn t overlap for very long, you were always willing to offer support and advice when I needed it. The passion, enthusiasm and friendship exhibited in the Mabury group will be missed. Thank you to all my friends and family that have supported me during my Ph.D. This would not have been possible without you. To my parents Stephanie and Bob, I cannot express my gratitude enough for everything you have done for me. Since I was a child, you taught and demonstrated to me the value in never giving up and overcoming adversity. Lastly, I would like to thank my wife Katie for your love and companionship. I am truly blessed to have married someone that encourages and supports me to pursue new and exciting challenges. You are my inspiration. v

6 Table of Contents CHAPTER ONE Introduction to FTACPs: The Largest Commercial Products of PFASs 1.1 Introduction to Perfluoroalkyl and Polyfluoroalkyl Substances Electrochemical Fluorination Telomerization Direct versus Indirect Sources Transport and Fate of PFCAs and PFSAs Fluorinated Polymers: Classification, Application and Degradation Fluoropolymers Perfluoropolyethers (PFPEs) Side-Chain Fluorinated Polymers Fluorinated Oxetane Polymers Fluorinated Urethane Polymers Fluorinated Acrylate Polymers Measuring the Degradation of FTACPs Indirect Analysis Direct Analysis MALDI Mass Spectrometry Sample Preparation for MALDI Principles of MALDI Principles of TOF Mass Spectrometry Goals and Hypotheses References 36 vi

7 CHAPTER TWO Influence of Fluorination on the Characterization of Fluorotelomer- Based Acrylate Polymers by Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry 2.1 Abstract Introduction Experimental Chemicals Synthesis of Fluorotelomer-based Acrylate Polymers (FTACPs) Synthesis of Decafluoroazobenzene (DFAB) Synthesis of 4,4-dihydroxyoctafluoroazobenzene (dihyofab) Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF) MALDI-TOF Sample Preparation Scanning Electron Microscopy (SEM) Results and Discussion FTACP Design and Solubility Influence of Fluorination on a Conventional Sample Preparation Towards a Fluorinated Sample Preparation Characterization of FTACPs Conclusions Acknowledgements References 76 vii

8 CHAPTER THREE Investigating the Biodegradability of a Fluorotelomer-Based Acrylate Polymer in a Soil-Plant Microcosm by Indirect and Direct Analysis 3.1 Abstract Introduction Experimental Chemicals Microcosm Materials FTACP Polymerization Residual Removal FTACP Characterization Biodegradation Experimental Design Extraction and Analysis Quality Assurance (QA) Results and Discussion FTACP Characterization Indirect Analysis of FTACP Biodegradation Direct Analysis of FTACP Biodegradation Indirect versus Direct Analysis Estimating a FTACP Biodegradation Half-Life Environmental Implications Acknowledgements References 104 viii

9 CHAPTER FOUR Matrix Normalized MALDI-TOF Quantification of a Fluorotelomer- Based Acrylate Polymer (FTACP) 4.1 Abstract Introduction Experimental Materials Sample Preparation MALDI-TOF Instrumentation Scanning Electron Microscopy (SEM) Extraction Method Results and Discussion Graphite Support Preparation Development of P N Method Inter- and Intra-Day Variability Testing the P N Method Environmental Implications Acknowledgements References 132 CHAPTER FIVE A Global Survey of Perfluoroalkyl Carboxylates (PFCAs) and Perfluoroalkane Sulfonates (PFSAs) in Surface Soils: Distribution Patterns and Mode of Occurrence 5.1 Abstract Introduction 140 ix

10 5.3 Materials and Methods Chemicals Sample Collection Extraction Method Instrumental Analysis and Quantification Quality Assurance and Quality Control Soil Characterization Statistical Analysis Results and Discussion Quality Metrics PFAS Concentrations Global PFCA and PFSA Distribution Principle Component Analysis (PCA) Inferred Mode of Occurrence Environmental Implications Acknowledgements References 160 CHAPTER SIX Summary, Conclusions and Future Directions 6.1 Summary and Conclusions Future Directions References 171 x

11 List of Tables Table 1.1 Relative isomeric composition of PFOSF derived materials. 4 Table 1.2 Critical surface tensions of hydrocarbon and fluorocarbon constituents. 19 Table 1.3 Possible ionization mechanisms in MALDI mass spectrometry. 33 Table 2.1 Table 3.1 Table 4.1 Table 4.2 Table 4.3 Table 4.4 Table 5.1 Relative degree of FTACP fluorination based on a calculated chain length ratio and their corresponding solubility in non-fluorinated and fluorinated solvents. Summed products for all FTACP potting conditions from 0 to 5.5 month along with the calculated 8:2 FTOH equivalent and estimated residual 8:2 FTOH and 8:2 FTAC levels. Normalized polymer response (P N ) for poly(8:2 FTAC-co-HDA) standards using dithranol concentrations of 20, 10 and 5 mg ml -1. A mixing ratio of 5:10:1 was used for each sample with NaTFA prepared at a concentration of 10 mg ml -1. Summary of external calibration curves obtained at various dithranol and NaTFA concentration. Summary of external calibration curves obtained over consecutive weeks (Inter-Day), and within a single day (Intra-Day). Method test for MALDI-TOF external calibration curves to quantify poly(8:2 FTAC-co-HDA) recovered from blank tube and aqueous media. Continental summary PFAS concentration ranges in pg/g dry weight with the continental geometric mean in parentheses xi

12 List of Figures Figure 1.1 ECF-based products derived from perfluoroalkane sulfonyl fluorides (PASF). 5 Figure 1.2 Fluorotelomer-based products derived from fluorotelomer iodide (FTI). 7 Figure 1.3 Figure 1.4 Figure 1.5 Figure 1.6 Outline of PFCA and PFSA sources to the environment emitted either directly or indirectly via the transformation of PFAS precursors. Atmospheric transformation for the production of PFCAs from FTOHs and FTACs. Fundamental structures of fluoropolymers, perfluoroethers and sidechain fluorinated polymers. (A) oxetane-based with n = 1, 2 or 4; (B) urethane-based with n = 4-16 and x = -CH 2 CH 2 - or -CH 2 CH 2 N(R)SO 2 - where R = -C m H 2m+1 (m = 0, 1, 2 or 4); (C) acrylate-based with n = 4-16 and x = -CH 2 CH 2 - or -CH 2 CH 2 N(R)SO 2 - where R = -C m H 2m+1 (m = 0, 1, 2 or 4). Diagram of contact angle for the wetting (<90 o ) and beading (>90 o ) of a surface Figure 1.7 Generalized structure of fluorinated acrylate polymers where m = 3, 5 15 and n = Figure 1.8 Degradation pathway of fluorinated acrylate polymers. 26 Figure 1.9 Chemical structures of several common MALDI matrices. 30 Figure 1.10 Diagram of the principle of MALDI-TOF. 33 Figure 2.1 Synthesized FTACPs having m = 5 or 7 and n = 3, 7 or Figure 2.2 MALDI-ToF mass spectra of (A) poly(8:2 FTAC-co-HDA) and (B) poly(8:2 FTAC-co-BA) using Dith as the matrix and NaTFA as the cationization agents prepared in THF with a mixing ratio of 10:5:1. 65 Figure 2.3 SEM images of (A and B) poly(8:2 FTAC-co-HDA) and (C and D) poly(8:2 FTAC-co-BA) using Dith as the matrix and NaTFA as the cationization agents. All samples were prepared in THF with a mixing ratio of 10:5:1 (A and C) and 10:1:1 (B and D). The images illustrate the differences between the FTACPs and mixing ratio upon solvent evaporation. The dark center in panel C represents the increased aggregation of the partially dissolved poly(8:2 FTAC-co-BA). 66 xii

13 Figure 2.4 Figure 2.5 Chemical structures of matrices of interest in this study: (A) dithranol (Dith), (B) 2-[(2E)-3-(4-tert-butylphenyl)-2-methylprop-2- enylidene]malononitrile (DCTB), (C) pentafluorobenzoic acid (PFBzA), (D) pentafluorocinnamic acid (PFCnA), (E) decafluoroazobenzene (DFAB) and (F) 4,4-dihydroxyoctafluoroazobenzene (dihyofab). MALDI-TOF mass spectra of poly(8:2 FTAC-co-BA) prepared in (A) TFT using DFAB as the matrix, (B) HCFC-225 using DFAB as the matrix and (C) TFT using DCTB as the matrix. All samples used NaTFA as the cationization agent and a mixing ratio of 10:5: Figure 2.6 SEM images of poly(8:2 FTAC-co-BA) prepared in (A) 95:5 TFT:MeOH using DFAB as the matrix and (B) 95:5 HCFC-225:MeOH using DFAB as the matrix. Both samples used NaTFA as the cationization agent and a mixing ratio of 10:5:1. 71 Figure 2.7 Figure 2.8 Characterization of poly(8:2 FTAC-co-HDA) based on MALDI-TOF results obtained using Dith and NaTFA prepared in THF with a mixing ratio of 10:5:1 Characterization of poly(8:2 FTAC-co-BA) based on MALDI-TOF results obtained using DCTB and NaTFA prepared in TFT with a mixing ratio of 10:5: Figure 2.9 Proposed chemical structures of the identified FTACP repeat patterns. 74 Figure 3.1 Proposed structure of commercial FTACPs where m = 5-13 and n = 1-17 (A) and structure of the unique FTACP used in this investigation containing hydrogen and hexadecyl thiol end groups (B). 93 Figure 3.2 Figure 3.3 Figure 3.4 Figure 4.1 Amount of FTACP degradation products observed in microcosm soil (A) and plant (B) for the FTACP/Plant condition. Inlaid pie charts showing the relative distribution of PFHxA and PFOA in soil and plant at 5.5 month. MALDI-TOF characterization of model FTACP in soil extracts for (A) FTACP/Soil, (B) FTACP/Plant and (C) FTACP/Plant/Biosolids conditions. Please note that these results are strictly qualitative. Relative peak intensity of model FTACP in soil extracts for (A) FTACP/Soil, (B) FTACP/Plant and (C) FTACP/Plant/Biosolids conditions. Please note that these results are strictly qualitative. Structure of poly(8:2 FTAC-co-HDA) characterized from a previous study xiii

14 Figure 4.2 MALDI-TOF mass spectra of (A) dithranol and (B) poly(8:2 FTAC-co- HDA). Inset panels show the reference matrix signal (659.2 m/z) used to normalize the poly(8:2 FTAC-co-HDA) signals. 120 Figure 4.3 Calibration curves obtained using (A) dithranol concentration of 20, 10 and 5 mg ml -1 and (B) NaTFA concentration of 10, 5 and 1 mg ml -1. For dithranol experiments (A), NaTFA was prepared at 10 mg ml -1. For NaTFA experiments (B), dithranol was prepared at 20 mg ml Figure 4.4 Figure 5.1 Figure 5.2 Figure 5.3 Figure 5.4 Figure 5.5 Calibration curves obtained (A) replicated over a three-week period and (B) replicated three times within a single day. For all experiments, dithranol and NaTFA were prepared at 20 and 10 mg ml -1, respectively. Overview of global PFCA and PFSA concentrations in North American (NA), European (EU), Asian (AS), African (AF), Australian (AU), South American (SA) and Antarctic (AN) surface soils. ΣPFCAs (A) and ΣPFSAs (B) plotted against latitude. Note that higher concentrations of both PFCAs and PFSAs >20 o in the northern hemisphere. Approximate North American sampling locations (A), and the longitudinal distribution of total PFCAs (B) and PFSAs (C) in North American (NA) surface soils. Solid black line represents the middle longitude of NA, while dashed red and blue lines represent Los Angeles, CA and New York City, NY. Principal component score plot of the global PFCA and PFSA surface soil concentrations. The inset shows the numerical ordering of the four quadrants. Ratio of PFOA to PFNA versus log scale of ΣPFCA concentrations for all sampling locations. Dashed lines represent gas phase oxidation of PFAS precursors between 1/1 and 5/1, and the lower stoichiometric bound for direct emission of PFCAs (Direct 8/1). Note that locations AN01, AU03 and EU09 were excluded because PFNA was either <LOQ or xiv

15 List of Appendices Appendix A Supporting information for Chapter Two 175 Appendix B Supporting information for Chapter Three 179 Appendix C Supporting information for Chapter Four 210 Appendix D Supporting information for Chapter Five 224 xv

16 Preface This thesis is organized as a series of manuscripts that have been published or are in preparation for submission to be published in peer-reviewed scientific journals. As such, repetition of introductory materials and methodology was inevitable. All manuscripts were written by Keegan Rankin with critical comments provided by Scott Mabury and all other co-authors. Contributors of all co-authors are provided in detail below. Chapter One Introduction to FTACPs: The Largest Commercial Products of PFASs Author list Keegan Rankin Contributions Prepared by Keegan Rankin with editorial comments provided by Scott Mabury. Chapter Two Influence of Fluorination on the Characterization of Fluorotelomer-Based Acrylate Polymers by Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry Published in Anal. Chimica. Acta 2014, 808, Author list Keegan Rankin and Scott A. Mabury Contributions Prepared by Keegan Rankin with editorial comments provided by Scott Mabury. Keegan Rankin performed all experimental work related to this project. Chapter Three Investigating the Biodegradability of a Fluorotelomer-Based Acrylate Polymer in a Soil-Plant Microcosm by Indirect and Direct Analysis. Published in Environ. Sci. Technol. 2014, 48, Author list Keegan Rankin, Holly Lee, Pablo J. Tseng and Scott A. Mabury Contributions Prepared by Keegan Rankin with editorial comments provided by Holly Lee, xvi

17 Pablo Tseng and Scott Mabury. Keegan Rankin developed and performed all extractions and MALDI-TOF analyses related to this project. Pablo Tseng synthesized the FTACP and designed the experiment. Holly Lee performed extraction and LC-MS/MS analyses related to this project. Chapter Four Matrix Normalized MALDI-TOF Quantification of a Fluorotelomer-Based Acrylate Polymer (FTACP) Submitted to Environ. Sci. Technol. (Manuscript ID: es v) Author list Keegan Rankin and Scott A. Mabury Contributions Prepared by Keegan Rankin with editorial comments provided by Scott Mabury. Keegan Rankin performed all experimental work related to this project. Chapter Five A Global Survey of Perfluoroalkyl Carboxylates (PFCAs) and Perfluoroalkane Sulfonates (PFSAs) in Surface Soils: Distribution Patterns and Mode of Occurrence To be submitted to Environ. Sci. Technol. Author list Keegan Rankin, John W. Washington, Thomas Jenkins and Scott A. Mabury Contributions Prepared by Keegan Rankin with editorial comments provided by John Washington and Scott Mabury. Keegan Rankin performed all extractions at the Environmental Protection Agency s National Exposure Research Laboratory (Athens, GA). Keegan Rankin and John Washington performed LC-MS/MS analyses at the University of Toronto and National Exposure Research Laboratory (Athens, GA), respectively. Chapter Six Summary, Conclusions and Future Work Author list Keegan Rankin Contributions Prepared by Keegan Rankin with editorial comments provided by Scott Mabury. xvii

18 Other Publications During Ph.D.: Washington, J.W.; Naile, E,J.; Rankin, K.; Jenkins, T.M. Decades-Scale Degradation of Commercial, Side-Chain, Fluorotelomer-based Polymers in Soils and Water. Environ. Sci. Technol. 2015, 49, Rankin, K.M.; Sproviero, M.; Rankin, K.; Sharma, P.; Wetmore, S.D.; Manderville, R.A. C 8 - heteroaryl-2`-deoxyguanosine adducts as conformational fluorescent probes in the Nar1 recognition sequence. J. Org. Chem. 2012, 77, xviii

19 List of Abbreviations This page provides a list of acronyms mentioned in this dissertation. θ γ lv γ sv γ sl λ ACN AF AFFF AIBA AN ARXPS AS BA CFC CFC-113 CHCA D DCTB DFAB DHB dihyofab Contact Angle Liquid-Vapor Interfacial Tensions Solid-Vapor Interfacial Tensions Solid-Liquid Interfacial Tensions Wavelength Acetonitrile Africa Aqueous Film Forming Foam 2,2-Azobis(2-methylpropionamide) Dihydrochloride Antarctica Angle-Resolved X-ray Photoelectron Spectroscopy Asia Butyl Acrylate Chlorofluorocarbons Trichlorotrifluoroethane α-cyano-4-hydroxycinnamic Acid Ion Path Length 2-[(2E)-3-(4-tert-Butylphenyl)-2-methylprop-2-enylidene]malononitrile Decafluoroazobenzene 2,5-Dihydroxybenzoic Acid 4,4-Dihydroxyoctafluoroazobenzene Dith Dithranol xix

20 DMF DMSO DSC ECF ECFACPs ECFURPs EU FASA FASACs FASE FBSA FTAC FTACP FTAL FTCA FTI FTICR FTO FTOH FTP FTS FTSH FTUCA FTURP GC-MS Dimethyl Formamide Dimethyl Sulfoxide Differential Scanning Calorimetry Electrochemical Fluorination Electrochemical Fluorination-Based Acrylate Polymers Electrochemical Fluorination-Based Urethane Polymers Europe N-Alkyl Perfluoroalkane Sulfonamides N-Alkyl Perfluoroalkane Sulfonamidoethyl Acrylate (FASACs) N-Alkyl Perfluoroalkane Sulfonamidoethanol Perfluorobutane Sulfonamide Fluorotelomer Acrylate Fluorotelomer-Based Acrylate Polymer Fluorotelomer Aldehyde Fluorotelomer Carboxylate Fluorotelomer Iodide Fourier Transform Ion Cyclotron Resonance Fluorotelomer Olefin Fluorotelomer Alcohol Fluorotelomer-Based Polymer Fluorotelomer Stearate Fluorotelomer Thiol Fluorotelomer Unsaturated Carboxylate Fluorotelomer-Based Urethane Polymer Gas Chromatography-Mass Spectrometry xx

21 GPC HCFC HCFC-225 HDA HFP LC-MS/MS LOD LOQ LRT m MALDI-TOF MCP M:A:Cat MeFBSEA MeFOSEA M i M n MTBE M w NA NEXAFS NMR OA PAF PAP Gel Permeation Chromatography Hydrochlorofluorocarbon Dichloropentafluoropropanes Hexadecyl Acrylate Hexafluoropropylene Liquid Chromatography Tandem Mass Spectrometry Limit of Detection Limit of Quantitation Long-Range Transport Ion Mass Matrix-assisted Laser Desorption/Ionization Time-of-Flight Micro-Channel Plate Matrix:FTACP:Cationization Agent Mixing Ratio N-methylperfluorobutanesulfonamidoethyl acrylate N-methylperfluorooctanesulfonamidoethyl acrylate Matrix Signal Intensity Number Average Molecular Weight tert-butyl Methyl Ether Weight Average Molecular Weight North America Near-Edge X-ray Absorption Fine Structure Nuclear Magnetic Resonance Octyl Acrylate Perfluoroalkanoyl Fluoride Polyfluoroalkyl Phosphate Ester xxi

22 PBDE PC PCA PCB PCDD/F PDI PFAI PFAL PFAS PFBzA PFCA PFCnA PFDA PFDoDA PFHpA PFHxA PFHxS PFNA PFOA PFOS PFPE PFPAs PFPiA PFPMIE PFPrA Polybrominated Diphenyl Ether Principle Component Principle Component Analysis Polychlorinated Biphenyl Polychlorinated Dibenzo-p-Dioxin and Dibenzofuran Polydispersity Index Perfluoroalkyl Iodide Perfluoroalkyl Aldehyde Perfluoroalkyl and Polyfluoroalkyl Substance Pentafluorobenzoic Acid Perfluoroalkyl Carboxylate trans-pentafluorocinnamic Acid Perfluorodecanoate Perfluorododecanoate Perfluoroheptanoate Perfluorohexanoate Perfluorohexanesulfonate Perfluorononanoate Perfluorooctanoate Perfluorooctanesulfonate Perfluoropolyether Perfluoroalkyl Phosphonates Perfluoroalkyl Phophinates Perfluoropolymethylisopropylether Perfluoropropinoate xxii

23 PFSA PFUnDA PFTeDA PFTrDA P i pk a P N POP PPCO PSAF PTFE PVDF PW R 2 R F /R H RSD SA SEM SFM SPE t t 1/2 TBAS TFA TFE Perfluoroalkyl Sulfonate Perfluoroundecanoate Perfluorotetradecanoate Perfluorotridecanoate Polymer Signal Intensity Acid Dissociation Constant Normalized Polymer Response Persistent Organic Pollutants Polypropylene Copolymer Perfluoroalkane Sulfonyl Fluoride Polytetrafluoroethylene Polyvinylidene Fluoride Polished Water Coefficient of Determination Ratio of Perfluorinated to Hydrogenated Carbon Relative Standard Deviation South America Scanning Electron Microscopy Scanning Force Microscopy Solid Phase Extraction Ion Flight Time Half-Life Tetrabutyl Ammonium Hydrogen Sulfate Trifluoroacetate Tetrafluoroethylene xxiii

24 TFT THF TOC V VDF WWTP XPS z α,α,α-trifluorotoluene Tetrathydrofuran Total Organic Carbon Voltage Vinylidene Fluoride Wastewater Treatment Plant X-ray Photoelectron Spectroscopy Ion Charge xxiv

25 1 CHAPTER ONE Introduction to FTACPs: The Largest Commercial Products of PFASs Keegan Rankin Contributions Prepared by Keegan Rankin with editorial comments provided by Scott Mabury.

26 2 1.1 Introduction to Perfluoroalkyl and Polyfluoroalkyl Substances Perfluoroalkyl and polyfluoroalkyl substances (PFASs) is a term used to describe fluorinated surfactants and surface protectants that possess a perfluorinated moiety (C n F 2n+1 ). 1 Historically, PFASs have primarily been synthesized by electrochemical fluorination (ECF) and telomerization, with the majority of PFASs being utilized for the production of surfactants and polymers. 2,3 When compared to hydrocarbon analogues, PFASs exhibit higher chemical and thermal stability, 3 making PFASs ideal for a variety of commercial applications. However, these same properties also render many PFASs or their degradation products persistent in the environment. The widespread detection of PFASs in humans 4 and wildlife 5 has not only raised concern about their potential toxicological effects, but has also about how they are released to the environment. With evidence that perfluoroalkyl carboxylates (PFCAs) and perfluoroalkanesulfonates (PFSAs) with 7 perfluorinated carbons accumulate in biota, 6,7 regulatory agencies have aimed to eliminate commercial PFAS materials bearing these longchain perfluorinated moieties. 8,9 Despite the knowledge that long-chain PFCAs and PFSAs are products from the degradation of commercial PFAS materials, very little is known about the environmental fate of fluorinated polymers. The goal of this thesis was to develop the necessary analytical methods to measure fluorinated polymers, specifically fluorotelomer-based acrylate polymers (FTACPs), and to evaluate FTACP degradation under different environmental conditions.

27 Electrochemical Fluorination Developed by Simons in the 1930s, ECF replaces the C-H bonds of a substrate with C-F, by applying a direct current (5-7 ev) to an anhydrous solution of hydrogen fluoride. The 3M Company began utilizing ECF in 1949 to manufacture a suite of fluorinated surfactants and polymers that were largely based on perfluorooctyl chemistry, 2 with 3M regarded as the largest manufacturer of PFASs from 1949 to 2002 until the company voluntarily phased out all perfluorooctyl products because of their bioaccumulation potential. 13 The company has since shifted to manufacturing ECF-based surfactants and polymers using perfluorobutyl chemistry. 14,15 The manufacturing of ECF-based material begins with a feedstock of linear aliphatic sulfonyl fluorides (Eq. 1.1) or acyl fluorides (Eq. 1.2), which in the presence of anhydrous hydrogen fluoride and a direct current, produces perfluoroalkane sulfonyl fluorides (PSAFs; C n F 2n+1 SO 2 F) and perfluoroalkanoyl fluorides (PAFs; C n F 2n+1 COF), respectively. C n H 2n+1 SO 2 F + (2n + 2)HF C n F 2n+1 SO 2 F + by-products (Eq. 1.1) C n H 2n+1 COF + (2n + 2)HF C n F 2n+1 COF + by-products (Eq. 1.2) 3M s manufacturing of fluorinated surfactants and polymers is predominantly derived from PSAFs, which are often reacted with primary amines such as methylamine, to produce N- alkyl perfluoroalkane sulfonamides (FASAs; where m = 1, 2 or 4). Alternatively, PSAFs can simply be subjected to base-catalyzed hydrolysis to produce PFSAs. The monosubstituted N-

28 4 alkyl FASAs can then be reacted with ethylene carbonate to produce N-alkyl perfluoroalkane sulfonamidoethanols (FASEs). Reaction with POCl 3 produces FASEs-based phosphate esters (FASAmPAP), which have been used as water and oil repellent in food packaging. The N-alkyl FASEs can also serve directly as a monomer for ECF-based urethane polymers (ECFURPs) if crosslinked with polyisocyanates. Functionalizing FASE with an acrylic acid to N-alkyl perfluoroalkane sulfonamidoethyl acrylates (FASACs) has served as primary monomers for ECF-based acrylate polymers (ECFACPs) such as ScotchGard. A schematic of several historical PSAF derived products is shown in Figure 1.1. Because ECF is a relatively aggressive process, many isomeric by-products are formed by internal radical migration. 16 For perfluorooctanesulfonate (PFOS), there are 89 proposed isomeric structures produced by ECF manufacturing. 17 However, only 11 isomers have been measured in 3M material, 18 with linear PFOS constituting ~70% of all possible isomers (Table 1.1). Table 1.1: Relative isomeric composition of PFOSF derived materials. 19 Type of Isomer Relative Isomeric Weight (%) Linear 69.1 ± 1.81 Monomethyl 17.2 ± 1.18 Isopropyl 9.90 ± 0.67 Alpha 2.72 ± 1.04 t-butyl 0.24 ± 0.02 Dimethyl 0.13 ± 0.01

29 Figure 1.1: ECF-based products derived from perfluoroalkane sulfonyl fluorides (PASF). 5

30 Telomerization Despite being discovered in the early 1940s by DuPont Company, 20,21 telomerization was apparently not used to manufacture PFASs until the 1970s. With the voluntary phase-out of perfluorooctyl products by 3M in 2002, 14 telomerization has emerged as the predominant manufacturing method of PFASs. DuPont has continued to manufacturer perfluorooctyl products, but is transitioning to products based on perfluorohexyl chemistry in agreement with the 2015 PFOA Stewardship program. 8,22 Telomerization begins with the reaction of tetrafluoroethylene (taxogen) with iodine pentafluoride in the presence of a catalyst, to produce pentafluoroethyl iodide (telogen) (Eq. 1.3). 3 The telogen is then reacted with the taxogen to produce perfluoroalkyl iodide (PFAI) (Eq. 1.4), which is referred to as Telomer A. 3 Unlike ECF, telomerization produces almost exclusively linear isomers. 5CF 2 =CF 2 + IF 5 + 2I 2 5CF 3 CF 2 I (Eq. 1.3) CF 3 CF 2 I + ncf 2 =CF 2 CF 3 CF 2 (CF 2 CF 2 ) n I (Eq. 1.4) As shown in Figure 1.2, there are four principle manufacturing routes from PFAI: 1) oxidation to produce PFCAs with an even number of carbons; 2) a Gringard reaction with CO 2 to produce PFCAs with an odd number of carbons; 3) reaction with phosphorus to produce perfluoroalkyl phosphinates (PFPiAs) and phosphonates (PFPAs); or 4) the addition of ethylene to produce (n:2) fluorotelomer iodide (n:2 FTIs), referred to as Telomer B. The production of

31 Figure 1.2: Fluorotelomer-based products derived from fluorotelomer iodide (FTI). 7

32 8 FTIs is the principle manufacturing route, as it is the main building block for most fluorotelomerbased surfactants and polymers. Reacting FTIs with thiourea produces fluorotelomer thiols (FTSHs), from which a variety of aqueous film forming foams (AFFF) can be derived. The mono-, di- and tri-substituted polyfluoroalkyl phosphates (PAPs) used in food-contact paper packaging and as leveling agents are produced by reacting FTIs with POCl 3. Hydrolysis of FTIs to fluorotelomer alcohols (FTOHs), followed by crosslinking with polyisocyanates, produces fluorotelomer-based urethane polymers (FTURPs). Functionalizing FTOH with acrylic acid produces fluorotelomer acrylates (FTACs), which is the principle monomer in fluorotelomerbased acrylate polymers (FTACPs). FTACPs are the largest fraction of commercial fluorotelomer products, 23,24 and constitute >80% of all fluorotelomer-based raw materials manufactured Direct versus Indirect Sources The ubiquity of PFCAs and PFSAs in the environment has resulted from decades of manufacturing and use of commercial PFAS materials. Despite clear evidence that PFCAs and PFSAs are present globally in humans, 4,25,26 new sources of PFCAs and PFSAs continue to emerge. This section aims to make a clear distinction between direct and indirect sources, while describing several significant sources. Direct source is a term used to describe PFCAs and PFSAs released to the environment as acids, anions or ammonium salts resulting from their manufacturing, use in the manufacturing of other commercial PFAS materials, and as ingredients or impurities in commercial PFAS materials. Indirect source is a term used to describe biotic or abiotic transformation of any PFAS

33 9 to PFCAs or PFSAs; these PFASs are commonly referred to as precursors. Indirect emission results primarily from the transformation of fluorotelomer- and ECF-based commercial materials, but also includes residual reactants that may be present in the commercial material. A simplified overview of the both direct and indirect sources is presented in Figure 1.3. Direct Sources Direct Sources Manufacturing Manufacturing of of PFCA and PFSA PFSA Indirect Sources Use Use in in the the Manufacturing Manufacturing of of Commercial Products Products Ingredient in in Commercial Products Products in Impurity in Commercial Products Products Indirect Sources Fluorotelomer-Based Commercial Fluorotelomer-Based Products Commercial Products ECF-Based Commercial ECF-Based Products Commercial Products Residuals in Commercial Residuals Products in Commercial Products Figure 1.3: Outline of PFCA and PFSA sources to the environment emitted either directly or indirectly via the transformation of PFAS precursors. Prior to the voluntary phase-out of PFASs with 7 perfluorinated carbons, PFCAs and PFSAs were directly used in several commercial applications worldwide. In particular, the eight and nine carbon PFCA congeners, perfluorooctanoate (PFOA) and perfluorononanoate (PFNA), were used as a processing aid in the manufacturing of fluoropolymers such as polytetrafluoroethylene (PTFE) and polyvinylidene fluoride (PVDF). The total amount of PFOA and PFNA released from the fluoropolymer industry was estimated to range from 2610 to 5720 tons between 1951 to 2004, 13 and is believed to represent the largest historical source of PFCAs.

