Biodegradation of Fluorinated Telomer Compounds As a Source of Perfluorinated Acid Formation in the Environment

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BIODEGRADATION OF FLUORINATED TELOMER COMPOUNDS AS A SOURCE OF PERFLUORINATED ACID FORMATION IN THE ENVIRONMENT

Mary Joyce Dinglasan-Panlilio

A thesis submitted in conformity

with the requirements for the degree

Doctor of Philosophy

Department of Chemistry

University of Toronto

395 Wellington Street 395, rue Wellington Ottawa ON K1A0N4 Ottawa ON K1A0N4 Canada Canada

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Doctor of Philosophy Degree 2008

Mary Joyce Dinglasan-Panlilio

Department of Chemistry, University of Toronto

ABSTRACT

The widespread detection of PFCAs in environmental matrices, along with their observed persistence and potential toxicity, has prompted investigation into their sources. The goal of this research was to determine whether biodegradation of various telomer compounds leads to formation of PFCAs and to assess the overall contribution of this class of compounds to the global contamination of PFCAs.

The biodegradation of the 8:2 FTOH (CF3(CF2)7CH2CH2OH) was investigated under aerobic conditions in a mixed microbial system. 8:2 FTOH was rapidly degraded forming perfluorooctanoic acid (PFOA) as the terminal metabolite. Other metabolites detected were the 8:2 telomer aldehyde (8:2 FTAL) along with the saturated and unsaturated telomer acids (8:2 FTCA and 8:2 FTUCA). It was proposed that a (3-oxidation type of mechanism maybe involved in the degradation.

Investigations into sources of FTOHs to the environment were also pursued. Residual unbound FTOHs in varying chain lengths (C6-C14) were measured in significant amounts from several commercially available and industrially applied fluorinated polymeric and surfactant materials. It was suggested that application of these materials is likely to release these compounds to the environment, which can degrade ultimately leading to PFCA production.

Additional sources of FTOH release to the environment were demonstrated from the biodegradation of ester type fluorinated telomer monomers. The telomer alcohol produced

from the aerobic degradation of these monomers was observed to further degrade forming previously determined FTOH metabolites including PFOA.

A model fluorotelomer acrylate polymer was synthesized and subjected to biodegradation

experiments to determine whether they are also potential sources of PFCAs to the

environment. The lack of FTOH and PFOA formation from these experiments suggest that these macromolecules are more stable than FTOHs and ester telomer monomers.

This research confirms the formation of PFCAs from the biodegradation of telomer

alcohols and monomers and suggests that these compounds make an important contribution to

the environmental contamination of PFCAs. Future experiments were proposed to better

investigate the biodegradation potential of telomer polymers.

- in - ACKNOWLEDGEMENTS

First and foremost, I would like to acknowledge my primary supervisor, Dr. Scott

Mabury, to whom I owe so much. For providing me with that initial spark of interest in research, for showing me the joys and rewards of teaching, and for always inspiring me to raise the bar in everything I do, for all of these I am truly grateful. I aspire to be the great educator and researcher that you are.

To my other supervisor, Dr. Elizabeth Edwards, your continued support and guidance have been integral in my years as a graduate student.

I would like to acknowledge members of the Mabury Group, past and present. It has been a privilege working with such a talented group of people. Thanks also goes to all the

students and alumnus of the Environmental Chemistry program, you have shared in this great adventure. To the soon to be Dr. Nana Kwamena, it has been a joy to collaborate with you.

Thank you for your never-ending support and above all, for your friendship. I would never have gotten through these past couple of years without you.

Thank you to the administrative and research staff of the Chemistry Department who have helped me along the way. Special thanks to Penny Ashcroft Moore, Dan Mathers, of the

ANALEST facility and Alex Young of the AIMS facility.

Funding support from National Science and Engineering Research Council and

OMNOVA foundation is greatly appreciated.

To my entire family, Mom, Dad, Allan and the boys, "Sparky and Philo", you've all

shaped who I am today. Thank you for loving me despite my faults and for being proud of what

I do. You have instilled in me a great love for education and a passion for learning. Despite all

my years studying, it is you who taught me that what matters most is family.

- iv - To my husband, Joel, you are my strength. Thank you for following me to Toronto and to our next adventure. This achievement is as much yours as it is mine. Your infinite love, support, patience and understanding are what got me through. You have always made me laugh when I needed it the most and you have always been there with a shoulder to cry on when frustration got to me. I can't wait to start the next chapter of our life together.

- v - PREFACE

This thesis is organized as a series of manuscripts that have been published, have been submitted to, or in preparation for submission to scientific peer reviewed journals. As a result, some repetition of introductory material and description of methods used in the series of studies is unavoidable. As identified by primary authorship, all manuscripts were prepared by Mary Joyce Dinglasan-Panlilio with critical comments by Elizabeth A. Edwards and Scott A. Mabury. The list of manuscripts along with contributions of authors is outlined below.

Chapter I - Biodegradation of Aliphatic Organohalogen Contaminants

Chapter II - Fluorotelomer Biodegradation Yields Poly- and Perfluorinated Acids

Published in - Environ. Sci. Technol. 2004, 38, 2857-2864

Author list - Mary Joyce A. Dinglasan, Yun Ye, Elizabeth A. Edwards and Scott A Mabury

Contributions - Mary Joyce Dinglasan carried out the experimental design and set-up along with gas chromatographic (GC) analysis of volatile analytes of interest. Yun Ye performed liquid chromatography tandem mass spectrometry (LCMSMS) analysis of non-volatile metabolites. Overall data analysis and interpretation was done by Mary Joyce Dinglasan. All versions of the manuscript were prepared by Mary Joyce Dinglasan with critical comments provided by Elizabeth A. Edwards and Scott A. Mabury.

Chapter III - Significant Residual Fluorinated Alcohols Present in Various Fluorinated Materials

Published in - Environ. Sci. Technol. 2006, 40, 1447-1453

Author list - Mary Joyce A. Dinglasan-Panlilio and Scott A Mabury

Contributions — Mary Joyce A. Dinglasan-Panlilio designed and conducted the experiments. All data analysis, interpretation and manuscript preparation were also carried out by Mary Joyce A. Dinglasan-Panlilio. Intellectual input and critical comments were provided by Scott A. Mabury.

- vi - Chapter IV - Biodegradation of Fluorotelomer Based Monomers as a Source of Fluorotelomer Alcohols

To be submitted to - Environ. Sci. Technol.

Author list - Mary Joyce A. Dinglasan, Elizabeth A. Edwards and Scott A Mabury

Contributions — Mary Joyce A. Dinglasan-Panlilio designed and conducted all experiments. Data analysis, interpretation and all versions of the manuscript was prepared by Mary Joyce A. Dinglasan-Panlilio. Scott A. Mabury and Elizabeth A. Edwards provided critical comments.

Chapter V - Investigation into the Biodegradation of Fluorinated Telomer Polymers

Chapter VI - Summary, Conclusions and Future Research Perspectives

- vn - Other publication during PhD:

MacDonald, M.M., Dinglasan-Panlilio, M.J.A, Mabury, S.A., Solomon, K.R. and Sibley, P.K. Fluorotelomer Acids are More Toxic than Perfluorinated Acids. Environ. Sci. Technol. Accepted.

Other publication in preparation for submission:

Dinglasan-Panlilio, M.J.A., Kwamena, N-O., Mabury, S.A. An Undergraduate Experiment to Measure Chemical Markers for Polluted Air. J. Chem. Ed. 2007, In preparation.

- vm - TABLE OF CONTENTS

CHAPTER I: Biodegradation of Aliphatic Organohalogen Contaminants

1.1 Introduction 2 1.2 Environmental Contamination of Organohalogen Compounds 3 1.2.1 Aliphatic Organochlorine Compunds 3 1.2.2 Aliphatic Organobromine Compounds 7 1.2.3 Aliphatic Organofluorine Compounds 9 1.3 Perfluoroalkyl Compounds in the Environment 11 1.3.1 Introduction to PFCs 11 1.3.2 Perfluorinated Carboxylic Acids (PFCAs) 14 1.3.2a Environmental Monitoring 16 1.3.2b Biological Monitoring 19 1.3.3 Perfluorosulfonic Acids and Polyfluorinated Sulfonamides 22 1.3.4 Fluorinated Telomer Compounds 22 1.3.4a Fluorotelomer Alcohols 23 1.3.4b Fluorotelomer Monomers 25 1.3.4c Fluorotelomer Polymers 25 1.3.4d Fluorotelomer Surfactants 27 1.4 Relevance of Fluorotelomer Compounds on PFCA Contamination 27 1.4.1 Direct Sources 27 1.4.2 Indirect Sources 29 1.4.2a Detection of FTOHs in the Atmosphere 30 1.4.2b Abiotic Transformation of FTOHs 31 1.4.2c Biotic Transformation of FTOHs 35 1.4.3 Sources of FTOHs 38 1.4.4 PFCA Source Patterns 40 1.5 Microbial Metabolism of Xenobiotics 41 1.5.1 Biological Factors Affecting Biodegradation 43 1.5.2 Physico-Chemical Factors Affecting Biodegradation 44

IX 1.5.3 Environmental Factors 45 1.6 Microbial Degradation of Aliphatic Organohalogen Contaminants 46 1.6.1 Biodegradation of Aliphatic Organochlorine Compunds 46 1.6.1a Oxidation 47 1.6.1b Substitution 48 1.6.1c Reductive Dehalogenation 49 1.6.2 Biodegradation of Aliphatic Organobromine Compounds 50 1.6.3 Biodegradation of Aliphatic Organofluorine Compounds 52 1.7 Goals and Hypothesis 57 1.8 Literature Cited 60

CHAPTER II: Fluorotelomer Alcohol Biodegradation Yields Poly- and Perfluorinated Acids

Mary Joyce A. Dinglasan, Yun Ye, Elizabeth A. Edwards and Scott A Mabury Environ. Set Technol. 2004, 38, 2857-2864.

2.1 Abstract 83 2.2 Introduction 84 2.3 Experimental Section 88 2.3.1 Media and Chemicals 88 2.3.2 Protein Analysis 88 2.3.3 Growth Conditions and Culture Preparation 89 2.3.4 GC/ECD and GC/MS Analysis of 8:2 FTOH 89 2.3.5 LC/MS/MS Analysis of Non-Volatile Metabolites 91 2.3.6 QA/QC 93 2.4 Results and Discussion 95 2.5 Acknowledgements 105 2.6 Literature Cited 106 2.7 Addendum to Chapter II 109 2.8 Literature Cited in Addendum 113

- x - CHAPTER III: Significant Residual Fluorinated Alcohols Present in Various Fluorinated Materials

Mary Joyce A. Dinglasan-Panlilio and Scott A. Mabury Environ. Sci. Technol. 2006, 40, 1447-1453

3.1 Abstract 116 3.2 Introduction 117 3.3 Materials and Methods 120 3.3.1 Chemicals and Standards 120 3.3.2 Preparation of Experimental Vessels 121 3.3.3 Instrumental Analysis and Quantification 122 3.3.4 Dry Mass Determination 123 3.3.5 Quality Control and Assurance 123 3.4 Results and Discussion 124 3.5 Acknowledgements 135 3.6 Literature Cited 136

CHAPTER IV : Biodegradation of Fluorotelomer-based Monomers as a Source of Fluorotelomer Alcohols

Mary Joyce Dinglasan-Panlilio, Elizabeth A. Edwards and Scott A. Mabury Environ. Sci. Technol. 2007, To be submitted

4.1 Abstract 140 4.2 Introduction 141 4.3 Materials and Methods 144 4.3.1 Media and Chemicals 144 4.3.2 Growth Conditions and Microcosm Preparation 144 4.3.3 GC/MS Analysis of Volatile Monomers and FTOHs 145 4.3.4 LC/MS/MS Analysis of Non-Volatiles 146 4.4 Results and Discussion 148 4.4.1 Experiments with an Ester Monomer 149

xi - 4.4.2 Experiments with an Ether Monomer 153 4.4.3 Experiments with a Urethane Monomer 154 4.5 Acknowledgements 158 4.6 Literature Cited 158