34 10 The fluoropolymer industry has since implemented technology to capture and recycle PFOA and PFNA, reducing direct emissions in 2006 by >90 and 67%, respectively. 27,28 The eight carbon PFSA congener, PFOS, has been used in AFFFs, hydraulic fluids, photolithography and semiconductors. 29,30 For PFSAs, PFOS was the principle PFSA homologue used in the manufacturing of ECF-based products, with direct emission estimates ranging from 280 to 2340 tons between 1957 to Although the major manufacturer, 3M, has since eliminated PFOS from its manufacturing process and product lines, 14 the use of PFOS continues for purposes exempt under Part III of Annex B of the Stockholm Convention. Direct emission of PFOS from ongoing manufacturing was estimated to range from 70 to 680 tons from 2003 to Previous emission estimates suggest that direct sources of both PFCAs and PFSAs are roughly 10 to 100 times greater than indirect sources. 13,31 In their emission estimates, Prevedouros et al. postulated that the transformation of fluorotelomer-based precursors was the largest indirect source, accounting for 6 to 130 tons of PFCAs from 1975 to This estimate was based exclusively on the transformation of FTOHs and fluorotelomer olefins (FTOs), and predicted that commercial fluorotelomer-based surface protectants do not transform to PFCAs. However, since the Prevedouros et al. study, there has been mounting evidence that commercial PFASs are significant indirect sources of PFCAs and PFSAs. For example, PAPs, common surface protectants used in food-contact papers, have been observed to transform to PFCAs in rats, 32,33 wastewater treatment plant (WWTP) mixed liquor, 34 and soils. 35 With the emergence of new indirect sources, estimating the total indirect sources becomes more challenging, but likely far exceeds previous indirect estimates of PFCA and PFSA. 13,31 Of great

35 11 interest is determination of the role that fluorinated polymers such as FTACPs, have on total indirect sources, as they are the largest class of all PFASs Transport and Fate of PFCAs and PFSAs The global distribution of PFCAs and PFSAs has been proposed to result from a combination of oceanic 13,31,36 and atmospheric long-range transport (LRT), with the mode of LRT dependent on the type of emission source. PFCAs and PFSAs have low acid dissociation constants (pk a ), negligible vapor pressures and reasonable water solubility, 40,41 which supports oceanic LRT. Indirect sources such as the volatile precursors FTOHs and FASAs, have atmospheric lifetimes of ~20 days, before being oxidized in the atmosphere to PFCAs, and PFSAs for sulfonamide-based materials. 42,43 A brief description of PFCAs and PFSAs LRT will be presented here, with a particular focus on the atmospheric LRT of FTOHs and FTACs. The distribution of PFCAs emitted directly from fluoropolymer manufacturing facilities has been postulated to be 12, 23 and 65% to land, air and water, respectively. 44 PFCAs and PFSAs emitted to land, presumably meaning their disposal to municipal landfills, would reside indefinitely unless leached into nearby watersheds. Atmospheric concentrations of PFOA have been measured near fluoropolymer manufacturing facilities, 45 and has been suggested as an avenue for the atmospheric LRT of PFCAs. However, the atmospheric lifetime of PFCAs largely depends on wet- and dry-deposition, 46 favoring regionalized atmospheric transport rather than LRT. This is consistent with an elevated concentration of PFCAs in precipitate collected nearer urban centers compared to rural locations. 47 Given their similar chemical structures and properties, an analogous assumption can also be made for PFSAs. PFCAs and PFSAs released

36 12 from fluoropolymer manufacturing facilities through wastewater discharge constitutes the primary avenue of direct emission. 44 The detection of PFCAs and PFSAs in municipal WWTP sludge 48 suggests a fraction of PFCAs and PFSAs are removed from the wastewater during the treatment process, and presents an alternative route of contamination from the application of WWTP sludge to farmland. 49,50 The detection of PFCAs and PFSAs in WWTP effluent, 51,52 suggests most PFCAs and PFSAs emitted in wastewater will enter into the regional watershed, and eventually be transported into the oceans. Once in the oceans, slow oceanic LRT would circulate PFCAs and PFSAs globally, which is consistent with their widespread detection in the Atlantic, Arctic and Pacific oceans Volatile precursors such as FTOHs and FASAs, are known indirect sources of PFCAs, and PFSAs for sulfonamide-based materials, 42,43 and have been measured globally in the atmosphere Understanding how atmospheric LRT of volatile precursors that contribute to the global distribution of PFCAs and PFSAs continues to be active area of research. Of particular interest are fluorotelomer-based materials, which became the largest class of PFASs produced worldwide after 3M s voluntary phase-out in FTOHs and FTACs are especially relevant in the present dissertation because they are known residuals in 61,62 and likely transformation products of surfactants and side-chain fluorinated polymers Because FTOHs and FTACs do not absorb actinic radiation, photolysis is not expected to be major loss pathways, 66,67 nor is wet or dry deposition with estimated atmospheric lifetimes for FTOHs of and 8 years, respectively. 37 Rather, the dominant atmospheric fate of FTOHs and FTACs will be oxidation with hydroxyl radicals (. OH). 37,67 Several smog chamber studies have probed the atmospheric fate of FTOHs 42,68,69 and FTACs, 67 along with FTIs 70 and fluorotelomer olefins (FTOs), 71,72 to demonstrate their reaction with atmospheric oxidants such

37 13 as chlorine atoms or hydroxyl radicals. As shown in Figure 1.4, the atmospheric transformation pathways for FTOH and FTAC are identical following the formation of fluorotelomer aldehyde (FTAL). The reaction of FTAL with oxidants or light produces perfluoroalkyl aldehyde (PFAL), 73 which is the primary intermediate for the atmospheric formation of PFCAs. Oxidation of FTAL to FTCA is also expected, 42 and is consistent with the detection of FTCA in rainwaters. 74 The transformation of PFAL is believed to be dominated by photolysis to a perfluoroalkyl radical (CF 3 (CF 2 ) n ), 75 but oxidation to a perfluoroalkyl acyl peroxy radical (CF 3 (CF 2 ) n C(O)OO ) is also possible. 42 Subsequent reaction of the perfluoroalkyl acyl peroxy radical with HO 2 produces odd PFCAs (CF 3 (CF 2 ) n C(O)OH) from fluorotelomer-based PFASs (ie. 8:2 FTOH PFNA), while reaction with an alkyl peroxy radical (RO 2 ) produces the perfluoroalkyl radical. 42 Reaction of the perfluoroalkyl radical with oxygen produces a perfluoroalkyl peroxy radical (CF 3 (CF 2 ) n OO ). The fate of the perfluoroalkyl peroxy radical depends on the presence of nitrogen oxide (NO x ). Under low NO x conditions, such as in rural areas, reaction of the perfluoroalkyl peroxy radical with an alkyl peroxy radical, followed by elimination and hydrolysis steps, produces even PFCAs (CF 3 (CF 2 ) n-1 C(O)OH) from fluorotelomer-based PFASs (ie. 8:2 FTOH PFOA). 42 In contrast, under high NO x conditions, such as in urban areas, the perfluoroalkyl peroxy radical would undergo a cyclical unzipping loss of carbonyl fluoride (COF 2 ) units, producing perfluoroalkyl peroxy radicals one carbon shorter (CF 3 (CF 2 ) m OO, where m = n-1, n-2 ). 42 The shorter perfluoroalkyl peroxy radicals would then produce a homologues series of PFCAs (ie. 8:2 FTOH perfluoroheptanoate (PFHpA), perfluoropropinoate (PFPrA) trifluoroacetate (TFA)). 42

38 14 FTOH FTAC FT Gly FTAL FTCA PFAL PFCA Unzipping PFCA Figure 1.4: Atmospheric transformation for the production of PFCAs from FTOHs and FTACs.

39 15 Based on atmospheric oxidation to PFCAs, the lifetime of FTOHs and FTACs in the troposphere is expected to be 20 days 37 and ~10 day 67, respectively. The lifetime of FTOHs and FTACs are sufficient to support atmospheric LRT to remote regions from indirect sources. 37,67 Once oxidized to PFCAs, wet- and dry-deposition would be the primary removal pathway. 46 Although the ultimate fate of PFCAs and PFSAs in the environment is not yet fully understood, it has been theorized that oceans are the dominant global sink, 13,31,36 and that freshwaters and sediment 58,79,80 are minor sinks. Prevedouros et al. estimated the distribution of PFOA in sediment, freshwater and ocean water is approximately 3, 3-7 and 90-94%, respectively. 13 However, these estimates are based on the assumption that direct sources are ~10 to 100 times greater than those from indirect sources. The atmospheric LRT of volatile precursors to remote locations is a known indirect source of PFCAs and PFSAs in sediment and freshwater, 58,81 and snow. 82 Detection of PFCAs and PFSAs in these remote environments suggests that the contribution from indirect sources is not fully appreciated in previous fate estimations. This also includes the role soils could contribute as a PFCA and PFSA sink given volatile precursors and products are transported across terrestrial environments. 1.2 Fluorinated Polymers: Classification, Application and Degradation. Since the accidental discovery of PTFE by Roy Plunkett in 1938, 83 fluorinated polymers have quickly evolved to be an integral part of everyday life. In 2010 the revenue from the fluorinated polymer industry was estimated to be approximately $5.8 billion. 84 The benefits of fluorinated polymers in commercial applications results from improved repellency, lubricity, and chemical and thermal stability. 85 Because fluorinated polymers can be prepared from a variety

40 16 of fluorinated and non-fluorinated monomers, the commercial applications of these polymers are diverse, examples of which include coatings for electrical wires, high-performance lubricants and surfactants, and surface protectants. Specific industries tailor the formulation of the fluorinated polymers to optimize the desired performance. Generally, fluorinated polymers are classified as fluoropolymers, perfluoropolyethers (PFPEs) or side-chain fluorinated polymers, based on their chemical structure, as shown in Figure 1.5. A brief discussion of each class of fluorinated polymer is presented in the following sections, with an emphasis on side-chain fluorinated polymers. Fluoropolymers Perfluoropolyethers Side-Chain A B C Figure 1.5: Fundamental structures of fluoropolymers, perfluoroethers and side-chain fluorinated polymers. (A) oxetane-based with n = 1, 2 or 4; (B) urethane-based with n = 4-16 and x = -CH 2 CH 2 - or -CH 2 CH 2 N(R)SO 2 - where R = -C m H 2m+1 (m = 0, 1, 2 or 4); (C) acrylate-based with n = 4-16 and x = -CH 2 CH 2 - or -CH 2 CH 2 N(R)SO 2 - where R = -C m H 2m+1 (m = 0, 1, 2 or 4).

41 Fluoropolymers Fluoropolymers are arguably the most well known class of fluorinated polymers as a result of DuPont s commercialization of Teflon (ie. PTFE). Fluoropolymers have since evolved to include numerous homopolymers or copolymers of per- and polyfluorinated monomers such as tetrafluoroethylene (TFE), vinylidene fluoride (VDF) and hexafluoropropylene (HFP). As shown in Figure 1.5, fluoropolymers possess a carbon backbone to which fluorine atoms are directly attached; a structure which lends to properties of high thermal and chemical stability, low coefficient of friction and dielectric properties. 85 Fluoropolymers are found in a variety of commercial applications, ranging from non-stick cookware to low-friction coatings in the precision tool industry. Although PTFE constitutes the largest fraction of the fluoropolymer market (~70% ), 86 novel copolymers with TFE or VDF are becoming increasingly popular as their properties can be modified to fit specific applications. Prior to the voluntary phase-out of PFOS related products, 87 and the concerted effort by fluorochemical companies to eliminate the production of PFASs having 7 perfluorinated carbon chains, 8,9 fluoropolymer manufacturing was the direct source of PFCAs and PFSAs. 13 Despite thermally destroying ~62% of the fluorosurfactants from curing of the crude fluoropolymer, ~38% (210 to 320 tons) of PFOA and PFNA was directly emitted to the environment from 1951 to ,88 These emissions have since been eliminated in agreement with the 2010/2015 PFOA Stewardship Program; 8 thus fluoropolymers are no longer a significant direct source of long-chain PFCAs and PFSAs. There is also some evidence that the thermal decomposition of fluoropolymers themselves yield PFCAs. 89,90

42 Perfluoropolyethers (PFPEs) Similar to fluoropolymers, PFPEs also have a fluorinated backbone, but differ due to the repeating perfluorinated methyl (-CF 2 -), ethyl (-CF 2 CF 2 -) and isopropyl (-CF 2 (CF 3 )CF 2 -) units that are separated by oxygen atoms (Figure 1.5). In general, PFPEs are copolymers of TFE and HFP that are synthesized by photo-initiated oxidation, which produces randomly distributed - CF 2 -, -CF 2 CF 2 - and -CF 2 (CF 3 )CF 2 - units. 91 Depending on the commercial application, PFPEs can have end-groups such as hydrogen or fluorine, or hydrophilic groups like carboxylates or phosphates. PFPEs, which are based on 3 perfluorinated carbons, have been marketed as potential alternatives to ECF- and fluorotelomer-based polymers based on 7 perfluorinated. 1 When functionalized with hydrogen and fluorine, PFPEs can be used as heat transfer fluids at lower molecular weight (<1000 Da), and as high-performances lubricants at higher molecular weight (>5000 Da). 92 Because PFPEs possess oxygen atoms inserted between the perfluorinated units, they typically exist as liquids with a high stability. The chemical inertness of PFPEs suggests they are attractive alternatives to cholorofluorocarbons (CFCs), but environmental concerns as a greenhouse gas have been raised. While the lack of chlorine atoms in PFPEs means they do not cause destruction to the ozone layer, in contrast to CFCs, the low molecular weight PFPE, perfluoropolymethylisopropylether (PFPMIE) has a long atmospheric lifetime (~800 years) and a global warming potential of 9000 relative to CO Greater attention has been placed on PFPEs functionalized with hydrophilic groups, which are used as grease proofing agents in food packaging. When applied to paper or cardboard, these PFPEs lower the surface energy and coefficient of friction, producing a highly oleophobic and hydrophobic surface. 85 These are attractive alternatives to previous grease

43 19 proofing agents, such as PAPs, which were demonstrated to degrade to long chain PFCAs under environmental conditions Side-Chain Fluorinated Polymers The term side-chain is used to describe a class of fluorinated polymers that possess polyfluorinated appendages covalently bonded to the polymer backbone. 1 Side-chain fluorinated polymers differ from fluoropolymers and PFPEs, as the polymer backbone does not contain any fluorine atoms. The commercial interest in side-chain fluorinated polymers centers on their ability to impart exceptional oil- and water-repellency. It is known that perfluorinated carbons have a reduced critical surface energy compared to hydrocarbons (Table 1.2). The critical surface of side-chain fluorinated polymers is optimized by positioning the fluorinated appendages nearly perpendicular to the air/material interface, thereby, increasing the number of CF 3 units at the surface. In contrast, fluoropolymers and PFPEs lie parallel to the air/material interface and have surface CF 2 units. 85 As a result, the critical surface energy of CF 3 (6 dyns/cm) is 3x lower than CF 2 (18 dyns/cm). 97 Table 1.2: Critical surface tensions of hydrocarbon and fluorocarbon constituents. 97 Surface Exposed Segment Critical Surface Tension (dyns/cm) -CH CH CF CF 3 6

44 20 Conceptually, it is easier to visualize the improved oil- and water-repellency offered by side-chain fluorinated polymers in terms of contact angles. Contact angle (θ) is defined by the mechanical equilibrium of the liquid-vapor (γ lv ), solid-vapor (γ sv ), and solid-liquid (γ sl ) interfacial tensions (Eq. 1.5). As shown in Figure 1.6, on an untreated surface, the liquid wets the surface with is <90 o, whereas on a treated surface, the liquid is repelled and beads with >90 o. 98 For side-chain fluorinated polymers, contact angles for water and oil are typically >100 o, and have even been reported up to 165 o for a block copolymer containing 8:2 FTAC. 103 This has led to three sub classes of side-chain fluorinated polymers; oxetane, urethane and acrylate, all of which have been marketed as highly effective surface protectants in the carpet, textile, upholstery and paper industries. 23 (Eq. 1.5) < 90 o > 90 o Wetting g sl g lv g sv Wetting Beading Figure 1.6: Diagram of contact angle for the wetting (<90 o ) and beading (>90 o ) of a surface.

45 Fluorinated Oxetane Polymers Poly(oxetanes) are a type of polymer that are prepared by the Lewis acid-catalyzed, cationic ring-opening polymerization of oxetane monomers Fluorinated oxetane polymers are prepared in the same manner, but first require the functionalization of the oxetane monomer with a short-chain fluorinated alcohol Following polymerization, the resulting polymer structure possesses a short-chain fluorinated appendage, 4 perfluorinated carbons, covalently bonded to the hydrocarbon backbone through an ether moiety (Figure 1.5). In the early 2000s, Omnova Solutions Inc. recognized the potential of fluorinated oxetane polymers as an alternative to side-chain fluorinated polymers that have 7 perfluorinated carbons, and developed and marketed a line of fluorinated oxetane polymers, PolyFox. 110 It has been demonstrated that fluorinated oxetane polymers have comparable surfactant properties to long-chain fluorosurfactants, such as 3M s Fluorad FC-129, 108,109 and are often used as surface tension lowering additives in fluorinated urethane and acrylate polymers Fluorinated Urethane Polymers For decades, poly(urethanes) were most commonly prepared by the reaction of an isocyanate (-N=C=O) with an alcohol (-OH), which forms a urethane linkage. Fluorinated urethane polymers are prepared through the reaction of a diisocyanate homopolymer with a fluorinated alcohol, such as FASE or FTOH, 111 to produce fluorinated appendages bonded to a hydrocarbon backbone through carbamate ester moieties (Figure 1.5). To optimize oil- and water-repellency, fluorinated urethane polymers are prepared with fluorinated alcohols having 7 perfluorinated carbons. 112 With the increasing evidence that commercial PFASs serve as an

46 22 indirect source of long-chain PFCAs and PFSAs, the stability of fluorinated urethane polymers has come into question. It is known that non-fluorinated polyurethanes are susceptible to biodegradation, likely resulting from the hydrolysis of the carbamate ester bond by esterase enzymes. 113 Given that fluorinated urethane polymers have the same carbamate esters as non-fluorinated polyurethanes, hydrolysis of fluorinated urethane polymers appears likely. However, in a 5 day abiotic hydrolysis study with a FTURP, no degradation products such as FTOHs and PFCAs, were observed. 114 But when a similar FTURP prepared from 8:2 FTOH was incubated for 2 years in aerobic soils, PFOA was observed above levels expected from the transformation of residual 8:2 FTOH, which suggested degradation of the FTURP. 115 The authors reported a biodegradation half-life of 102 years (range of 28 to 241 years), and estimated that FTURPs contributed at least 14 tonnes of PFOA indirect emissions from 1985 to To date, no other studies have studied fluorinated urethane polymers as an indirect source of PFSAs Fluorinated Acrylate Polymers Fluorinated acrylate polymers are the dominant surface protectant derived from both ECF- and fluorotelomer-based materials. Polymerization of fluorinated acrylate monomers, such as FASACs and FTACs, produces fluorinated appendages bonded to a hydrocarbon backbone through ester moieties (Figure 1.5). Additional nonfluorinated monomers such as hydrocarbon acrylates, are copolymerized with the fluorinated acrylate to produce a side-chain fluorinated polymer that is a highly effective surface protectants in the carpet, textile, upholstery and paper industries. 23 Similar to FTURPs, fluorinated acrylates with 7 perfluorinated carbons were

47 23 widely used because of improved surface repellency; 102,116,117 this has also raised concern as to whether fluorinated acrylate polymers are a significant indirect source of long-chain PFCAs and PFSAs. Until the phase-out of the original formulation in 2002, 3M used N- methylperfluorooctanesulfonamidoethyl acrylate (MeFOSEA) as the principle building block for ScotchGard. The company has since shifted to a new formulation based on N- methylperfluorobutanesulfonamidoethyl acrylate (MeFBSEA). 118 Very little is known about the degradation of ECFACPs, with only a single study reporting the degradation of model ECFACPs. 65 The authors reported the transformation of pre-2002 and post-2002 model ECFACPs to FOSA and perfluorobutane sulfonamide (FBSA) in vitro using rat liver microsomes, 65 but did not report a biodegradation half-life. Interestingly, several metabolites observed in previous in vitro studies of similar ECF-based surfactants, 119,120 were not detected by Chu and Letcher, nor was there any loss of the parent compound. This makes it is difficult to determine if the model ECFACPs are an indirect source of PFSAs, but further investigation of ECFACPs biodegradation are warranted. Developed by DuPont late 1980s, 23,121 FTACPs are the largest class of commercial fluorotelomer products, 23,24 and are estimated to constitute >80% of all fluorotelomer-based raw materials produced worldwide. 13 FTACPs are typically prepared by free radical polymerization of fluorotelomer acrylates (ie. 8:2 FTAC), and other non-fluorinated monomers such as hydrocarbon acrylates and vinylidene chloride. 121,122 A generalized chemical structure of FTACPs is shown in Figure 1.7.

48 24 Figure 1.7: Generalized structure of fluorinated acrylate polymers where m = 3, 5 15 and n = The degradation of FTACPs in the environment has been a widely contested subject, given the potential of FTACPs as the largest indirect source of PFCAs. Two potential FTACP degradation pathways from the cleavage of the ester or urethane linkage, or breakage from the carbon-carbon backbone, have been proposed. 63 Cleavage of the ester moiety is the most probable pathway (Pathway A in Figure 1.8), and results in the release of fluorotelomer appendages such as FTOHs. Breaking the FTACP backbone is less likely (Pathway B in Figure 1.8), but has been reported for polyacrylic acid. 123 Pathway B would produce smaller oligomeric species, which could subsequently degrade by Pathway A. A proposed scheme for the degradation of FTACPs with 8:2 fluorotelomer appendages to PFCAs is shown in Figure 1.8. Microbial hydrolysis of ester moieties of fluorotelomer-based materials, such as PAPs and fluorotelomer stearate monoester (FTS), has been previously observed in aerobic environments. 34,35,124 Similar to the transformation of PAPs and FTS, the first product of FTACP degradation via Pathway A is 8:2 FTOH. The transformation of FTOHs to PFCAs has been well documented, and first involves oxidation to 8:2 FTAL, which is then be rapidly transformed to 8:2 FTCA. 133 There are two possible degradation pathways of 8:2 FTCA; 1) - oxidation to PFNA as reported with different mammalian hepatocytes, 127,134 or 2) elimination of

49 25 HF to 8:2 FTUCA. 132 Although the exact mechanism of 8:2 FTUCA transformation is not completely clear, -oxidation to PFOA via the 7:2 sftoh intermediate can occur, and oxidation to PFHpA and PFHxA via the 7:3 FTUCA intermediate occurs, as recently reviewed by Butt et al. 135 The degradation of commercial FTACPs has previously been studied in soils, by measuring the intermediates and terminal PFCAs consistent with 8:2 FTOH biotransformation. 63,64 The authors reported respective biodegradation half-lives ranging and years based on polymer mass. Washington et al. have suggested that the half-life could be closer to years if corrected to the FTACP particle surface area. 64 This discrepancy in biodegradation half-lives has raised a debate as to whether FTACP are an indirect source of PFCAs. A common issue plaguing the validity of both studies is the presence of residual FTOHs and FTACs because they are known indirect sources of PFCAs, and have been reported in commercial FTACPs. 61,62 As described previously, the transformation of FTOHs to PFCAs is known to occur, and would be similar for the transformation of FTACs to PFCAs, which has been reported in vivo with rainbow trout, 136 in vitro with rainbow trout liver and stomach S9 fractions, 137 and in soils. 138 Thus, the detection of target analytes must be above levels attributed to residual FTOHs and FTACs in order to suggest FTACP biodegradation, 64 which is a difficult task if there is a large background signal.

50 26 Pathway A Pathway B FTACP Fragments 8:2 FTOH 8:2 FTAL PFOA 8:2 FTCA PFNA 7:2 sftoh 8:2 FTUCA 7:3 FTCA PFHxA 7:3 FTUCA PFHpA Figure 1.8: Degradation pathway of fluorinated acrylate polymers.

51 Measuring the Degradation of FTACPs Indirect Analysis Knowledge of the environmental fate of FTACPs has largely relied on the indirect measurement of presumed degradation products, such as FTOHs and PFCAs, by gas chromatography mass spectrometry (GC-MS) and liquid chromatography tandem mass spectrometry (LC-MS/MS). 63,64 Although these indirect analyses provide valuable information regarding the degradation of FTACP, there still remains a degree of uncertainty as to whether the products result from degradation of FTACPs or residual PFASs present in the polymeric material, such as FTOHs or FTACs. These residuals have been measured up to 5% in commercial FTACP products, 61,62 and are known to transform to the presumed products of FTACP degadation. 125, ,138,139 Because fluorinated materials have a tendency to co-associate, a property referred to as fluorophilicity, removing residuals from FTACPs is extremely challenging. The most effective way of removing residuals is to heat the FTACP at an elevated temperature to drive off volatile residuals, followed by serial extractions with an organic solvent. 140 However, FTACPs, particularly those used commercially, are often insoluble in non-fluorinated solvents due to their high degree of fluorination, 141 making it difficult to remove any remaining residuals and nonvolatile impurities such as PFCAs. While the amount of residuals and impurities present in FTACPs can be greatly reduced, it is difficult to eliminate them completely. Therefore, investigators are often left to measure FTACP degradation products above a high background signal.

52 Direct Analysis The application of techniques to directly measure FTACPs was previously believed to be impossible. 64 Techniques such as near-edge X-ray absorption fine structure (NEXAFS), 100, X-ray photoelectron spectroscopy (XPS), 100,145,146 angle-resolved XPS (ARXPS), 100,147 and scanning electron and force microscopy (SEM and SFM), 100,141 are commonly used to provide surface chemistry and morphology of FTACPs upon curing to a surface, while some success obtaining molecular weight information has been achieved by gel permeation chromatography (GPC) using a fluorinated solvent such as hydrochlorofluorocarbons (HCFCs) or trifluorotoluene (TFT) In addition, studies have reported the application of matrixassisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry to characterize side-chain fluorinated polymers, but not FTACPs. Given that MALDI-TOF is often used to characterize synthetic polymers, providing both molecular weight and structural information, it is an attractive technique for the measurement of FTACP degradation. The MALDI-TOF analysis of a synthetic polymer produces a mass spectrum with a characteristic repeat pattern. Changes to this repeat pattern, such as mass shifts and a decrease in signal intensity, suggests an alteration in the principle polymer structure. MALDI-TOF analysis has been used to evaluate the biodegradation of non-fluorinated polymers, such as poly(vinylpyrrolidone) and poly(ethylene glycol). 158,159 Development and application of a direct MALDI-TOF method for FTACP degradation studies would eliminate any uncertainty associated with indirect analyses. With the exception of the direct MALDI-TOF analysis reported in this dissertation, there are no other established methods to directly analyze FTACP degradation.

53 MALDI Mass Spectrometry Since introduced in 1988, MALDI mass spectrometry has become one of the primary methods for producing intact gas-phase ions of large non-volatile compounds, including proteins and synthetic polymers. For synthetic polymers, MALDI mass spectrometry provides specific structural characterization, which can be used to determine weight average molecular weight (M w ), number average molecular weight (M n ), and polydispersity index (PDI) As will be explained in the following sections, MALDI mass spectrometry can be broken into three fundamental components: 1) sample preparation, 2) desorption/ionization and 3) detection Sample Preparation for MALDI The first step in analyzing any analyte by MALDI mass spectrometry is selection of an appropriate sample preparation procedure. This is commonly referred to as the black art of MALDI sample preparation because it often requires a trial and error approach. The overall objective is to obtain intimate contact between the analyte and matrix molecules, 167 increasing the odds of observing the desired signals. Given the chemical diversity of synthetic polymers, selecting appropriate matrix, cationization agent and deposition method (or plating method) is not always straightforward. Ensuring compatibility between the matrix and polymer can be made easier with prior knowledge about the relative hydrophobicity and polarity of the matrix and polymer. 168,169 For most polymers, common matrices such as those shown in Figure 1.9, will yield successful results. However, for more novel polymers, a customized approach may be required. 170 Once a matrix has been selected, the ionization of the polymer then needs to be considered. Ionization

54 30 of the polymer often requires a cationization agent, as most polymers have a low proton affinity. Polymers possessing a number of heteroatoms atoms, such as polyethers and polyacrylates, are readily ionized using an alkali salt, such as lithium or sodium trifluoroacetate. Polymers with no heteroatoms are difficult to ionize, unless they have a degree of unsaturation, such as polystyrenes and polybutadienes, and require a metal that interacts strongly with -bonds, such as silver or copper. Figure 1.9: Chemical structures of several common MALDI matrices. It is extremely advantageous if the analyte, matrix and cationization are soluble in a single organic solvent, allowing conventional solvent-based sample preparation, such as the dried droplet method, to be used. This will increase the probability that the polymer and matrix

55 31 undergo co-crystallization upon solvent evaporation. If the analyte and matrix are not soluble in a common solvent, then a modification to the solvent-based sample preparation will be required. Multi-layering techniques have been investigated to overcome solvent incompatibility, and involve separate deposition of the analyte and matrix. Alternatively, solvent-free sample preparations are available for insoluble polymers, but are often laborious and timeconsuming. The dried-droplet method is arguably the most common deposition method used, but other methods such as thin- 177,178 and seed-layering, 179 and electrospray have been demonstrated to further enhance sample homogeneity Principles of MALDI Although the exact mechanism of MALDI ion formation is not fully understood, it is known to involve both desorption and ionization processes. To desorb polymer molecules, a pulsed laser, typically a N 2 ( = 337 nm) or Nd:YAG ( = 355 nm) laser, is irradiated at the surface for a short duration (<1 to 5 nanoseconds). The matrix molecules are rapidly heated via the absorption of high-energy photons causing localized sublimation, which generates a plume of neutral and charged species into the gas phase. 186 Through this desorption process, neighboring polymer molecules are transferred intact into the gas phase, as illustrated in Figure Thus, it is not only critical that there is intimate contact between the matrix and polymer molecules, but also that the matrix itself absorbs the photons emitted from the laser.

56 32 Reflectron Detector Linear Detector Desorption Desorption Ionization + Ionization Irradiation Irradiation Desorption Electrodes Source Analyte Ionization Matrix Irradiation Analyte Source Matrix Extraction GridElectrodes Source Reflectron Detector Linear Detector Figure 1.10: Diagram of the principle of MALDI-TOF. While ionization of molecules is still a debated process of MALDI mass spectrometry, it is generally accepted to occur via a combination of different mechanisms. A list of accepted ionization mechanisms are summarized in Table 1.3. The most commonly accepted ionization mechanism occurs from gas-phase proton or cation transfer Gas-phase proton transfer results from the photoionized matrix molecules, whereas gas-phase cation transfer, which is more relevant for synthetic polymers, results from free cations generated from a cationization

57 33 agent. Regardless of the exact mechanism of ionization, ionization of the analyte occurs rapidly after irradiation of the laser, and before the ions are accelerated towards the mass analyser. Table 1.3: Possible ionization mechanisms in MALDI mass spectrometry. Desorption of preformed ions Ionization by energy pooling Rupture of matrix and statistical charging of the resulting cluster/particles Gas-phase proton transfer Gas-phase cation transfer Electron transfer Principles of TOF Mass Spectrometry While a variety of mass spectrometers, including Fourier transform ion cyclotron resonance (FT-ICR), quadrupole ion trap and Orbitrap instruments, have been coupled with MALDI, it is TOF instruments that are most commonly used. This is attributed to the high sensitivity, unlimited mass range, good resolving power of TOF mass spectrometers and affordability. Upon desorption of the analyte from the MALDI plate, ions are accelerated from the source towards a field free region by a drop in potential (V) between the source and extraction grid shown in Figure As a result, ions entering and exiting the field free region have the same kinetic energy, as expressed in Eq. 1.6, where m and z are the mass and charge of the ion,

58 34 and v is the velocity of the ion exiting the source. The ions are then separated in the field free region based on their m/z. To see this, v can be expressed as the length of the field free region (D), divided by the time (t) for the ion to reach the detector (Eq. 1.7). Substituting Eq. 1.7 into Eq. 6, the flight time of the ion is inversely proportional to the square root of its mass-to-charge ratio, as expressed by Eq Given that ion flight time requires a starting point, TOFs are operated in a pulsed fashion, and couple well with the pulsed laser. Irradiation of the laser on the surface of the MALDI plate represents the starting point, generating a discrete packet of ions that are then separated based on their masses. Depending on the desired resolving power of the mass spectrometer and the polymer s molecular weight, MALDI-TOF instruments can be operated in a linear or reflectron mode. In linear mode, the ions travel a linear flight path to the detector, whereas in reflectron mode, a series of parallel electrodes (ion mirrors) are used to redirect the ions in the opposite direction towards another detector (Figure 1.10). Reflectron mode increases the distance ions travel

59 35 before reaching the detector, allowing for greater separation. In addition, the parallel electrodes help focus ions of a particular m/z, but with slightly different kinetic energies. Ions with a greater kinetic energy will penetrate deeper into the parallel electrodes than ions of lower kinetic energy, allowing the ions to reach the detector simultaneously. These features greatly improve the resolving power of TOFs operated in reflectron (up to 100,000) compared to linear mode (~10,000). Typically, reflectron mode is used for polymers having a molecular weight <20 kda, and linear mode for polymers >20 kda because at higher molecular weights it becomes difficult to achieve isotopic resolution regardless of the operation mode. 1.4 Goals and Hypotheses The overall goal of this dissertation was to develop and apply MALDI-TOF methods capable of directly analyzing FTACP degradation. This approach would presumably eliminate the uncertainty of FTACP degradation attributed to the transformation of residual FTOHs and FTACs, which plagued previous studies. 63,64 The focus of Chapter 2 is the impact FTACP fluorination has on MALDI-TOF sample preparation and analysis. By synthesizing a variety of FTACPs with differing ratios of perfluorinated to hydrogenated carbons, we examined the success of MALDI-TOF analysis when conventional or novel fluorinated sample preparations were employed. In Chapter 3, novel fluorinated extraction and sample preparation techniques were investigated to directly measure the biodegradation of a model FTACP by MALDI-TOF in a soil-plant microcosm. We hypothesized that changes to signal intensity and relative signal distribution of the characteristic FTACP mass spectrum would provide direct evidence of FTACP biodegradation. To test this

60 36 hypothesis, FTACP biodegradation was investigated directly based on the MALDI-TOF signal and relative intensities, and indirectly by the formation of presumed transformation products. To improve upon the qualitative MALDI-TOF analysis used in Chapter 3, the development of a quantitative MALDI-TOF method for FTACPs was also investigated in Chapter 4. We hypothesized that the matrix used in the sample preparation could itself be used as a pseudo internal standard. The hypothesis was tested using a model FTACP to generate a reliable calibration method, and was tested by quantify the model FTACP in various aqueous media. Further application of this method was investigated in a FTACP hydrolysis study in Chapter 5. Successful development and application of this method would allow FTACP degradation to be directly quantified. FTOHs are distributed globally in the atmosphere contributing to ubiquity of PFCAs in the environment, and are the first degradation product of FTACPs. Off gassing of FTOHs from the degradation of FTACPs could represent a significant indirect source of PFCAs to the terrestrial environment. In Chapter 6, the global distribution of PFASs in surface soils was investigated using samples collected from all continents. We hypothesized that soils would be a significant sink of PFCAs and PFSAs from the atmospheric LRT of volatile precursors. This hypothesis was tested using soils collected removed from obvious human activity, which minimized the impact from direct sources. 1.5 References (1) Buck, R. C.; Franklin, J.; Berger, U.; Conder, J. M.; Cousins, I. T.; de Voogt, P.; Jensen, A. A.; Kannan, K.; Mabury, S. A.; van Leeuwen, S. P. Perfluoroalkyl and polyfluoroalkyl substances in the environment: Terminology, classification, and origins. Integr. Environ. Assess. Manage. 2011, 7,

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77 53 CHAPTER TWO Influence of Fluorination on the Characterization of Fluorotelomer-Based Acrylate Polymers by Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry Keegan Rankin and Scott A. Mabury Published In Anal. Chimica. Acta 2014, 808, Contributions Prepared by Keegan Rankin with editorial comments provided by Scott Mabury. Keegan Rankin performed all experimental work related to this project. Reprinted with permission from Analytica Chimica Acta. Copyright 2014, Elsevier Inc.

78 Abstract The relative degree of fluorotelomer-based acrylate polymers (FTACPs) fluorination was demonstrated to influence the sample preparation protocol for matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. A homologous series of FTACPs were synthesized from fluorotelomer and hydrocarbon acrylates of different chain lengths, which varied the ratio of perfluorinated to hydrogenated carbons (R F /R H ). The solubility of FTACPs in tetrahydrofuran (THF) and chloroform was observed to decrease for highly fluorinated FTACPs (R F /R H >0.5) promoting FTACP aggregation. No dependence on the degree of fluorination was observed for the solubility of FTACPs in the fluorinated solvents trifluorotoluene (TFT) or dichloropentafluoropropanes (HCFC-225). For FTACPs with a low degree of fluorination such as poly(8:2 FTAC-co-HDA) (R F /R H = 0.375), MALDI-TOF analysis was successful using a conventional sample preparation protocol with THF, and dithranol (Dith) matrix. Conversely, the poor solubility of the highly fluorinated poly(8:2 FTAC-co-BA) (R F /R H = 1.5) in THF resulted in mass discrimination. Several fluorinated sample preparation protocols were evaluated for poly(8:2 FTAC-co-BA) using TFT and HCFC-225, and decafluoroazobenzene (DFAB) or 2-[(2E)-3-(4-tert-butylphenyl)-2-methylprop-2- enylidene]malononitrile (DCTB) matrices. The high volatility of HCFC-225 decreased FTACP pooling during solvent evaporation in comparison to the less volatile TFT, and improved the quantity of detectable signals. MALDI-TOF analysis of poly(8:2 FTAC-co-BA) in a 95:5 HCFC-225:Methanol with DCTB being the best sample preparation protocol for highly fluorinated FTACPs in this study producing the highest number of observable signals. Employing a fluorinated sample preparation offers the capability of analyzing other highly fluorinated polymers that are not compatible with conventional sample preparations.

79 Introduction Fluorotelomer-based acrylate polymers (FTACPs) are a group of fluorinated polymers belonging to the class of side-chain fluorinated polymer that also includes urethane and oxetane materials. 1 Like all fluorinated polymers, the attractiveness of FTACPs stems from the improved repellency, lubricity, and chemical and thermal stability when the hydrogen atoms are replaced by fluorine atoms. 2 However, unlike other classes of fluorinated polymers such as fluoropolymers (ie. polytetrafluoroethylene (PTFE)) and perfluoropolyethers (PFPEs), FTACPs do not possess a fluorinated backbone, but rather fluorinated appendages covalently bound by an ester moiety to a hydrocarbon backbone. When applied to a material, the fluorinated appendages are thought to orientate perpendicular to the surface, which optimizes the number of surface-cf 3 units and reduces the critical surface energy. 2,3 This reduction in critical surface energy makes FTACPs highly effective surface protectants in the carpet, textile, upholstery and paper industries. 4 The term fluorotelomer-based denotes the incorporation of material derived from the telomerization process developed by the Du Pont Company in the early 1940s. 5-7 For FTACPs, this refers to the polymerization of fluorotelomer acrylates [CH 2 =CHC(O)OCH 2 CH 2 (CF 2 ) n CF 3, n = 1-17]. The preparation of FTACPs is often carried out by free radical polymerization in an aqueous emulsion using a combination of fluorotelomer and hydrocarbon acrylates, and often other non-fluorinated monomers. 8,9 By varying the ratio and chain length of the fluorotelomer and hydrocarbon acrylates, the wettability and tackiness can be optimized for a specific function Generally, the desirable hydrophobic and oleophobic properties are achieved when the fluorotelomer acrylate has 8 perfluorinated carbons and the hydrocarbon acrylate has 10 hydrogenated carbons. 10,13,14 Additional non-fluorinated monomers, such as hydroxyl acrylates,

80 56 are often employed as minor constituents to improve the solubility, offer cross-linking capability, and facilitate stain and dirt removal during laundering The diversity of monomer composition has led to FTACPs becoming the largest fraction of commercial fluorotelomer products, 4,22 constituting >80% of all fluorotelomer-based raw materials produced worldwide. 23 The high degree of fluorination and copolymerization of multiple monomers poses several challenges for the characterization of FTACPs. Techniques such as near-edge x-ray absorption fine structure (NEXAPS), x-ray photoelectron spectroscopy (XPS), 24,28,29 angleresolved XPS (ARXPS), 24,30 and scanning electron and force microscopy (SEM and SFM), 19,24 provide information regarding the surface morphology of side-chain fluorinated polymers including FTACPs, but lack molecular weight information. The estimation of weight average and number average molecular weight (M w and M n ) are traditionally carried out by gel permeation chromatography (GPC). However, M w and M n can be misrepresented for FTACPs due to their poor solubility in conventional GPC solvents such as chloroform and tetrahydrofuran (THF). 19 GPC analyses using fluorinated solvents such as hydrochlorofluorocarbons (HCFCs) or trifluorotoluene (TFT) have been reported to overcome solubility issues associated with highly fluorinated side-chain polymers. Although this approach can offer a better estimation of molecular weight, it does not provide structural information such as endgroups and monomer distribution. Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry has evolved into an integral technique for synthetic polymer characterization, which provides both molecular weight properties and structural information Establishing an appropriate sample preparation protocol largely dictates the success of the MALDI-TOF

81 57 analysis, and can be influenced by the solubility of the target polymer. 41,42 FTACPs provide a unique challenge as their relative degree of fluorination can render conventional sample preparations ineffective. Presently, a handful of studies have explored the MALDI-TOF analysis of side-chain fluorinated polymers, however none were fluorotelomer-based In these studies, polymers having a relatively low degree of fluorination were soluble in non-fluorinated solvents and were successfully analyzed using the MALDI matrices 2,5-dihydroxybenzoic acid (DHB) 44 and dithranol (Dith). 45,46 On the other hand, polymers having a higher degree of fluorination were insoluble in THF, and required a multi-layer sample preparation where the polymers were prepared in the now banned trichlorotrifluoroethane (CFC-113). 43 It is generally accepted that single-solvent systems are the preferred sample preparation protocols as they reduce the likelihood of sample segregation that can occur with multi-solvent systems. 47,48 Our interest in establishing a sample preparation protocol for the MALDI-TOF analysis of FTACPs stems from their potential to serve as sources of perfluorinated carboxylates (PFCAs). 49,50 PFCAs are persistent environmental contaminants that have been demonstrated to accumulate in biota with increasing perfluorinated chain length (>6 CF 2 ). 51,52 Although FTACPs have not yet been confirmed as a PFCA source, other fluorotelomer-based commercial products, such as polyfluoroalkyl phosphate esters (PAPs) 53 and fluorotelomer stearate monoester (FTS), 54,55 are known to biodegrade to PFCAs. In the present work, we investigated the influence of FTACP fluorination on the MALDI-TOF sample preparation to facilitate a future biodegradation study of FTACPs. A homologous series of FTACPs were synthesized for this study. Scanning electron microscopy (SEM) was used to supplement the MADLI-TOF results by providing a surface distribution image for the various sample preparation protocols.

82 Experimental Chemicals 2,2-azobis(2-methylpropionamide) dihydrochloride (AIBA, 98%), 2,3,4,5,6- pentafluoroaniline (99%), 2-[(2E)-3-(4-tert-butylphenyl)-2-methylprop-2- enylidene]malononitrile (DCTB, 98%), acetone-d 6 (99.9%), butyl acrylate (BA, 99%), dithranol (Dith, 98.5%), sodium trifluoroacetate (NaTFA, 98%), sodium hypochlorite, tert-butyl methyl ether (MTBE, 99%), tetrabutylammonium hydrogen sulfate (n-bu 4 NHSO 4, 97%), tetrahydrofuran (THF, 99%) and trifluorotoluene (TFT, 99%) were all purchased from Sigma Aldrich (St. Louis, MO). Chloroform ( 99.9%) and ethyl acetate ( 99.9%) were purchased from Fisher Scientific (Pittsburgh, PA). 1H,1H,2H,2H-perfluorooctyl acrylate (6:2 FTAC, 97%), 1H,1H,2H,2H-perfluorodecyl acrylate (8:2 FTAC, 97%), dichloropentafluoropropanes (HCFC-225ca/cb, >99%), pentafluorobenzoic acid (PFBzA, 99%) and trans-pentafluorocinnamic acid (PFCnA, 98%) were all purchased from Synquest Labs Inc. (Alachua, FL). Hexadecyl acrylate (HDA) was purchased from Monomer-Polymer and Dajac Labs Inc. (Feasterville, PA). Octyl acrylate (OA) was purchased from Scientific Polymer Inc. (Ontario, NY). Methanol (>99%) was purchased from EMD Chemicals Inc. (Mississauga, ON) Synthesis of Fluorotelomer-based Acrylate Polymers (FTACPs) Polymerization of the FTACPs was carried out in a 3-neck round bottom flask equipped with a condenser and a magnetic stir bar. The reaction vessel containing ethyl acetate was first purged with nitrogen for 60 minutes. Equimolar amounts of fluorotelomer acrylate (ie. 8:2 FTAC) and hydrocarbon acrylate (ie. BA) were added to the reaction vessel, and the reaction

83 59 initiated by the addition of 0.5-1% aqueous AIBA. The contents were gradually brought to 70 o C over 60 minutes and held for ~15 hours using a IKA-Werke RCT hot plate with a IKA-Werke ETS-D4 temperature controller (IKA Werke, Staufen, DE). Upon completion, all contents were transferred to a single neck round bottom flask, and concentrated by rotary evaporation. The crude product was sonicated in methanol, dried, and heated at 75 o C for several days to remove any unreacted monomers Synthesis of Decafluoroazobenzene (DFAB) 56,57 A suspension of pentafluoroaniline (10 g, 54.6 mmol) in 360 ml of sodium hypochlorite (10-15% free chlorine) was stirred vigorously at room temperature overnight. The solution was extracted using MTBE (3 x 150 ml), and the combined extracts were washed with HPLC grade water (5 x 100 ml), dried with MgSO 4 and concentrated by rotary evaporation. The crude product was purified by recrystallization in ethanol yielding red-orange crystals (20%), mp o C. 19 F NMR (376 MHz, acetone-d 6, relative to 4-trifluoromethoxyacetanilide (4- TFMeAC)): to (m, 4F), to (m, 2F), to (m, 4F) Synthesis of 4,4-dihydroxyoctafluoroazobenzene (dihyofab) 56,57 To a stirred solution of DFAB (1g, 2.76 mmol) in toluene (15 ml) cooled in a 10 o C water bath, a solution of NaOH (7.5 g) in water (15 ml) was added followed by n-bu 4 NHSO 4 (1.87 g, 5.52 mmol). The reaction contents were stirred for 20 minutes at room temperature, followed by the addition of toluene (15 ml) and water (15 ml) resulting in the separation of a dark

84 60 brown/red precipitate. The precipitate was recovered by filtration, washed with toluene (10 ml) and 2-propanol (10 ml), and recrystallized in 2-propanol yielding the n-bu 4 NHSO 4 salt of dihyofab. To obtain the free phenol of dihyofab, the n-bu 4 NHSO 4 salt (1 g) was partitioned between 1 M H 2 SO 4 (12 ml) and MTBE (24 ml). The organic phase was separated and washed with 1 M H 2 SO 4 (2 x 12 ml) and water (2 x 12 ml). After evaporation of the organic phase, the contents were recrystallized in ethanol:water (1:2) yielding a fine orange powder (30%), mp o C. 19 F NMR (376 MHz, acetone-d 6, relative to 4-TFMeAC): to (m, 4F), to (m, 4F) Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF) Mass spectra were acquired using a Waters Micromass MALDI micro MX TOF mass spectrometer (Waters Corporation, Milford, MA) equipped with a nm nitrogen laser. The instrument was operated in positive reflectron mode with a 12 kv source voltage, 5.2 kv reflectron voltage, 1.9 kv pulse voltage and 2.1 kv detector voltage. The laser strength was varied depending on the matrix selected. A total of 50 mass spectra per sample were acquired, and summed using Waters Mass Lynx V4.1 mass spectrometry software MALDI-TOF Sample Preparation FTACP solutions were prepared in THF, TFT or HCFC-225 at 10 mg ml -1 solution. Dith was prepared in THF at 20 mg ml -1. DFAB and DCTB were either prepared in solutions of

85 61 95:5 TFT:MeOH or 85:15 HCFC-225:MeOH at 20 mg ml -1. dihyofab was prepared in a 70:30 HCFC-225:MeOH solution at 20 mg ml -1. Sodium trifluoroacetate was prepared in either THF or MeOH at 10 mg ml -1. Each sample was then prepared with a specific mixing ratio of matrix:ftacp:cationization agent (M:A:Cat). A 1 L aliquot was then spotted onto a stainless steel MALDI target plate and allowed to air dry Scanning Electron Microscopy (SEM) Sample preparation protocols were evaluated using a Quanta FEG 250 Environmental SEM (FEI Company, Hillsboro, OR). SEM images were acquired using a large field detector (LFD) under low vacuum (130 Pa) with the electron beam operated with a 10 kv voltage and a current of 5 na. 2.4 Results and Discussion FTACP Design and Solubility A total of six fluorotelomer-based acrylate polymers (FTACPs) were prepared from 6:2 or 8:2 fluorotelomer acrylate (FTAC) and either butyl acrylate (BA), octyl acrylate (OA) or hexadecylacrylate (HDA) producing material that ranged from a sticky gel to a solid wax (Table A1). The architecture of the FTACPs was intended to mimic those used commercially, but excluded other minor constituents such as hydroxyl acrylates and vinylidene chloride. 8,9 Incorporation of the fluorotelomer and hydrocarbon acrylates yielded FTACPs having two distinct repeat units with different ratios of perfluorinated and hydrogenated carbons (Figure

86 62 2.1). Varying this ratio changes the relative degree FTACP fluorination and is reported in this study as R F /R H (Table 2.1). Figure 2.1: Synthesized FTACPs having m = 5 or 7 and n = 3, 7 or 15. The solubility of FTACPs was studied by preparing 10 mg ml -1 solutions in both nonfluorinated and fluorinated solvents. In tetrahydrofuran (THF) and chloroform, the FTACP solutions were observed to be transparent for R F /R H <0.5 and becoming cloudy at R F /R H >0.5 (Table 2.1). The cloudiness presumably results from the formation of aggregates where the fluorinated appendages of FTACPs face inwards similar to micelle formation as reported for other fluorinated materials. 58,59 Thus, the solubility of FTACPs in non-fluorinated solvents increase with decreasing fluorination. Conversely, when the fluorinated solvents trifluorotoluene (TFT) and dichloropentafluoropropanes (HCFC-225) were employed the solubility of FTACPs was observed to be independent of R F /R H (Table 2.1). The preferential solubility of highly fluorinated analytes in fluorinated solvents has been the subject of theoretical studies, and is often referred to as fluorophilicity. Selecting an appropriate solvent is an important consideration for the sample preparation of FTACPs for matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) analysis.

87 63 Table 2.1: Relative degree of FTACP fluorination based on a calculated chain length ratio and their corresponding solubility in non-fluorinated and fluorinated solvents. Polymer R F /R H THF Chloroform TFT HCFC-225 Poly(6:2 FTAC-co-BA) 1 slightly soluble slightly soluble soluble soluble Poly(6:2 FTAC-co-OA) 0.5 moderately soluble moderately soluble soluble soluble Poly(6:2 FTAC-co-HDA) 0.25 soluble soluble soluble soluble Poly(8:2 FTAC-co-BA) 1.5 slightly soluble slightly soluble soluble soluble Poly(8:2 FTAC-co-OA) 0.75 moderately soluble moderately soluble soluble soluble Poly(8:2 FTAC-co-HDA) soluble soluble soluble soluble *R F /R H was calculated from the number of perfluorinated carbons in the acrylate chain minus its two hydrocarbons (ie. 8 2 = 6) then dividing by the number of hydrocarbons in the hydrocarbon acrylates (ie. 6/16 = 0.375).

88 Influence of Fluorination on a Conventional Sample Preparation The influence of the degree of FTACP fluorination on MALDI-TOF sample preparation was explored for poly(8:2 FTAC-co-HDA) and poly(8:2 FTAC-co-BA) having respective R F /R H of and 1.5. Poly(8:2 FTAC-co-HDA) and poly(8:2 FTAC-co-BA) were prepared in THF with dithranol (Dith) and NaTFA. Dith was selected as the matrix based on its suitability for hydrophobic polymers 63 and reported use for other side-chain fluorinated polymers. 45,46 Several alkali and transition organic salts were explored as cationization agents and NaTFA demonstrated the most efficient ionization in this study. With these parameters, a mixing ratio of 10:5:1 (M:A:Cat) was found to be the preferred sample preparation based on the quantity of observable MALDI-TOF signals. The mass spectrum for poly(8:2 FTAC-co-HDA) showed an abundance of intense signals spanning from 750 to 8000 Da (Figure 2.2A), from which the weight average molecular weight (M w ), number average molecular weight (M n ) and polydispersity index (PDI) were calculated to be 3080, 2370 and 1.3, respectively. In contrast, considerably fewer signals were observed in the mass spectrum for poly(8:2 FTAC-co-BA) spanning from 750 to 2500 Da (Figure 2.2B), and having M w = 1232, M n = 1119 and PDI = 1.1. The substantial difference between the mass spectra led us to consider whether the high degree of fluorination for poly(8:2 FTAC-co-BA) was resulting in mass discrimination. Formation of poly(8:2 FTAC-co-BA) aggregates in THF could result in reduction of the desorption efficiency favoring the more soluble and lower molecular weight poly(8:2 FTAC-co-BA) molecules. The broader distribution observed for poly(8:2 FTAC-co-HDA) results from its lower degree of fluorination promoting a homogeneous distribution independent of molecular weight.

89 65 Figure 2.2: MALDI-TOF mass spectra of (A) poly(8:2 FTAC-co-HDA) and (B) poly(8:2 FTAC-co-BA) using Dith as the matrix and NaTFA as the cationization agents prepared in THF with a mixing ratio of 10:5:1. The examination of the distribution of FTACPs and Dith on the MALDI plate upon solvent evaporation was performed using scanning electron microscopy (SEM). In the presence of FTACP, a series of ring-like deposits resulted from crystallization as the solvent front retracted (Figure 2.3). For poly(8:2 FTAC-co-HDA), there was no observable difference between SEM results when the fraction of FTACP was decreased from 10:5:1 to 10:1:1 (Figures 2.3A and 2.3B). In contrast, the SEM results for poly(8:2 FTAC-co-BA) prepared at 10:5:1 had several dark deposits not observed at 10:1:1 (Figures 2.3C and 2.3D), nor for poly(8:2 FTAC-co-

90 66 HDA). The dark deposits could represent poly(8:2 FTAC-co-BA) aggregates causing a significant reduction in signal intensity. Thus, for highly fluorinated FTACPs conventional nonfluorinated sample preparation protocols are not appropriate. A B C D Figure 2.3: SEM images of (A and B) poly(8:2 FTAC-co-HDA) and (C and D) poly(8:2 FTACco-BA) using Dith as the matrix and NaTFA as the cationization agents. All samples were prepared in THF with a mixing ratio of 10:5:1 (A and C) and 10:1:1 (B and D). The images illustrate the differences between the FTACPs and mixing ratio upon solvent evaporation. The dark center in panel C represents the increased aggregation of the partially dissolved poly(8:2 FTAC-co-BA).

91 Towards a Fluorinated Sample Preparation An alternative approach is to take advantage of fluorophilicity and implement a fluorinated sample preparation for FTACPs that are insoluble in non-fluorinated solvents. Poly(8:2 FTAC-co-BA) was found to be soluble in TFT and HCFC-225; however, the solubility of Dith in these solvents was quite poor and required a more applicable matrix. Marie et al. reported the characterization of perfluoropolyethers (PFPEs) by MALDI-TOF using two fluorinated matrices, pentafluorobenzoic acid (PFBzA, Figure 2.4C) and pentafluorocinnamic acid (PFCnA, Figure 2.4D). 64 Although this novel approach improved desorption/ionization of PFPEs over non-fluorinated matrices, PFBzA and PFCnA did not absorb photons emitted at 337 nm strongly. Decafluoroazobenzene (DFAB, Figure 2.4E) and 4,4- dihydroxyoctafluoroazobenzene (dihyofab, Figure 2.4F) are two highly conjugated compounds that have been utilized as matrices for the MALDI-TOF analysis of several insoluble but non-fluorinated synthetic polymers. 57,65,66 Their application as matrices for the analysis of poly(8:2 FTAC-co-BA) was investigated in our effort to establish a fluorinated sample preparation protocol.

92 68 A B C D E F Figure 2.4: Chemical structures of matrices of interest in this study: (A) dithranol (Dith), (B) 2- [(2E)-3-(4-tert-butylphenyl)-2-methylprop-2-enylidene]malononitrile (DCTB), (C) pentafluorobenzoic acid (PFBzA), (D) pentafluorocinnamic acid (PFCnA), (E) decafluoroazobenzene (DFAB) and (F) 4,4-dihydroxyoctafluoroazobenzene (dihyofab). Poly(8:2 FTAC-co-BA) was first analyzed using dihyofab, as it had reported to be a more affective matrix than DFAB. 57 The presence of two hydroxyl groups reduced the solubility of dihyofab in both TFT and HCFC-225 requiring it to be prepared in a 70:30 solution of TFT:Methanol (MeOH) or HCFC-225:MeOH. The final sample preparation protocol involved the preparation of poly(8:2 FTAC-co-BA) in either 95:5 TFT:MeOH or 95:5 HCFC-225:MeOH with NaTFA in MeOH at a mixing ratio of 10:5:1. However, MALDI-TOF analyses with dihyofab produced consistently poor results. The repeat pattern previously observed for poly(8:2 FTAC-co-BA) with Dith (Figure 2.2B) was never observed with dihyofab. Poly(8:2

93 69 FTAC-co-BA) is insoluble in MeOH, and the high fraction required to dissolve dihyofab could explain the poor MALDI-TOF results with this sample preparation protocol. The sample preparation protocol was modified for DFAB by dissolving it along with poly(8:2 FTAC-co-BA) in 95:5 TFT:Methanol solutions with NaTFA in MeOH at a ratio of 10:5:1. The MALDI-TOF result for poly(8:2 FTAC-co-BA) showed no observable signals above 2000 Da (Figure 2.5A), and having M w = 1270, M n = 1153 and PDI = 1.1. This result showed no improvement over the result obtained for poly(8:2 FTAC-co-BA) when the nonfluorinated sample preparation using Dith was employed (Figure 2.2B). An important observation made using this fluorinated sample preparation protocol was the slow evaporation of TFT. Generally, solvents having a higher rate of evaporation are preferred to obtain a more homogeneous matrix/sample distribution, and is the reason why solvents such as dimethyl sulfoxide (DMSO) and dimethyl formamide (DMF) should be avoided. 67 To put this into context, the vapor pressure of TFT along with HCFC-225 were compared to several nonfluorinated solvents using predicted values obtained by AOPWin (Table A2). 68 At 4.5 kpa (at 25 o C) the vapor pressure of TFT is an order of magnitude higher than DMF (0.47 kpa at 25 o C), but roughly five times lower than THF (23 kpa at 25 o C). The distribution of poly(8:2 FTAC-co- BA) and DFAB molecules was determined by SEM and showed a dark ring shape deposit at the center of the well (Figure 2.6A). Driven by fluorophilicity, the poly(8:2 FTAC-co-BA) molecules appear to concentrate in the slowly evaporating TFT droplet until saturated, at which point they deposit at the droplet s edge. The use of TFT with this sample preparation protocol reduces the desorption efficiency and can explain the poor MALDI-TOF result of poly(8:2 FTAC-co-BA).

94 Figure 2.5: MALDI-TOF mass spectra of poly(8:2 FTAC-co-BA) prepared in (A) TFT using DFAB as the matrix, (B) HCFC-225 using DFAB as the matrix and (C) TFT using DCTB as the matrix. All samples used NaTFA as the cationization agent and a mixing ratio of 10:5:1. 70

95 71 A modification to the sample preparation protocol was made by replacing the slow evaporating TFT with the highly volatile HCFC-225 (40 kpa at 25 o C) there was a modest enhancement in the number of observable poly(8:2 FTAC-co-BA) signals (Figure 2.5B), and having M w = 1305, M n = 1183 and PDI = 1.1. The sample preparation required DFAB to be dissolved in a 85:15 HCFC-225:MeOH solution to achieve the desired 20 mg ml -1, and was mixed with a 95:5 HCFC-225:MeOH poly(8:2 FTAC-co-BA) solution with NaTFA in MeOH at a ratio of 10:5:1. SEM imaging revealed many small poly(8:2 FTAC-co-BA) deposits dispersed throughout the matrix crystals (Figure 2.6B), which suggests that the evaporation rate of HCFC- 225 prevents the pooling of poly(8:2 FTAC-co-BA) molecules. This sample preparation resulted in more efficient desorption and subsequent ionization of poly(8:2 FTAC-co-BA) than when TFT was employed (Figure 2.5A). However, a major drawback for both DFAB and dihyofab is high laser energy (<20% attenuation) required for desorption, which could result in significant fragmentation of FTACP molecules. A B Figure 2.6: SEM images of poly(8:2 FTAC-co-BA) prepared in (A) 95:5 TFT:MeOH using DFAB as the matrix and (B) 95:5 HCFC-225:MeOH using DFAB as the matrix. Both samples used NaTFA as the cationization agent and a mixing ratio of 10:5:1.

96 72 The non-fluorinated matrix 2-[(2E)-3-(4-tert-butylphenyl)-2-methylprop-2- enylidene]malononitrile (DCTB, Figure 2.4B) has a couple of features that make it suitable for this study. Firstly, DCTB is soluble in both TFT and HCFC-225, and secondly it requires low laser energy for desorption. 69 Several different sample preparation protocols were tried with DCTB, but the best results were achieved when DCTB and poly(8:2 FTAC-co-BA) were prepared in 95:5 HCFC-225:MeOH with NaTFA in MeOH at a mixing ratio of 10:5:1. The distribution of signals in the mass spectrum was larger than observed with any other matrices, and spanned from 750 to 4000 Da (Figure 2.5C). In addition, the molecular weights of M w = 2225, M n = 1837 and PDI = 1.2 were nearly double those reported for the sample preparation with Dith. A comparison of detectable signals from 2000 to 3000 Da for poly(8:2 FTAC-co-BA) illustrates the importance of selecting an appropriate sample preparation (insets of Figure 2.5). Signals not observable when DFAB was prepared in TFT were weakly detected when HCFC- 225 was used and became readily detectable when DCTB was employed. Unfortunately, SEM imaging for this sample required a different set of instrumental conditions and were therefore omitted. Nonetheless, the application of a highly volatile fluorinated solvent in combination with DCTB was clearly demonstrated to be the best sample preparation protocol for the MALDI- TOF analysis of highly fluorinated FTACPs in this study Characterization of FTACPs The MALDI mass spectra of poly(8:2 FTAC-co-HDA) and poly(8:2 FTAC-co-BA) were used to obtain structural information, and were acquired with their respective optimized sample preparation protocols. A close examination of the poly(8:2 FTAC-co-HDA) mass spectrum using Dith and NaTFA showed a major series of repeating signals from 1200 to 1775 Da having

97 73 either a 518 or 296 Da transition, which corresponds to different ratios of 8:2 FTAC (X) to HDA (Y), respectively (Figure 2.7). This ratio was an exact integer for the ratio of X to Y implying there was no additional endgroup functionality. Results for poly(8:2 FTAC-co-BA) using DCTB and NaTFA showed a similar series repeating signals from 1025 to 1625 Da having either a 518 or 128 Da transition corresponding to 8:2 FTAC (X) to BA (Y), respectively (Figure 2.8). The major series for both poly(8:2 FTAC-co-HDA) and poly(8:2 FTAC-co-BA) presumably contained a FTACP structure having a terminal alkene moiety as illustrated in Figure 2.9. Similar structures have been proposed for a linear polyacrylate, 70 and as a minor constituent of perfluoroalklyl acrylic oligomers. 71 In addition, several less abundant repeating signals were observed in both mass spectra (U), and had a peak spacing of 296 or 128 Da corresponding to the masses of HDA and BA, respectively. The exact compositions of these minor series have not yet been identified at this point, but could be further explored with tandem mass spectrometry. Figure 2.7: Characterization of poly(8:2 FTAC-co-HDA) based on MALDI-TOF results obtained using Dith and NaTFA prepared in THF with a mixing ratio of 10:5:1.