CHAPTER V : Investigation into the Biodegradation of Fluorotelomer-based Polymers

5.1 Abstract 163 5.2 Introduction 164 5.3 Materials and Methods 167 5.3.1 Chemicals 167 5.3.2 Polymer Synthesis 167 5.3.3 Polymer Characterization 169 5.3.4 Biodegradation Experiments 170 5.3.4a Purge and Trap Experiment 170 5.3.4b Activated Sludge Experiment 171 5.3.5 Instrumental Analysis and Quantification 172 5.3.5a GC/MS Analysis of Purge and Trap Experiment Samples 172 5.3.5b LC/MS/MS Analysis of Non-Volatile Metabolites 172 5.4 Results and Discussion 173 5.4.1 Polymer Characterization 174 5.4.2 Biodegradation of Model Telomer Polymer 179 5.5 Acknowledgements 186 5.5 Literature Cited 186

CHAPTER VI: Summary, Conclusions and Future Perspectives 6.1 Summary and Conclusions 192 6.2 Future Research Perspectives 197 6.2.1 Additional Polymer Degradation Experiments 197 6.2.2 Degradation of Other Telomer based Compounds 198

xn 6.2.3 Degradation Under Various Conditions and Matrices 198 6.2.4 Mechanistic and Enzymatic Studies on FTOH Biodegradation 199 6.2.5 Reactivity of FTOH Intermediates 199 6.2.6 Method Development on Measuring Precursors in 200 Environmental Samples 6.3 Literature Cited 200

- xm - LIST OF FIGURES

CHAPTER ONE

Figure 1-1 Electrochemical fluorinated reaction of methane 13

Figure 1-2 Synthesis of perfluorinated carboxylic acids from telomerization 13

Figure 1-3 Chemical structure of perfluorooctanoic acid (PFOA) 15

Figure 1-4 Chemical structure of perfluooctane sulfonic acid (PFOS) 22

Figure 1-5 Chemical structure of the 8:2 fluorotelomer alcohol 24

Figure 1-6 Chemical structure of 2(perfluorooctyl) ethyl acrylate 25

Figure 1-7 Representative structure of a fluorotelomer based copolymer 26

Figure 1-8 Representative structure of Zonyl® UR, a phosphate telomer surfactant 27

Figure 1-9 Mechanism of oxidation of organochlorine aliphatic compounds. 48

Figure 1-10 Mechanism of subsitution reactions of chlorinated aliphatics. 49

Figure 1-11 Reductive dechlorination mechanisms 50

Figure 1-12 Chemical structures of polyfluorinated sulfonamides. 56

Figure 1-13 Proposed mechanism for the enzymatic defluorination of 57 3 -fluoropyruvate

CHAPTER TWO

Figure 2-1 Typical GC/ECD chromatogram of 8:2 FTOH and allylic 8:2 FTOH 94

Figure 2-lb LCMSMS chromatogram of non-volatile metabolites of 8:2 FTOH 94

Figure 2-2 Confirmation of metabolites observed from the degradation of 8:2 FTOH 95

Figure 2-3 Typical transformation kinetics and mass balance of 8:2 FTOH 97 Biodegradation

Figure 2-4 GC/MS extracted chromatograms of synthesized 8:2 FTAL standard 99 and 8:2 FTAL detected in active samples.

xiv Figure 2-4b LC/MS/MS chromatogram of blank, sterile control, and an active 99 samples in 8:2 FTOH degradation study

Figure 2-5 Proposed biodegradation pathway and products of 8:2 FTOH 104

CHAPTER III

Figure 3-1 Stylized schematic of postulated steps leading to the production of 120 telomer-based polymers

Figure 3-2 GCMS chromatogram of sample obtained from purging of Teflon™ 128 Advance Rug and Carpet Protector

Figure 3-3 Profiles of residual unbound polyfluorinated alcohols in the seven 130 fluorinated materials.

Figure 3-4 Cumulative residual alcohols extracted in Zonyl™ FSO 100 131

CHAPTER IV

Figure 4-1 Sample chromatograms of an active vessel vs sterile control in 148 8:2 methacrylate biodegradation experiment at T=0 and T=3 days.

Figure 4-2 Degradation of ester monomer, 8:2 methacrylate in active vessels and 151 sterile control.

Figure 4-3 Degradation of ester monomer, 8:2 acrylate in active vessels and 152

sterile control

Figure 4-4 Ether monomer degradation in active vessels and sterile control 153

Figure 4-5 Urethane monomer biodegradation in active vessels and sterile controls 156

CHAPTER V

Figure 5-1 Schematic representation of fluorotelomer based polymer synthesis. 168

Figure 5-2 JH NMR spectra of monomers used in polymer synthesis 174

Figure 5-2c !H NMR of synthesized Polymer I 175

Figure 5-3 19F NMR of 8:2 acrylate monomer and synthesized Polymer I. 176

- xv - Figure 5.4 Expanded MALDI-MS spectra from mass range 4,000 to 6,000 178 of Polymer I

Figure 5.5 Removal of residual 8:2 FTOH and 8:2 acrylate monomer from 180 Polymer II spiked vessels.

Figure 5-6 Cumulative FTOH produced from active and control vessels spiked 181 with Polymer I and Polymer II.

Figure 5-7 PFOA production in pure activated sludge experiment. 184

- xvi - LIST OF TABLES

CHAPTER ONE PAGE

Table 1-1: Common chlorinated aliphatics in the environment 5

Table 1-2: General structure of chlorinated paraffins 7

Table 1-3: Common brominated aliphatics in the environment 9

Table 1-4: Structures and abbreviations of perfluoroalkyl chemicals of interest 12

Table 1-5: Physical properties of perfluorooctanoic acid (PFOA) 15

Table 1-6: Physical properties of the 8:2 fiuorotelomer alcohol (8:2 FTOH) 24

Table 1-7: Summary of measured concentrations of fiuorotelomer carboxylie acids 34 in various environmental matrices.

CHAPTER II

Table 2-1: Acronym, Structure and Molecular Weight of Perfiuorinated Compounds 86 of Interest

Table 2-2: Optimized MS/MS Conditions for Metabolite Confirmation 92

CHAPTER III

Table 3-1: Total residual polyfluorinated telomer alcohols and sulfonamides removed 132 from various fluorinated polymeric and surfactant materials.

CHAPTER IV

Table 4-1: Structures and molecular ions of analytes telomer monomers investigated. 143

CHAPTER V

Table 5-1 Molecular weights of synthesized polymers based on GPC analysis. 177

Table 5-2 Summary of measured 8:2 FTOH metabolites from polymers sludge 185 experiment.

- xvii - LIST OF APPENDICES

Appendix A: Supporting Information for Chapter II 203

Appendix B: Supporting Information for Chapter III 206

Appendix C: Supporting Information for Chapter V 212

- xvm - CHAPTER I

BLODEGRADATION OF ALIPHATIC ORGANOHALOGEN CONTAMINANTS 2

1.1 INTRODUCTION

Large amounts of synthetic chemicals have been introduced into the environment for the past several decades. Release of chemicals into the environment may be deliberate as in the case of pesticide application or accidental as is the case with spills.

Chemical discharge to the environment also occurs from release of byproducts as a function of use of treated materials, such as the case with polybrominated diphenyl ethers

(PBDEs). Degradation of compounds in both terrestrial and aquatic environments by microorganisms is an important fate of contaminants (7). The ability of microorganisms to breakdown chemicals has long been exploited in the treatment of industrial and domestic wastewater in sewage treatment plants and has recently been gaining momentum as a way to treat contaminated sites, a process known as bioremediation (2).

Many chemicals find their way into wastewater as a consequence of their method of disposal or as a function of their use (ie. chemicals used as cleaners or additives in personal care products etc.) (J). Many chemicals in current production are also halogenated. In a recent compilation of persistent chemicals with potential for bioaccumulation, 30% were organohalogens, while another list identifying persistent chemicals with potential for long range atmospheric transport contained 80% organohalogens (4). This chapter reviews what is known about the biodegradation of aliphatic organohalogen contaminants. The focus of the thesis however, is on the biodegradation of a class of organofluorine compounds known as fluorinated telomers and how it relates to the perfluorocarboxylic acid contamination in the environment. 3

1.2 ENVIRONMENTAL CONTAMINATION OF ORGANOHALOGEN COMPOUNDS

Many environmentally relevant xenobiotics introduced for industrial or commercial use are halogenated and halogenation has often been implicated as a contributor to persistence (5). Although some halogenated compounds are labile under reductive conditions, each electronegative element on a compound withdraws electrons from the ring or chain making it resistant to electrophilic reactions such as oxidations (6), contributing to its recalcitrance. Also, addition of chlorine or bromine increases the size of molecules and provides steric hindrance toward any incoming reagent on the molecule.

The list of aliphatic organohalogen compounds that have been detected in the environment is extensive and is only briefly discussed in this chapter.

1.2.1 Aliphatic Organochlorine Compounds

Chlorinated organic molecules constitute the largest single group of compounds on the list of priority pollutants compiled by the United States Environmental Protection

Agency (U.S. EPA) (7). Chlorinated compounds have been well studied and monitored in the environment since problems associated with l,l,l-trichloro-2,2 bis(p- chlorophenyl)ethane or DDT along with other polychlorinated biphenyls (PCBs) were highly publicized in Rachel Carlson's book, Silent Spring, first published in 1962.

Occurrence of cyclic aliphatic chlorinated compounds in the environment is well documented (8-16). Many of these compounds are chlorinated pesticides such as lindane, heptachlor, and dieldrin applied intentionally and consequently remain present in the environment despite not being used for decades. Aliphatic chlorinated compounds such as tetrachloroethylene (PCE), trichloroethylene (TCE), vinylchloride (VC) and 4 chlorinated ethanes (Table 1-1), commonly used solvents in industry, are ubiquitous groundwater contaminants and have been detected in drinking water aquifers (5, 17, 18).

Moran et al. (19) surveyed groundwater samples from the entire United States between

1985-2002 for the presence of PCE, TCE, 1,1,1-trichloroethane and methylene chloride

(also known as dichloromethane) and found one or more of the compounds to be present in 17% of the samples analyzed, PCE being the most frequently detected. Concentrations of these aliphatic organochlorines ranged from 0.02 p,g L"1 to 4800 (j,g L"1, PCE again was the most abundant solvent detected. In addition to the above compounds, other chlorinated CI and C2 hydrocarbons such as chloroform, 1,1-dichloroethane, 1,2- dichloroethane were also detected worldwide in marine waters at various concentrations

(20). Chlorinated solvents are typically used as degreasing and dry cleaning agents and have entered the environment through leakage from storage tanks and irresponsible disposal in the past (21). Additionally, vinyl chloride and 1,2-dichloroethane are bulk chemicals used as intermediates in the production of polyvinyl chloride plastics (PVC)

(20) and are likely released in the environment as a consequence of their high industrial use.

Inherent physical properties of these chlorinated aliphatics allow them to

diseminate easily in groundwater. This class of compounds has high vapour pressures, high water solubilities, low organic partitioning coefficients, and low viscosities and

interfacial tension (19). Unlike their aromatic counterpart, they are not likely to partition

into biota due to their low octanol water partitioning coefficients (Kow) (20). These

compounds continue to be produced in the United States and in Europe at 78 to 8000 kt y"

1 (21). 5

Table 1-1 Common Short Chain Chlorinated Aliphatics in the Environment.

Class Common Name Acronym Structure H Dichloromethane or methyl I Chloromethane DCM H—C—CI chloride I CI

H H I I 1,1 -dichloroethane 1,1 -DCA CI C—C—H I I CI H

CI CI I I 1,2 -dichloroethane 1,2-DCA H—C—C—H I I H H Chloroethanes CI H I I 1,1,1-TCA CI C—C—H 1,1,1 -trichloroethane I I CI H CI CI I I 1,1,2-TCA CI C—C—H 1,1,2-trichloroethane I I H H

1,1,2,2-tetrachloroethylene PCE /c~\ CI CI

1,1,2-trichloroethene TCE C,\-/'

Chloroethenes CI H

C'\-/Cl cis-1,2-dichloroethene cw-DCE H H

Vinyl Chloride VC C'\-/' H H 6

Longer chain chlorinated aliphatics have also been detected widespread in the environment and have been the subject of several reviews (22-27). Also known as chlorinated paraffins (CPs), this class of chlorinated aliphatics has a general formula

CxH(2x+2)-yCly and is produced by the chlorination of different «-alkane fractions derived from petroleum distillation (24). These chemicals are divided into groups designated by their chain lengths (Table 1-2): short chain CPs (SCCPs) refer to compounds with 10-13 carbon atoms, medium chain CPs (MCCPs) are those with 14-17 carbon atoms and long- chain CPs (LCCPs) comprise of 18 or more carbon atoms (24). The level of chlorination of CPs are reported to vary anywhere from 30 to 72% by weight (28). The many possible positions for the chlorine atoms and presence of chiral carbon atoms lead to a large number of potential isomers, enantiomers and diastereoisomers for this class of compounds. CPs have been produced since the 1930s for use as additives in lubricants and cutting fluids for metal-working and applied as plasticizers and flame retardants in plastics, sealants and leather (24). CPs have been measured in various environmental matrices including seawater and freshwater samples (29, 30), in air at concentrations ranging from 1130 to 3040 pg m"3 (31, 32) and in sediments from the Czech Republic

(33) as well as sediments from Canadian lakes (28). It has also been detected in a wide range of biota samples (22, 30, 34) as well as in human blood (35). Detection in biota of these compounds were expected due to their physical properties, including the octanol water partitioning (log Kow) reported to be greater than 5 (26). Hence, unlike the short chain aliphatic chlorinated solvents, these compounds are likely to bioaccumulate and biomagnify in biota. Release of these compounds to the environment has been attributed to high production volumes, reported to range from 7,900 to 12,700 tonnes in 1998 in North America alone (30), wide range of usage as well as improper disposal of products that incorporate these chemicals (24).

Table 1-2 General Structure of Chlorinated Paraffins

Class Acronym General Structure

Short Chain Chlorinated SCCP Ci0H(22-y)Cly - Ci3H(28-y)Cly Paraffins

Medium Chain Chlorinated MCCP Ci4H(3o-y)Cly - Ci7H(36-y)Cly Paraffins

Long Chain Chlorinated LCCP Ci8H(38-y)Cly - C>i8H(>38-y)Cly Paraffins

1.2.2 Aliphatic Organobromine Compounds

Organobromines receive much less attention than organochlorines in the environment, until recently when environmental monitoring studies on organobromine contaminants have focused on polybrominated diphenyl ethers (PBDEs) and other brominated flame retardants (BFRs). These aromatic substances have been detected in air, soil and water samples as well as in fish and animal tissues worldwide (36). A primary concern for regulators is their detection in human blood serum and breast milk worldwide with concentrations doubling every five years (37-40).

Hexabromocyclododecane (HBCD) is a cyclic aliphatic organobromine compound that 8 has been a subject of several studies also due to their widespread detection in the environment and in humans (41, 42) Fewer studies however, have investigated aliphatic organobromine compounds. Brominated alkanes such as methyl bromide, 1,2- dibromoethane, 1,2-dibromoethene and propargyl bromide have been used worldwide in agriculture as fumigants and nematicides (43) as well as to control weeds (44). These compounds are integral in the cultivation of numerous agricultural products including strawberries and tomatoes (44). These agricultural applications have resulted in them becoming common groundwater contaminants. It is important to note that brominated methanes are also naturally produced by marine algae and phytoplankton (45, 46) which can add to the burden of these compounds in the environment. Goodwin et al. (45) have estimated that biogenic and anthropogenic emissions are of similar magnitudes. Methyl bromide has been banned as part of the Montreal Protocol due to its potential for stratospheric ozone depletion (47). There are no current reports of environmental detection of longer chain brominated aliphatic compounds in the published scientific literature. 9

Table 1-3 Common Brominated Aliphatics in the Environment

Class Common Name Structure H Methyl Bromide I Br—C—H I H

Br Dibromomethane I Bromomethanes Br C—H I H

Br Bromoform I Br C—H I Br

Br Br Bromoethanes 1,2 -dibromoethane I I H C—C—H I I H H

Br Br Bromoethenes 1,2-dibromoethene \ / /c=c\ H H

1.2.3 Aliphatic Organofluorine Compounds

Fluorinated organic compounds have a wider array of industrial and commercial applications than organobromine compounds, however up until recently, both classes of compounds received less attention when compared to their chlorinated analogues. The limited attention organofluorines has received was attributed to the inherent challenges in measuring them in environmental matrices (48), their lack of regulation and their biological inertness that was perceived to have limited impact to human health and the environment (49). This same "inertness" however, leads to resistance to degradation and 10 accumulation. With the aid of advances in the field of analytical chemistry, specifically the development of the electrospray ionization technique coupled with mass spectrometry, came the detection of previously unknown organofluorine contaminants that has garnered much interest because of their environmental persistence and ubiquity.

Perfluorinated alkyl chemicals (PFCs) is a large family of synthetic organofluorine contaminants recently found to be ubiquitous in the environment (48, 50). Investigating a possible source of a class of PFCs known as perfluorinated carboxylic acids (PFCAs) is the focus of this thesis. It is also worthwhile to note the existence of other long known aliphatic organofluorine contaminants.

Carbon tetrafluoride (CF4) is the most abundant perfluorocarbon in the earth's atmosphere (51) and was measured in stored air at 74 parts per trillion (pptv) in 1997 of which 40 pptv are from natural emission and the rest from aluminum and semi conductor industries (51). Perfluoroethane (C2F6) and perfluoropropane (C3F8) were also measured in the same study at significantly lower concentrations of 2.9 and 0.9 pptv respectively

(51).

An important class of fluorinated aliphatic organics detected in the environment are the chlorofluorocarbons (CFCs) used as refrigerants and aerosols in industrial processes and domestic products. Their widespread use led to their detection in the atmosphere and implication as agents in stratospheric ozone depletion and global warming (52, 53). Worldwide production of CFCs has since been banned under the

Montreal Protocol of 1989 and its succeeding amendments. Some CFCs were also used as degreasing agents akin to the chlorinated solvents and similarly detected in groundwater (54). Hydrochlorofluorocarbons (HCFCs) and hydrofluorocarbons (HFCs) were marketed as replacements for the banned CFCs (55, 56). These compounds are one 11 or two carbon aliphatics, similar in structure as the CFCs but are hydrogenated. The presence of hydrogen atoms on these compounds allow them to be susceptible to atmospheric tropospheric oxidation thus reducing their potential to enter the stratosphere and cause damage to ozone. However, these oxidation reactions had led to the production of trifluoroacetate (TFA) in the environment, a highly persistent compound

(Section 1.6.3) known to inhibit plant growth (55, 56).

1.3 PERFLUOROALKYL COMPOUNDS IN THE ENVIRONMENT

1.3.1 Introduction to PFCs

Perfluoroalkyl chemicals (PFCs) are compounds prevalently used in various

aspects of industry and commerce for the past 50 years (57). These chemicals are

characterized by a hydrophobic perfluorinated alkyl chain and a hydrophilic end group

with the general structure F(CF2)nR- The perfluorinated chain can vary in lengths

typically n= 4 to 17 and the type of hydrophilic end group (ie. carboxylate, sulfonate,

phosphate etc.) is typically used to classify these compounds. The various PFCs of

interest in this thesis are summarized in Table 1-3 and described in sections 1.3.2 - 1.3.4. 12

Table 1-4 Structural Formula and Acronyms of Perfluoroalkyl Chemicals of Interest Class Common Name Acronym Structural Formula

PFHpA CF (CF ) COOH Perfluorinated Perfluoroheptanoic Acid 3 2 6 PFOA CF (CF ) COOH Carboxylic Acids Perfluorooctanoic Acid 3 2 7 Perfluorononanoic Acid PFNA CF3(CF2)8COOH

Polyfluorinated Perfluorooctane Sulfonate PFOS CF3(CF2)8S03 Sulfonates

N-Ethyl-Perfluorooctane N-EtFOSE CF3(CF2)8S02N(CH2CH2)CH2CH2OH Perfluorinated Sulfonamidoethanol Sulfonamides N-Ethyl-Perfluorooctane N-EtFOSA CF3(CF2)8S02N(CH2CH3)H Sulfonamide

6:2 Fluorotelomer Alcohol 6:2 FTOH CF (CF ) CH CH OH Fluorotelomer 3 2 5 2 2 ., . ,,.,,, , 8:2 Fluorotelomer Alcohol 8:2 FTOH CF3(CF2)7CH2CH2OH Alcohols/Aldehyde ^ Fluorotelomer Akohol 10:2 FTOH CF3(CF2)9CH2CH2OH 8:2 Fluorotelomer 8:2 FTAL CF3(CF2)7CH2COH Aldehyde

8:2 Fluorotelomer 8:2 FTCA CF3(CF2)7CH2COOH Carboxylic Acid Fluorotelomer 8:2 Fluorotelomer 8:2 FTUCA CF3(CF2)6CF=CHCOOH Carboxylic Acids Unsaturated Carboxylic Acid

8:2 FTOH Acrylate 8:2 ACY CF3(CF2)7CH2CH2OCOCHCH2 8:2 FTOH Methacrylate :2 META CF3(CF2)7CH2CH2OCOC(CH3)CH2 Fluorotelomer 8:2 FTOH Oxetane 8:2 OXE CF3(CF2)7CH2CH2OCH2C(CH2OCH2)CH3 Monomers 8:2 FTOH Urethane 8:2 BAL CF3(CF2)7(CH2)2OCONH(CH2)6NHCO(CH2) 2(CF2)7CF3

Synthesis. PFCs are primarily synthesized using two distinct methods: electrochemical fluorination and telomerization.

Electrochemical fluorination (ECF) was first invented by Simons in 1948 (58) and involves the organic substance to be fluorinated be dispersed in hydrogen fluoride (HF) while passing an electric current with a voltage between 5-7 V (58). Hydrogen is evolved on the cathode and the organic substance is fluorinated. Typically all hydrogen atoms in the molecule are replaced by fluorine but some functional groups such as carboxylic 13

acids are retained. An ECF reaction scheme for methane is shown in Figure 1-1. A

consequence of this process is the fragmentation of the carbon chain resulting in branching of the products (59, 60).

-ei"- -WH +

CH4 • CH3ads • CH3ads

-ee~" -H-FT+ -e F" + CH3Fads • CH2Fads • CH2Fads •

-2e, -H+, F -2e", -H+, P F"

CH2F2ads • CHF3ads • CF4ads •

Figure 1-1 Electrochemical fluorination reaction sequence of methane (Reproduced from (58))

Telomerization is a process first developed by the DuPont company involving the

reaction between a compound, called a telogen, with an ethylenically unsaturated

molecule referred to as a taxogen and catalyzed by SbF3, AICI3, ZrCU or organic

peroxides such as bis(trichloroacryloyl) peroxides (58, 61) (Figure 1-2). This reaction

produces a perfluoroalkyl iodide that can be oxidized under extreme conditions to form

PFCAs or hydrolyzed to form alcohols and other PFCs. PFCs that are synthesized by

telomerization are primarily linear in structure given that a linear telogen is typically used

in the reaction (58-60).