98 74 Figure 2.8: Characterization of poly(8:2 FTAC-co-BA) based on MALDI-TOF results obtained using DCTB and NaTFA prepared in TFT with a mixing ratio of 10:5:1. Figure 2.9: Proposed chemical structures of the identified FTACP repeat patterns.

99 Conclusions FTACPs can be characterized by MALDI-TOF, but their relative degree of fluorination is an important consideration when selecting an appropriate sample preparation protocol. Application of a fluorinated sample preparation can improve the analysis of highly fluorinated FTACPs, which are poorly soluble in non-fluorinated solvents. These solubility issues can be diminished using fluorinated solvents that have appreciable vapor pressures. The highly volatile HCFC-225 appears to be the best solvent for the sample preparation of highly fluorinated FTACPs. The fluorinated matrix DFAB was explored, but only a modest improvement was observed over Dith. The non-fluorinated matrix DCTB prepared in HCFC-225 increased the number of observable signals and their intensity. Establishing sample preparation protocols for FTACPs with a broad degree of fluorination allowed for their characterization by MALDI-TOF mass spectrometry. 2.6 Acknowledgements The authors would like to thanks Dr. Mark Nitz (University of Toronto) for providing access to the MALDI-TOF system, and Dr. Neil Coombs and Ilya Gourevich (University of Toronto) for their assistance with the SEM. The presented study is supported by Natural Science and Engineering Research Council of Canada (NSERC), and the Ontario Ministry of Training, Colleges and Universities for an Ontario Graduate Scholarship (OGS).

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106 82 CHAPTER THREE Investigating the Biodegradability of a Fluorotelomer-Based Acrylate Polymer in a Soil-Plant Microcosm by Indirect and Direct Analysis Keegan Rankin, Holly Lee, Pablo J. Tseng and Scott A. Mabury Published in Environ. Sci. Technol. 2014, 48, Contributions Prepared by Keegan Rankin with editorial comments provided by Holly Lee, Pablo Tseng and Scott Mabury. Keegan Rankin developed and performed all extractions and MALDI-TOF analyses related to this project. Pablo Tseng synthesized the FTACP and designed the experiment. Holly Lee performed extraction and LC-MS/MS analyses related to this project. Reprinted with permission from Environmental Science and Technology Copyright 2014, American Chemical Society

107 Abstract Fluorotelomer-based acrylate polymers (FTACPs) are a class of side-chain fluorinated polymers used for a variety of commercial applications. The degradation of FTACPs through ester hydrolysis and/or cleavage of the polymer backbone could serve as a significant source of perfluoroalkyl carboxylates (PFCAs). The biodegradation of FTACPs was evaluated in a soilplant microcosm over 5.5 months in the absence/presence of wastewater treatment plant (WWTP) biosolids, using a unique FTACP determined to be a homopolymer of 8:2 fluorotelomer acrylate (8:2 FTAC). Though structurally different from commercial FTACPs, the unique FTACP possesses 8:2 fluorotelomer side chain appendages bound to the polymer backbone via ester moieties. Liberation and subsequent biodegradation of the 8:2 fluorotelomer appendages was indirectly determined by monitoring for PFCAs of varying chain lengths (C6- C9) and known fluorotelomer intermediates by liquid chromatography tandem mass spectrometry (LC-MS/MS). A FTACP biodegradation half-life range of years was inferred from the 8:2 fluorotelomer alcohol (8:2 FTOH) equivalent of the unique FTACP and the increase of degradation products. The progress of FTACP biodegradation was also directly monitored qualitatively using matrix-assisted laser desorption/ionization time-of-flight (MALDI- TOF) mass spectrometry. The combination of indirect and direct analysis indicated that the model FTACP biodegraded predominantly to perfluorooctanoate (PFOA) in soils, and at a significantly higher rate in the presence of a plant and WWTP biosolids. 3.2 Introduction Fluorotelomer-based polymers (FTPs) are commonly used surface protectants in the carpet, textile, upholstery and paper industries. 1 The manufacture of FTPs constitutes >80% of

108 84 all fluorotelomer-based raw materials produced worldwide. 2 Recent concerns have been raised over the role of FTPs as perfluoroalkyl carboxylate (PFCA) precursors. 3,4 Residual fluorotelomer alcohols (FTOHs) are known to be present in FTP materials, 5,6 and have been demonstrated to readily biodegrade to PFCAs in aerobic soil and waste water treatment plant (WWTP) media At levels <5% by mass of commercial FTP materials, 5 however, residual FTOHs and other fluorotelomer material such as fluorotelomer acrylates (FTACs) are only one of the sources of PFCAs. A major uncertainty surrounding FTPs is whether the degradation of the polymer itself can serve as a significant source of PFCAs. To date, only a few peer-reviewed studies have aimed at evaluating the degradability of FTPs. 3,4,13 The polymerization of FTPs is typically carried out in an aqueous emulsion, which yields a fluorotelomer appendage covalently bound to a hydrocarbon backbone through either an ester or urethane linkage. 14,15 FTPs can therefore be sub-classified as fluorotelomer-based acrylate polymers (FTACPs) having an ester linkage or fluorotelomer-based urethane polymers (FTURPs) having a urethane linkage. FTACPs account for the largest distribution of commercial fluorotelomer production, 1,16 and are the focus of this investigation. The hydrophobic and lipophobic nature of all FTPs is attributed to the fluorotelomer appendage and its number of perfluorinated carbons. It can be inferred from several studies that a minimum of eight perfluorinated carbons are required for FTACPs to impart sufficient surface protectant properties Consequently, the degradation of the FTPs could serve as an indirect source of long-chain PFCAs (>7 CF 2 ), which have a demonstrated ability to accumulate in biota. 20,21 Russell et al. 3 proposed two potential degradation pathways of FTPs: 1) cleavage of the ester or urethane linkage or 2) breakage of the carbon-carbon backbone. Cleavage of the linking moiety would release the bound fluorotelomer appendage as FTOHs that then degrade to PFCAs.

109 85 Recent studies demonstrated that pathway 1 could occur via microbial hydrolysis of ester linkages of fluorotelomer-based material under aerobic environments, such as polyfluoroalkyl phosphate esters (PAPs) and fluorotelomer stearate monoester (FTS). 22,23 Alternatively, breaking the polymer backbone would yield smaller oligomeric species, which could more readily biodegrade because of the lower molecular weight. If the linking ester moieties are inaccessible to microbes, then pathway 2 could occur via -oxidation of the polymer backbone as reported for polyacrylic acid. 24 The approach for assessing the biodegradability of FTPs used to date, was to measure both intermediates and terminal PFCA products that result from the biotransformation of FTOHs rather than analytically probing the polymer directly. This indirect approach was used in the three FTP biodegradation studies. 3,4,13 However, the presence of intermediates and PFCAs does not directly confirm FTP degradation, as the PFCAs themselves may be present in commercial products or result from the degradation of other fluorotelomer-based residual materials. As such, the detection of target analytes above residual levels was generally used to identify FTP degradation, 4 a difficult task if there is a large background signal. In two parallel studies, the biodegradation half-lives for FTACPs and FTURPs were calculated to be and years respectively, based on polymer mass. 3,13 In a separate study, Washington et al. 4 reported a biodegradation half-life for FTACPs of years based on polymer mass, but also suggested that the half-life could be closer to years when normalized to the polymer particle surface area. The discrepancy between these half-lives is a reason the biodegradability of FTPs remains widely debated. The objective of this study was to evaluate the biodegradation of a unique FTACP in a soil-plant microcosm over 5 months. The unique FTACP was synthesized in-house using 8:2

110 86 fluorotelomer acrylate (8:2 FTAC) as the primary monomer, and exhaustively purified by removing volatile FTACs and/or FTOH residuals prior to incubation. Biodegradation of the model FTACP was evaluated under several different soil conditions, including soils amended with WWTP biosolids and sown with the alfalfa species, Medicago truncatula. Quantification of degradation products by liquid chromatography tandem mass spectrometry (LC-MS/MS) served as an indirect means to determine FTACP biodegradation and were used to estimate FTACP biodegradation half-lives. We also report herein, the first direct evidence of FTACP biodegradation using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry to analyze the polymer itself. 3.3 Experimental Chemicals A complete list of chemicals used in this study can be found in Appendix B for details Microcosm Materials Sandy loam soil was collected from an agricultural farmland (Northumberland County, ON; 44 o 05 N, 78 o 01 W) in 2009 and sieved with a 2 mm stainless steel mesh. Soil characterization was performed by SGS AgriFood Laboratories (Guelph, ON) and reported as follows: ph 5.5; 1.8% organic matter; cation exchange capacity of 96 µmol/g; 49 mg/kg of NaHCO 3 -extractable P; 63% sand, 32% silt and 5% clay. WWTP biosolid material (30% solids) was obtained from the North Toronto WWTP (Toronto, ON) in The alfafa plant species

111 87 Medicago truncatula was grown from seeds donated by the Stinchcombe research group (Department of Ecology and Evolutionary Biology, University of Toronto, ON) FTACP Polymerization Multiple approaches were attempted to synthesize a FTACP possessing three repeating monomers, but were unsuccessful. A unique FTACP homopolymer was synthesized in-house by aqueous dispersion following two commercial patents, 14,15 and is detailed in Appendix B. Briefly, 8:2 FTAC and butyl acrylate were added to an aqueous solution of dodecyl amine hydrochloride and hexadecylthiol in a round bottom flask equipped with a magnetic stirrer and dry ice condenser. The solution was purged with nitrogen for 2.5 hours at 5 o C and then a cold solution of vinylidene chloride and acetone was added. The polymerization was initiated with 2,2 -azobis(2-methylpropionamide) dihydrochloride (AIBA) and proceeded for 15 hours at 80 o C. Upon completion, the aqueous dispersion was filtered and the opaque FTACP material was collected Residual Removal Post-polymerization, a series of wash and heat purging steps were used to remove residual FTOH and FTAC impurities. The FTACP material was placed under vacuum while periodically washed with a 80:20 methanol:water solution for 14 days. The FTACP material was then melted using a 95 o C oil bath while continuously purged with carbon-filtered air for 20 days. A fraction of the FTACP material was then heated in the same manner for 3 days during which any volatile compounds were collected in XAD cartridges. The cartridges were extracted with

112 88 ethyl acetate and analyzed by gas chromatography-mass spectrometry (GC-MS). Although this approach differs from the method recently reported by Washington et al., 25 our method was also found to exhaustively extract volatile FTOHs and FTACs. The purified FTACP was determined to contain 4.71 and 2.63 nmole residual 8:2 FTOH and 8:2 FTAC per gram of FTACP representing 2.2 x 10-4 and 1.4 x 10-4 wt%, respectively. Low PFCA levels, 4.7 x 10-7 wt%, were observed as impurities in the purified FTACP, and explain the PFCAs concentrations observed at 0 months FTACP Characterization The unique FTACP was characterized by differential scanning calorimetry (DSC) and MALDI-TOF mass spectrometry. DSC procedures and results are presented in Appendix B. MALDI-TOF characterization was carried out using a Waters Micromass MALDI micro MX TOF mass spectrometer (Waters Corporation, Milford, MA) equipped with a nitrogen laser ( = nm) operated at a frequency of 5 Hz and rastered in a pre-set pattern. Mass spectra were acquired in positive ion and reflectron modes using a flight tube voltage of 12 kv. A total of 100 mass spectra were acquired per sample, and summed using Waters Mass Lynx V4.1 mass spectrometry software. FTACP samples were prepared for MALDI-TOF analysis using the two-layer dried droplet method. 26,27 Dithranol served as the matrix and lithium trifluoroacetate as the cationization agent, and were co-dissolved in trichloromethane (CHCl 3 ) at 10 mg/ml and 1 mg/ml, respectively. FTACP samples were prepared in -trifluorotoluene (TFT) at 10 mg/ml. Deposition of 1 L of the matrix:cationization agent solution onto a stainless steel

113 89 Waters MALDI target plate served as the first layer. A 1 L aliquot of the FTACP solution was deposited as the second layer on top of the dried matrix:cationization agent solution Biodegradation Experimental Design The five experimental variables were prepared as follows: (1) Soil Control soil without biosolids (n = 1 per timepoint); (2) Plant/Biosolids Control biosolids-amended soil sown with plant seeds (n = 3 per timepoint); (3) FTACP/Soil soil without biosolids mixed with 50 mg FTACP (n = 3 per timepoint); (4) FTACP/Plant soil without biosolids sown with plant seeds mixed with 50 mg FTACP (n = 3 per timepoint); (5) FTACP/Plant/Biosolids biosolidsamended soil sown with plant seeds mixed with 50 mg FTACP (n = 3 per timepoint). Biosolids-amended soils were prepared at a mixing rate of 16 g biosolids/kg of soil (~8.7 metric dry tons/ha) in an Odjob TM concrete mixer (Scepter Corporation, Toronto, ON). This rate is similar to the 5-year maximal application rate of 8 tons/ha permitted in Ontario. 28 Approximately 600 g soil and biosolids were transferred into each pot. Between 5 10 Medicago truncatula seeds were planted in each pot for the three conditions, Plant/Biosolids Control, FTACP/Plant and FTACP/Plant/Biosolids, followed by inoculation of cultured rhizobia. Preparation of the rhizobia culture is described in Appendix B. Catch plates were placed under each pot to capture some, though not all, target analytes that may have leached from the soil during watering of the plants. All pots were kept in a greenhouse (Earth Sciences Centre, University of Toronto, ON) for 5.5 months under natural sunlight and supplementary illumination (200 µmol/m 2 /sec) at a temperature regime of 25/21 o C day/night, and watered daily.

114 90 Soils were sampled at 1.5, 3.5 and 5.5 months by sacrificing the entire pot, and then immediately mixed with mg of sodium azide (NaN 3 ) to halt microbial activity. Initial concentrations of target analytes were measured in soil using one pot (n = 1) of all five conditions prior to the addition of the model FTACP. Soil was sampled in triplicate (n = 3) from each of the three pots in all conditions at each time point, except for the Soil Control in which soil was sampled from 1 pot. For Plant/Biosolids Control, FTACP/Plant and FTACP/Plant/Biosolids, the plant shoots and roots were harvested, cleaned of soil particles and archived together. Catch plates were also archived. All archived microcosm compartments were stored at 4 o C until analysis Extraction and Analysis Soil extractions was performed on 2 g soil samples by sonication at 60 o C for minutes in 5 ml of a basic methanol (1% (v/v) ammonium hydroxide). Following centrifugation at 6000 rpm, the supernatant was decanted into a new polypropylene tube, and the soil extracted a second time. The supernatants were combined and blown to dryness. Plant matter was lyophilized and homogenized finely using a mortar pestle, and 2 g samples extracted twice in 10 ml basic methanol sonication at 60 o C for minutes. The extracts were cleaned using ENVI Carb cartridges (Supelclean, 1 ml/100 mg) and blown to dryness. Catch plates were rinsed with 10 ml basic methanol and blown to dryness. Soil, plant and catch plate extracts were reconstituted with 2 ml methanol, passed through 0.2 µm Nylon filters, and then analyzed using an Agilent 1100 high pressure liquid chromatography (HPLC) coupled to an Applied Biosystems/MDS Sciex API4000 triple quadrupole MS (Concord, ON) operated in negative electrospray ionization mode. Chromatographic separation was performed using a GeminiNX

115 91 C18 column (4.6 x 50 mm, 3 µm; Phenomenex, Torrance, CA). Detailed instrumental parameters used be found in Appendix B. The unique FTACP was extracted from soils using -trifluorotoluene (TFT). For each pot, 2 x 5 g soil samples were each extracted with 5 ml of TFT by sonication and vortexing for 5 minutes at room temperature. The two extracts per pot were combined and the solids removed. The extract was then blown to dryness under nitrogen and re-constituted in 1 ml of TFT. MALDI-TOF analysis was carried out using the procedures described above Quality Assurance (QA) Quantitation of the PFCAs, fluorotelomer carboxylates (FTCAs) and fluorotelomer unsaturated carboxylates (FTUCAs) was performed using mass-labeled internal standards except for those analytes where no corresponding mass-labeled standards were available at the time of the experiment. In these few cases, quantification was performed using structurally similar internal standards as surrogate standards (Table B1). Spike and recoveries from soil, plant and catch plate ranged from , and %, respectively. Additional QA information is provided in Appendix B. 3.4 Results and Discussion FTACP Characterization Structural characterization of the unique FTACP was performed by MALDI-TOF producing a characteristic pattern indicative of a synthetic polymer with repeating signals having

116 92 a spacing of 518 Da for both major and minor series shown in Figure B2. The number average molecular weight (M n, Eq. 3.1), weight average molecular weight (M w, Eq. 3.2) and polydispersity index (PDI, Eq. 3.3) were calculated from the major repeating series to be 3007 and 3747 Da, and 1.25, respectively. These values are below the ~40000 molecular weight postulated by Russell et al. for commercial FTACPs, 3 which could result in our unique FTACP being more susceptible to biodegradation. (Eq. 3.1) (Eq. 3.2) (Eq. 3.3) As the intended FTACP structure was to contain three repeating units and resemble the suggested structure of commercial FTACPs (Figure 3.1A), 3 the monomeric species 8:2 FTAC, butyl acrylate and vinylidene chloride were all included in the polymerization. However, after close inspection of the mass spectrum (Figure B2) and a comparison of the experimental and theoretical isotopic pattern for the 1819 m/z signal (Figure B3), it was determined that neither butyl acrylate nor vinylidene chloride were incorporated into the FTACP. The peak spacing of 518 Da corresponds to the mass of 8:2 FTAC and confirms the successful polymerization of a FTACP. Our unique FTACP was determined to be solely a homopolymer of 8:2 FTAC containing hydrogen and hexadecylthiol end groups (Figure 3.1B), and have primarily between 2 to 16 fluorotelomer appendages. Though the two FTACPs shown in Figure 3.1 have structural differences, our unique FTACP (Figure 3.1B) possesses certain features that make it a suitable

117 93 surrogate to investigate the stability of commercial FTACPs. Firstly, the MALDI-TOF results demonstrated that our FTACP contains 8:2 fluorotelomer appendages covalently bound to the hydrocarbon backbone through an ester linkage. FTACs are the principal monomer used in the preparation of commercial FTACPs, 14,15 and have the same bonding of fluorotelomer appendages to the polymer backbone. Secondly, the side-by-side configuration of the 8:2 fluorotelomer appendages in our FTACP could render the ester moiety more sterically constrained than a commercial FTACP, which have additional interspersed non-fluorinated monomers. The presence of non-fluorinated monomers could affect the lability of the ester moieties making commercial FTACPs more susceptible to microbial hydrolysis. Thus, our unique FTACP likely represented a suitable experimental probe for assessing the lability of FTPs having moderate molecular weights. (A) (B) Figure 3.1: Proposed structure of commercial FTACPs where m = 5-13 and n = 1-17 (A) and structure of the unique FTACP used in this investigation containing hydrogen and hexadecyl thiol end groups (B).

118 Indirect Analysis of FTACP Biodegradation The observed intermediate and product trends for FTACP/Soil, FTACP/Plant and FTACP/Plant/Biosolids are consistent with the biotransformation of 8:2 FTOH to PFOA previously reported in aerobic soil, 10,11 which suggests either degradation of the unique FTACP or residual 8:2 FTOH or 8:2 FTAC. From the 50 mg of unique FTACP spiked into each pot, the 8:2 FTOH equivalence was calculated to be 8.88 x 10 4 nmole using the M n and M w values as outlined in Appendix B. As described earlier, residual 8:2 FTOH and 8:2 FTAC in the exhaustively purified FTACP material were estimated to be 4.71 and 2.63 nmole per gram of FTACP, which equates to as much as and nmole residual 8:2 FTOH and 8:2 FTAC per pot (Table 3.1). For the control pots, Soil Control and Plant/Biosolids Control, PFOA was observed to have the highest background level at 5.87 and 36.5 nmole, respectively. Therefore, the detection of intermediates and products, as much as 1800 nmole at 5.5 months, presumably resulted from the biotransformation of the unique FTACP and not from the conversion of residuals. FTACP biodegradation was inferred from the observed intermediates, 8:2 FTCA and 7:3 FTCA, 8:2 FTUCA and 7:3 FTUCA, and PFCA (C6-C9) products detected in soil, plant and catch plate (Tables B6-B11). The inclusion of the stable intermediate 7:3 FTCA and terminal PFCAs C6-C8 expands upon the conceptual model used by Russell et al. in their FTACP biodegradation study, 3 which solely considered the primary biotransformation of 8:2 FTOH to PFOA via 8:2 FTOH 8:2 FTCA 8:2 FTUCA 7:2 secondary FTOH (sftoh) PFOA. Unfortunately, the analysis of 8:2 FTOH was omitted from our investigation as any volatile products released from the soil and/or plants could not be quantified because the pots were exposed to the open atmosphere of the greenhouse; thus the overall proportion of products, as determined through indirect measurement, from FTACP degradation is likely reported here.

119 95 Table 3.1: Summed products for all FTACP potting conditions from 0 to 5.5 month along with the calculated 8:2 FTOH equivalent and estimated residual 8:2 FTOH and 8:2 FTAC levels. Summed Products (nmole) 8:2 FTOH Equivalent Time FTACP/Soil FTACP/Plant FTACP/Plant/ (nmole) (month) Biosolids 8.88 x x x x 10 1 Residuals (nmole) x x x :2 FTOH 2.36 x x x x :2 FTAC 1.32 x x x x 10 3 Incubation of the unique FTACP in all soil conditions resulted in the accumulation of perfluorohexanoate (PFHxA), perfluoroheptanoate (PFHpA) and PFOA concurrently with the reduction of 8:2 FTCA and 8:2 FTUCA as shown in Figure 3.2 for FTACP/Plant; similar trends are illustrated in Appendix B Figure B5 (FTACP/Soil) and B6 (FTACP/Plant/Biosolids). As expected, PFOA was the dominant product constituting 57, 70 and 80% of products in all microcosm compartments in FTACP/Soil, FTACP/Plant and FTACP/Plant/Biosolids, respectively (Table B13). The formation of the stable intermediate 7:3 FTCA is consistent with transformation pathways of 8:2 FTCA and 8:2 FTUCA observed in aerobic microbial degradation. 29 Subsequent dealkylation and defluorination steps of 7:3 FTCA presumably explains the production of PFHxA and PFHpA. 30 The accumulation of 7:3 FTCA, PFHxA and PFHpA varied depending on the experimental conditions, but all were observed to be minor products. The -oxidation of 8:2 FTOH to PFNA has been reported with different mammalian hepatocytes, but at 1% of the total stable products; PFNA was only observed within background levels in this study.

120 Amount of Analyte (nmole) Amount of Analyte (nmole) 96 (A) PFHxA PFHpA PFOA PFNA Soil 8:2 FTCA 8:2 FTUCA 7:3 FTCA 7:3 FTUCA Time (months) (B) PFOA 11% PFHxA Plant 40 89% 29% 71% months Soil Plant Time (months) Figure 3.2: Amount of FTACP degradation products observed in microcosm soil (A) and plant (B) for the FTACP/Plant condition. Inlaid pie charts showing the relative distribution of PFHxA and PFOA in soil and plant at 5.5 month. Measured concentration of target analytes in soil varied substantially amongst FTACP/Soil, FTACP/Plant and FTACP/Plant/Biosolids (Table 3.1) and detailed in Appendix B (Table B6 and B9), which suggests an influence between the degree of FTACP biodegradation and probable microbial activity. At 5.5 months, the summed analytes level in FTACP/Soil, FTACP/Plant and FTAC/Plant/Biosolids were determined to be 2.54 x 10 2 nmole, 6.94x 10 2 nmole and 1.80 x 10 3 nmole, respectively. Enhanced microbial activity has been demonstrated to increase with plant production, 34 and is consistent with the increase in analytes for

121 97 FTACP/Plant and FTACP/Plant/Biosolids. PFCAs and PFCA precursors arising from the WWTP biosolids themselves were accounted for using a control experiment in biosolid amended soil and an alfalfa plant (Plant/Biosolids Control), as described in Appendix B. The observed concentrations in these controls were significantly lower than those reported for FTACP/Plant/Biosolids. Target analytes were also observed to increase in alfalfa plants throughout incubation. Uptake into plants has previously been reported for PFOA and PFOS, consistent with the increase of PFHxA, PFHpA and PFOA levels in the plant as observed in this study (Figure 3.2B and Figure B6B). Plant PFOA levels were observed up to 44.0 ± 12.7 nmole and 49.3 ± 12.9 nmole for PFOA at 5.5 months for FTACP/Plant and FTAC/Plant/Biosolids (Table B7). The relative percentage of PFCAs that translocated into the plant decreased with increasing PFCA chain length (Table B12). Most intermediates in the plants fell below the LOD or were detected at levels <1 nmole (Table B10). Uptake presumably occurred from transpiration of soil water into plant compartments through vegetative transport tissues such as the xylem, which is consistent with higher PFOA and PFOS concentrations in vegetative compartments such as leaves and stalks, rather than storage compartments such as fruits, tubers, and grains, as previously reported. 35,36,38 The results presented here are consistent with an increased mobility of shorter chain PFCAs from soil into plants, which is supported by previous published reports. 39,40 After taking into consideration the translocation of the target analytes into the alfalfa plant there remains an unexplained reduction from 3.5 to 5.5 months. For example, a reduction of PFOA levels of 78, 29 and 13% was observed for FTACP/Soil, FTACP/Plant and FTACP/Plant/Biosolids, respectively. Leaching of analytes from the soil during watering is the

122 98 most plausible explanation; however, exact concentrations cannot be determined because the total volume of catch plate overflow was unknown. The observed PFOA reduction trends for FTACP/Soil, FTACP/Plant and FTACP/Plant/Biosolids presumably resulted from the higher organic matter content in FTACP/Plant and FTACP/Plant/Biosolids pots, which may have retained more PFCAs via sorption to the organic matter. Alternatively, the reduction in PFOA level could result from the degradation of PFOA to 2H-PFOA; as was previously proposed by Washington et al. 4 This pathway has not yet been corroborated by other studies, and attempts to detect 2H-PFOA in this study were unsuccessful (data not shown) Direct Analysis of FTACP Biodegradation Qualitative MALDI-TOF data of the unique FTACP extracted from soils, including peak signal intensity and relative intensity, are summarized in Appendix B Tables B14-B16. Previous aerobic biodegradation studies of other synthetic polymers such as poly(vinylpyrrolidone) and poly(ethylene glycol), utilized MALDI-TOF to evaluate structural changes of the polymer through mass shifts and decrease of signal intensity of the characteristic mass spectrum repeat pattern. 41,42 In our investigation, both mean and relative signal intensities were examined qualitatively to determine structural alterations to our unique FTACP. Prior to this study, the direct analysis of FTACP biodegradation was suggested to be impossible. 4 Microcosm soil extracts were first evaluated by comparing the observed signal intensities as a function of the number of 8:2 fluorotelomer appendage units. When the FTACP was incubated in soil (FTACP/Soil) the signal intensity from 1.5 to 5.5 months was observed to decrease by ~65% (Figure 3.3A). For pots containing an alfalfa plant (FTACP/Plant, Figure

123 Intensity (a.u.) Intensity (a.u.) Intensity (a.u.) B) and WWTP biosolids (FTACP/Plant/Biosolids, Figure 3.3C) a greater decrease in signal intensity of ~85% and ~80% was observed, which may indicate a greater degree of FTACP biodegradation. However, sorption of FTACP to organic matter could also explain the reduction in signal intensity upon addition of a plant and biosolids (ie. signal intensity FTACP/Soil > FTACP/Plant FTACP/Plant/Biosolids) (A) (B) (C) Figure 3.3: MALDI-TOF characterization of model FTACP in soil extracts for (A) FTACP/Soil, (B) FTACP/Plant and (C) FTACP/Plant/Biosolids conditions. Please note that these results are strictly qualitative.

124 100 The MALDI-TOF results were then re-plotted as a function of relative intensity with respect to the most abundant signal; 1301 m/z. In FTACP/Soil pots, the relative signal distribution 1.5, 3.5 and 5.5 months did not differ significantly (Figure 3.4A), whereas, an increased contribution from the higher order fluorotelomer units resulted in a change in the relative signal distribution from 1.5 to 3.5 and 5.5 months for FTACP/Plant and FTACP/Plant/Biosolids pots (Figure 3.4B and 3.4C). Additionally, a greater increase in the M n and M w was calculated from 1.5 to 3.5 and 5.5 months for FTACP/Plant and FTACP/Plant/Biosolid pots (Table B17). Thus, an increase in the alteration of FTACPs having a lower molecular weight (<6 fluorotelomer units) was observed in FTACP/Plant and FTACP/Plant/Biosolids pots when compared to the results in FTACP/Soil pots. This suggests that FTACPs of lower molecular weight are more rapidly degraded than FTACPs of higher molecular weight (ie. decreasing the number of fluorotelomer units increases the rate of degradation). For higher molecular weight FTACPs, it is conceivable that the degradation occurs via a two-step process with an initial slow fragmentation to lower molecular weight FTACPs followed by a faster degradation of the lower molecular weight FTACPs to terminal PFCAs. Because FTACPs have a tendency to aggregate with the fluorotelomer units facing inwards in a micelle-like fashion, FTACPs with a greater number of fluorotelomer units likely shield the ester moieties from microbes, whereas, FTACPs having fewer fluorotelomer units may leave the ester moieties more accessible to microbes. Evaluating the relative distribution over the duration of the experiment suggests that the reduction in signal intensity cannot be solely from sorption. Rather, there appears to be an increase in FTACP biodegradation in microbially enriched soils with biosolids, which was not explicitly considered in previous studies. 3,4 These results provide the first direct evidence of FTACP biodegradation, and is an important step towards determining their environmental fate. Current efforts are in progress to further improve

125 Relative Intensity (%) Relative Intensity (%) Relative Intensity (%) 101 our direct MALDI-TOF analysis by developing a quantitative method, which will help bridge the gap between qualitative MALDI-TOF and indirect analyses. 100 (A) (B) (C) Figure 3.4: Relative peak intensity of model FTACP in soil extracts for (A) FTACP/Soil, (B) FTACP/Plant and (C) FTACP/Plant/Biosolids conditions. Please note that these results are strictly qualitative.