SbF3 F2C=CF2+ CF3CF2I • CF3CF2(CF2CF2)rll Taxogen Telogen

oleum, 100-180C, PreSS re 1 CF3CF2(CF2CF2)nl " * CF3(CF2CF2)n- CF2COOH + l2

Pd[P(C6H5)3]4 1 CF3CF2(CF2CF2)nl + C02 • CF3(CF2CF2)n- CF2COOH

(1)DMF, (2)H20

Figure 1-2 Synthesis of perfluorinated carboxylic acids by telomerization 14

Application. A unique characteristic of these compounds is the chemical stability of the fluorocarbon chain to strong acids, oxidizing agents, and concentrated alkalis that make them ideal in chemical applications where traditional hydrocarbons will decompose

(58). Perfluorinated and polyfluorinated chemicals are known for their surface-active properties and have been used as additives in adhesives to promote wetting and penetration of the substances being joined and have also been incorporated in cement to reduce shrinkage, hard surface cleaners to enhance cleaning power; cosmetics such as shampoos and conditioners to act as emulsifiers; polishes and waxes to help eliminate streaks and enhance gloss on floor finishes (58). They have also been used as antifogging agents on metal, plastic or glass surfaces and as antistatic agents on various substrates

(59). The electronic industry has made use of PFCs in electroplating of metals, insulators for wires and cables and electropolishing. PFCs have been incorporated in aqueous film- forming foams (AFFF) to improve foam stability (62). Fluoropolymer industries have used PFCs as emulsifiers in the emulsion polymerization of their polymers to improve physical properties of the polymer and increase the rate of polymerization (63).

1.3.2 Perfluorinated carboxylic acids (PFCAs)

Perfluorinated carboxylic acids (PFCAs) are perfluoroalkyl chemicals composed of a carboxylate end group and generally given the chemical formula RfCOOH where Rf denotes the perfluorinated portion of the molecule (58). These compounds can have various chain lengths ranging from 1 perfluorinated carbon up to at least 17 carbon chains

(58). Long chain analogues are anionic surfactants widely used in industry.

Perfluorooctanoic acid (PFOA), the 8 carbon version of this class of compounds shown in

Figure 1-3, is commonly detected in environmental matrices and has been of recent 15 regulatory concern (50). Some of the measured physical properties of PFOA are summarized in Table 1-4. Concerns for this class of perfluorinated compounds are due not only to their widespread contamination but also to their potential toxicity and their persistence. In addition, PFCAs with greater than 9 carbons have been shown to bioaccumulate in the environment.

Figure 1-3 Chemical structure of perfluorooctanoic acid (PFOA). A perfluorocarboxylic acid with 7 perfluorinated carbons.

Table 1-5 Physical properties of perfluoroctanoic acid (PFOA)

Physical Property Measurement Reference

Molar Mass (g/mol) 414.07

Acidity (pKa) 2.8 ±0.03 (64)

Water Solubility (g/L) 9.5 at 25°C (65) 3.4 (66) 4.1at22°C (67)

Boiling Point (°C) 189 °C (736mm) (65) Vapour Pressure (Pa) 4.19at25°C (68) 1720 (69)

Sorption (KQC) (L/kg) 0.99 (70) 2.06 (71) 1.90-2.17 (72)

Bioconcentration (BCF) 4.0 (Rainbow Trout) (73)

Bioaccumulation Factor (BAF) 0.038 (Rainbow Trout) (73) 3.2 (Lake Trout field (74) measurement) 16

1.3.2a Environmental Monitoring

Detection and quantification of PFCAs in various compartments in the environment has been well investigated in the past several years.

Atmosphere. PFOA has been detected in atmospheric particles in air samples collected from England at maximum concentrations of 828 pg m"3 (75) and even higher concentrations of 3,000-110,000 pg m"3 (76) measured around the vicinity of a fluoropolymer manufacturing facility. Particulates analyzed from air samples obtained in the Arctic in a recent study by Stock et al. measured various chain lengths of PFCA including the C8 to C14 homologues, PFOA being the dominant compound detected at average concentrations of 1.4 pg m"3 (77). The inherent physical properties of these compounds render them unlikely to be found in the vapour phase.

Precipitation. An entire suite of PFCAs (C2-C10) were detected in precipitation collected from 9 sites in North America over a span of two years (78). TFA (C2) was the highest PFCA measured (3-2400 ng L"1) while (C3-C7) followed at concentrations ranging from 0.1-120 ng L"1. PFOA and PFNA were generally found at lower concentrations ranging from 0.1 to 37 ng L"1. Precipitation samples from remote locations in Canada also had similar levels of the C2-C8 homologues at concentrations ranging from 0.5 to 140 ng/L (79).

Aquatic Systems. PFCAs (C6-C8) were first detected by Moody et al. (80) in groundwater contaminated by aqueous fire fighting foam (AFFF) at total concentrations ranging from 124 to 7090 jag L"1. These contaminants were also detected in groundwater taken from a fire-training area in a Michigan air force base at concentrations of 3 to 110

|j,g L"1 (81). Surface waters worldwide have been sampled and analyzed for PFCA contamination. PFCAs were first detected in samples taken from a tributary creek of 17

Lake Ontario after an accidental spill of 20,000 L of AFFF (82, 83). PFCA homologues

C5-C8 were detected in these samples but only PFOA was quantified up to 11.3 |ug L"1.

Presence of these acids however, was not entirely related to the spill given that analysis of samples upstream of the spill also led to detection of these compounds.

River waters in North America namely the Tennessee (84), Niagara and Hudson

Rivers (85); the Rhine River, Moehne River and Ruhr River in Europe (86); and rivers in

Japan (87) as well as the Nan-Kan and Tour-Chyan rivers in Taiwan (88) all had

detectable levels of PFOA at nanogram per litre concentrations. Taiwan river samples

also had detectable levels of perfluorodecanoic acid (PFDA) in addition to PFOA at

concentrations up to 21 ng L"1 (88). Samples from the Rhine River, Moehne River and

Ruhr River also had measurable levels of shorter chain PFCAs including the C4-C7

homologues.

Water samples from lakes worldwide have been analyzed for PFCA

contamination. Levels of PFCAs in the Great Lakes and the surrounding area have been

investigated (61, 79, 85, 89-91). Boulanger et al. reported PFOA levels at 27 to 50 ng L"

\ Other Great Lakes studies have also detected the presence of other shorter and longer

chain PFCAs (C7-C12) at low nanogram per litre levels (79, 90, 91), an order of

magnitude less than reported by Boulanger et al. The study by Boulanger et al. had been

disputed (92) and was recently questioned in a study by Furdui et al. (93). Furdui et al.

measured PFCAs in water samples from the Great Lakes at concentrations ranging from

0.1 to 6.7 ng L"1. A recent study by Stock et al. have measured various PFCAs in remote

lakes in the Canadian Arctic (77). Concentrations measured in this study range from

non-detect to 5 ng L"1 in samples obtained from Amituk and Cornwallis Island, while

much higher concentrations were measured in Resolute and Meretta Lakes amounting to 18

5.6 to 69 ng L"1 even higher than observed in the Great Lakes. Stock et al. have suggested that these concentrations were likely due to a point source such as wastewater discharge or application of aqueous fire fighting foam from the Resolute Bay airport near the Resolute and Meretta lakes. A study in Europe found PFCAs (C7-C12) in Lake

Magiorre in northern Italy at 0.3 to 2.4 ng L"1 with PFOA being the most abundant (94).

Detection of these contaminants in surface and ground waters is of concern as these are sources of drinking water and thus a probable route of human exposure to these chemicals.

Advances in sample preparation and analytical techniques had paved the way for trace detection of PFCAs in parts-per-quadrillion (pg L"1) levels in sea and ocean waters worldwide. Yamashita et al. surveyed global oceanic waters for PFOA and PFNA and detected as low as 15 pg L"1 and 1.0 pg L"1 concentrations respectively in samples taken from surface waters of Central to Eastern Pacific ocean (95). Higher concentrations of

PFOA and PFNA measured at nanogram per litre levels were detected in coastal seawater samples from various parts of Asia (88, 95, 96) which was attributed to contamination input from nearby industrialized areas along the coast. In addition, Yamashita et al. measured trace levels of PFOA (45 to 56 pg L"1) in deep sea water samples taken from up to 4000 m deep (97).

Sediments. Sorption of PFCAs have been investigated and modeled in natural sediment by Higgins et al. (71, 98) where they found that perfluorocarbon chain length was the dominant structural feature that affects sorption. As such, detection of PFCAs in sediment samples was expected. Sediments taken from the San Francisco Bay area contained PFCAs of various chain lengths (C8-C13) from non-detect levels up to 0.8 ng g"1 of sediment, PFOA being the highest homologue detected (92). Stock et al. measured 19

PFCAs in sediments taken from Lake Ontario (91) and remote areas such as lakes in the

Canadian Arctic (77). PFCAs from C7 to C14 were detected in surface sediment samples from Lake Ontario at concentrations ranging from 0.019 to 9.8 ng g"1 dry weight

(91). Sediments obtained from Arctic lakes also contained PFCAs ranging from C7 to

C12 at concentrations of 0.059 to 7.5 ng g"1 (77). High concentrations detected in sediments taken from two of the Arctic lakes sampled, Resolute and Meretta Lakes, were attributed to local contamination.

1.3.2b Biological Monitoring

Wildlife. The first global detection of PFCs in wildlife was in 2001 by Giesy and

Kannan (48). There has since been numerous studies that monitored these organofluorine contaminants in various species of wildlife worldwide and which were recently reviewed by Houde et al. (50). Some of these studies are highlighted here.

Long chain PFCAs were first identified in fish samples taken from Etobicoke

Creek after an accidental spill of AFFF in 2002 (82). This study monitored PFCAs (C5-

C13) both upstream and downstream from the spill. Measurable levels of all PFCAs

were found upstream of the spill as well as in adjacent streams not impacted by the spill

indicating an alternative source of PFCA contamination. The detection of similar long

chain PFCAs (C9-C15) in various biota samples from the Canadian Arctic (99) had a

significant impact in opening up the debate as to sources of these compounds to this

remote region. Several other studies have detected PFCAs in biota samples from the

Arctic including livers of polar bears (60, 100-102), ring seals (103, 104) glaucous gulls

from the Norweigian Arctic (105) and various sea birds (106). Martin et al. had measured the bioaccumulation factors (BAFs) for PFCAs in laboratory studies using rainbow trout {Oncorhynchus mykiss). Their study determined that PFCAs with chain length >8 to be bioaccumulative (107) and that dietary exposure does not lead to biomagnification (73). Bioaccumulation in the field has since been investigated for these compounds in Lake Ontario food web (108), Eastern Arctic marine food web (109) and in the Bottlenose Dolphin (Tursiops truncatus) food web from

Florida and South Carolina in the United States (110). A critical finding in the Lake

Ontario food web study was the high PFCA concentrations observed in benthic organisms indicating that sediments is a major source of these contaminants to this food web.

Humans. Evidence first emerged of the presence of organofluorine compounds in human blood (111, 112) in 1968. Analytical techniques at the time however were not capable of identifying these compounds. In the past decade, as research into the environmental contamination of PFCAs became more prolific and as a direct result of advances in analytical instrumentation, PFCAs were found to be widespread in human blood and serum samples worldwide (113-123). Specifically, PFOA have been detected in historical human serum samples from 1983-1999 and 2003-2004 in Japan (115).

Kannan et al. measured PFOA in human blood, serum and plasma taken from several

countries including the US, Belgium, Italy, Poland, India, Colombia, Brazil, Malaysia

and Korea (116). A pilot study investigating concentrations of PFOA along with other

PFCAs in human serum taken from non-occupationally exposed Canadians (119) found

similar levels with samples from the US (124). In contrast, a recent study done in

Germany had lower concentrations than those from the US and Canada (122). Blood

samples from China had detectable levels of C6, C8-C11 PFCAs (123). Olsen et al. 21 compared human blood concentrations of several PFCs including PFOA in a large group of volunteer participants from the years 1974 to 1989 and found a statistically significant increase in concentrations but no increase from samples taken from 1989 to 2001 (121).

There was no observed dependence in age for PFOA concentrations (113, 125-127) but

gender differences were observed in some studies where males were reported to have

higher amounts of the compounds of interest (113, 115, 128). Most studies have focused

on the analysis of liver and blood; Maestri et al. (129) in addition, reported detection of

PFOA in other human tissues including lung, kidney, thyroid, adipose tissue, brain, basal

ganglia, skeletal muscle, pancreas and gonads. A recent concern is the detection of

various chain lengths of PFCAs in seminal fluid (C6-C11) at low nanogram-per-millilitre

concentrations indicating presence within the reproductive system (50) as well as (C8-

Cl 1) in human breast milk which may pose a potential risk of transfer of these

compounds to infants (130). Karrman et al. (131) determined that possible transfer of

PFCAs to infants is likely by lactation based on their data that matched concentrations of

maternal serum with breast milk.

In general, PFOA concentrations are higher in humans than in wildlife (50).

Human exposure to these compounds is of interest primarily due to their potential

toxicity (132-134). Detection of PFCAs of various chain lengths (C7-C13) in food

products such as fish, meat, fast food and popcorn (135) along with measurable amounts

of PFOA in dust collected from homes(73(5) suggests that there are likely multiple

exposure routes. 22

1.3.3 Perfluorosulfonic Acids and Polyfluorinated Sulfonamides

Although not the focus of this thesis, another important class of PFCs to be noted is perfluorosulfonic acids (PFSAs). These compounds are also anionic surfactants, characterized by the general structure F(CF)nSC>3H where n=4,6,8 or 10 and are typically manufactured by electrochemical fluorination (ECF) leading to production of both linear and branched forms. ECF production of PFSAs also results in several breakdown products including PFCAs (C2, C4, C5, and C8) which leads to PFCAs being impurities in commercial formulations (137). Perfluorooctane sulfonic acid (PFOS) shown in

Figure 1-4 is the most common PFSA analogous to PFOA of the PFCA class. PFOS is also detected widely in environmental, biota and human blood samples similar to PFCAs, often times at much higher concentrations (48, 50, 138). PFOS was also found to be more bioaccumulative than PFOA (107). These concerns have prompted the major manufacturer of these compounds to voluntarily phase out their C8 based chemistry and replace it with shorter chained substances (C4) (139, 140)

F.FFFF.FF.F

&$&&• Figure 1-4. Chemical structure of perfluorooctane sulfonic acid (PFOS).

1.3.4 Fluorinated Telomer Compounds

Fluorinated telomer compounds or fluorotelomers are a family of organofiuorine compounds named after the telomerization process from which they are produced. These compounds are primarily characterized by an even number of perfluorinated carbons 23 along with a hydrocarbon moiety. The manufacture of fiuorotelomer chemicals is a billion dollar industry shared by companies such as Asahi Glass Co. Ltd., Clariant,

Daikin Industries Ltd. and Dupont {141). These fluorinated compounds have been used in various applications. They are key ingredients in making fire fighting foams and coatings because of their unique surface-active properties. They are considered intermediates in the manufacture of stain, and water resistant additives for carpet, textiles, paper and other surfaces (141). Fiuorotelomer compounds are also marketed as both anti- foamers and foaming agents as well as coatings for food packaging to render them grease proof (142, 143). They have been used in producing weather resistant paint formulations

(144) and as protective treatments for stone materials (145) as well as in waxes and polishes (142). Fluorotelomers have also been used in the cosmetics industry specifically in the production of hair creams and rinses to keep hair from becoming oily

(58). There are many classes of fluorotelomers, four of which are of interest in this thesis project and briefly discussed below:

1.3.4a Fiuorotelomer Alcohols.

Fiuorotelomer alcohols (FTOHs) are telomer compounds with the general

structure F(CF2)nCH2CH20H, where n is typically an even number. The nomenclature used for these compounds denotes the number of perfluorinated carbons in relation to the

number of hydrogenated carbons they possess, an example of which is the 8:2 FTOH

(Figure 1-5). Analogous to the PFCAs, different chain lengths of FTOHs have been

produced ranging from the 4:2 FTOH (C6) up to 20:2 FTOH (C22) (146). Information

on physical properties of this class of compounds is limited although the 8:2 FTOH 24 homologue is the most studied. Some physical properties measured for this class of compounds are summarized in Table 1-5. F. F F. F R. F E. F

CH2CH2OH F F F F F

Figure 1-5 Structure of the 8:2 fluorotelomer alcohol (8:2 FTOH).

Table 1-6 Physical properties of fluorotelomer alcohols (FTOHs)

Physical Property 4:2 FTOH 6:2 FTOH 8:2 FTOH 10:2 FTOH Reference

Molar Mass (g/mol) 264 364.1 464.12 564.14

Melting Point (°C) 50 92-93 MSDS (Oakwood products)

Boiling Point (°C) 137.5 173.8 171.5-173.8 201.3-202 (147)

Vapour pressure (Pa) 216 18 4 0.2 (147) (Extrapolated at 25°C 464 113 35 10 (74) unless indicated) 1670 876 227 53 (148) 29 (45°C) (149)

Water solubility 0.71 ± 0.0466 0.137 ±0.053 (150) (mg/L at 25°C) 0.194 ±0.032 (151)

Henry's Law 1.83 ±0.19 1.66 ±0.24 1.31 ±0.32 (148) Constant (log KAW) -1.52 -0.56 0.58 1.60 (152) (Extrapolated at 25°C unless indicated) Octanol-Air 3.26 + 0.25 3.56 + 0.25 4.17 + 0.26 4.83 + 0.27 (148) Partitioning (log KoA) 4.8 5.26 5.56 (152)

Octanol-Water 1.97 4.88 2.91 (153) Partitioning (log KQW) 3.28 + 0.3 4.70 ± 0.3 6.14 ±0.3 7.57 ±0.3 (154)

Sorption (log KoC) 4.13 ±0.16 (757) 25

1.3.4b Fluorotelomer Monomers.

Fluorotelomer monomers are building blocks of the telomer based polymers and are likely found as impurities in these materials. These are FTOH based compounds marketed to produce polymers and oligomers with very low surface energies and a highly fluoro-functionalized surface (155). Monomers with different linkages may be synthesized from the FTOHs via various reactions (146, 156, 157). As part of this research project, biodegradation of these compounds was investigated in Chapter 4.

There are currently no physical property data available for these compounds. An example of this class of fluorotelomer compounds is the 2(perfluorooctyl) ethyl acrylate shown below.

CH2CH20—C—C=C ¥ f > F* V H H

Figure 1-6 Chemical structure of 2(perfluorooctyl) ethyl acrylate.

1.3.4c Fluorotelomer Polymers.

Fluorotelomer based polymers make up 80% of the telomer chemical industry

(141). They are distinct from fluoropolymers such as polyvinylidene fluoride (PVDF) and (polytetrafluoroethylene (PTFE) or Teflon™ where fluorine is attached to majority of the carbon atoms that make up the backbone (755). A fluorotelomer based polymer alternatively consists of a hydrocarbon backbone to which fluorinated alkyl chains are appended (158) (Figure 1-7). The fluoroalkyl chains on these polymers can be appended on the hydrocarbon backbone via ester, urethane, amide or ether linkages (156, 159, 160). 26

Fluorotelomer polymers can be homopolymers where a single type of fluorotelomer monomer was used in its synthesis. Alternatively, copolymers are polymers where non- fluorinated monomers were used in addition to the fluorotelomer monomers during its synthesis. Addition of the non-fluorinated monomers in the polymerization imparts beneficial repellency properties along with enhanced solubility of the compounds (161,

162). These are typically high molecular weight materials (>5,000 amu) that are currently of interest to regulators in both Canada and the US due to their potential to degrade in the environment forming the persistent PFCAs (163). A model telomer based polymer was synthesized and its potential to biodegrade under aerobic conditions was investigated as part of this research project and is discussed in Chapter 5.

CH, CH,

H2CN H,C

/ o=cx o=c( 0=C \ CH CH \ ^ \-./ V/CH - OH2 CH2

Figure 1-7 Representative structure of a fluorotelomer based copolymer 27

1.3.4d Fluorotelomer Surfactants.

Fluorotelomer surfactants are estimated to make up 20% of the telomer chemical industry (141). These materials are low molecular weight compounds (<1000 amu) consisting of a perfluorinated chain, ethyl spacer and a hydrophilic group. These surfactants are manufactured as anionic types that are phosphate, phosphonate, phosphonites, carboxylate or sulfonate based as well as nonionic types such as ethoxylates or betaines (58, 159).

| F F F F / F F' % F F F F •

Figure 1-8 Representative structure of a phosphate telomer surfactant, a component of the Zonyl® UR mixture.

1.4 RELEVANCE OF FLUOROTELOMER COMPOUNDS ON PFCA CONTAMINATION

As studies continue to emerge investigating the pervasive contamination of

PFCAs in the environment, including in remote regions such as the Arctic, it is apparent that research into potential sources of these contaminants is needed. It has been

hypothesized that PFCAs are released to the environment from direct and indirect

sources.

1.4.1 Direct Sources.

Direct sources of PFCAs refer to the discharge of these compounds from

manufacturing processes of the chemicals themselves as well as from discharges of the 28 fluoropolymer manufacturing processes that employ PFCAs, in addition to chemical disposals and spills. It was reported that PFOA emissions in 2000, from the largest manufacturing facility employing electrochemical fluorination (ECF) located in the US, amounted to 20 t corresponding to 5-10% of the total annual production (164, 165).

Industry wide historical emissions were estimated to be between 400 and 700 t for the years 1951-2004 (165). These emission rates decreased in 1999 to 45 t and to 15 t in

2004. With the cessation of production of the C8 chemistry by the ECF manufacturing sector, emissions are expected to further decrease to 7 t in 2006 (165). An accidental spill of AFFF was directly linked to significant amounts of PFCAs in water samples from

Etobicoke creek (82) and from groundwater around the vicinity of areas known to have routine fire fighting activities employing AFFF (62, 80, 81). Evidently direct input of

PFCAs to the environment is likely due to point sources.

In addition to direct release from manufacturing processes and AFFF spills,

PFCAs such as PFOA and PFNA are present as residuals or unintended byproducts in consumer articles that contain fluorotelomer based polymers or fluoropolymers and thus may be released from their application, use and disposal (166-169). The widespread

application of PFCAs as a processing aid in fluoropolymer industries has been attributed

as the largest single known direct source of PFCA emissions. Industry wide

fluoropolymer production data from 1951 to 2003 reported global PFOA and PFNA

emissions from fluoropolymer manufacture to be in the range of 2400 - 5400 t (165),

with PFOA dominating PFNA. Not only are these compounds emitted from the

manufacturing sites of polymers, they are also present as impurities in the commercial

formulation itself. Samples of a commercial based formulation of polytetraethylene

(PTFE) had measurable amounts of PFOA at approximately 45 ug L"1 (170). Several 29

fluorotelomer based products including a phosphate surfactant and acrylate and urethane type polymers contained 1 to 52 ug g"1 of PFOA (169). Washburn et al. measured PFOA

in finished consumer products such as carpeting, fluorotelomer polymer treated apparel

and upholstery at concentrations ranging from 0.2 to 50 ng/ cm2 of treated product (166).

PFOA and PFOS were also measured in a commercial surface protection product making

up 82% and 1% respectively of the total impurities measured in the product (167).

Recently, PFOA was detected in microwave popcorn bags, PTFE based dental floss and

dental tape and in vapours generated from routine use of non-stick cookware (143, 168).

Dissemination of PFCAs globally from direct sources was proposed to be aided

by atmospheric transport of PFCAs themselves or as part of marine aerosols and also

possibly by oceanic water transport (165). Aside from modeling studies that supports

oceanic transport as a significant pathway of PFCAs to the Arctic (777, 772), there has

been limited direct evidence to support these transport pathways.

1.4.2 Indirect Sources.

The detection of PFCAs in remote regions where there is an absence of

manufacturing facilities and/or places where the application of fluorinated treated

consumer products is minimal led to an alternate hypothesis that considers the likelihood

of indirect sources. Indirect sources refer to the production of PFCA in the environment

from the degradation of fluorinated precursor compounds. The first evidence of an

indirect source of PFCAs was shown by Ellis et al. where long and short chain PFCAs

were produced from the thermolysis of fluoropolymers (7 73). Recently, it has been

suggested that volatile precursor compounds with the capability of long-range

atmospheric transport such as the fluorotelomer alcohols may also degrade in the 30 environment to produce PFCAs. This hypothesis has been referred to as the

Polyfluorinated Alcohol Atmospheric Reaction and Transport (PAART) theory (77).

Evidence supporting this theory have recently been demonstrated in several studies and are briefly summarized below.