126 Indirect versus Direct Analysis Indirect LC-MS/MS and direct MALDI-TOF analyses served as complementary methods to unequivocally confirm biodegradation of the unique FTACP. The quantification of intermediates and products by indirect analysis were consistent with the biotransformation of 8:2 FTOH, while structural changes to the FTACP throughout incubation were observed by direct analysis. Results of the two analyses suggest microbial hydrolysis of the ester moiety liberating 8:2 FTOH from the polymer backbone followed by subsequent biotransformation. Both methods indicated an increase in FTACP biodegradation in the presence of an alfalfa plant and WWTP biosolids. These conditions suggest a relationship between microbial activity and rate of FTACP biodegradation Estimating a FTACP Biodegradation Half-Life Biodegradation rates and half-lives were calculated with first-order kinetics using the 8:2 FTOH equivalent and the summation of all intermediates and products. Additionally, the halflife calculations assume that the biotransformation of 8:2 FTOH to PFOA occurs faster than FTACP to 8:2 FTOH. First-order rate constants calculated by both including and excluding the data at 5.5 months are reported in Table B5. If the data from 5.5 months was included, the halflife range was observed to be from years. However, if the first-order constants took into consideration the reduction in stable products from 3.5 to 5.5 months likely due to watering loss, then a more realistic approximation of first-order rate constants would exclude the data at 5.5 months. Using the 8:2 FTOH mole equivalent as the initial concentration (C o = 8.8 x 10 4 nmole) and 8:2 FTOH mole equivalent minus the total transformation products at t = 1.5 and 3.5 month,

127 103 a FTACP biodegradation half-life range of 8-18 years was calculated. For both half-life ranges, the FTACP biodegradation rate constants increased upon the addition of the alfalfa plant and WWTP biosolids with the following trend: FTACP/Soil < FTACP/Plant < FTACP/Plant/Biosolids. The low end of our estimation is close to the half-life of years approximated when Washington et al. corrected for FTACP particle-size surface area Environmental Implications The aim of the soil-plant microcosm was to simulate the biodegradation of FTACPs that may occur in farmlands, especially those amended with WWTP biosolids. This is an important consideration, as the discharge of FTACPs from manufacturing facilities, or release through laundering into wastewater, would ultimately lead to their accumulation in WWTP biosolids. Biodegradation of FTACPs, in addition to other fluorotelomer-based materials such as FTSAs and PAPs present in WWTP biosolids, 43,44 could be a contributor to the high concentrations of PFCAs and PFSAs reported in WWTP biosolids and sludge. 43,45-47 In the present study, the biodegradation of a unique FTACP was observed in three soil conditions (FTACP/Soil, FTACP/Plant and FTACP/Plant/Biosolids) using conventional indirect LC-MS/MS and a novel direct MALDI-TOF analysis. The indirect results showed elevated PFHxA, PFHpA and PFOA levels in the presence of a plant and WWTP biosolids with noteworthy fractions observed in the plants, which could be an important route of human exposure to PFCAs. The calculated FTACP biodegradation half-lives ranged from years assuming the transformation of 8:2 FTOH to PFOA is faster than FTACP to 8:2 FTOH. Although strictly qualitative, our MALDI-TOF method provided the first direct evidence of FTACP biodegradation with half-lives potentially on the order of months instead of years. Changes in the characteristic repeat pattern implied that

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133 109 CHAPTER FOUR Matrix Normalized MALDI-TOF Quantification of a Fluorotelomer-Based Acrylate Polymer (FTACP) Keegan Rankin and Scott A. Mabury Submitted to Environ. Sci. Technol. (Manuscript ID: es v) Contributions Prepared by Keegan Rankin with editorial comments provided by Scott Mabury. Keegan Rankin performed all experimental work related to this project.

134 Abstract The degradation of fluorotelomer-based acrylate polymers (FTACPs) has been hypothesized to serve as a source of the environmental contaminants, perfluoroalkyl carboxylates (PFCAs). Studies have relied on indirect measurement of presumed degradation products to evaluate the environmental fate of FTACPs; however, this approach leaves a degree of uncertainty. The present study describes the development of a quantitative matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry method as the first direct analysis method for FTACPs. The model FTACP used in this study was poly(8:2 FTAC-co- HDA), a copolymer of 8:2 fluorotelomer acrylate (8:2 FTAC) and hexadecyl acrylate (HDA). Instead of relying on an internal standard polymer, the intensities of 40 poly(8:2 FTAC-co-HDA) signals ( Da) were normalized to the signal intensity of a matrix-sodium cluster (659 Da). We termed this value the normalized polymer response (P N ). By using the same dithranol solution for the sample preparation of poly(8:2 FTAC-co-HDA) standards, calibration curves with coefficient of determinations (R 2 ) typically >0.98 were produced. When poly(8:2 FTACco-HDA) samples were prepared with the sample dithranol solution as the poly(8:2 FTAC-co- HDA) standards, quantification to within 25% of the theoretical concentration was achieved. This approach minimized the sample-to-sample variability that typically plagues MALDI-TOF, and is the first method developed to directly quantify FTACPs. 4.2 Introduction Fluorinated polymers are the largest class of commercial fluorochemical products. Despite this fact, there is little known about their environmental fate and potential impact due to

135 111 difficulty in developing analytical methods to directly measure changes in the formal polymer structure. Fluorotelomer-based acrylate polymers (FTACPs) are a class of fluorinated polymers widely used as antiwetting and antistaining agents in the textile, upholstery, carpet and paper industries. 1 Specifically, FTACPs are copolymers prepared from fluorotelomer acrylates (FTACs), hydrocarbon acrylates, and often other non-fluorinated monomers. 2,3 Similar to other fluorinated polymers, FTACPs benefit from improved repellency, lubricity, and chemical and thermal stability through the replacement of hydrogen with fluorine. 4 Consequently, the properties that make FTACPs ideal for industrial applications have also raised concern about their environment fate. It is believed that degradation of FTACPs lead to the ubiquitous and persistent perfluoroalkyl carboxylates (PFCAs). 5 Because long-chain PFCAs (>7 perfluorinated carbons) have been demonstrated to accumulate in biota 6,7 and the desired antiwetting and antistaining properties of the original FTACP formulation required fluorotelomer acrylates having 8 perfluorinated carbons, 8-10 there is significant interest in directly assessing the environmental fate of FTACPs. Previous efforts have aimed at evaluating the degradation of FTACPs indirectly by measuring the transformation products (i.e., PFCAs) by high performance liquid chromatography tandem mass spectrometry (LC-MS/MS). 5,11 However, PFCAs are also known transformation products of other fluorotelomer-based material such as FTACs and fluorotelomer alcohols (FTOHs), which have been reported as residuals in the crude FTACP material at levels <5% (m/m). 18 Thus, indirect analysis FTACPs degradation often requires the measurement of transformation products above a high background signal. Alternatively, degradation of FTACP itself could be monitored using a direct analysis method. Our group recently developed a qualitative matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry method to investigate the degradation of FTACPs as a

136 112 complementary method to conventional LC-MS/MS. 19 Although qualitative, the MALDI-TOF results clearly indicated alterations to the FTACP s chemical structure caused by microbial degradation. Besides this qualitative MALDI-TOF method, there doesn t appear to be another direct analysis methods for FTACPs. MALDI-TOF is a technique often used to estimate molecular distributions (weight average molecular weight, M w, number average molecular weight, M n, and polydispersity, PD) and provide structural characterization of synthetic polymers Despite having been introduced in the 1980s, 24,25 MALDI-TOF has remained primarily a qualitative analytical technique because of poor sample-to-sample reproducibility. Successfully obtaining a MALDI mass spectrum first requires a sample preparation that is compatible with the synthetic polymer properties to ensure intimate contact with the matrix; 26 considering their chemical diversity, this is not trivial. Conventional solvent-based sample preparations, such as the dried droplet method, rely on proper matrix and solvent selection to increase the likelihood of co-crystallization of the polymer and matrix upon solvent evaporation. To some degree, an appropriate matrix can often be selected with prior knowledge regarding the relative hydrophobicity and polarity, 27,28 but may require tailoring of unique sample preparation for more problematic polymers. 29,30 It is preferable to use a single or azeotropic solvent system that allows the polymer, matrix and cationization agent to be prepared together In addition, the rate of solvent evaporation is a contributing factor to polymer and matrix co-crystallization with faster evaporating solvents improving the sample homogeneity. 34,35 Modifications to the deposition method using thin- 36,37 and seed-layered, 38 and electrospray have been reported to further enhance sample homogeneity and improve the shot-to-shot reproducibility. However, even with these improvements, MALDI-TOF remains difficult to use for quantification with sufficient precision.

137 113 A select number of studies have recently emerged reporting quantitative MALDI-TOF analysis of microcystins, 42 polymer additives, 43 saccharides, 44 peptides, 45,46 proteins 47,48 and synthetic polymers For synthetic polymers, use of an internal standard polymer is possible if it has similar chemical properties to the target polymer to ensure no discrimination during sample preparation and ionization. In addition, the mass spectrum of the internal standard polymer cannot overlap with that of the target polymer. The signal intensities or peak areas of the target polymer are then normalized to those of the internal standard polymer. The result minimizes sample-to-sample variability caused by differences in desorption, ionization and crystallization. Although studies have shown this to be an effective approach of quantifying synthetic polymers, processing two overlaid polymer mass spectra can be rather tedious and time consuming. This makes the work of Ahn et al. on the MALDI-TOF quantification of peptides using the matrix itself as a pseudo internal standard an appealing alternative. 52 The authors demonstrated that a linear calibration curve can be generated by taking the ratio of peptide to matrix signal intensities if the peptides concentration does not cause matrix suppression. If applicable for synthetic polymers, this approach would obviate the need for an internal standard polymer because the matrix itself would serve as an internal standard. The aim of the present study was to develop a MALDI-TOF method using the matrix signal as an internal standard to direct quantify FTACPs. Future application of this method would provide quantitative information regarding the environmental fate of FTACP and related fluorinated polymers. The model FTACP used in this study was poly(8:2 FTAC-co-HDA) copolymerized from 8:2 fluorotelomer acrylate (8:2 FTAC) and hexadecyl acrylate (HDA), with its chemical structure shown in Figure 4.1. Using a similar approach to Ahn et al., 52 the intensity of an apparent matrix-cation cluster was used to normalize poly(8:2 FTAC-co-HDA) signal intensities to minimize the sample-to-sample variability. Calibration curves were generated

138 114 using a series of poly(8:2 FTAC-co-HDA) standards, which allowed for reliable MALDI-TOF quantification whenever the same matrix solution was used to prepare both the poly(8:2 FTACco-HDA) standards and samples. To supplement our understanding of sample homogeneity, scanning electron microscopy (SEM) was used to provide surface distribution images following crystallization. Figure 4.1: Structure of poly(8:2 FTAC-co-HDA) characterized from a previous study Experimental Materials 1,8,9-Anthacenetriol (dithranol, 98.5%), methyl tert-butyl ether (MTBE, 99%), sodium trifluoroacetate (NaTFA, 98%) and tetrahydrofuran (THF, 99%) were all purchased from Sigma Aldrich (St. Louis, MO). Graphitic pencils (8B, 4B, 2B and HB) were purchased from Faber-Castell USA Inc. (Cleveland, OH). The model FTACPs, poly(8:2 FTAC-co-HDA), was synthesized in-house as outlined in a previous study 35 and are provided in Appendix C Sample Preparation Poly(8:2 FTAC-co-HDA) prepared as standards and samples were dissolved in THF at concentrations of 1, 2, 5, 10, 15, 20 or 25 mg ml -1. Dithranol served as the matrix and was

139 115 prepared in THF at concentrations of 5, 10 or 20 mg ml -1. NaTFA served as the cationization agent and was prepared in THF at concentrations of 1, 5 or 10 mg ml -1. Each sample was then prepared with a FTACP:matrix:cationization agent (A:M:Cat) mixing ratio of 5:10:1. A 1 L aliquot of the prepared poly(8:2 FTAC-co-HDA) standard or sample was then deposited onto a MALDI target plate using the dried droplet method, and allowed to dry under ambient conditions. For graphite support preparation, a layer of graphitic pencil lead (8B, 4B, 2B and HB) was applied to the MALDI plate. The poly(8:2 FTAC-co-HDA) standard or sample was then deposited onto the modified MALDI plate MALDI-TOF Instrumentation All mass spectra were acquired using an AB SCIEX 4800 MALDI TOF/TOF mass spectrometer (AB SCIEX, Framingham, MA) equipped with a 355 nm Nd:YAG operated in positive ion reflector mode. The mass range studied was ,000 Da with a focus mass of 2000 Da. Each mass spectrum was acquired by randomly rastering the laser over a uniform area of the poly(8:2 FTAC-co-HDA) standard or sample with a total of 8000 laser shots per spectrum. The summed data were processed using AB SCIEX Data Explore V4.9 mass spectrometry software Scanning Electron Microscopy (SEM) A Quanta FEG 250 Environmental SEM (FEI Company, Hillsboro, OR) was used to evaluate surface distribution. SEM images were acquired at 250x magnification using a large

140 116 field detector (LFD) under low vacuum (130 Pa) with the electron beam operated with a 10 kv voltage and a 5 na current Extraction Method Poly(8:2 FTAC-co-HDA) (n = 3) was extracted from 2.5 ml aqueous solutions by vortexing for 1 minute and sonicating for 15 minutes with 5 ml of MTBE. The extraction was repeated three times, and then the combined MTBE extracts were blown to dryness under a gentle stream of nitrogen. 4.4 Results and Discussion Graphite Support Preparation Prior to developing the quantitative MALDI-TOF method, the application of a graphite support was investigated as a means to improve sample homogeneity, shot-shot reproducibility, and enhanced signal intensity. Gorka et al. previously reported an 8-fold enhancement in signal intensity for peptides and proteins when graphite flakes were applied to the surface of the MALDI target plate. 53 Graphite flakes were first investigated as a graphite support in this study; however, the application of graphitic pencil lead (8B, 4B, 2B and HB) to the MALDI target plate were observed to produce a more even coating compared to graphite flakes. The relative percentage of graphite in the graphitic pencil leads is defined by their degree of Blackness = B and Hardness = H with 8B pencils having a high percentage and HB having a low percentage. Although all four graphitic pencil leads produced a homogeneous layer of small dithranol crystals (Figure C1), 8B lead was used exclusively because it was significantly less abrasive.

141 117 Crystallization of dithranol and poly(8:2 FTAC-co-HDA) in the absence and presence of a graphite support was investigated by SEM at a magnification of 250x, as shown in Figure C2. When dithranol was prepared with NaTFA at a mixing ration of 10:1 (respective concentration of 20 and 10 mg ml -1 ), dithranol crystals were observed with diameters up to 200 m in the absence of the graphite support (Figure C2A). Whereas, in the presence of the graphite support, the diameter of dithranol crystals were at least four times smaller at <50 m (Figure C2B). When a poly(8:2 FTAC-co-HDA) sample was prepared with dithranol and NaTFA at 5:10:1 (respective concentration of at 25, 20 and 10 mg ml -1 ) was deposited onto the MALDI target plate in the absence of a graphite, co-crystallization was observed in a spherical rings upon solvent evaporation (Figure C2C). In the presence of the graphite support, co-crystallization of poly(8:2 FTAC-co-HDA) and dithranol was observed to be more homogeneous, and spherical rings were absent (Figure C2D). Rather, there appeared to be a reduction in crystal size with an overall increase in the number of crystals per surface area. The diameter of crystals observed in Figures C2C and C2D are not reported because they tended to decrease in diameter moving from the perimeter towards the center of the MALDI target plate. It is apparent that the presence of a graphite support resulted in a more homogeneous distribution and higher number of crystals per surface area, which is consistent with the work of Gorka et al. who reported a thinner and more homogeneous layer of -cyano-4-hydroxycinnamic acid (CHCA) crystals. 53 Formation of smaller crystals likely resulted from the graphite particles acting as nucleation centers for matrix molecules. 54 This increases the rate of solvent evaporation, and in turn prevents growth of larger crystals. Producing smaller crystals is advantageous because it improves reproducibility, sensitivity and enhance signal intensity. 39,55,56

142 118 In the present investigation, the signal intensity of the observed dithranol-sodium cluster at 659 Da was ~4.5-fold higher (1.5 x 10 4 to 6.8 x 10 4 a.u.) in the presence of a graphite support (Table C1). Similarly, when a poly(8:2 FTAC-co-HDA) sample was analyzed in the presence of a graphite support, the signals associated with poly(8:2 FTAC-co-HDA) were observed to be ~2.5-fold higher (ie. 9.5 x 10 3 to 2.3 x 10 4 a.u. for a single poly(8:2 FTAC-co-HDA) signal at 1429 Da), and the dithranol-sodium cluster was ~3.5-fold higher (2.2 x 10 4 to 7.8 x 10 4 a.u.) (Table C1). Although the increase in signal intensity wasn t as significant as reported by Gorka et al., 53 the application of a graphite support to the MALDI target plate at least doubled the poly(8:2 FTAC-co-HDA) signal intensities. More importantly, there was an improvement in sample homogeneity and an increase in the number of crystals per surface area when a graphite support was used Development of P N Method The mass spectrum of poly(8:2 FTAC-co-HDA) was observed to have an abundance of signals ranging from ,000 Da as shown in Figure C3. In our previous work, the structural characterization of poly(8:2 FTAC-co-HDA) was determined based on the signal spacing of 518 and 296 Da corresponding to the molecular weight of 8:2 FTAC and HDA. 30 The M w, M n and PDI reported in that work were 3080, 2370 and 1.3, respectively. The approach used to quantify poly(8:2 FTAC-co-HDA) by MALDI-TOF in the present study was to normalize the signal intensities of poly(8:2 FTAC-co-HDA) to the signal intensity of a dithranol-sodium cluster at 659 Da. Because poly(8:2 FTAC-co-HDA) had a wide polydispersity, development of the calibration curve focused on 40 of the strongest signals from Da. As shown in Figure 4.2, the dithranol-sodium cluster at 659 Da signal was observed both in the absence (Figure

143 A) and presence of poly(8:2 FTAC-co-HDA) (Figure 4.2B). For both Figures 4.2A and 4.2B, NaTFA served as the cationization agent. In the absence of NaTFA, the signal at 659 Da was no longer observed (Figure C4), which suggests it is a dithranol-sodium cluster. This dithranolsodium cluster was consistently observed in all mass spectra obtained in this study, and was investigated as a means of normalizing the poly(8:2 FTAC-co-HDA) signals to reduce sampleto-sample variability. The intensities of the 40 poly(8:2 FTAC-co-HDA) signals ( Da) were normalized individually to the intensity of the dithranol-sodium cluster at 659 Da (Eq. 4.1). Specifically, the normalized polymer response (P N ) shown in Eq. 4.1 was calculated from the polymer signal intensity (P i ) and matrix signal intensity (M i ). Summation of the 40 individually normalized polymer signals was defined as P N, and was determined for each poly(8:2 FTAC-co- HDA) mass spectrum. Our working hypothesis was that using the same dithranol solution to prepare a set of poly(8:2 FTAC-co-HDA) standards a calibration curve of P N vs. poly(8:2 FTACco-HDA) concentration could be achieved. If confirmed, then the calibration curve would allow for quantification of poly(8:2 FTAC-co-HDA) in a sample. { } (Eq. 4.1)

144 % Intensity Relative Intensity (%) % Intensity % Intensity Relative Intensity (%) % Intensity Intensity (a.u.) Intensity (a.u.) Reflector Spec #1[BP = 519.0, 64812] Reflector Spec #1[B P = 519.0, 64812] x10 4 A 6.5E+4 6.5x M ass (m/z) m/z 4700 Reflector Spec #1[BP = 659.2, 86580] Mass (m/z) x E+4 B E Reflector S pec #1[BP = 659.2, 86580] M ass (m/z) 8.7x E Mass (m/z) Figure 4.2: MALDI-TOF mass spectra of (A) dithranol and (B) poly(8:2 FTAC-co-HDA). Inset panels show the reference matrix signal (659.2 m/z) used to normalize the poly(8:2 FTAC-co- HDA) signals. When P N was plotted vs. poly(8:2 FTAC-co-HDA) standard concentrations (1, 2, 5, 10, 15, 20 and 25 mg ml -1 ), a non-linear behavior was observed (Figure C5). The corresponding P N for each poly(8:2 FTAC-co-HDA) standard are presented in Table 4.1. Ahn et al. previously reported that the degree of matrix signal depends on the analyte concentration, and the degree of

145 121 matrix suppression moves from normal to anomalous. 52 Within this normal region (<70% matrix suppression), reliable quantification of peptides was achieved using the signal intensity ratio of matrix-to-analyte. These results suggested that the MALDI analysis in this study was also suffering from matrix suppression at higher poly(8:2 FTAC-co-HDA) concentrations. However, when the matrix signal intensities were compared in this study, only a moderate reduction in the 659 Da signal was observed for poly(8:2 FTAC-co-HDA) standards prepared with a 5 mg ml -1 dithranol solution (Figure C6). Poly(8:2 FTAC-co-HDA) standards prepared with 20 and 10 mg ml -1 dithranol solutions showed no obvious decrease in the 659 Da signal intensity. The complete list of the matrix signal intensities are presented in Table C2. If the nonlinear behavior between the P N and poly(8:2 FTAC-co-HDA) concentration was caused by matrix suppression, a reduction in the matrix signal intensity would have been expected for all three dithranol concentrations. When the data was re-plotted as P N vs. Ln[Poly(8:2 FTAC-co- HDA) concentration], the results fit a logarithmic regression with acceptable coefficient of determinations (R 2 ) >0.97 (Table 4.2) independent of dithranol concentration. Although the concentration of dithranol did not impact the non-linear behavior, it had a clear impact on the magnitude of P N response observed, as shown in Figure 4.3A and corresponding equations for the regression lines presented in Table 4.2. When corrected for the weight of dithranol used in the sample preparation, the P N for each poly(8:2 FTAC-co-HDA) standards was observed to increase as a function of dithranol concentration (5 < 10 < 20 mg ml - 1, Table C3), which suggests the desorption efficiency of poly(8:2 FTAC-co-HDA) molecules was decreasing. It is known that desorption depends on the rapid sublimation of matrix molecules transferring intact analyte molecules into the gas phases. Because FTACPs have a tendency to aggregate, 30 decreasing the molar ratio of dithranol to poly(8:2 FTAC-co-HDA) would favor aggregation, and reduce the number of poly(8:2 FTAC-co-HDA) molecules

146 122 Table 4.1: Normalized polymer response (P N ) for poly(8:2 FTAC-co-HDA) standards using dithranol concentrations of 20, 10 and 5 mg ml -1. A mixing ratio of 5:10:1 was used for each sample with NaTFA prepared at a concentration of 10 mg ml -1. Dithranol Concentration 20 mg ml mg ml -1 5 mg ml -1 Poly(8:2 FTAC-co-HDA) Concentration (mg ml -1 ) Normalized Polymer Response RSD (%) Normalized Polymer Response RSD (%) Normalized Polymer Response RSD (%) desorbed into the gas phase. Consequently, P i would decrease causing a reduction in the magnitude of P N. To illustrate how the molar ratio of dithranol to poly(8:2 FTAC-co-HDA) differed, the molar ratio was plotted as a function of poly(8:2 FTAC-co-HDA) concentration (Figure C7). As shown in Figure C7, the dithranol to poly(8:2 FTAC-co-HDA) molar ratio decreased with increasing poly(8:2 FTAC-co-HDA) concentration following the derivative of Ln[x] (Eq. 4.2). Therefore, if decreasing molar ratio favors poly(8:2 FTAC-co-HDA) aggregation, then a reduction in desorption efficiency with increasing poly(8:2 FTAC-co-HDA) concentrations likely explains the non-linear behavior observed.

147 123 (Eq. 4.2) However, there are other possible explanations for the non-linear behavior, which could have occurred during the ionization process. Similar to most synthetic polymers, FTACPs required a cationization agent to form a charged species. In this study, NaTFA served as the cationization agent because of its solubility in THF and the abundance of ester moieties in the poly(8:2 FTAC-co-HDA) structure (Figure 4.1). To explore the impact of NaTFA, a series of poly(8:2 FTAC-co-HDA) standards were analyzed using three different concentrations of NaTFA (1, 5 and 10 mg ml -1 ). As shown in Figure 4.3B, all three calibration curves were similar and showed little dependence on NaTFA concentration, and therefore is not likely the explanation for the observed non-linear behavior. The corresponding equations for the regression lines are presented in Table 4.2. Table 4.2: Summary of external calibration curves obtained at various dithranol and NaTFA concentration. Sample Slope X = 1 R 2 Dithranol 20 mg ml Dithranol 10 mg ml Dithranol 5 mg ml NaTFA 10 mg ml NaTFA 5 mg ml NaTFA 1 mg ml

148 Normalized Polymer Response (P N ) Normalized Polymer Response (P N ) Matrix 20 mg/ml 10 mg/ml 5 mg/ml A Ln[Poly(8:2 FTAC-co-HDA) Concentration] Cat. Agent 10 mg/ml 5 mg/ml 1 mg/ml B Ln[Poly(8:2 FTAC-co-HDA) Concentration] Figure 4.3: Calibration curves obtained using (A) dithranol concentration of 20, 10 and 5 mg ml -1 and (B) NaTFA concentration of 10, 5 and 1 mg ml -1. For dithranol experiments (A), NaTFA was prepared at 10 mg ml -1. For NaTFA experiments (B), dithranol was prepared at 20 mg ml -1. An alternative explanation relates to the total number of poly(8:2 FTAC-co-HDA) ions entering the TOF mass spectrometer. Once the pulsed photons strike the MALDI target plate, a

149 125 plume of neutrals and charged species are accelerated and focused by a series of short voltage pulses (i.e., time-lag focusing) prior to entering the TOF field-free region. In this method, these voltages, along with the laser parameters, were unchanged in order to maintain a consistency throughout all analyses. It is therefore conceivable that these operating voltages restricted a maximum number of total number of poly(8:2 FTAC-co-HDA) ions entering the field-free region. If true, this would presumably be reflected in a flattening of the signal response similar to the non-linear behavior reported herein. Also, the ability to detect the correct number of poly(8:2 FTAC-co-HDA) ions is another possible explanation for the non-linear behavior, and could result from saturation of the micro-channel plate (MCP) detector. The consequence of which can result in mass discrimination caused by a lower detection efficiency. 57 Although the mass range in this study was up to 10 kda, MCP saturation tends to occur when analyzing with a mass ranges over 10s of kda, such as proteins. 58 A comparison of the mass spectra acquired for 1 to 25 mg ml -1 poly(8:2 FTAC-co-HDA) standards showed no obvious differences, and suggests MCP saturation did not occur under these conditions. Based on the observation made in the present study, a decrease in desorption efficiency was the most plausible explanation for the non-linear behavior observed between P N and poly(8:2 FTAC-co-HDA) standards. The results for all calibration curves obtained at various dithranol and NaTFA concentrations indicated reasonable fitness to the linear regressions (R 2 >0.97; Table 4.2). The calculated P N for each poly(8:2 FTAC-co-HDA) standard had relative standard deviations (RSDs) <10% with few exception (Tables 4.1 and C3).

150 Inter- and Intra-Day Variability The reproducibility of P N calibration curves were investigated over several weeks (interday) and multiple replicates within a single day (intra-day). The results are presented graphically in Figure 4.4 and numerically in Table 4.3. For the inter-day experiment (Figure 4.4A), three different sets of poly(8:2 FTAC-co-HDA) standards (1, 2, 5, 10, 15, 20 and 25 mg ml -1 ) were analyzed on separate days over three weeks. Each set of standards was prepared separately with dithranol (20 mg ml -1 ) and NaTFA (10 mg ml -1 ) at a mixing ratio of 5:10:1. The slopes of the linear regressions were observed to range from (Table 4.3). Although there were differences in the regression slopes between weeks 1, 2 and 3, the fitness for each week were consistent with R 2 >0.97 and RSD for each poly(8:2 FTAC-co-HDA) standard <10% (Table C4). The range in regression slopes likely resulted from minor differences in the sample preparations or behavior of the instrument on a week-to-week basis. Table 4.3: Summary of external calibration curves obtained over consecutive weeks (Inter-Day), and within a single day (Intra-Day). Sample Slope Intercept R 2 Week 1 (Inter-Day) Week 2 (Inter-Day) Week 3 (Inter-Day) Replicate 1 (Intra-Day) Replicate 2 (Intra-Day) Replicate 3 (Intra-Day)

151 Normalized Polymer Response (P N ) Normalized Polymer Response (P N ) Inter-Day Week 1 Week 2 Week 3 A Ln[Poly(8:2 FTAC-co-HDA) Concentration] Intra-Day Replicate 1 Replicate 2 Replicate 3 B Ln[Poly(8:2 FTAC-co-HDA) Concentration] Figure 4.4: Calibration curves obtained (A) replicated over a three-week period and (B) replicated three times within a single day. For all experiments, dithranol and NaTFA were prepared at 20 and 10 mg ml -1, respectively. The intra-day experiment was performed by analyzing a single set of poly(8:2 FTAC-co- HDA) standards (1, 2, 5, 10, 15, 20 and 25 mg ml -1 ) using three different dithranol (20 mg ml -1 ) and NaTFA (10 mg ml -1 ) solutions within a single day. As shown in Figure 4.4B, the linear

152 128 regressions slopes from the three replicates were similar and ranged from (Table 4.4). The magnitude of P N was similar amongst the three replicate of the each standard, and once again all measured P N had RSD <10% (Table C5). Similar to the inter-day experiment, the linearity of the regressions were consistent with R 2 >0.98. The ability to obtain a fairly reproducible calibration curves while using separate dithranol solutions suggests that the P N method can mitigate variable sample preparations and MALDI-TOF analyses within a single day. Because the precision of the individual inter-day P N calibration curve was deemed acceptable, any variation appears to be systematic, and repeated analyses of a single set of poly(8:2 FTAC-co-HDA) standards within a given day (intra-day) yielded similar P N calibration curves. The ability to obtain linear calibration curves between P N vs. Ln[Poly(8:2 FTAC-co- HDA) concentration] reproducibly suggests the P N method could be used quantify poly(8:2 FTAC-co-HDA) samples with appropriate calibration in a given day by preparing both the standards and samples with the same dithranol solution Testing the P N Method The P N method was first tested using a set of three poly(8:2 FTAC-co-HDA) samples (5, 10 and 20 mg ml -1 ) and quantified using four separate calibration curves over several days. The poly(8:2 FTAC-co-HDA) samples (n = 3) were weighed and then dissolved in THF giving theoretical concentrations of 5.0, 10.9 and 20.4 mg ml -1 (Table C6). Preparation of the samples and each set of poly(8:2 FTAC-co-HDA) standards was performed using the same dithranol solution on the given day of analysis. Thus, calculation of P N values would be consistent between the samples and standards for each calibration curve. Calibration curves had acceptable linearity with R 2 >0.98, and are presented along with their corresponding regression equations in

153 129 Table C7. With each calibration curve, analysis of individual samples was replicated nine times. The sample concentrations determined from each of the four calibration curves were then combined, and are reported as measured concentration in Table C6. The measured concentrations were 4.0 (RSD 11%), 13.2 (RSD 17.3%) and 22.6 mg ml -1 (RSD 6.50%), and were within 20% of the theoretical concentrations. To further test the P N method, poly(8:2 FTAC-co-HDA) samples (5, 10 and 20 mg ml -1 ) were spiked into deionized and river (collected from the Etobicoke Creek in Brampton, ON) water, along with a poly(8:2 FTAC-co-HDA) control in the absence of an aqueous media. Following extraction with MTBE, the extracts were prepared for analysis with a set of poly(8:2 FTAC-co-HDA) standards using the same dithranol solution as described above. The calibration curves used to validate the P N method had acceptable linearity with R 2 >0.98, and are presented along with their corresponding regression equations in Table C8. Each sample was extracted in triplicate (n = 3), and then analyzed three times. The mean poly(8:2 FTAC-co-HDA) concentrations ranged from , and mg ml -1, and were typically measured within 25% of the theoretical concentrations of 4.67, 10.6 and 22.2 mg ml -1 (Table 4), with recoveries ranging from %. In addition to the above tests samples, a poly(8:2 FTAC-co-HDA) sample (n = 5) from an on going hydrolysis study was measured to be 22.9 ± 2.45 mg ml -1, and was within 9% of the theoretical concentrations of 25.1 mg ml -1 (Table 4.4). The results from the different test samples demonstrated the reasonable effectiveness and reliability of the P N method to quantify poly(8:2 FTAC-co-HDA) samples having different concentrations.