1.4.2a Detection of FTOHs in the Atmosphere

The large production volumes of xenobiotics are not, by themselves, sufficient cause for concern until they are detected in the environment. The industrial production of

FTOHs that began in 1970 (165) has grown to an estimated 12 000 metric tonnes globally by 2004 corresponding to approximately $700 million in revenue (141). In 2002, these compounds (C8-C14) were first detected in the atmosphere at concentrations ranging from 20 to 85 pg m"3 at a rural sampling location (Long Point, ON) and from 7 to 393 pg nf3 at an urban sampling location (Toronto, ON) (174, 175) with 8:2 FTOH as the predominant compound measured at both locations.

The first study outside of North America to detect these compounds was from

Germany in 2007 (176). This study found little difference between concentrations measured from a metropolitan sampling location (28-119 pg m"3) and that of a rural site

(7.2-75 pg m"3). It was also the first study to detect the shortest FTOH homologue, 4:2

FTOH, in addition to other previously detected homologues. A latitudinal gradient study that investigated concentrations of FTOHs from Germany to South Africa demonstrated that these fluorinated compounds may be restricted in the northern hemisphere. Peak concentrations of 8:2 FTOH were measured at 176 pg m"3 at the northern most sampling location and much lower concentrations of approximately 2 pg m" were found in the 31 southern most site (177). FTOHs were also detected in air samples from England at 9 to

326 pg m"3 (75).

Recently, analysis of Arctic air samples contained FTOHs (C8-C12) at concentrations ranging from 2.8 to 14 pg m"3 (77). Shoeib et al. measured different homologues of FTOHs (6:2 FTOH tol0:2 FTOH) in Arctic air samples as well in both the gaseous and particulate phase at similar concentrations, ranging from 2.65 to 11.4 pg m"3 and 0.8 to 3.5 pg m"3 respectively. They had compared these measured concentrations to urban samples taken from the same study, and found the Arctic concentrations to be 3 x lower (178). Detection of these compounds in Arctic samples is strong confirmation of the long atmospheric lifetime of these compounds. Ellis et al. (179) reported the atmospheric lifetime of FTOHs to be approximately 20 days irrespective of chain length.

Presence in the Arctic atmosphere also confirms the capability of long range atmospheric transport and widespread hemispheric distribution (179) of these aliphatic fluorinated compounds. Finally, concentrations measured corroborate a modeling study that predicted FTOH concentrations to be 5 times lower in the Arctic than in source regions

(180).

1.4.2b Abiotic Transformation of FTOHs

In addition to detection in the atmosphere, there are a myriad of smog chamber studies that demonstrate FTOHs could degrade via atmospheric oxidation to form various homologues of PFCAs in low NOx environments such as the Arctic (179, 181-184).

Intermediates formed in the degradation experiments include the fluorotelomer aldehyde

F(CF2)nCH2COH and the fluorotelomer carboxylic acid (F(CF2)nCH2COOH (184). The atmospheric degradation pathway of FTOHs under these conditions shows that equal 32 yields of the odd and even PFCAs are expected products. This trend was evident in a recent study by Young et al. (185) of High Arctic ice caps. This study reported similar concentrations of PFOA and PFNA(C8 and C9) as well as of PFDA and PFUnA (CIO and CI 1) from these samples that point to atmospheric oxidation of FTOHs occurring in this remote region. In addition, because High Arctic ice caps only receive contamination from atmospheric sources and ratios of PFCAs do not correlate with sodium concentrations detected in these samples that would indicate marine aerosol input, it was suggested that the input of PFCAs is primarily from atmospheric oxidation of volatile precursors like the FTOHs followed by wet or dry deposition (755). These studies strongly suggest that detection of PFCAs in arctic wildlife may also be primarily from this source.

In non-Arctic regions, the presence of PFCAs in rainwater samples from both urban and rural locations further suggests PFCA formation from FTOHs. The detection

of intermediate compounds identified by Ellis et al. (184) in the atmospheric oxidation of

FTOHs in surface waters and precipitation is further evidence of FTOH as an indirect

source. Fluorotelomer carboxylic acids were first measured in rain samples from a

Canadian location by Loewen et al.(186) at low nanogram-per-litre concentrations (Table

1-6). 8:2 FTCA, 8:2 FTUCA, 10:2 FTCA and 10:2 FTUCA were detected. Scott et al

reported the presence of similar compounds in precipitation samples from North

American cities along with PFCAs at concentration ranges of 0.07 to 8.6 ng L" (78).

Surface water concentrations for these intermediates have been detected in both remote

and urban locations such as Arctic lakes (77) and Tokyo and Tomakomai Bays in Japan

(187) (Table 1-6). 33

In natural waters, a likely fate of compounds is to undergo direct or indirect photolysis. A study by Gauthier and Mabury (188) investigated the aqueous photolysis

of FTOHs and found PFCAs as terminal products in indirect photolysis reactions using

Lake Ontario water and various compositions of synthetic field waters, establishing

FTOHs yet again as probable indirect sources of these contaminants to the environment.

The half-life of the 8:2 FTOH in Lake Ontario water was measured as 93 hours and half-

lives varied in synthetic field waters (SFW) ranging from 0.8 to 163 hours. Synthetic

field water studies demonstrated that OH radicals were the primary degradation agent, nitrate promoted photolysis rates and dissolved organic matter had an inhibitory effect

(188). They found no evidence of transformation under direct photolysis conditions.

Analogous to atmospheric oxidation reactions, intermediate compounds identified from

this study were the fluorotelomer aldehyde (FTAL) and fluorotelomer carboxylic acids

(8:2 FTCA and 8:2 FTUCA). 34 Table 1-7 Summary of measured concentrations of fiuorotelomer carboxylic acids in various environmental matrices.

Environmental Compounds Concentration References Matrix Samples

Atmosphere Arctic atmospheric particles 8:2 FTUCA 0.06 pg/m (mean) (77) 10:2 FTUCA 0.07 pg/m3 (mean)

Rain water from Winnipeg, 8:2 FTCA 1.0±0.8ng/L (186) Canada 8:2 FTUCA 0.12 ± 0.02 ng/L 10:2 FTCA 0.3 ± 0.04 ng/L 10:2 FTUCA 0.12 ±0.01 ng/L

Rain water from North 8:2 FTCA <0.07 - 8.6 ng/L (78) American sites 8:2 FTUCA <0.07 - 0.9 ng/L 10:2 FTCA <0.07-1.3 ng/L 10:2 FTUCA <0.07 - 0.8 ng/L Water Arctic lake water 8:2 FTUCA Non detect-15 ng/L (77) 10:2 FTUCA Non detect-1 lng/L

Surface Waters (Tokyo Bay, 8:2 FTCA

Thick Billed Murres 8:2 FTUCA 0.001- 0.08 ng/g (w.w.) (106) 10:2 FTUCA <0.20 ng/g (w.w.) Northern Fulmars 8:2 FTUCA <0.02 ng/g (w.w.) 10:2 FTUCA <0.20 - 24.6 ng/g (w.w.)

Arctic Ringed Seals 8:2 FTCA <4.6 ng/g (w.w.) (103) 8:2 FTUCA O.04- 0.13 ng/g (w.w.) 10:2 FTCA Detected but not Biota 10:2 FTUCA quantified <0.70 - 9.2 ng/g (w.w.)

Free Ranging Bottlenose 8:2 FTUCA 0.3-1.3 ng/g (w.w.) (189) Dolphins 10:2 FTUCA <0.4-1.4 ng/g (w.w.)

Lake Trout from Great Lakes 8:2 FTUCA O.004-0.18 ng/g (w.w.) (74) 10:2 FTUCA 0.001-0.05 ng/g (w.w.)

Arctic sediment 8:2 FTUCA Non detect-0.92 ng/g (77) 10:2 FTUCA (dry wgt) Sediment Non detect-1.9 ng/g (dry wgt) Lake Ontario sediment 8:2 FTUCA O.072-0.13 ng/g (dry (Pi) 10:2 FTUCA wgt) Wastewater Effluent 8:2 FTCA <2.5 - 7 ng/L (.190) Facilities 8:2 FTUCA <2.5-29 ng/L 35

1.4.2c Biotic Transformation of FTOHs.

PFCAs were also shown as products of biotic reactions of FTOHs. FTOHs were first reported to be biotransformed in rats in a study by Hagen et al. (191) as early as

1981. Using gas chromatography coupled with helium microwave plasma detection along with 19F NMR, metabolites identified in blood samples were the 8:2 FTCA and

PFOA. The formation of the unsaturated telomer carboxylic acid (8:2 FTUCA) was suggested but not confirmed. The authors proposed that a p-oxidation mechanism may be involved in the transformation of the detected intermediates leading to PFOA. Martin et al. (192) confirmed these results in studies involving both whole rats and isolated rat hepatocytes. In addition to previously determined intermediates, other metabolites identified in this study include the glucoronide and sulfate conjugates of FTOH, the fluorotelomer aldehyde (8:2 FTAL) reported to be short lived as it quickly formed its unsaturated version (8:2 FTUAL) via loss of HF, the tetrahydroperfluorodecanoate,

(CF3(CF2)6CH2CH2C02~) and dihydroperfluorodecenoate (CF3(CF2)6CH=CHC02"). The production of perfluorononanoate (PFNA) at very low yields was also reported which suggests that an a-oxidation mechanism may also be implicated in the overall metabolism of FTOHs by rats (192). This study also alluded to the reactivity of the electrophilic unsaturated intermediates detected. Compounds such as the unsaturated telomer aldehyde (8:2 FTUAL) could bind to endogenous nucleophiles in biological systems, such as proteins and nucleic acids, leading to potentially more toxic effects than the terminal metabolite, PFOA. Kudo et al. (193) also reported the accumulation of

PFOA in the liver that led to peroxisome proliferation after feeding 8:2 FTOH to rats.

8:2 FTCA and PFNA were also identified as metabolites. Several environmental 36 monitoring studies have identified the presence of FTOH intermediates in diverse species

from various locations including aquatic birds and ringed seals from the Canadian Arctic

(103, 106), free ranging bottlenose dolphins from the U.S. East coastal waters (189) and

lake trout from the Great Lakes (74). Measured concentrations of fluorotelomer

carboxylic acids in various biota samples are summarized in Table 1-6. These studies provide direct evidence that FTOHs are metabolized by different species in the

environment and may likely be an important route for exposure to PFCAs by humans.

As part of this thesis project, biodegradation of the 8:2 FTOH by a mixed culture

and activated sludge under aerobic conditions was investigated (Chapter 2) to determine

whether microorganisms are capable of degrading this class of organofiuorine

compounds. Under these conditions, the 8:2 FTOH was degraded forming the telomer

aldehyde and telomer carboxylic acids, 8:2 FTCA and 8:2 FTUCA as intermediates and

PFOA as the terminal metabolite (194). This was the first study to report the formation

of the telomer aldehyde (8:2 FTAL) as an intermediate in biotic reactions of FTOHs. A

degradation pathway was proposed based on these detected metabolites. PFNA was also

monitored during the experiment but was not detected, indicating that unlike in rats, a-

oxidation may not be a plausible degradation mechanism for microorganisms. In

contrast, an earlier screening study of a mixture of different chain lengths of FTOHs

(195) exposed to municipal wastewater treatment sludge showed some evidence of oc-

oxidation based on minor odd chain acids detected in their samples. A study that

simulated microbial degradation using the Catabolite Software Engine (CATABOL) was

used to predict biodegradation products of several PFCs including 8:2 FTOH (196). This

modeling study had predicted that PFOA is generated from microbial metabolism of 8:2 37

FTOH via 8:2 FTCA and 8:2 FTUCA. Two other studies by Wang et al. (197, 198) examined the fate of 14C-labelled 8:2 FTOH when amended to aerobic sewage sludge.

They reported a metabolite, CF3(CF2)6CF£2CH2COOH (7:3 Acid) not previously observed in other studies and in their follow up study reported three other metabolites:

CF3(CF2)6CHOHCH3 (7:2-sFTOH), CF3(CF2)6CF2CH=CHCOOH (7:3 unsaturated acid)

and the CF3(CF2)CH=CHCONH2 (7:2 u amide). Inoculum used in this investigation was acclimated on 5 to 10 mg L"1 of 8:2 FTOH, which may explain the alternative degradation pathways observed. The authors presented data showing evidence that perfluorocarbon chains defluorinate leading to telomer alcohol mineralization (198).