154 130 Table 4.4: Method test for MALDI-TOF external calibration curves to quantify poly(8:2 FTAC-co-HDA) recovered from blank tube and aqueous media. Theoretical (mg ml -1 ) Control Recovery (mg ml -1 ) n = 3 Milli-Q Recovery (mg ml -1 ) n = 3 River Recovery (mg ml -1 ) n = 3 Hydrolysis Sample (mg ml -1 ) n = ± ± ± ± ± ± ± ± ± ± 2.45 Recovery (%)

155 Environmental Implications The results from this study demonstrated the quantitative MALDI-TOF analysis of poly(8:2 FTAC-co-HDA) using our P N method to develop reproducible calibration curves. By using the same dithranol solution in the sample preparation for samples and standards, P N values were calculated from the ion abundance ratio of poly(8:2 FTAC-co-HDA) to a dithranol-sodium cluster can be directly compared. This approach minimized the sample-to-sample irreproducibility of MALDI-TOF by normalizing with the same dithranol solution, and did not require an internal standard polymer. Considering MALDI-TOF is not as precise as other analytical methods such as GC-MS and LC-MS/MS, quantifying a poly(8:2 FTAC-co-HDA) sample within 25% is promising. Application of our P N method to future FTACP degradation studies could provide direct quantitative information about their environmental fate. For example, if poly(8:2 FTAC-co- HDA) degrades there will be specific changes in MALDI-TOF mass spectrum. The P N method would quantify the magnitude of the poly(8:2 FTAC-co-HDA) degradation based on a reduction in intensity to any of the 40 signals, and have a corresponding smaller calculated P N. Resulting degradation products, such a smaller FTACP oligomers, would be excluded from the calculation of P N because they have a different m/z not being monitored. Therefore, the P N method could quantify concentration change of a FTACP over time, and provide the first-ever unequivocal information about the environmental fate of FTACPs.

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162 138 CHAPTER FIVE A Global Survey of Perfluoroalkyl Carboxylates (PFCAs) and Perfluoroalkane Sulfonates (PFSAs) in Surface Soils: Distribution Patterns and Mode of Occurrence Keegan Rankin, Scott A. Mabury, Thomas M. Jenkins and John W. Washington Submitted to Environ. Sci. Technol. Contributions Prepared by Keegan Rankin with editorial comments provided by John Washington and Scott Mabury. Keegan Rankin performed all extractions at the Environmental Protection Agency s National Exposure Research Laboratory (Athens, GA). Keegan Rankin and John Washington performed LC-MS/MS analyses at the University of Toronto and National Exposure Research Laboratory (Athens, GA), respectively.

163 Abstract The global distribution of 32 perfluoroalkyl and polyfluoroalkyl substances (PFASs) in surface soils was determined in both urban and rural locations from all continents (North America n = 33, Europe n = 10, Asia n = 6, Africa n = 5, Australia n = 4, South America n = 3 and Antarctica n = 1) using ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) systems. Quantifiable levels of perfluoroalkyl carboxylates (PFCAs; PFHxA- PFTeDA14) were observed in all samples with total concentrations ranging from pg/g. Perfluoroalkane sulfonates (PFSAs; PFHxS, PFOS and PFDS) were detected in all samples but one, ranging from <LOQ-3270 pg/g. Perfluorooctanoate (PFOA) and perfluorooctanesulfonate (PFOS) were the most commonly detected analytes at concentrations up to 2670 and 3100 pg/g, respectively. Interestingly, the concentration of all PFCAs minus PFOA was observed to be roughly 2 higher than the concentration of PFOA. The geometric mean PFCA and PFSA concentrations were observed to be higher in the northern hemisphere (930 and 170 pg/g) compared to the southern hemisphere (190 and 33 pg/g). Samples from the continental USA were found to have a longitudinal trend of increasing PFCA and PFSA concentrations eastwardly from mid-continent. Principal component analysis (PCA) of PFCA and PFSA compositions were similar amongst most locations with only a few locations having statistical differences, which were found to be related to the proximity to urban and point sources. These results suggest PFCAs and PFSAs are globally distributed in surface soils and indicate that soils could be a significant perfluoroalkylate reservoir on the global scale.

164 Introduction Perfluoroalkyl and polyfluoroalkyl substances (PFASs) are widely used as surfactants and surface protectants possessing a perfluorinated moiety (C n F 2n+1 ) with many analogues exhibiting high chemical and thermal stability. 1,2 Consequently, the inherent properties that make PFASs ideal for commercial applications also render many persistent and ubiquitous in the environment. Perfluoroalkyl carboxylates (PFCAs) and sulfonates (PFSAs) are two groups of PFASs that have received considerable attention due to their widespread detection in the precipitate, 3 fresh and sea water, 4-6 sediment, 7,8 and wildlife and humans. 9,10 Historically, PFCAs and PFSAs have been emitted to the environment directly, or indirectly through the biotic and abiotic transformation of polyfluorinated precursors. The global distribution of PFCAs and PFSAs in the terrestrial environment likely results from a combination of gas- and particle-phase atmospheric long-range transport (LRT) Volatile precursors such as fluorotelomer alcohols (FTOHs) and perfluoroalkyl sulfonamides (FASAs), are known residuals in 14 and likely degradation products of surfactants and side-chain fluorinated polymers, and have been measured globally in the atmosphere Both FTOHs 23 and FASAs 24 undergo atmospheric oxidation to produce PFCAs, and PFSAs for sulfonamide-based materials. 23,24 Evidence of PFCAs and PFSAs in rural and urban precipitation 25,26 support LRT of volatile precursors as a mode of global dissemination. Because PFCAs and PFSAs have low acid dissociation constants (pk a ), negligible vapor pressures and relatively high water solubility, 27,28 it has been posited that oceans are the dominant global PFCA and PFSA reservoir. 29 Despite the knowledge that PFCAs and PFSAs also exhibited a moderate affinity for sorbing to solid matrices such as sediments, 30,31 little is known about their fate in the terrestrial environment.

165 141 Soils are known to be an important sink for other persistent organic pollutants (POPs) such as polybrominated diphenyl ethers (PBDEs), polychlorinated biphenyls (PCBs) and polychlorinated dibenzo-p-dioxins and dibenzofurans (PCDD/Fs) In addition to locally contaminated soils, atmospheric LRT has led to numerous POPs being globally distributed in background soils, making remote background soils a key component in global mass-balance estimates of POPs as well. 34 Despite having been detected globally, little is known about PFCAs and PFSAs in soils, and to date, studies largely have focused on soils impacted by either the application of PFAS-contaminated wastewater treatment biosolids to agricultural fields or nearby fluorochemical manufacturing facilities, 40 which have PFCA and PFSA concentrations in excess of pg/g. These concentrations are significantly higher than <5000 pg/g of PFCAs and PFSAs previously reported with a few background soils in China, South Korea and USA Strynar et al. 46 recently performed a study on 60 surface soils from 6 countries (China, Greece, Japan, Mexico, Norway and USA). Unfortunately, only 28 and 48% of the soils investigated had quantifiable levels of PFOA and PFOS, respectively. Soils having quantifiable levels of PFOA and PFOS were reported to have PFCA and PFSA concentrations up to and pg/g respectively, suggesting significant impact from local sources. The objective of the present study was to evaluate the global distribution of PFCAs in background surface soils, which were distant from obvious human activity. A collection of 62 samples was obtained from 22 countries, representing all continents, and analyzed for a suite of 32 PFASs that included 9 PFCAs, 3 PFSAs, 10 FTCAs (fluorotelomer carboxylates) and 10 FTUCAs (unsaturated fluorotelomer carboxylates), with a limit of quantitation (LOQ) 10 lower than reported by Strynar et al. Comparison of PFCA and PFSA concentrations were used to determine geographical differences amongst the locations to postulate on the impact of urbanization and local sources.

166 Materials and Methods Chemicals A detailed list of all chemicals can be found in Appendix D Sample Collection Soil samples were graciously collected by colleague scientists, who were chosen with the objective of obtaining a wide geographic sample distribution. Sample collectors were requested to sample the top 10 cm from a location near them that they judged to have limited recent human impact, using a sampling kit we supplied and following instructions supplied with the kit (detailed in Appendix D). Each sampling location was assigned an arbitrary alpha numerical ID based on the continent where they were obtained. A detailed list of all sampling locations can be found in Tables D1-D3 with corresponding maps in Figures D1-D Extraction Method The extraction method used in this study was based on previous methods. 41,47,48 Each soil was extracted in triplicate with 5 g (wet weight) samples transferred into methanol-washed polypropylene copolymer (PPCO) centrifuge tubes and sealed with PPCO caps. The soil samples were spiked with 2000 pg 13 C 8 mass-labeled perfluorooctanoate (M8PFOA) recovery standard and vortexed for seconds. A 400 µl aliquot of 2 M sodium hydroxide prepared in polished 18 MΩ water (PW; SI) and an 8.5 ml aliquot of a 90:10 acetonitrile (ACN):PW solution were mixed into the soils by vortexing for seconds, sealed with Parafilm and then

167 143 sonicated in an ice bath for 60 minutes. Next the samples were mounted onto a LabQuake rotisserie mixer and rotated for ~15 hours at 8 revolutions per minute then centrifuged at 36.6 kg (17,500 rpm) and o C for 15 minutes. The supernatants were decanted into glass vials and a second round of 90:10 ACN:PW extraction performed on the soils. The two supernatants were combined in the glass vial and blown to near dryness under filtered air in a solid-phaseextraction (SPE) manifold described in the (Figure D7). The extracts were dissolved in 4 ml tetrabutyl ammonium hydrogen sulfate (TBAS) ion-pairing solution, extracted into 5 ml of methyl-tert-butyl ether (MTBE) by vortexing, and then stored in a freezer overnight. The TBAS solution was then extracted again with a second 5 ml aliquot of MTBE. The MTBE fractions were decanted into pre-weighed glass vials and blown to dryness in the SPE assembly at room temperature. The glass vials were re-weighed and the dried extracts reconstituted with a 1 ml aliquot of 60:40 ACN:PW containing 100 pg/g mass-labeled surrogate standards described in Appendix D. The glass vials were weighed a final time prior to filtering with 0.2 µm Nylon filters Instrumental Analysis and Quantification Two separate Waters liquid chromatograph tandem mass spectrometry (LC-MS/MS) systems (Waters Corporation, Milford, MA) were used in the present study. The soil extracts were analyzed for PFCAs and fluorotelomer acids at the USEPA (Athens, GA) using an Acquity UPLC coupled to a Quattro Premier triple-quadrupole mass spectrometer operated in negative electrospray ionization mode. Chromatographic separation was performed using an Acquity BEH C18 column (1.7 m, 2.1 x 100 mm) at 35 o C with a Waters Frit guard disc (0.2 m, 2.1

168 144 mm) with instrumental parameter, methods, calibration and internal standards largely the same as that described in our earlier work. 48 The soil extracts were then shipped overnight to the University of Toronto (Toronto, ON) for PFSA quantitation, and qualitative analyses for % branch chains of PFOA and PFOS. Here, analyses were performed using an Acquity UPLC coupled to a Xevo-TQ-S MS/MS operated in negative electrospray ionization mode. Chromatographic separation was performed using an Acquity BEH C18 column (1.7 m, 2.1 x 75 mm) at 60 o C. A detailed description of both chromatography methods can be found in Appendix D Quality Assurance and Quality Control Field blanks consisting of clean Ottawa sand were deployed with each sample kit, and exposed to sampling equipment and the environment at the sample location, to detect contamination arising from sample collection or transit back to the laboratory. In the lab, each sample was stored in a locked box in a cooler at ~4 o C until extractions were initiated. Samples were prepared by: i) sieving through a methanol-washed, stainless-steel, 2-mm sieve, and mixing with a methanol-washed spatula before withdrawing aliquots for extraction; and ii) extracting three separate aliquots in parallel to characterize the combined effects of any remaining heterogeneity in the soil as well as analytical uncertainty. Process blanks were included with each extraction round to characterize analyte concentrations arising from the extraction procedure and associated laboratory activities. All samples and blanks were spiked with M8PFOA as a recovery standard.

169 145 The limit of detection (LOD) and LOQ were determined using a two-mean Student s - test having common, but unknown variance: 49 ( ) where t is the test statistic used to define LOD and LOQ, is the soil-sample mean, is the process-blank mean, is the pooled sample variance, and numbers of observations are given by n 1 = 3 soil-sample replicates and n 2 = 10 process-blanks. The pooled sample variance is defined as: To define LOQ and LOD, we compared our calculated values of t to critical t values ( ) for a one-tailed -test abbreviated, where is the specified significance level, (1) signifies one-tailed, and v is the degrees of freedom (v = n 1 + n 2-2 = = 11). The values we chose were at and at. So we defined LOQ as > and LOD as >, meaning there is a respective 99.9% and 95% certainty the observed sample concentration statistically exceeds the process blank levels. Sample values exceeding the LOQ are reported herein as blank corrected, i.e., reported soil concentrations are analytical concentrations minus mean process-blank values.

170 Soil Characterization Soil samples were submitted to the University of Georgia s Agricultural and Environmental Services Laboratories for total organic carbon (TOC) analysis. Because no relationship was found between TOC and total concentrations of PFCA or PFSA, nor with respect to homologue chain length, further TOC discussion and tabulated data are supplied (Tables D1-D3) Statistical Analysis Principal component analysis (PCA) was used to evaluate PFAS distributions amongst all sampling locations. The PFAS data were preprocessed by subtracting process-blank means from location means and dividing by location standard deviations as a qualitative correction for heteroscedasticity in the data. The FTCA and FTUCA data were excluded from PCA because many locations had non-detectable levels. PCA was performed using a correlation matrix with Statistica 12 (StatSoft Inc., Tulsa, OK). 5.4 Results and Discussion Quality Metrics As discussed in detail in Appendix D, most field sand blanks and laboratory process blanks returned low parts-per-trillion detections, typically <20 pg/g, supporting the reliability of these data. Three field sand blanks (NA23, AF03 and AF05) out of 61 (Table D7), and one laboratory process blank (TB1) out of 11 (Table D8) did return anomalous PFOA concentrations

171 147 of ± 10.1, ± 31.2, ± 7.4 and ± 35.4 pg/g, respectively. As discussed in Appendix D, examination of all other quality metrics for the samples associated with these blanks did not suggest loss of integrity, and most analytical results for these samples did not fall near either extreme in the data distribution, so we report these samples here. Nevertheless, results for the eight samples associated with these blanks (AF03, AF05, NA06, NA09, NA11, NA19, NA23, AS01) should be regarded with caution, as we note in the tabulated results PFAS Concentrations Every soil sample in our global survey had quantifiable concentrations of at least three PFCAs, with total PFCAs ( PFCAs) ranging from pg/g. All samples but one, from rural Estonia (EU09), had quantifiable PFSAs, with PFSAs ranging from <LOQ-3270 pg/g (Table 5.1). Congener profiles of PFCAs and PFSAs for each sampling location are presented in Figure 5.1. Detailed lists of all sample locations and their PFCAs and PFSAs are provided in Tables D1-D3, and individual analytes are tabulated in Tables D12-D23. Generally, the most abundant congeners were PFOA and PFOS with concentrations up to PFOA = 3440 pg/g for a sample from Japan (AS04) and PFOS = 3130 pg/g for a sample from Copenhagen, Denmark (EU01). Both PFOA and PFHxA were detected in all samples, and PFOS was detected in all samples except for one Estonian sample (EU09). The ubiquity of detections in our survey is remarkable given the remote isolation of some of our sample locations. For example, Lake Bonney, Antarctica (AN01) had PFOA = 48 pg/g and PFOS = 7 pg/g, Mapunguwe National Park, South Africa (AF02) had PFOA = 14 pg/g and PFOS = 4 pg/g, Vehendi (Lake Vortsjarv), Estonia (EU09) had PFOA = 15 pg/g and PFOS <LOQ, Inuvik (Northwest Territories), Canada (NA15) had PFOA = 270 pg/g and PFOS = 18 pg/g.

172 148 Table 5.1: Continental summary PFAS concentration ranges in pg/g dry weight with the continental geometric mean in parentheses. Continent ΣPFCA ΣPFSA North America (NA) * (1820.4) (410.3) Europe (EU) Asia (AS) Africa (AF) Australia (AU) South America (SA) (1001.4) (4714.8) (548.2) (673.1) (137.7) (808) (183.1) (67.5) (154.1) (36.4) Antarctica (AN) *Includes a sample from Waimea, Hawaii (NA19).

173 149 AN01 SA03 SA02 SA01 AU04 AU03 AU02 AU01 AF05 AF04 AF03 AF02 AF01 AS06 AS05 AS04 AS03 AS02 AS01 EU10 EU09 EU08 EU07 EU06 EU05 EU04 EU03 EU02 EU01 NA33 NA32 NA31 NA30 NA29 NA28 NA27 NA26 NA25 NA24 NA23 NA22 NA21 NA20 NA19 NA18 NA17 NA16 NA15 NA14 NA13 NA12 NA11 NA10 NA09 NA08 NA07 NA06 NA05 NA04 NA03 NA02 NA01 PFSAs PFCAs PFHxA PFHpA PFOA PFNA PFDA PFUnDA PFDoDA PFTrDA PFTeDA PFHxS PFOS PFDS Concentration (pg/g) Figure 5.1: Overview of global PFCA and PFSA concentrations in North American (NA), European (EU), Asian (AS), African (AF), Australian (AU), South American (SA) and Antarctic (AN) surface soils.

174 150 Addressing isomeric distribution (Tables D24-D26), the detection of PFOA and PFOS isomers is expected to result from the atmospheric LRT of electrochemical fluorination (ECF)- based precursors, such as FASAs and polyfluorinated amides (PFAMs). While FASAs were intended for commercial products, PFAMs such as N-methylperfluorooctanamide (MeFOA) and N-ethylperfluorooctanamide (EtFOA), are byproducts in the synthesis of polyfluorinated sulfonamides, and could be an important indirect source of ECF-based PFOA. 50 Both FASAs and PFAMs are predicted to have atmospheric lifetimes between 2-20 days, 24,50 which is sufficient to deliver isomeric PFOA and PFOS to remote regions. In the present study, the mean proportion of linear PFOA and PFOS in soils were qualitatively observed to be ~70 and 75%, and are consistent with the ~78 and 70% linear reported in technical-grade PFOA 51 and PFOS. 52 The proportion of linear PFOA (Figure D8) and PFOS (Figure D9) increased in a statistically significant trend, with increasing total compound concentration. While both PFOA and PFOS show positive trends in linearity with increasing concentration, the slope of this trend is significantly steeper for PFOA than PFOS (Figure D8 vs. D9). Among possible causes for the PFOA trend, this might reflect an early global distribution of PFOA during ECF production, followed by increase in telomer-based PFOA in regions where it was produced. With the exception of a few locations having detectable concentrations of FTCA ( pg/g) and FTUCA ( pg/g), most of the samples were (Tables D12-D23). Considering FTCAs are products of FTOH atmospheric oxidation 23 and have been detected along with FTUCAs in rain water, 25,26 detection of FTCAs and FTUCAs in soils was expected. It is known that FTCAs and FTUCAs are rapidly transformed to PFCAs in saturated soils, 53 which may contribute to the lack of detectable levels in most background soils analyzed. Of the background soils having detectable levels, locations AS01, AS04, NA02, NA13, NA20, NA28-31 and NA33 were most noteworthy having FTCA and/or FTUCA concentrations >20 pg/g.

175 151 Although it is difficult to explain the elevated FTCAs and FTUCAs at these locations, contribution from a nearby fluorotelomer source is possible. Further discussion of several locations is presented in the following text Global PFCA and PFSA Distribution At the hemispheric scale, PFCAs and PFSAs showed sharply contrasting distributions north vs. south of the equator, with a relatively wide range of values evident in the northern hemisphere, but only comparatively low values in the southern hemisphere (Figures 5.2A and 5.2B). For the northern hemisphere, the geometric mean values for PFCAs and PFSAs were 930 and 170 ng/g (ranges of and pg/g), much in excess of the southern hemisphere which had geometric means of 190 and 30 ng/g (ranges of and pg/g), respectively. Similar hemispheric dependence for PFCAs and PFSAs has been reported in surface ocean waters 5,6 as well as the atmosphere for the neutral and volatile precursors, FTOHs, FASAs and perfluoroalkyl sulfonamide ethanols (FASEs). 54,55 Like these earlier studies in water and air, our soil results are consistent with the majority of historical direct and indirect PFCA and PFSA sources being emitted from northern-hemisphere continents. 29,56,57 Addressing the continental scale, for both PFCAs and PFSAs, the geometric mean concentrations descended as follows: (Asia, North America) > (Europe, Australia) > (Africa, South America, Antarctica) (Table 5.1). This pattern is consistent with the hemispheric pattern in Asia and North America, which have the highest geometric means, are in the northern hemisphere. South America, Antarctica and much of Africa, which occupy the lowest geometric mean grouping, are in the southern hemisphere. Also noteworthy is that, for every continent, the

176 152 Total PFCA Concentration (pg/g) NA EU (A) AS AF AU 8000 SA AN Latitude Total PFSA Concentration (pg/g) NA (B) EU AS AF AU SA AN Latitude Figure 5.2: PFCAs (A) and PFSAs (B) plotted against latitude. Note that higher concentrations of both PFCAs and PFSAs >20 o in the northern hemisphere. geometric mean PFCA concentrations were considerably larger than the PFSA (Table 5.1). Whereas graphing PFASs as a function of latitude reveals a distinct hemispherical contrast

177 153 (Figure 5.2A and 5.2B), plots of PFSAs and PFCAs against longitude generally render few noteworthy patterns (Figures D10-D12) except for North America. Total concentrations of PFCAs and PFSAs for North American soil samples (Figure 5.3A), increased longitudinally from west to east (Figures 5.3B and 5.3C; a latitudinal comparison is presented in Figure D13). Most PFCA soil concentrations, ranging from the west coast (approximately -125 o ) to central North America (approximately -100 o ), were 1000 pg/g. A sharp increase in the frequency of higher PFCA concentrations, up to ~6000 pg/g, was observed east of -100 o, particularly for samples on the northeast seaboard (Figure 5.3B). The highest values of PFSA also were located in the east (Figure 5.3C) but, with only two such high values, our dataset does not support a statistically significant PFSA longitudinal pattern for North America. This contrast in PFCA vs. PFSA longitudinal patterns is consistent with a trend previously observed for atmospheric measurements of neutral precursors on-board a ship during a sampling campaign off the eastern seaboard. 58 The authors reported a sharper increase in FTOH concentration compared to FASE and FASA concentrations moving northeast from the Gulf of Mexico to Boston, MA, 58 which the authors attributed to elevated neutral precursor emissions nearer urban locations than remote locations. 19,22,59-61 Air masses moving westerly would accumulate precursors emitted from the heavily urbanized eastern USA, and be the primary source for elevated PFCA and PFSA concentrations in rural locations. For example, the sample from Whipple Dam State Park, PA (NA11), located 250 km northwest of Philadelphia, PA, had PFCA and PFSA concentrations of 2360 and 577 pg/g, respectively; the sample was collected off trail in a long-term forest with no urban or suburban development for miles in any direction. Similarly, the sample from Holderness, NH (NA17), in the rural White Mountains of New Hampshire, had PFCA and PFSA concentrations of 4240 and 1840 pg/g, respectively; this sample was collected off-trail at the very apex of a completely wooded hill. Given that both of

178 154 these samples were collected from wooded areas, regionally remote from the nearest densely populated areas, the high PFCA and PFSA concentrations likely result from high precursor atmospheric concentrations previously documented for the northeast US region. 58 Figure 5.3: Approximate North American sampling locations (A), and the longitudinal distribution of total PFCAs (B) and PFSAs (C) in North American (NA) surface soils. Solid black line represents the middle longitude of NA, while dashed red and blue lines represent Los Angeles, CA and New York City, NY Principal Component Analysis (PCA) The primary sources of variance in our PFCA and PFSA data are depicted in a principalcomponent score plot (Figure 5.4), the quadrants of which have been labeled numerically 1-4 for discussion purposes. Of the total variance, PC1 contributed 42.59% with PFNA, PFDA,

179 155 PFUnDA, PFDoDA, PFTrDA and PFTeDA having the highest loading factors, while PC2 contributed 17.99% with the highest loading factors from PFHxA, PFHpA, PFOA, PFHxS and PFOS (Table D27). So the primary component of variation (PC1) was dominated by PFCA homologues longer than PFOA and the secondary component (PC2) accounted for variation in short-chain PFCAs and PFSAs. Principal Component 2: 17.99% NA 4 1 EU 3 AS04 2 AS EU01 AF EU04 NA02 AU AS01 SA EU03 AS03 NA17 AN NA30 NA28 NA29 NA Principal Component 1: 42.59% Figure 5.4: Principal component score plot of the global PFCA and PFSA surface soil concentrations. The inset shows the numerical ordering of the four quadrants. The majority of samples showed little deviation from one another with 37 of the 62 samples tightly clustered between quadrants 3 and 4 (dashed circle in Figure 5.4) and 13 samples falling just outside. The remaining 12 samples had a more pronounced separation from the cluster with 8 samples (AS01, AS03, AS04, EU01, EU03, EU04, NA02 and NA17) in quadrant 1 and 4 samples in quadrant 2 (NA28-NA31). These 12 PCA outliers all fall among the highest

180 156 PFCA samples in our dataset suggesting that the dominant sources of variation are attributable to samples having elevated PFCAs, presumably due to local or regional sources. The outlier samples in quadrant 1 were collected within or near large urban areas and had elevated levels of PFOA and PFOS with the exception of the Asian samples AS01 and AS04, which had elevated PFHxA and PFHpA (Figure 5.1). The outlier samples from quadrant 2 (NA28-NA31) were collected from in and near the municipal water-supply well fields for Penns Grove (NJ), USA, and showed the highest levels of PFNA-PFTeDA (Figure 5.1). These Penns Grove samples are located less than 10 km east of Wilmington, DE, where fluorotelomer-based products were manufactured in the past Inferred Mode of Occurrence Given the remote location of many samples (e.g., Antarctica; Mapunguwe National Park, South Africa; Vehendi, Estonia; Inuvik, Canada), the ubiquitous detection of PFCAs and PFSAs indicates LRT as a significant contributor to global PFAS dissemination. While it is known that some PFASs are subject to LRT, some uncertainty remains regarding the principal mode of LRT, but a significant proportion from a combination of gas- and particle-phase transport of volatile precursors in concert with atmospheric oxidation and deposition In previous smog chamber studies, 23,62 atmospheric FTOHs were subject to gas-phase oxidation by OH radicals, with the dominant products generally being roughly equimolar n-1 and n PFCAs for n:2 FTOH reactants. For example, 8:2 FTOH would presumably form roughly equimolar PFOA (n-1) and PFNA (n), and is consistent with a PFOA to PFNA ratio of 1.1/1 observed in Arctic glacial ice. 63 Given the results of the smog chamber, the gas-phase oxidation

181 157 of fluorotelomer-based precursors under environmental conditions is expected to yield a similar product distribution of PFCAs in soils. Deviation from the predicted equimolar even to odd PFCA distribution may suggest differences in the concentrations of atmospheric reagents (ie. NO x and OH) compared to the controlled smog chamber studies. Alternatively, the heterogeneous photooxidation of FTOHs on particulate matter proposed by Styler et al. 64 could result in up to 6x higher distribution of even than odd PFCAs. However, beyond this study little is known about the role particulate matter has on the atmospheric transformation of neutral fluorotelomer precursors. When the ratio of PFOA/PFNA was plotted as a function of PFCAs (Figure 5.5), only a few soils had a PFOA/PFNA ratio of 1/1. Prominent among these are the four samples from Penns Grove, NJ (NA28-NA31), which were unique in the PCA plot lying far from the origin in quadrant 2 (Figure 5.4). Given the close proximity of samples NA28-NA31 to a major fluorotelomer manufacturing facility, the near equimolar n and n-1 PFCAs observed (Table D12) likely result from the gas-phase oxidation of fluorotelomer-based precursors similar to previous laboratory studies. 23,62 In contrast, the majority of the samples had a PFOA/PFNA ratio between 1/1 and 5/1 (Figure 5.5), and are consistent with PFCA distributions previously observed in rainwater from rural and remote locations, 26 and Arctic glacial ice 63 and lakes. 20 Given the remoteness of these previous samples and many of the soils in the present study, LRT of neutral fluorotelomer precursors followed by gas-phase oxidation, and wet or dry deposition was likely the source of PFCAs detected.

182 158 Figure 5.5: Ratio of PFOA to PFNA versus log scale of PFCA concentrations for all sampling locations. Dashed 1/1 and 5/1 lines are assumed to represent gas phase oxidation of PFAS precursors, and the lower stoichiometric bound for direct emission of PFCAs (Direct 8/1). Note that locations AN01, AU03 and EU09 were excluded because PFNA was either <LOQ or. Regarding direct PFCA emission, Prevedouros et al. 29 reported that the direct global emissions in the year 2000 yielded a PFOA/PFNA ratio of at least 8/1. Several soils from the present study had PFOA/PFNA ratios greater than 5/1, and may not be consistent with gas-phase oxidation suggesting a potential additional contribution from nearby direct sources. Many of these soils were collected close to urban regions, which supports the short-lived atmospheric lifetime of PFCAs directly emitted. Some investigators have hypothesized that ocean transport of PFCAs and PFSAs can serve as a source to remote regions through the marine-aerosol formation and deposition. 29,56 In an effort to examine the possible role of ocean transport, we plotted the PFOA/PFNA ratio for the Antarctic sample (AN01), and the 12 samples having PFCAs lower than AN01 against

183 159 distance from ocean water (Figure D14). Four samples were located within 100 km of marine waters had PFOA/PFNA ratio >8/1, suggesting possible impact from marine-aerosol. Addressing the long-chain PFCAs, 58 of 62 samples in our survey had detectable levels of one or more PFDA-PFTeDA (Table D12, D16 and D20), with approximate PFDA/PFUnDA and PFDoDA/PFTrDA ratios of 2 and 4, respectively. Based on the low historical direct production rate of these compounds, this observation also supports oxidation of precursors as the major mode of occurrence for these PFCAs, and were consistent with the detection of PFDA- PFTeDA in rainwater, 26 and Arctic glacial ice 63 and lakes 20 in remote locations. 5.5 Environmental Implications Results from this study demonstrated that PFASs are ubiquitous in background soils with PFCAs and PFSAs concentrations up to and 3200 pg/g, respectively. Locations nearer urban areas or suspected point sources had elevated PFCA and PFSA concentrations when compared to rural and remote locations. In contrast, our sample set included representatives from some of the most undeveloped and farthest reaches of terrestrial Earth, every surface soil that we tested had quantifiable concentrations of at least two PFASs. Given that roughly 30% of Earth s surface is terrestrial, the data we present here confirm that soils are a major repository for PFASs at the global scale. The ratios of n-1/n PFCAs observed are generally consistent with the gas-phase oxidation of volatile precursors as the dominant mode of LRT, albeit, perhaps with some influence from coastal regions. This finding of a dominant atmospheric mode of occurrence may have important implications for potential future PFAS releases to the environment because landfills commonly combust/vent generated

184 160 gases to the atmosphere by design. To the extent fluorotelomer compounds generated from landfilled waste are vented like other landfill gases, disposed consumer goods could constitute a large source term of PFASs to the environment in the decades to come. 5.6 Acknowledgements The authors extend their sincere thanks to colleague scientists around the world for their sampling efforts; due to space limitations, we reserve acknowledging them by name for the Supporting Information. The present study is supported by the USEPA and the Natural Science and Engineering Research Council of Canada (NSERC), and the Ontario Ministry of Training, Colleges and Universities for an Ontario Graduate Scholarship (OGS). The authors would also like to thank the Department of Chemistry at the University of Toronto for a Special Opportunity Graduate Travel Fellowship for travel expenses. 5.7 References (1) Kissa, E. Fluorinated surfactants and repellents; Hubbard, A. T., Ed.; 2nd ed.; Marcel Dekker, Inc.: New York, 2001; Vol. 97. (2) Buck, R. C.; Franklin, J.; Berger, U.; Conder, J. M.; Cousins, I. T.; de Voogt, P.; Jensen, A. A.; Kannan, K.; Mabury, S. A.; van Leeuwen, S. P. Perfluoroalkyl and polyfluoroalkyl substances in the environment: Terminology, classification, and origins. Integr. Environ. Assess. Manage. 2011, 7, (3) Taniyasu, S.; Yamashita, N.; Moon, H.-B.; Kwok, K. Y.; Lam, P. K. S.; Horii, Y.; Petrick, G.; Kannan, K. Does wet precipitation represent local and regional atmospheric transportation by perfluorinated alkyl substances? Environ. Int. 2013, 55, (4) Yamashita, N.; Kannan, K.; Taniyasu, S.; Horii, Y.; Petrick, G.; Gamo, T. A global survey of perfluorinated acids in oceans. Mar. Pollut. Bull. 2005, 51,

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190 166 CHAPTER SIX Summary, Conclusions and Future Directions Keegan Rankin Contributions Prepared by Keegan Rankin with editorial comments provided by Scott Mabury.