Breaking of carbon-fluorine bonds by microorganisms in perfluorinated aliphatics however, is uncommon (Section 1.5.3) and rarely reported (49).

Wastewater treatment plants (WWTP) may serve as point sources of PFCAs to the environment by means of wastewater effluents, septic discharge or sludge application to agricultural lands (199). Studies that monitored mass flows in WWTPs confirm the

presence of different PFCA homologues and also show increases in influent versus

effluent concentrations. Schultz et al. (200) reported as high as 352% increase in PFOA

concentrations in 7 of 10 WWTPs effluent samples studied. Similar trends were

observed by Sinclair et al. with increase in PFOA and PFNA of 211% and 345%

respectively. Environmental input of PFCAs attributed to WWTPs is likely combined

contribution from both direct and indirect sources. The presence of PFCAs in influent

samples suggest PFCA input from direct sources while the increase in effluent

concentrations point to likely degradation of FTOHs or other precursor compounds.

These reports in addition to the detection of FTOH intermediates, 8:2 FTCA (<2.5 - 7 ng 38

L"1) and 8:2 FTUCA (<2.5-29 ng L"1) in waste water treatment effluent (190) and sediments (77, 91) is suggestive of FTOH biodegradation occurring in the environment.

1.4.3 Sources of FTOHs

FTOH input to the environment has been attributed to possible release of these compounds from the application of products that incorporate them. FTOHs have been found as impurities in several commercial and industrial products in significant amounts

(168, 169, 201). As part of this thesis project, several fluorotelomer based materials such as surfactants and polymers, were analyzed and found to contain 0.04-3.8% residual

FTOHs on a fluorinated alcohol per dry mass basis (Chapter 2) (201). This was the first study to detect and quantify FTOHs from commercial and industrial products. It was suggested that residuals could be a major source of FTOH input to the environment if an estimate is performed using current production rates combined with data from this study and the calculated flux reported by Ellis et al. (179) in their investigation of the atmospheric lifetimes of FTOH. Larsen et al. (169) determined residuals of the 6:2, 8:2 and 10:2 FTOHs from samples of urethane and ester type telomer polymers.

Concentrations reported for both the 6:2 and 8:2 FTOH ranged from 11 to 1200 \xg g"1 polymer extracted, and 5 to 650 ug g"1 for the 10:2 FTOH (169). Direct comparison of these reported values with the study presented in this thesis (Chapter 2) is difficult due to the limited information provided by the authors regarding the composition of the polymers they measured (ie. were the polymers investigated solids or aqueous dispersions?). However, if it were assumed that polymers analyzed by Larsen et al. were aqueous dispersions that contained up to 90% water (based on dry mass determination by 39

Dinglasan-Panlilio et al.), residuals measured would correspond to 0.005 to 1.2% of the polymers and are well within the range determined by Dinglasan-Panlilio et al. (201). A recent study by Sinclair et al. (168) examined release of telomer alcohols from non stick cookware under normal cooking conditions as well as from microwave popcorn bags.

They had measured mean concentrations of 6:2 FTOH at <15 to 204 pg cm" (area of pan) and 8:2 FTOH at <15 to 625 pg cm"2 and decreasing concentrations were measured from the pans after continued use. Microwave popcorn bags had measurable quantities of both alcohols before and after cooking at levels of <1.6 to 3.4 ng cm"2.

Few studies have examined indirect sources of FTOHs. It was reported that telomer based phosphate surfactants have the potential to seep into food from treated paper packaging and may be a probable route of exposure for humans to perfluorinated compounds (143). D'eon et al. had recently shown that these polyfluoroalkyl phosphates

(PAPs) were transformed by rats producing PFOA as a metabolite (202). Although they did not measure the production of FTOHs directly, the detection of known FTOH metabolites was indicative of FTOH formation and subsequent transformation. Another potential source of FTOHs are the telomer monomers used widely in the telomer polymer industry. The biodegradation potential of fluorotelomer monomers with ester, ether and urethane linkages were investigated as part of this research project (Chapter 3) (203).

2(Perfluorooctyl) ethyl acrylate (8:2 acrylate) and 2(Perfluorooctyl) ethyl methacrylate

(8:2 methacrylate) are ester type monomers were found to yield FTOHs when added to microcosms inoculated with wastewater sludge. These biotic reactions are additional sources of FTOH as these monomers are probable impurities also present in telomer based polymers. Finally, it has been hypothesized that telomer based polymers have the 40 potential to biodegrade forming FTOHs and ultimately PFCAs (50, 169, 194, 201). This hypothesis is investigated and discussed in Chapter 5. A model acrylate type telomer based polymer was synthesized, characterized and subjected to biodegradation

experiments using inoculum taken from a wastewater treatment facility.

1.4.4 PFCA Source Patterns

The observed odd even patterns of carbon chain lengths of PFCAs may be

suggestive of potential sources. The observation of greater odd versus even carbon chain pattern (C8

said to be consistent with an FTOH based source. This trend could be explained by the

atmospheric oxidation of FTOHs which appears to yield similar amounts odd and even

chains clearly evident from analysis of High Arctic ice caps (185) and rainwater samples

(78), but because the longer chain (odd) bioaccumulates more, it is detected at higher

concentrations (184). Contrasting patterns were observed in WWTP samples where even

chains dominate the odd (92, 190, 199, 200). This pattern is expected since PFCAs

detected in WWTPs are also attributed to FTOH based sources. It has been shown that

biodegradation of FTOHs leads to the primary production of even chains (194, 197).

Further to the odd-even pattern, investigation of isomer profiles of PFCAs in

polar bears (60) and humans may also point to potential sources (205). Based on the

findings that linear chained PFCAs dominate in these samples, it was suggested that this

could be due to a primarily linear source such as the FTOHs as suppose to it being an

ECF signature. Telomerization, the process by which FTOHs are made, primarily

produces linear chains given that linear precursors are used in the process. In contrast, 41 the ECF process produces a series of isomers or branched chains. However, definitive conclusions regarding source can not be drawn based on isomer distribution since studies have shown biological discrimination of branched isomers where they are readily absorbed and/or eliminated (205, 206). This observation however, may be dose or concentration dependent and is currently being investigated (205).

1.5 MICROBIAL METABOLISM OF XENOBIOTICS

Biodegradation is the breakdown of substrates as a consequence of microbial activity. The contaminants serve as the substrate or food source for the microorganisms and the degradation of the contaminants often leads to production of energy that can be used for microbial growth. This occurrence however is somewhat uncommon under typical environmental conditions. Under incidental environmental conditions cometabolism is known to occur more frequently. Cometabolism is where the energy obtained from the degradation of the compound is unable to support growth.

Biodegradation of xenobiotics often involves a series of steps or a pathway that may ultimately lead to mineralization. Complete biodegradation or mineralization refers to the oxidation of compounds to carbon dioxide and water, and if compounds contain nitrogen, sulfur, phosphorus, or halogen - it is accompanied by the release of ammonium or nitrite, sulfate, phosphate or halide (207). In contrast, some compounds are only partially degraded or biotransformed where the main skeleton of the substrate remain intact but are only modified slightly through hydroxylation, oxidation or reduction (208). 42

Biodegradation proceeds primarily via two steps: uptake of the substrate followed by the metabolism of the substrate. There are three possible modes of uptake for organic

compounds: passive or active diffusion of solubilized organic compound into the cell,

direct contact of cells with the organic compound, aided possibly by cell modifications

such as fibriae or cell surface hydrophobicity which increase attachment of the cell to the

organic compound and also direct contact with fine submicrometer size substrate droplets

dispersed in the aqueous phase (209). The mode of uptake depends primarily on the water solubility of the compound. There are however, only two possible modes of uptake

of insoluble organics into the cell: direct contact with the substrate or utilization of the

solubilized substrate. Microorganisms require a high water activity (>96) for active

metabolism (210) and substrates with low water solubility poses a challenge to support

growth. Water solubility of a substrate of compound also dictates the mode of uptake by

cells.

Each step in the biodegradation of a substrate is catalyzed by a specific enzyme

produced by the degrading cell. Enzymes are typically produced inside the cell requiring

a compound to partition into the cell membrane prior to being degraded. There are also

enzymes produced outside the cell to catalyze degradation reactions referred to as

extracellular enzymes (209). These enzymes are critical in the degradation of

macromolecules such as the natural polymer cellulose. Macromolecules are broken down

into smaller sub units outside the cell. These smaller subunits are then transported into

the cell for further degradation. Degradation by either internal or extracellular enzymes

will cease at any step if the appropriate enzyme is not present. This lack of enzyme is a

common reason for recalcitrance of xenobiotics and is often the case for compounds with 43 unusual structures (211). Alternatively, the chemical architecture is a vital reason why

enzymes don't work on many xenobiotics. Hence, compounds that resemble natural

substrates are easily broken down and those that are dissimilar are often degraded slowly,

if at all. Some enzymes catalyze very specific reactions (212), while others have wide

specificities. Many studies have reported degradation of xenobiotics by isolates in

laboratory studies (212), whereby one organism is capable of complete mineralization of

a substrate. In the environment, degradation of xenobiotics is attributed to consortium of

organisms. Various other factors affect microbial activity, which directly affect the potential for xenobiotics to be degraded in the environment. Factors can be classified as:

biological, physico-chemical and environmental.

1.5.1 Biological Factors affecting biodegradation

Adaptation of Microbes and Genetic Potential. As discussed earlier, degradation of

contaminants is dependent on the presence of appropriate enzymes. If indigenous

microorganisms do not possess enzymes capable of breaking down contaminants then

degradation will not proceed. Often degradation of xenobiotics proceeds after a period of

adaptation or acclimation of indigenous microbes (212), the length of this period is often

related to the chemical structure. Previous exposure to a contaminant through repeated

releases or frequent spills will create an environment in which the biodegradation

pathway is maintained within an adapted community. Adaptation of microbes results in

induction of enzymes capable of degradation is followed by an increase in the population

of biodegrading organisms (213). 44

Toxicity. High contaminant concentration have been reported to inhibit biodegradation at contaminated sites (214). A common mechanism of microbial toxicity by organic contaminants has been disruption of cell membrane permeability (214). There are instances where toxicity is demonstrated by reactive intermediates rather than parent compounds (208, 211) such as in the case of TCE and its intermediate vinyl chloride as well as DDE and DDT. (214)

1.5.2 Physico-Chemical Factors Affecting Biodegradation

Bioavailability. Contaminants that have low water solubility and are strongly sorbed to soil or sediment are said to have limited bioavailability. This low bioavailability decreases the contaminant's potential for biodegradation. Contaminants can have different sorption mechanisms to soil depending if they are capable of weak

(Vanderwaals, H-bonding, hydrophobic interactions) or strong (covalent bonding) intermolecular forces. Sorption of weakly bound organics typically characterized by non- covalent bonding is reversible allowing contaminants to be released back into solution, freeing it for microbial utilization (275). Bioavailability is also reduced as contaminants diffuse into soil matrix microsites that are inaccessible to microbes due to pore size exclusion (216). It has been reported that reversible contaminant residues in soil or sediment decreases with time, hence biodegradation is expected to decrease as contaminants age and become more sequestered into these matrix microsites (217). Also, contaminants can be made non-bioavailable as they are incorporated into soil organic matter, a process called humification. This process is known to be irreversible, involving formation of covalent bonds and is said to be a part of the ageing process (218). 45

Chemical Structure. The chemical structure of contaminants is highly relevant in

determining their potential to be degraded by microorganisms. Steric effects resulting

from branching or presence of functional groups may affect the chemistry at the reaction

site of enzymes thus resulting in slow degradation. These steric effects can hinder the

contact between the contaminant substrate and enzyme (214). Electronic effects can also

hinder association of enzyme and contaminant substrate. Functional groups of

contaminants can be either electron donating or withdrawing and thus directly change the

electron density at the reaction site (209). If degradation is driven by oxidation reactions,

functional groups that add to the electron density of the reaction site increase biodegradation rates and those that reduce the electron density decrease the degradation

rates, thus, fluorination is expected to result in long-lived PFCAs. The opposite is

observed if the mechanism of degradation is primarily a reductive process (ie. faster

degradation rates observed with decreased electron density).