191 Summary and Conclusions This dissertation investigated the development and application of matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry to directly analyze the degradation of fluorotelomer-based acrylate polymers (FTACPs), which we hypothesized to be a significant indirect source of PFCAs. Historically, FTACPs have accounted for the largest fraction of commercial fluorotelomer products, 1,2 with >80% of all fluorotelomer-based raw materials produced worldwide directed towards manufacturing of FTACP. 3 FTACPs are excellent surface protectants, and have been used in textile, upholstery and paper industries. 1 Although the fluorotelomer industry has shifted to FTACPs bearing fluorinated appendages with <7 perfluorinated carbons, FTACPs with fluorinated appendages >7 perfluorinated carbons were used for decades on commercial products, and could be the largest indirect source of long-chain PFCAs (>7 perfluorinated carbons) to the environment. To directly analyze FTACP degradation, MALDI-TOF methods were developed. Chapter 2 outlines the trials and tribulations encountered when preparing and analyzing FTACPs with different degrees of fluorination. Using a homologous series of FTACPs with different ratios of perfluorinated and hydrogenated carbons (R F /R H ), non-fluorinated and fluorinated sample preparations were examined by MALDI-TOF and scanning electron microscopy (SEM). FTACPs with a low degree of fluorination (R F /R H <0.5) were readily analyzed using conventional non-fluorinated sample preparations, while FTACPs with a higher degree of fluorination (R F /R H >0.5), required a novel fluorinated sample preparation. Establishing an appropriate sample preparation is critical to directly analyze FTACPs by MALDI-TOF. The application of MALDI-TOF to directly measure FTACP degradation was explored in Chapter 3 by investigating the biodegradation of a model FTACP, poly(8:2 FTAC), in a

192 168 soil:plant microcosm. Over 5.5 months, the signal intensities of the poly(8:2 FTAC) characteristic repeat pattern were observed to decrease for the three soil conditions studied: 1) FTACP/soil; 2) FTACP/plant; and 3) FTACP/plant/biosolids. The reduction in signal intensity was initially hypothesized to result from the biodegradation of poly(8:2 FTAC), but presumably also resulted from sorption of poly(8:2 FTAC) to organic matter. Interestingly, a shift in the relative distribution of the characteristic repeat pattern was observed in the FTACP/plant and FTACP/plant/biosolids conditions, which suggested biodegradation of the lower molecular poly(8:2 FTAC) oligomers. This observation was consistent with an increase of biodegradation products (ie. PFHxA, PFHpA and PFOA) measured indirectly by liquid chromatography tandem mass spectrometry (LC-MS/MS). The biodegradation half-life of poly(8:2 FTAC) ranged from 8 to 111 years, and are ~10 lower than estimated for commercial FTACPs. 4,5 The difference in degradation rates likely resulted from the lower molecular weight of poly(8:2 FTAC) when compared to the commercial FTACPs. Although qualitative, the MALDI-TOF results represented the first direct evidence of FTACP degradation. The direct analysis of FTACPs was further investigated in Chapter 4, with the development of a quantitative MALDI-TOF method. The approach was largely based on previous MALDI-TOF work quantifying peptides, 6 in which the matrix itself served as a pseudo internal standard. Normalizing the signal intensities of 40 poly(8:2 FTAC-co-HDA) signals from 911 to 4612 Da to the signal intensity of a dithranol-sodium cluster was observed to minimize the sample to sample irreproducibility, which often plagues MALDI-TOF. When the same dithranol solution was used to prepare a set of poly(8:2 FTAC-co-HDA) standards, calibration curves typically had coefficient of determinations (R 2 ) >0.98. Testing the method showed that poly(8:2 FTAC-co-HDA) samples in aqueous media could be directly quantified to within 25% of the theoretical concentrations.

193 169 In Chapter 5, surface soils from all continents were observed to have quantifiable levels of long-chain PFCAs and PFSAs. Given that most soils were collected from locations with little human activity, the PFCAs and PFSAs detected likely resulted from the atmospheric LRT of volatile precursors, such as fluorotelomer alcohols (FTOHs) and perfluoroalkyl sulfonamides (FASAs). This represents a significant PFCAs and PFSAs sink not considered in previous fate models. 3,7,8 FTOHs have been measured as residuals in commercial FTACPs, 9,10 and are known products from the degradation of commercial fluorotelomer-based materials such as polyfluoroalkyl phosphates (PAPs). 11,12 Off gassing of residual FTOHs, and as the first degradation product of commercial fluorotelomer-based materials, particularly FTACPs, could support the atmospheric LRT of volatile precursors for decades to come. The atmospheric oxidation of these precursors to PFCAs and PFSAs, followed by wet- and dry-deposition to soils should be considered as an important environmental sink. 6.2 Future Directions The principle goal of this dissertation was to directly analyze FTACP degradation. Qualitative MALDI-TOF results strongly suggest that FTACPs will degrade under environmental conditions, and will likely be a significant indirect source of PFCAs. With the development of a direct quantitative method, there are several areas that warrant further investigation. The decrease in the signal intensity observed in the soil:plant microcosm study could have resulted from the sorption of poly(8:2 FTAC) to organic matter. The sorption of PFASs to solid matrices has been reported, and is an intriguing question with respect to FTACPs for a

194 170 couple of reasons. Firstly, if FTACP sorb to solid, then the ester moieties could be less accessible to the microbes, thereby reducing the degradation rate of FTACP. Thus, FTACPs would be an indirect source of PFCAs over centuries rather than decades. Secondly, FTACPs are applied to the textiles, upholsteries and paper as aqueous emulsion, 18,19 and were likely discharged in wastewater from these industries. Sorption of FTACPs to wastewater treatment plant (WWTP) biosolids could be an indirect route of human exposure to PFCAs through the application of WWTP biosolids to farmland. PFCA precursors have been reported in farmland that have be amended with WWTP biosolids, as observed in the widespread PFCA and PFSA contamination in farmland around Decatur, AL after it received PFAS contaminated WWTP biosolids Uptake of PFASs into vegetation has been reported, and could be an important indirect route of PFCA human exposure if agricultural farmland are amended with FTACP contaminated WWTP biosolids. Although the biodegradation of poly(8:2 FTAC) was measured directly by MALDI-TOF and indirectly by LC-MS/MS, the direct measurements were strictly qualitative. Applying the direct quantitative method to investigate the biodegradation of FTACPs in different media would enhance our understanding environmental fate of FTACPs. The degradation studies presented in this dissertation have involved model rather than commercial FTACPs. Directly investigating the degradation of commercial FTACPs could reduce the uncertainty surrounding the half-lives determined by indirect analysis previously reported. 4,5 Although both our model and commercial FTACPs have 8:2 FT appendages covalently bonded to the polymer backbone, commercial FTACPs have additional monomers, and molecular weights of ~40,000 Da. 4,18,19 The properties of commercial FTACPs render most insoluble in non-fluorinated solvents, and would likely require a novel fluorinated sample

195 171 preparation, similar to those described in Chapter 3, for direct MALDI-TOF analysis. Attempts were made to analyze commercial FTACPs by MALDI-TOF using a variety of non-fluorinated and fluorinated sample preparations, but to date no mass spectra have been successful obtained. If commercial FTACPs are to be directly analyzed by MALDI-TOF, further efforts to develop a compatible sample preparation are required. As FTACP manufacturers comply with the 2015 PFOA Stewardship Program, 27 new formulations that offer similar surface protectant properties, but do not contain fluorinated appendages with 7 perfluorinated carbons will be developed. This includes novel fluorinated acrylate polymers prepared from acrylate monomers bearing branched, cyclic and linear appendages with 6 perfluorinated carbons Identifying the environmental and health impact these new fluorinated acrylate polymer could become important in the future. Application of the direct MALDI-TOF methods presented in this dissertation could be an approach used in assessing potential environmental fate studies of new fluorinated acrylate polymers. 6.3 References (1) Rao, N. S. Textile Finishes & Fluorosurfactants. In Organofluorine Chemistry. Principles and Commercial Applications; Banks, R. E.; Smart, B. E.; Tatlow, J. C., Eds.; Plenum Press: New York, 1994; pp (2) Telomer Research Program Update. Presentation to the U.S. EPA. Public Docket AR , (3) Prevedouros, K.; Cousins, I.; Buck, R. Sources, fate and transport of perfluorocarboxylates. Environ. Sci. Technol. 2006, 40, (4) Russell, M. H.; Berti, W. R.; Szostek, B.; Buck, R. C. Investigation of the biodegradation potential of a fluoroacrylate polymer product in aerobic soils. Environ. Sci. Technol. 2008, 42,

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197 173 (16) Ahrens, L.; Yeung, L.; Taniyasu, S.; Lam, P.; Yamashita, N. Partitioning of perfluorooctanoate (PFOA), perfluorooctane sulfonate (PFOS) and perfluorooctane sulfonamide (PFOSA) between water and sediment. Chemosphere 2011, 85, (17) Zhou, Q.; Deng, S.; Zhang, Q.; Fan, Q.; Huang, J.; Yu, G. Sorption of perfluorooctane sulfonate and perfluorooctanoate on activated sludge. Chemosphere 2010, 81, (18) Greenwood, E. J.; Lore, A. L.; Rao, N. S. Oil- and water-repellent copolymers. US 4,742,140, (19) Raiford, K. G.; Greenwood, E. J.; Dettre, R. H. Water-and oil-repellent fluoro (meth) acrylate copolymers. US 5,344,903, (20) Washington, J. W.; Yoo, H.; Ellington, J. J.; Jenkins, T. M.; Libelo, E. L. Concentrations, distribution, and persistence of perfluoroalkylates in sludge-applied soils near Decatur, Alabama, USA. Environ. Sci. Technol. 2010, 44, (21) Yoo, H.; Washington, J. W.; Ellington, J. J.; Jenkins, T. M.; Neill, M. P. Concentrations, distribution, and persistence of fluorotelomer alcohols in sludge-applied soils near Decatur, Alabama, USA. Environ. Sci. Technol. 2010, 44, (22) Lindstrom, A. B.; Strynar, M. J.; Delinsky, A. D.; Nakayama, S. F.; McMillan, L.; Libelo, E. L.; Neill, M.; Thomas, L. Application of WWTP biosolids and resulting perfluorinated compound contamination of surface and well water in Decatur, Alabama, USA. Environ. Sci. Technol. 2011, 45, (23) Stahl, T.; Heyn, J.; Thiele, H.; Hüther, J.; Failing, K.; Georgii, S.; Brunn, H. Carryover of perfluorooctanoic acid (PFOA) and perfluorooctane sulfonate (PFOS) from soil to plants. Arch. Environ. Contam. Toxicol. 2009, 57, (24) Lechner, M.; Knapp, H. Carryover of perfluorooctanoic acid (PFOA) and perfluorooctane sulfonate (PFOS) from soil to plant and distribution to the different plant compartments studied in cultures of carrots (Daucus Carotassp. Sativus), potatoes (Solanum Tuberosum), and cucumbers (Cucumis Sativus). J. Agr. Food Chem. 2011, 59, (25) Felizeter, S.; McLachlan, M. S.; de Voogt, P. Uptake of perfluorinated alkyl acids by hydroponically grown lettuce (Lactuca sativa). Environ. Sci. Technol. 2012, 46, (26) Blaine, A. C.; Rich, C. D.; Sedlacko, E. M.; Hundal, L. S.; Kumar, K.; Lau, C.; Mills, M. A.; Harris, K. M.; Higgins, C. P. Perfluoroalkyl acid distribution in various plant compartments of edible crops grown in biosolids-amended soils. Environ. Sci. Technol. 2014, 48,

198 174 (27) 2010/2015 PFOA Stewardship Program; US Environmental Protection Agency, Ed.; (28) Wang, Y.; J. J, Fitzgerald. Polymeric extenders for surface effects. US 7,652,112, (29) Yamamoto, I.; Masutani, T. Surface-treating agent comprising fluoropolymer , (30) van Buskirk, G.; Casella, V. M. Fabric treatment for stain release , (31) Shenoy, S.; Pollino, J. M.; Raghavanpillai, A.; Rosen, B. M.; Wysong, E. B. Process for producing fluorinated copolymers of (meth)acrylates and (meth)acrylic acid amine complexes. 2012/ , 2012.

199 175 APPENDIX A SUPPORTING INFORMATION FOR CHAPTER TWO Influence of Fluorination on the Characterization of Fluorotelomer-Based Acrylate Polymers by Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry

200 176 f1 (ppm) Figure A1: 19 F NMR spectrum obtained for decafluoroazobenzene.

201 177 f1 (ppm) Figure A2: 19 F NMR spectrum obtained for 4,4-dihydroxyoctafluoroazobenzene.

202 178 Table A1: Synthesized FTACPs and their respective appearance. Polymer Poly(6:2 FTAC-co-BA) Poly(6:2 FTAC-co-OA) Poly(6:2 FTAC-co-HDA) Poly(8:2 FTAC-co-BA) Poly(8:2 FTAC-co-OA) Poly(8:2 FTAC-co-HDA) Physical Appearance Viscous Transparent Gel Viscous Yellow Gel Yellow/Opaque Waxy Paste Viscous Transparent Gel Viscous Yellow Gel Yellow/Opaque Waxy Solid Table A2: Predicted vapor pressures for non-fluorinated and fluorinated organic solvents. Solvent Molecular Formula Vapor Pressure at 25 o C (kpa)* Dimethyl sulfoxide(dmso) C 2 H 6 OS Dimethylformamide DMF) C 3 H 7 NO 0.47 trifluorotoluene (TFT) C 7 H 5 F Tetrahydrofuran (THF) C 4 H 8 O 23 Chloroform CHCl 3 25 Dichloropentafluoropropanes (HCFC-225) C 3 HCl 2 F 5 40 *Predicted vapor pressures using AOPWin obtained from the USEPA.

203 179 APPENDIX B SUPPORTING INFORMATION FOR CHAPTER THREE Investigating the Biodegradability of a Fluorotelomer-Based Acrylate Polymer in a Soil- Plant Microcosm by Direct and Indirect Analysis

204 180 Experimental Chemicals. Butyl acrylate ( 99%), 1,1-dichloroethylene ( 99%), dodecyl amine ( 99%), hexadecyl thiol ( 95%), 2,2 -azobis(2-methylpropionamide) dihydrochloride (AIBA, 97%), 1,8,9-anthracenetriol (dithranol, 98.5%), lithium trifluoroacetate (98%) and trifluorotoluene (TFT, 99%) were purchased from Sigma Aldrich (St. Louis, MO). Dodecyl amine hydrochloride ( 97%) was purchased from Alfa Aesar (Ward Hill, MA). Sodium azide (NaN 3 ) was purchased from Anachemia Sciences (Montreal, ON). Glacial acetic acid was purchased from Caledon Labs (Georgetown, ON). Chloroform (ACS reagent, 99%) was purchased from Fischer Scientific (Fair Lawn, N). Methanol (Omnisolv, >99%), water (Omnisolv, >99%) and ammonium hydroxide (30%) were purchased from EMD Chemicals, Inc. (Mississauga, ON). 7:3 fluorotelomer carboxylate (7:3 FTCA, >97%) and 8:2 fluorotelomer acrylate (8:2 FTAC, >97%) were purchased from SynQuest Labs, Inc. (Alachua, FL). Perfluorohexanoate (PFHxA, >99%), perfluoroheptanoate (PFHpA, >99%), perfluorooctanoate (PFOA, >99%), perfluorononanoate (PFNA, >99%), 8:2 fluorotelomer carboxylate (FTCA, >98%), 8:2 fluorotelomer unsaturated carboxylate (FTUCA, >98%), and the mass-labeled internal standards 13 C 2 -PFHxA (>99%), 13 C 4 -PFOA (>99%), 13 C 5 -PFNA (>99%), 13 C 2 -PFDA (>99%) and 13 C 2-8:2 FTUCA (>98%) were all graciously donated by Wellington Laboratories (Guelph, ON). FTACP Polymerization. Polymerization was carried out in a three-neck round bottom flask equipped with a magnetic stirrer and dry ice condenser. An aqueous solution containing dodecyl amine hydrochloride and hexadecylthiol was prepared in Barnstead E-pure water, and transfered to the reaction vessel. To this solution, 15.0 g of 8:2 FTAC and 8.0 g butyl acrylate were added, emulsified and purged with nitrogen at 5 o C for 2.5 hours. After which, a solution of

205 g vinylidene chloride in dry ice cooled acetone was added. The polymerization was initiated using 1.0 g of AIBA in Barnstead E-pure water. The contents of the reaction vessel were gradually brought to 80 o C over 1 hour and held for 15 hours. The resulting opaque FTACP solids were collected by filtration. Preparation of Rhizobia Inoculum. Six different Sinorhizobium strains were cultured on agar plates. The agar media containinging 18 g/l agar, 60 g/l urea and 29 g/l sodium chloride in distilled water was autoclaved at 120 o C for 20 minutes then poured onto 6 petri plates, and allowed to solidify. Each rhizobium strain was added to each plate and incubated at 30 o C for 5 days. Single colonies of the 6 rhizobium strains were transferred and incubated in autoclaved liquid YG growth media containing 5 g/l tryptone (Difco, Bioshop), 3 g/l yeast extract (Difco, Bioshop), and 1.5 g/l calcium chloride (Sigma), and then adjusted to ph 7. Cell density of the rhizobia inoculum mixture was adjusted to ~10 8 cells/ml (based on optical density of 600 nm) by diluting with distilled water. Target Analyte Analysis. All samples were analysis by high pressure liquid chromatography tandem mass spectrometry (HPLC-MS/MS), using an Agilent 1100 HPLC coupled to an Applied Biosystems/MDS Sciex API4000 triple quadrupole MS (Concord, ON) operated in negative electrospray ionization mode. Chromatographic separation was performed using a GeminiNX C18 column (4.6 x 50 mm, 3 µm; Phenomenex, Torrance, CA). Three HPLC gradient methods were used for the analysis of the target analytes. Analysis of PFHxA, PFHpA, PFOA, 7:3 and 8:2 FTCAs and FTUCAs was performed on 25 µl injections using a gradient method at 500 µl/minutes with HPLC grade methanol and water prepared into 50 mm ammonium acetate mobile phases. The gradient parameters were as follows: at t = 0 minutes 80:20 water:methanol changing to 10:90 at t = 3.0 minutes and held

206 182 until t = 5.0 minutes, then returning 80:20 water:methanol at t = 5.5 minutes and re-equilibrate until t = 7 minutes. For the analysis of PFNA and PFDA, the samples were injected as 25 µl injections and analyzed by the following gradient method at 500 µl/minutes, using the same mobile phases as above: the initial solvent composition at t = 0 minutes was 25:75 water:methanol, changing to 5:95 over a period of 2 minutes at t = 2.0 minutes and held for 2 minutes to t = 4.0 minutes, before returning to the initial composition of 25:75 water:methanol at t = 4.5 minutes. The column was allowed to re-equilibrate for 1.5 minutes for a total run time of 6 minutes. Though PFDA was monitored, it was not expected as a biodegradation product and was not detected of background levels. A list of the analyte-specific multiple reaction monitoring (MRM) transitions for all target analytes and their corresponding mass-labeled internal standards is provided in Table B1. Quality Assurance. Spike and recovery experiments in soil were performed in triplicate (n = 3) by adding 100 ng of 8:2 FTCAs and FTUCAs, 7:3 FTCAs, and C6 C10 PFCAs to 1 g of control soil. Plant recovery experiments were performed in triplicate (n = 3) by adding 100 ng of the FTCAs and FTUCAs, and 10 ng of the C6 C10 PFCAs to 2 g of control plant material. Extraction and analysis was performed as previously described. Spike and recovery experiments were also performed in triplicate (n = 3) in the catch plates by adding 100 ng of the FTCAs and FTUCAs, and 10 ng of the C6 C10 PFCAs to clean catch plates containing 5 ml of MTBE. The analytes were mixed with MTBE in the plates, and the MTBE was allowed to evaporate overnight. Extraction was performed the following day as previously described. Matrix recoveries for each analyte are listed in Table B1.

207 183 FTACP Characterization Differential Scanning Calorimetry. Physical properties were determined on a 2920 Modulated DSC V2.6A from TA Instruments (New Castle, DE). The temperature scanining range was -30 o C to 180 o C with a heating cycle of 10 o C/minutes, followed by a cooling cycle at the same rate. The model FTACP was determined to have a melting point of 67.8 o C and a crystallization point at 55.3 o C (Figure B3). There was no detection of a glass transition temperature (T g ), which could fall below the lower limit of this instrument. Matrix-Assisted Laser Desorption/Ionization-Time-of-Flight. Structural characterization of the model FTACP was challenging due to a high degree of fluorination, which reduces their solubility in conventional hydrocarbon solvents. The analysis of similar FTACPs has been reported by both electrospray ionization time-of-flight (ESI-TOF) 1,2 and matrix-assisted desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. 1 We chose to use MALDI-TOF over ESI-TOF because of the solvent required to dissolve the model FTACP and to reduce the possibility of multiply charged ions. The analysis of synthetic polymers by MALDI-TOF often requires compound specific sample preparations methods. For the unique FTACP a multi-layer approach was determined to be the most affective method for overcoming their low solubility in tetrahydrofuran (THF) and chloroform (CHCl 3 ). Specifically, a two-layer sample preparation method was employed with the first layer comprising of the matrix and cationization agent prepared in CHCl 3 followed by the second layer containing the FTACP in trifluorotoluene (TFT). A graphite layer was applied to the MALDI sample plate as it has been shown to enhance the signal quality for certain synthetic polymers. 3 Changes to this sample preparation method yielded poor results. The results showed a dominant repeating pattern of 518 Da (*) that corresponds to the mass of 8:2 FTAC (Figure B2). As mentioned, 8:2 FTAC, butyl acrylate and vinylidene chloride

208 184 were present in the polymerization, and could have been incorporated into the model FTACP. The exact composition of the FTACP was determined by evaluating all possible monomer and end group combinations. For example, the signal at 1819 m/z could correspond to 2 x hydrogen end groups, 8 x vinylidene chloride and 2 x 8:2 FTAC, or the homopolymerization of 3 x 8:2 FTAC with hydrogen and hexadecyl thiol end groups. Comparing the theoretical and experimental isotope distribution at 1819 m/z it was evident that 16 chlorine atoms were not present (data not shown) eliminating the first possible composition. The theoretical and experimental isotope distribution presented in Figure B3 and Table B3 were nearly identical. This implied that the model FTACP was a homopolymer of 8:2 FTAC, and was further confirmed by comparing multiple theoretical and experimental masses of multiple repeat signals, as shown in Table B4. Additionally, when 8:2 FTAC was polymerized in the absence of butyl acrylate, the MALDI-TOF mass spectrum shown in Figure B8 had the same major signals as observed in Figure B2. Inspection of the isotopic distribution confirmed the absence of chlorine atoms, and provides further validation that our unique FTACP was a homopolymer of 8:2 FTAC. When a narrow mass range from Figure B2 is enhanced, it becomes apparent that additional signals are present between the major repeat pattern. These signals correspond to minor FTACPs that possess different monomer and end group combinations. For example, the most prominent minor repeat pattern (+) was determined to be the homopolymerization of 8:2 FTAC, but possessing two hexadecyl thiol end groups (Figure B2). Minor FTACPs constitute a very small fraction of the model FTACP material, and were not monitored in the microcosm study because their signals were unable to be resolvable from background signals. The number average molecular weight (M n ) for the model FTACP was calculated using the following equation: M n = M i N i N i Eq. B1

209 185 Where M represents the mass and N represents the observed intensity from MALDI-ToF analysis. The weight average molecular weight (M w ) was determined as follows: M n = M i 2 N i M i N i Eq. B2 The M n and M w were determined to be 3007 and 3747 Da respectively, and the polydispersity index (M n /M w ) to be Microcosm Control Experiments All target analytes present in the soil or arising from the WWTP biosolid were quantified using two control conditions. Relative amounts of PFCAs (C6-C10), 8:2 FTCA, 8:2 FTUCA, 7:3 FTCA and 7:3 FTUCA in the microcosm compartment are summarized in Tables B6-B11. Background levels of PFCAs in aerobic soil (Soil Control) prior to incubation ranged from 0.02 ± 0.02 nmole for PFHpA to 0.4 ± 0.02 nmole for PFOA. After incubation the PFCA levels ranged from 0.2 ± 0.06 nmole for PFHpA to 1.0 ± 0.8 nmole for PFHxA. Catch plate levels were <0.005 nmole or below the LOD. Control soils amended with WWTP biosolid (Plant/Biosolids Control) were found to have substantially higher levels of PFCAs ranging from 2.8 ± 0.1 nmole for PFHxA to 23.3 ± 0.5 nmole for PFOA again the most abundant. Nanomolar levels are consistent with previously reported levels in WWTP biosolids. 4-8 Levels were observed to peak at t = 3.5 month and decreased at t = 5.5 month. Translocation into the plant was observed for all PFCAs, but at low levels ranging from 0.02 ± 0.09 nmole for PFHpA to 2.0 ± 0.7 nmole for PFHxA. Loss to the catch plate was <0.1 nmole for all PFCAs. All other target analytes were below the LOD except for 7:3 FTCA, which ranged from 1.7 ± 0.2 to 0.5 ± 0.1 nomle at t = 0 and 5.5 month, respectively.

210 186 Expectedly, the MALDI-TOF analysis of control soils showed no mass spectral evidence consistent with the characteristic pattern of our model FTACP (data not shown). Calculation of 8:2 FTOH Equivalent The 8:2 FTOH equivalent of the model FTACP was calculated by averaging the M n and M w, and the corresponding average number of 8:2 FTOH appendages at that molecular weight (5.96). From the calculations shown below, it was determined that the 8:2 FTOH equivalent is 8.88 x 10 4 nmole. Avg. Molecular Weight of FTACP = g/mole Avg. Number of 8:2 FTOH Appendages = 5.96 Mass of FTACP/Microcosm Pot = 0.05 g = nmole Avg. Molecular Weight of FTACP g/mole nmole FTACP x 5.96 mole 8:2 FTOH Appendages = nmole mole of FTACP 8:2 FTOH Equivalence = 8.88 x 10 4 nmole Biodegradation Rate Constant and Half-Life Calculations The FTACP biodegradation rates and half-lives were calculated from the 8:2 FTOH equivalent and summation of all intermediates and products using first-order kinetics where k 1 : k 1 = -ln(c/c o ) t Eq. B3 The concentration of FTACP at t = 0 (C o ) equals the 8:2 FTOH mole equivalent, and the concentration at t = 1.5, 3.5 and 5.5 month is the 8:2 FTOH equivalent minus the summation of

211 187 the four biodegradation products. The loss of biodegradation products from 3.5 to 5.5 month led us to use two separate plots to evaluate the FTACP biodegradation rates and half-lives. A halflife range of 8-18 year was calculated when the results from 5.5 month were excluded. Alternatively, omitting the results at 3.5 month is a more conservative half-life estimation of year, but does not consider the total amount of stable products generated. References (1) Latourte, L.; Blais, J.; Tabet, J.; Cole, R. Desorption behavior and distributions of fluorinated polymers in MALDI and electrospray ionization mass spectrometry. Anal. Chem. 1997, 69, (2) Romack, T. J.; Danell, A. S.; Cottone, T. M.; Dutta, S. K. Characterization of perfluoroalkyl acrylic oligomers by electrospray ionization time-of-flight mass spectrometry. Rapid Commun. Mass Spectrom. 2008, 22, (3) Berger-Nicoletti, E.; Wurm, F.; Kilbinger, A.; Frey, H. Pencil lead as a matrix for MALDI-ToF mass spectrometry of sensitive functional polymers. Macromolecules 2007, 40, (4) Higgins, C. P.; Field, J. A.; Criddle, C. S.; Luthy, R. G. Quantitative determination of perfluorochemicals in sediments and domestic sludge. Environ. Sci. Technol. 2005, 39, (5) Schultz, M. M.; Higgins, C. P.; Huset, C. A.; Luthy, R. G.; Barofsky, D. F.; Field, J. A. Fluorochemical mass flows in a municipal wastewater treatment facility. Environ. Sci. Technol. 2006, 40, (6) Sinclair, E.; Kannan, K. Mass loading and fate of perfluoroalkyl surfactants in wastewater treatment plants. Environ. Sci. Technol. 2006, 40, (7) Loganathan, B.; Sajwan, K.; Sinclair, E. Perfluoroalkyl sulfonates and perfluorocarboxylates in two wastewater treatment facilities in Kentucky and Georgia. Water Res. 2007, 41, (8) D'eon, J. C.; Crozier, P. W.; Furdui, V. I.; Reiner, E. J.; Libelo, E. L.; Mabury, S. A. Observation of a Commercial Fluorinated Material, the Polyfluoroalkyl Phosphoric Acid Diesters, in Human Sera, Wastewater Treatment Plant Sludge, and Paper Fibers. Environ. Sci. Technol. 2009, 43,

212 188 Additional Figures and Tables Figure B1: Differential scanning calorimetry characterization of the model FTACP.

213 Relative Intensity (%) % % * 518 Da * * Poly(82 FTAc-co-butyl-co-VC 1) Reflec ( Dith + LiTFA) 0 0 * * * m/z m/z m/z Figure B2: Characteristic MALDI-TOF mass spectrum of the model FTACP obtained in positive ion mode. Major repeating pattern (*) corresponds to additional fluorotelomer acrylate (518 Da) having a hydrogen and hexadecyl thiol end groups. Inlaid panel highlights both major and minor (+) patterns observed.

214 190 A B Figure B3: Theoretical (A) and experimental (B) isotopic distribution of C 55 H 55 O 6 F 51 SLi corresponding to the signal observed at 1819 m/z.

215 ln(c/co) Time (months) FTACP/Soil FTACP/Plant FTACP/Plant/Biosolids Figure B4: Biodegradation rates for the model FTACP. Dashed lines represent the inclusion of 5.5 months and solid lines represent the exclusion of 5.5 months.

216 Amount of Analyte (nmole) PFHxA PFHpA PFOA PFNA 8:2 FTCA 8:2 FTUCA 7:3 FTCA 7:3 FTUCA Time (months) Figure B5: Amount of FTACP degradation products observed in microcosm soil for FTACP/Soil.

217 Amount of Analyte (nmole) Amount of Analyte (nmole) PFHxA PFHpA PFOA PFNA 8:2 FTCA 8:2 FTUCA 7:3 FTCA 7:3 FTUCA (A) Time (months) (B) Time (months) Figure B6: Amount of FTACP degradation products observed in microcosm soil (A) and plant (B) for FTACP/Plant/Biosolids.

218 Relative Intensity (%) Relative Intensity (%) Relative Intensity (%) m/z 5.5 months m/z 3.5 months m/z 1.5 months Figure B7: MALDI-TOF analysis of soil extracts at 1.5, 3.5 and 5.5 months obtained for Condition 4.

219 Relative Intensity (%) % Relative Intensity (%) % 195 PJT A m/z 0 m/z FTAc + Vinylidene Chloride B m/z 1900 m/z Figure B8: MALDI-TOF mass spectrum of poly(8:2 FTAC) polymerized in the presence (A) and absence (B) of butyl acrylate.

220 196 Table B1: Multiple reaction monitoring (MRM) transitions for all target analytes and internal standards and their matrix recoveries from the three microcosm compartments. Recovery (%) (n = 3) Target Mass Internal Mass Catch Analyte Transition Standard Transition Soil Plant Plate Fluorotelomer Saturated and Unsaturated Carboxylate (FTCA and FTUCA) 8:2 FTCA 477.0> 459.0> C :2 FTUCA ± 8 87 ± ± 7 7:3 FTCA 441.0> 459.0> C :2 FTUCA ± :2 FTUCA 457.0> 459.0> 135 ± C :2 FTUCA 100 ± ± 2 7:3 FTUCA 439.0> 459.0> C :2 FTUCA Perfluorocarboxylate (PFCA) PFHxA 312.8> 314.8> C PFHxA ± ± ± 60 PFHpA 362.8> 417.0> C PFOA ± ± 2 73 ± 21 PFOA 413.0> 13 C 4 -PFOA 417.0> 73 ± ± 5 76 ± 13 PFNA PFDA > > C 5 -PFNA 13 C 2 -PFDA > > ± ± ± 9 94 ± ± 5 83 ± 2

221 197 Table B2: Limits of detection (LODs) and limits of quantitation (LOQs) in soil and plants for target analytes. Target Analyte Soil Method Plant Method LOD (ng/g) LOQ (ng/g) LOD (ng/g) LOQ (ng/g) Fluorotelomer Saturated and Unsaturated Carboxylate (FTCA and FTUCA) 8:2 FTCA :3 FTCA :2 FTUCA :3 FTUCA Perfluorocarboxylate (PFCA) PFHxA PFHpA PFOA PFNA PFDA

222 198 Table B3: Relative intensities of theoretical and experimental isotopic distributions. Relative Intensity (%) Mass/Charge (m/z) Theoretical C 55 H 55 O 6 F 51 SLi Experimental C 55 H 55 O 6 F 51 SLi

223 199 Table B4: Observed and theoretical mass spectrometry signal for the model FTACPs corresponding to 2-5 fluorotelomer units. Fluorotelomer Units Observed m/z Theoretical m/z Table B5: Calculated biodegradation rates and half-lives for the model FTACP. Condition Time Point Omitted (months) Degradation Rate (months -1 ) Half-Life (years) x x x N/A 7.0 x N/A 1.6 x N/A 4.2 x R 2

224 200 Table B6: Calculated moles of PFCAs (C6-C10) in soil for all microcosm conditions. Analyte Time (mth) Soil Control Plant/Biosolids Control FTACP/Soil FTACP/Plant FTACP/Plant/Biosolids nmole SE nmole SE nmole SE nmole SE nmole SE PFHxA x x PFHpA x x x x x PFOA x x x x x x x x x x x PFNA x x x x x PFDA

225 201 Table B7: Calculated moles of PFCAs (C6-C10) in plant for all microcosm conditions. Analyte Time (mth) Soil Control Plant/Biosolids Control FTACP/Soil FTACP/Plant FTACP/Plant/Biosolids nmole SE nmole SE nmole SE nmole SE nmole SE PFHxA x x PFHpA x x x x x x x PFOA x x x x PFNA x x x x x x x x x x x x x PFDA x x x x x x x x x x x x x x x 10-2

226 202 Table B8: Calculated moles of PFCAs (C6-C10) in catch plate for all microcosm conditions. Analyte Time (mth) Soil Control Plant/Biosolids Control FTACP/Soil FTACP/Plant FTACP/Plant/Biosolids nmole SE nmole SE nmole SE nmole SE nmole SE PFHxA x 3.60 x 3.90 x x x 3.50 x x x x x x x x 10-2 PFHpA x 5.20 x 7.30 x x x x x x x x x x x PFOA x x x x x x x x x 10-2 PFNA x 2.00 x 2.00 x x x x x x x x x x x x x x x x x 10-3 PFDA x 5.00 x x x x x x x x x x x x x 10-3

227 203 Table B9: Calculated moles of intermediates in soil for all microcosm conditions. Analyte Time (mth) Soil Control Plant/Biosolids Control FTACP/Soil FTACP/Plant FTACP/Plant/Biosolids nmole SE nmole SE nmole SE nmole SE nmole SE 8: FTCA : FTUCA : FTCA x : FTUCA x 8.00 x x x x x x x x x x 10-2

228 204 Table B10: Calculated moles of intermediates in plant for all microcosm conditions. Analyte Time (mth) Soil Control Plant/Biosolids Control FTACP/Soil FTACP/Plant FTACP/Plant/Biosolids nmole SE nmole SE nmole SE nmole SE nmole SE 8: FTCA x : FTUCA x x x x x : FTCA x x x x : FTUCA x

229 205 Table B11: Calculated moles of intermediates in catch plate for all microcosm conditions. Analyte Time (mth) Soil Control Plant/Biosolids Control FTACP/Soil FTACP/Plant FTACP/Plant/Biosolids nmole SE nmole SE nmole SE nmole SE nmole SE 8: FTCA x 1.40 x 4.60 x x x x x x x x x x : FTUCA x 3.10 x 5.70 x x x x x x x x x x x x : FTCA x 1.30 x 4.00 x x x x x 1.00 x 4.20 x x x x x x x x x x : FTUCA

230 206 Table B12: Percent distribution of PFCAs in the three microcosm compartments. Analyte Time (mth) FTACP/Soil FTACP/Plant FTACP/Plant/Biosolids Soil Catch Plate Soil Plant Catch Plate Soil Plant Catch Plate PFHxA PFHpA PFOA PFNA PFDA

231 207 Table B13: Percent contribution of stable products summed for all three compartments at 5.5 month. Analyte FTACP/Soil FTACP/Plant FTACP/Plant/Biosolids PFHxA PFHpA PFOA PFNA :3 FTCA Table B14: MALDI-TOF data for FTACP/Soil. Please note these results are strictly qualitative. Fluorotelomer Peak Intensity (a.u.) Relative Intensity (%) Units 1.5 mth 3.5 mth 5.5 mth 1.5 mth 3.5 mth 5.5 mth ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 9 10 ± ± ± 7 8 ± ± 8 10 ± 6 7 ± ± 7 9 ± 5 6 ± ± 5 8 ± 5 5 ± ± 4 7 ± 4 5 ± 4

232 208 Table B15: MALDI-TOF data for FTACP/Plant. Please note these results are strictly qualitative. Fluorotelomer Peak Intensity (a.u.) Relative Intensity (%) Units 1.5 mth 3.5 mth 5.5 mth 1.5 mth 3.5 mth 5.5 mth ± ± ± ± ± ± ± ± ± ± ± ± ±20 46 ± ± ± ± ± ± ± ± ± ± ± ± 8 33 ± ± ± 7 28 ± ± ±6 25 ± ± ± 5 20 ± ± ± 4 18 ± ± ± 3 17 ± 11 9 ± 4

233 209 Table B16: MALDI-TOF data for FTACP/Plant/Biosolids. Please note these results are strictly qualitative. Fluorotelomer Peak Intensity (a.u.) Relative Intensity (%) Units 1.5 mth 3.5 mth 5.5 mth 1.5 mth 3.5 mth 5.5 mth ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 8 21 ± ± ± ± ± ± 5 15 ± ± ± 5 12 ± 8 14 ± ± 3 10 ± 7 12 ± ± 3 9 ± 6 10 ± 7 Table B17: Observed changes in the number and weight average molecular during FTACP incubation. Condition Time (month) M n M w PDI

234 210 APPENDIX C SUPPORTING INFORMATION FOR CHAPTER FOUR Matrix Normalized MALDI-TOF Quantification of a Fluorotelomer-Based Acrylate Polymer

235 211 Synthesis of Poly(8:2 FTAC-co-HDA) Polymerization of the poly(8:2 FTAC-co-HDA) was carried out in a 3-neck round bottom flask equipped with a condenser and a magnetic stir bar. The reaction vessel containing ethyl acetate was first purged with nitrogen for 60 minutes. Equimolar amounts of fluorotelomer acrylate (ie. 8:2 FTAC) and hydrocarbon acrylate (ie. BA) were added to the reaction vessel, and the reaction initiated by the addition of 0.5-1% aqueous AIBN. The contents were gradually brought to 70 o C over 60 minutes and held for ~15 hours using a IKA-Werke RCT hot plate with a IKA-Werke ETS-D4 temperature controller (IKA Werke, Staufen, DE). Upon completion, all contents were transferred to a single neck round bottom flask, and concentrated by rotary evaporation. The crude product was sonicated in methanol, dried, and heated at 75 o C for several days to remove any unreacted monomers.

236 212 A B C D Figure C1: SEM images at a magnification of 250x of dithranol crystallized after application of HB (A), 2B (B), 4B (C) and 8B (D) graphitic pencil lead onto the MALDI target plate.

237 213 A B C D Figure C2. SEM images of (A and B) dithranol and (C and D) poly(8:2 FTAC-co-HDA) at a magnification of 250x. (A) And (C) show the dried droplet crystallization on an unmodified MALDI plate. (B) And (D) show the dried droplet crystallization after coating a MALDI plate with graphitic lead from an 8B pencil.

238 % Intensity Relative Intensity (%) % Intensity Intensity (a.u.) Reflector Spec #1[BP = 659.2, 86580] 8.7x E Reflector S pec #1[BP = 659.2, 86580] Mass (m/z) 4.4 x E Mass (m/z) Figure C3: MALDI-TOF mass spectrum of poly(8:2 FTAC-co-HDA). Inset shows the repeat patterns used for characterization.

239 % In Relative Intensity (%) % In ten sity Intensity (a.u.) Reflector Spec #1[BP = 477.0, 79789] 8.0x E Reflector Spec #1[BP = 477.0, 79789] 8.0 E M a s s (m /z ) M m/z a s s (m ) Figure C4: MALDI-TOF mass spectrum of dithranol acquired in the absence of NaTFA. Inset shows the absence of a signal at 659 Da.

240 Normalized poly(8:2 FTAC-co-HDA) signal intensities mg/ml 10 mg/ml 5 mg/ml Concentration of poly(8:2 FTAC-co-HDA) (mg/ml) Figure C5: Calibration curves obtained using dithranol concentration of 20, 10 and 5 mg ml -1 when plotting P N vs. poly(8:2 FTAC-co-HDA) concentrations.

241 Intensity (a.u.) Matrix 20 mg/ml 10 mg/ml 5 mg/ml Concentration of Poly(8:2 FTAC-co-HDA) (mg ml -1 ) Figure C6: Variability in the inherent 659 Da signal intensity during analysis of poly(8:2 FTACco-HDA) when using dithranol at concentrations of 20, 10 and 5 mg ml -1.

242 Molar Ratio of Dithranol to Poly(8:2 FTAC-co-HDA) Matrix 20 mg/ml 10 mg/ml 5 mg/ml Concentration of Poly(8:2 FTAC-co-HDA) (mg ml -1 ) Figure C7: Molar ratio of dithranol to poly(8:2 FTAC-co-HDA) obtained with dithranol at concentrations of 20, 10 and 5 mg ml -1. Note: the moles of poly(8:2 FTAC-co-HDA) was calculated using the molar mass (2725 g/mole) the average of M w and M n.

243 219 Table C1. Observed signal intensities of a matrix cluster at 659 Da and poly(8:2 FTAC-co- HDA) at 1429 Da in the absence and presence of a graphite support (n = 3). Dithranol samples were prepared at 20 mg ml -1 with a NaTFA at 10 mg ml -1 and a mixing ratio of 10:1 Poly(8:2 FTAC-co-HDA) samples were prepared at 25 mg ml -1 with dithranol at 20 mg ml -1 and NaTFA at 10 mg ml -1 and a mixing ratio of 5:10:1. Sample Mass (Da) Signal Intensity without Graphite (a.u.) RSD (%) Signal Intensity with Graphite (a.u.) RSD (%) Dithranol x x Poly(8:2 FTAC-co-HDA) x x x x Table C2: Signal intensities of the 659 Da matrix cluster observed for poly(8:2 FTAC-co-HDA) standards using dithranol concentrations of 20, 10 and 5 mg ml -1. A mixing ratio of 5:10:1 was used for each sample with NaTFA prepared at a concentration of 10 mg ml -1. Poly(8:2 FTAC-co-HDA) Concentration (mg ml -1 ) Dithranol Concentration 20 mg ml mg ml -1 5 mg ml -1 Signal Intensity (a.u.) RSD (%) Signal Intensity (a.u.) RSD (%) Signal Intensity (a.u.) RSD (%) x x x x x x x x x x x x x x x x x x x x x

244 220 Table C3: Normalized polymer response (P N ) for poly(8:2 FTAC-co-HDA) standards using NaTFA concentrations of 10, 5 and 1 mg ml -1. A mixing ratio of 5:10:1 was used for each sample with dithranol prepared at a concentration of 20 mg ml -1. Poly(8:2 FTAC-co-HDA) Concentration (mg ml -1 ) NaTFA Concentration 10 mg ml -1 5 mg ml -1 1 mg ml -1 Normalized Polymer Response RSD (%) Normalized Polymer Response RSD (%) Normalized Polymer Response RSD (%) Table C4: Normalized polymer response (P N ) for poly(8:2 FTAC-co-HDA) standards observed over a three week period. A mixing ratio of 5:10:1 was used for each sample with dithranol and NaTFA prepared at respective concentrations of 20 and 10 mg ml -1. Poly(8:2 FTAC-co-HDA) Concentration (mg ml -1 ) Normalized Polymer Response Week 1 Week 2 Week 3 RSD (%) Normalized Polymer Response RSD (%) Normalized Polymer Response RSD (%)

245 221 Table C5: Normalized polymer response (P N ) for poly(8:2 FTAC-co-HDA) standards replicated three times within a single day. A mixing ratio of 5:10:1 was used for each sample with dithranol and NaTFA prepared at respective concentrations of 20 and 10 mg ml -1. Poly(8:2 FTAC-co-HDA) Concentration (mg ml -1 ) Replicate 1 Plate 1 Replicate 2 Plate 1 Replicate 3 Plate 2 Normalized Polymer Response RSD (%) Normalized Polymer Response RSD (%) Normalized Polymer Response RSD (%) Table C6: Theoretical poly(8:2 FTAC-co-HDA) sample concentrations in comparison to their measured concentrations determined using four external calibration curves (n = 3) Mass of Poly(8:2 FTAC-co-HDA) (g) Theoretical Concentration of Poly(8:2 FTAC-co-HDA) (mg ml -1 ) Measured Concentration of Poly(8:2 FTAC-co-HDA) (mg ml -1 ) ± ± ± 1.47 Table C7: Summary of external calibration curves obtained over several days for method validation. Calibration Curve Slope X = 1 R 2 Trial Trial Trial Trial

246 222 Table C8: Summary of external calibration curves used to quantify extracted poly(8:2 FTAC-co- HDA) to validate the P N method. Calibration Curve Slope X = 1 R 2 Trial Trial Table C9: Observed signal intensities of a matrix cluster at 659 Da at NaTFA concentrations of 10, 5 and 1 mg ml -1 (n = 5). A mixing ratio of 5:10:1 was used for each sample with dithranol prepared at a concentration of 20 mg ml -1. Poly(8:2 FTAC-co-HDA) Concentration (mg ml -1 ) NaTFA Concentration 10 mg ml -1 5 mg ml -1 1 mg ml -1 Signal Intensity (a.u.) RSD (%) Signal Intensity (a.u.) RSD (%) Signal Intensity (a.u.) RSD (%) x x x x x x x x x x x x x x x x x x x x x

247 223 Table C10: Observed signal intensities of a matrix cluster at 659 Da over a three week period (n = 5). A mixing ratio of 5:10:1 was used for each sample with dithranol and NaTFA prepared at respective concentrations of 20 and 10 mg ml -1. Poly(8:2 FTAC-co-HDA) Concentration (mg ml -1 ) Signal Intensity (a.u.) Week 1 Week 2 Week 3 RSD (%) Signal Intensity (a.u.) RSD (%) Signal Intensity (a.u.) RSD (%) x x x x x x x x x x x x x x x x x x x x Table C11: Observed signal intensities of a matrix cluster at 659 Da replicated three times within a single day (n = 5). A mixing ratio of 5:10:1 was used for each sample with dithranol and NaTFA prepared at respective concentrations of 20 and 10 mg ml -1. Poly(8:2 FTAC-co-HDA) Concentration (mg ml -1 ) Replicate 1 Plate 1 Replicate 2 Plate 1 Replicate 3 Plate 2 Signal Signal Signal RSD RSD RSD Intensity Intensity Intensity (%) (%) (%) (a.u.) (a.u.) (a.u.) x x x x x x x x x x x x x x x x x x x x x

248 224 APPENDIX D SUPPORTING INFORMATION FOR CHAPTER FIVE A Global Survey of Perfluoroalkyl Carboxylates (PFCAs) and Perfluoroalkane Sulfonates (PFSAs) in Surface Soils: Distribution Patterns and Mode of Occurrence

249 225 Experimental Chemicals. Unless noted, all chemicals used in this study were of the highest purity offered by the suppliers, uniformly 97% purity. Unlabeled and labeled perfluorocarboxylic acids and telomer acids all were purchased as certified standards from Wellington Laboratories through TerraChem (Shawnee Mission, KS, USA). Tetrabutylammonium hydrogen sulfate (TBAHS) and sodium carbonate were purchased from Aldrich Chemical (Milwaukee, WI, USA). Acetonitrile (ACN), glacial acetic acid, methanol (MeOH) and methyl tert-butyl ether (MTBE) were purchased from Fisher Chemical (Fairlawn, NJ, USA). Oasis HLB solid-phase extraction (SPE) cartridges, 35-cm 3 capacity, were purchased from Waters (Milford, MA, USA). For the ion-pairing agent, a TBAHS mixture (TBA-mix) was prepared by slowly combining two parts 0.25 M Na 2 CO 3 solution and one part 0.50 M TBAHS solution by volume to avoid spillage caused by CO 2 generation. The resulting mixture was polished by passage through HLB cartridge to remove PFOA (as detailed below for polished water), which we observed to be present in the TBAHS product as purchased. Sample-Collection Details. Most sample collectors were known to the authors, but to achieve a better geographic distribution, some were solicited based on their status as authors of published papers and/or university faculty profiles available on the internet. Scientists in geoscience, soils, chemistry, and engineering departments were preferred due to expected training in collecting uncontaminated samples, but presented with special opportunities to choice regions, a medical doctor and a professor of mathematics also agreed to collect samples for us. Identical sampling kits, which contained instructions and everything needed to sample, were prepared at the EPA/Athens laboratory and were shipped to each location. Sampling kits were prepared entirely with new, unused materials and included: 1) placing nitrile gloves in a zip-lock baggie; 2) placing a methanol-washed stainless-steel trowel in a zip-lock baggie; 3)

250 226 placing Ottawa sand, purchased from a laboratory supplier, in a polypropylene co-polymer (PPCO) methanol-washed sample container as a field collection blank; 4) adding a second PPCO, methanol-washed sample container as the sample container; 5) return postage and labeling; and 6) sampling and return instructions. In the instructions, collectors were asked to obtain a surface-soil sample from a nearby location they deemed to have limited recent human impact following the provided written instructions: 1) don nitrile gloves; 2) clear natural, unhumified, litter from the sampling location; 3) open zip-lock baggie and remove the trowel without touching the blade; 4) open the sample bottle containing the Ottawa sand, pour all the sand onto the trowel blade, pour the sand back into the blank sample bottle and close the sample bottle (collection blank); 5) sample the soil from the surface to about 10-cm depth, placing the soil into the sample bottle, taking care to touch the soil only with the trowel blade and the sample-bottle lip and close the sample bottle; 5) return the sample bottles and trowel to the zip-lock baggie, seal the baggie, and return the baggie to the mailing box; and 6) seal the box and mail back to the Office of Research and Development (ORD) of the United States Environmental Protection Agency (USEPA) in Athens, GA by the return-delivery method organized by the EPA. In all cases, the EPA provided the most expeditious mode of delivery that could be established; this varied in some cases due to availability of courier services and/or export laws of the country of origin. The sampling instructions did not request the sampler to record the exact sample location because Federal managers determined that such a request would constitute a breach of the Federal Paperwork Reduction Act of Despite this, in many instances, the samplers volunteered GPS coordinates, a map or a description of the sampling location. In the remainder, the authors used the best available information to assign the sample location.

251 227 Once the sample was received at the laboratory, large particulates were removed from all soil samples using a methanol-washed 2-mm stainless steel sieve. Soil samples then were returned to their original containers and stored at 4 o C until extraction. Polished Water. Polished water was achieved by passing 18 MΩ water through an Oasis 35 cc HLB cartridge into a two-liter Erlenmeyer flask that was purchased, methanol washed, then dedicated solely to this use. Once a total of 6 L of the 18 MΩ water was passed through the HLB cartridge it was replaced. SPE Manifold. Soil extracts were blown to dryness using a solid phase extraction (SPE) manifold as depicted in Figure S7. The samples were placed in the manifold and placed under vacuum. Air was passed through nylon filters and directed into the sample vials with methanol rinsed stainless steel needles. In order to increase the rate of evaporation of the 90:10 acetonitrile(acn):polished water(pw), a heating pad was fastened to the manifold. EPA LC-MS/MS Parameters. The strong needle, weak needle and seal washes were as follows: 60:40 ACN:PW, PW and 10:90 ACN:PW. A 20 µl aliquot of the extract was injected onto the BEH C18 column and separated with using ACN and PW adjusted to ph 3 with acetic acid as the mobile phases. University of Toronto LC-MS/MS Parameters. All parameters were the same as above except the injection volume was 5 µl. Dry-Weight Determination. Extracted soils were left in the PPCO tubes and placed in a vacuum desiccator for several days. The tubes were re-weighed until a constant weight was obtained, which was then used to calculate the dry weight PFCA and PFSA concentrations.

252 228 Additional Discussion Quality Metrics: The Ottawa sand field blanks were quantitated for the two dominant analytes we detected in this study, PFOA and PFOS (Table D4-D6). Most sand blanks were found to contain low, and relatively constant concentrations of both analytes, suggesting no sample-collection or transit contamination. The sand blanks for three samples of remote origin, Buea, Camaroon (AF03), Mabira, Uganda (AF05) and El Yunque, Puerto Rico (NA23), returned anomalous PFAS concentrations suggesting the potential for collection or transit contamination of these samples (Table D7). Reviewing the data for these three samples, in all cases analyte concentrations fell toward the low end of our database, but they generally did not fall at the lowest limits. There were no obvious packaging anomalies upon delivery receipt of these samples, nor did we discern unusual concentrations or homologue distributions for the samples themselves. Based on these observations that, by all metrics other than the sand blanks, these samples were unexceptional, we retained these samples in our study, but results for these samples should be regarded with a degree of caution. As expected given the sensitivity of our instruments, low levels of PFASs, <50 pg/g (parts per trillion) solvent, were detected in the procedural blanks (Table D8-D11). Of the 11 procedural blanks, prepared and analyzed interspersed among all samples run over the course of the investigation, one blank returned PFCA concentrations up to ~100x greater than all other blanks (Table D8). This blank was the first item handled when extraction activities were transferred from one team member to another. Because the second blank prepared by this team member was similar to the other blanks (Table D8), and because the five samples prepared with this blank (NA06, NA09, NA11, NA19, AS01) had lower concentrations of most analytes than this blank, we omitted this blank from detection-limit definition and retained the five samples in the study, so these five samples should be regarded with caution.

253 229 For all data, we subtracted the mean concentrations of the ten process blanks from the soil values we report herein. Regarding recovery, spiked M8PFOA ranged from % with an average of 108% for all samples (Table D1-D3). We report numerical concentrations only if they exceeded LOQ; values falling below LOQ, but above LOD, are reported as <LOQ, and values below LOD are reported as. Finally, all eight samples associated with anomalous sand (AF03, AF05, NA23) or process blanks (NA06, NA09, NA11, NA19, AS01), which should be regarded with caution, are annotated as such in the SI tables. Relationship with total organic carbon (TOC). As sorption of PFCAs and PFSAs in solid matrices is believed to be driven by partitioning to OC, 1-3 TOC was measured for each soil sample and ranged from 0.1 for the Antarctic sample (AN01) to 38.9% for Akumal, Mexico (NA06) with data presented in Tables D1-D3. Unlike previous studies that reported an increase in sorption with the number of perfluorinated carbons ( 7 C F s), 1-3 no chain length dependency was observed in this study. Since volatility decreases with the number of perfluorinated carbons, the flux of longer chain congeners into the soil will depend on distance from local or regional sources. Gellrich et al. 4 recently demonstrated that shorter chain congeners are only eliminated from a soil column in the presence of long chain congeners. These two factors may preclude any relationship between TOC and the number of perfluorinated carbons. In addition, unlike POPs such as PBDEs and PCBs that have been demonstrated to have a correlation with TOC in background soils, 5,6 no relationship was observed between TOC and total concentrations of PFCA or PFSA. Several factors such as the proximity to emission sources, precipitate and other soil properties differ significantly amongst sampling locations, which may preclude any direct comparison with TOC. Locations nearer emission sources have higher total concentrations of PFCA and PFSA, but may have a low fraction of TOC such as NA28-31 with TOC values ranging from 0.44 to 2.54%. Whereas several rural or remote

254 230 locations could have higher TOC, but significantly lower PFCA and PFSA concentrations such as NA15 having a TOC value of 3.26%. The volume of precipitation could also impact the retention of PFCAs and PFSAs in the surface layer due to their water solubility. Davis et al. 7 demonstrated that PFOA emitted from a nearby fluorochemical manufacturing facility was deposited in the surface soil, but was transported into the groundwater with increasing precipitation. However, further studies are needed to fully understand the impact precipitation has on the retention of PFCAs and PFSAs in the surface layer. Lastly, there are inherent differences in soil properties such as ph and cation exchange capacity (CEC), because of the geographical differences between sampling locations, which could suppress the sorption of PFCAs and PFSAs to TOC. For example, the bridging of anionic PFCAs and PFSAs with cations such as Fe 3+ and Ca 2+ to the soils having net negative surfaces has been proposed as an important sorption mechanism for PFOS to sediment. 2 Unfortunately, due to limited amounts of sample, further soil analyses were not performed in this study, but this is an mportant area of research to completely understand the fate of PFCAs and PFSAs in soils. Acknowledgements and sincere thanks to the samplers. We express our deep gratitude to the following people who were kind enough to collect samples for us. They agreed to do this for us despite that in some cases, they did not know us, they were on busy travel schedules, they were in primitive and austere conditions. These individuals and their affiliations include: Anonymous scientist, Don Betowski (USEPA), Robert Dobos (USDA-NRCS), Stephen Duirk (USEPA), Jackson Ellington (USEPA), Mark Ferrey (Minnesota Pollution Control Agency), Walter Frick (USEPA), Bob Gilkes (University of Western Australia), Janet Hergt (The University of Melbourne, Australia), Julio Haberland (Universidad e Chile), Ed Heithmar (USEPA), Chris Hickey (National Institute of Water and Atmospheric Research, New Zealand), Said Hillal (USEPA), Lisa Hoferkamp (University of Alaska Southeast), Peter Jeffers (State

255 231 University of New York, Cortland), Thomas Jenkins (SEEP/USEPA), Sharon Katz (Aurora Research Institute, Canada), Scott Korom (University of North Dakota), Ming Lai (University of Georgia), Don Macalady (Colorado School of Mines), Bruce Mathews (University of Hawaii, Hilo), Mirta Mihovolovic (Duke University), Marirosa Molina (USEPA), Shoji Nakayama (National Institute for Environmental Studies, Japan), Valentine Nzengung (University of Georgia), Vincent O Malley (Dublin, Ireland), Carlos Perdomo (Montevideo, Uruguay), Laura Rivera-Rodriguez (Centro de Investigaciones Biologicas del Noreste, S.C., Mexico), Felix Roman (University of Puerto Rico, Mayaguez), Arthur Rose (Penn State University), Vladimir Samarkin (University of Georgia), Lidia Samarkina (SEEP/USEPA), Mary Scholes (University of Witwatersrand, South Africa), Kaye Spark (University of Queensland, Australia), B.T. Thomas (USEPA), Arvo Tuvikene (Estonian University of Life Sciences), John Wilson (USEPA), Charles Wong (University of Alberta, Canada), Gary Ziegler (Penns Grove, NJ) References (1) Higgins, C.; Luthy, R. G. Sorption of perfluorinated surfactants on sediments. Environ. Sci. Technol. 2006, 40, (2) You, C.; Jia, C.; Pan, G. Effect of salinity and sediment characteristics on the sorption and desorption of perfluorooctane sulfonate at sediment-water interface. Environ. Pollut. 2010, 158, (3) Ahrens, L.; Yeung, L.; Taniyasu, S.; Lam, P.; Yamashita, N. Partitioning of perfluorooctanoate (PFOA), perfluorooctane sulfonate (PFOS) and perfluorooctane sulfonamide (PFOSA) between water and sediment. Chemosphere 2011, 85, (4) Gellrich, V.; Stahl, T.; Knepper, T. P. Behavior of perfluorinated compounds in soils during leaching experiments. Chemosphere 2012, 87, (5) Nam, J. J.; Gustafsson, O.; Kurt-Karakus, P.; Breivik, K.; Steinnes, E.; Jones, K. C. Relationships between organic matter, black carbon and persistent organic pollutants in European background soils: Implications for sources and environmental fate. Environ. Pollut. 2008, 156,

256 232 (6) Liu, W.; Li, W.; Xing, B.; Chen, J.; Tao, S. Sorption isotherms of brominated diphenyl ethers on natural soils with different organic carbon fractions. Environ. Pollut. 2011, 159, (7) Davis, K. L.; Aucoin, M. D.; Larsen, B. S.; Kaiser, M. A.; Hartten, A. S. Transport of ammonium perfluorooctanoate in environmental media near a fluoropolymer manufacturing facility. Chemosphere 2007, 67,

257 Figure D1: Approximate sampling locations in Europe (EU). 233

258 Figure D2: Approximate sampling locations in Asia (AS). 234

259 Figure D3: Approximate sampling locations in Africa (AF). 235

260 Figure D4: Approximate sampling locations in continental Australia (AU). 236

261 Figure D5: Approximate sampling locations in South America (SA). 237

262 Figure D6: Approximate sampling locations in Antarctica (AN). 238

263 Figure D7: SPE manifold adapted for solvent evaporation. 239