1.5.3 Environmental Factors.

There are a plethora of environmental factors that affect the potential of

contaminants to be degraded by microbes or the rates at which they are degraded.

Oxygen availability is considered the primary factor that affects degradation rates in the

environment. In general aerobic degradation is much faster than anaerobic degradation

(209). Organic matter has more complex effects on biodegradation of xenobiotics. There

is a correlation between organic matter and microbial populations. Surface soils that are

high in organic matter are also known to have higher numbers of microorganisms thus a

greater potential for contaminant degradation to occur. There is a decreasing trend of 46 organic matter and microbial populations with depth in terrestrial ecosystems (219).

Higher organic matter content however, also leads to decrease in bioavailability of hydrophobic contaminants (214). Other environmental factors that affect biodegradation rates of contaminants are nutrient availability such as nitrogen and phosphorus, temperature and acidity (220). Degradation has been reported to occur at a broad range of temperature since microorganisms are capable of adapting to temperature extremes to maintain metabolic activity. However, seasonal temperature fluctuations in the natural environment have been shown to affect the rate at which biodegradation occurs (221,

222). Degradation of hydrocarbon contaminants has been reported to proceed faster at higher alkaline conditions than in acidic conditions (209).

1.6 MICROBIAL DEGRADATION OF ALIPHATIC ORGANOHALOGEN CONTAMINANTS

As biodegradation of an organofluorine class of compounds is the focus of this thesis, a brief description of the known mechanisms of degradation is warranted. It is

suggested that biodegradation mechanisms of organohalogens in general parallel each

other (207); the discussion will highlight the similarities and some differences of how

microbial organisms metabolize aliphatic organochlorine, organobromine and

organofluorine compounds.

1.6.1 Biodegradation of Aliphatic Organochlorine Compounds

Biodegradation of aliphatic chlorinated organics in enrichment and pure cultures,

as well as in the natural environment and engineered systems is well studied and has been 47 the subject of several reviews (5, 17, 18, 21, 208, 223-225). A small number of studies are highlighted here as it relates to degradation mechanisms.

The principle mechanisms that microbes have developed for degrading organochlorine compounds involve removal of halogens, which can be broadly categorized as follows:

1.6.1a Oxidation

Oxidation reactions involve the direct incorporation of oxygen onto the substrate being degraded. If the oxygen is incorporated on the same carbon as the chloro group, the resulting intermediate is chemically unstable and will spontaneously convert to an aldehyde and release HC1. Oxidation of chloroethenes such as the case for trichloroethene (TCE) (225) results in the formation of unstable epoxides that decompose to form chloroacetic acids (21) (Figure 1-9). These reactions are catalyzed by monooxygenase or dioxygenase enzymes. These are non-substrate specific enzymes and

are often involved in cometabolic degradation. Rates of degradation via this mechanism

decrease with increasing chlorine substitution leading to a higher degree of recalcitrance

under aerobic conditions (211). 48

H OH I 02 I R—C—CI > R—C—CI -• R—C=0 + HCI

H H H HCI H a Cl \ O CI \ x o2 \ A / J\ II I /C=\ -^ /°—\ " H—C—C—H H H H H i

Figure 1-9 Mechanism of oxidation of organochlorine aliphatic compounds. R = rest of molecule

1.6.1b Substitution

Substitutions are nucleophilic reactions in which the chlorine on a mono- or di- substituted substrate is replaced by nucleophiles such as hydroxyl, methyl, or sulfuryl groups (Figure 1-10) (21). Each is mediated by specific enzymes such as haloalkane dehydrogenase, glutathione transferase and methyl transferase. These mechanisms are commonly employed by aerobic microorganisms and have been confirmed as the mechanism involved in the biodegradation of 1,2 dicloroethane by pure cultures of

Xanthobacter autotrophicus strain GJ10 (226). Dechlorination of dichloromethane

(DCM) by facultative methylotrophic bacteria is catalyzed by inducible glutathione S- transferases in a thiolytic substitution reaction (18). 49

A) H Haloalkane M I Dehalogenases | R—C—CI + H20 • R—C—OH + HCI

H H

B) H H I Gluthathione I R—C—CI + R2—SH • R—C—SR2 + HCI

H H

y Methyl ^ I Transferases I H—C—CI + R—H • R—C—H + HCI H H

Figure 1-10 Mechanism of subsitution reactions of chlorinated aliphatics. A) hydrolytic B) Thiolytic C) Methyl Transferase. Adapted in part from Field and Sierra-Alvarez (2004).

1.6.1c Reductive Dehalogenation

Reductive dehalogenation is the primary mechanism of degradation of highly chlorinated aliphatic compounds under anaerobic conditions (209, 225). This reaction involves a two-electron-transfer releasing a chloride ion that is replaced by hydrogen (7).

There are a number of studies that link metabolism of chlorinated aliphatics via reductive dechlorination under methanogenic, sulfate-reducing and denitrifying conditions (17, 18,

225, 227). This type of degradation has been shown in the complete reductive dechlorination of tetrachloroethene or perchloroethene (PCE) to ethene by pure cultures of Dehalococcoides ethenogenes strain 195 where H2 was used as the electron donor

(228). Three common mechanisms are shown below: 50

A) M H I 2e-, 2H+ I R—C—CI • R—C—H + HCI H H

B) CI CI 2e-, 2H+ \ / R—C—C—H • C=C + 2 HCI H H H H

C) CO + 2 HCI CI H2°

H—C—CI : • 2 HCI + R—C I I HON 01 2 CI - C00H + 2 HCI Figure 1-11 Reductive dechlorination mechanisms. A) Reductive Hydrogenolysis B) Reductive Dihaloelimination C) Hydrolytic Reduction. Adapted from Field and Sierra- Alvarez (2004).

1.6.2 Biodegradation of Aliphatic Organobromine Compounds

Investigations into the biodegradation pathways of aliphatic bromides are limited

(229). Brominated methanes, dibromomethane (CH^B^) and methyl bromide (CHsBr) were reported to be completely mineralized to CO2 by a seawater enrichment culture under aerobic conditions in contrast to bromoform (CHBr3) which was resistant to degradation (46). Similar observations were made in substrate studies of various methane oxidizing bacteria, where complete debromination was observed for CH^Br but a lack of degradation for CHBr3 (44, 230). In addition, Streger et al. (44) also observed complete dehalogenation of 1,2-dibromoethene in methanogenic cultures. Mineralization of brominated ethanes has also been observed under aerobic conditions. 1,2- bromoethane was degraded by an enrichment culture that was also capable of using bromoethane, bromoethanol and bromoacetate as substrates (231). The authors of this 51

study inferred that the ability of the enrichment culture to also rapidly degrade bromoethanol and bromoacetate made it a likely candidate as an intermediate, thus

suggesting that a hydrolytic or oxidative pathway was involved. Degradation of longer

chain mono brominated alkanes was investigated using Pseudomonas strains by Shochat

et al. (229). This study tested C2 to C18 homologues and found one strain to be capable

of dehalogenation of all chain lengths while another was only able to debrominate the C7

to C12 homologues. Studies using 1-bromooctane yielded 1-octanol as a product which

indicated the existence of hydrolytic dehalogenation mechanism (229).

Under strict anaerobic conditions, methane producing bacteria were capable of

degrading bromoethane, 1,2-dibromoethane and 1,2-dibromoethylene to ethane, ethene

and acetylene respectively (232). Involvement of reductive dihaloelimination and

reductive hydrogenolysis were suggested as plausible degradation mechanisms. Many

studies that show degradation of brominated aliphatics also demonstrate capability of

degrading their chlorinated analogues. Enzymatic studies have shown that a similar

family of enzymes catalyzes these reactions leading to similar mechanisms of

degradation (43).

Recently, hexabromocyclododecane (HBCD), a cyclic aliphatic organobromine

widely used as a flame retardant, was observed to undergo biodegradation in digester

sludge and freshwater aquatic sediment mixtures under both aerobic, low redox and

anaerobic conditions (233, 234). A degradation mechanism proposed by Davis et al.

involves sequential debromination of HBCD via dihaloelimination where a loss of two

bromines from vicinal carbons occurs at each step with subsequent formation of a double 52 bond between the adjacent carbon atoms (233). The occurrence of complete

dcbromination of HBCD in the environment was suggested by the study.

1.6.3 Biodegradation of Aliphatic Organofluorine Compounds

In general, the removal of halogens from substrates follow the order of I > Br > CI

> F (207). Clearly, the challenge in removing fluorine is due to the C-F bond having one

of the largest bond energies in nature. When compared to the C-Cl bond, the C-F bond

was determined to be 25 kcal mol"1 stronger (49). This bond energy imparts unique

stability to fluorinated chemicals that is lacking in its chlorine or bromine counterparts.

Despite this inherent stability, there have been reports of defluorination for some

fluorinated aliphatics (207, 235, 236).

Fluoromethane (CH3F), the simplest organic fluorine compound was oxidized in

pure cultures of Nitrosomonas europaea, producing formaldehyde as a result. This

reaction was mediated by the enzyme ammonia monooxygenase (237). Trifiuoroethene

(CF2=CHF) was metabolized to glyoxylate, difluoroacetate and the rearranged product

trifluoroacetaldehyde by the methanotroph Methylosinus trichosporium strain OB3b

(238), a strain also reported capable of degrading trichloroethene. Biodegradation studies

of hydrofluorocarbons (HFCs) and chlorofluorocarbons (CFCs) have shown

dechlorination under reductive environments without the evidence for reductive

replacement of fluorine (207). Lesage et al. (54) investigated the degradation of CFC-

113 (1,1,2 trichloro, 1,2,2-trifluoroethane) under anaerobic conditions and observed loss

of chlorine from the parent compound but without any fluorine loss. The product

detected in this investigation was HCFC-133b (2-chloro, 1,1,2 trifluoroethane). 53

Microbial defluorination of monofluoroacetate (MFA), a naturally produced organofluorine, has been shown in several studies (236, 239, 240). In pseudomonads, defluorination via a hydrolytic attack of the carbon-fluorine bond led to production of fluoride and glycolate, catalyzed by an enzyme described as a haloacetate halidohydrolase (241). Goldman et al. (239) used H2180 in their degradation experiments to demonstrate that the source of oxygen introduced to the parent compound originated from water. In contrast, trifluoroacetic acid (TFA) has been found to be highly persistent in the environment. This haloacetic acid (HA) has been investigated in numerous studies due to its ubiquity, persistence and toxicity (242). Visscher et al. (243) had initially reported rapid degradation of 14C labeled TFA under both anoxic and oxic conditions in sediment. Results of this study however have been called into question and could not be replicated by subsequent researchers (244). A study by Kim et al. (245) had showed evidence of fluoride release and acetic acid production from TFA degradation by an ethanol degrading mixed culture although under extreme anaerobic conditions and at elevated temperatures (245). Other microbial degradation studies under anaerobic conditions have shown no evidence of TFA degradation in field microcosms (242), wetland vernal pool systems (246), sewage sludge, cattle rumen and freshwater and saltwater sediments (244).

Polyfluorinated aliphatics are generally resistant to defluorination and are only attacked at non-fluorinated side chains. This resistance is attributed to the rigid perfluorinated backbone that interferes with molecule-enzyme interactions in biological

systems (49). In a study of perfluoroalkyl sulfonates by Key et al. (235), fully fluorinated

compounds such as trifluoromethyl sulfonate (CF3SO3H) and perfluorooctane sulfonate 54

(CF3(CF2)7S03H) were not degraded. Difluoromethane sulfonate however, was degraded producing stoichiometric equivalents of fluoride. H-PFOS (CF3(CF2)5CH2CH2S03H), a fluorotelomer based surfactant typically incorporated in aqueous fire fighting foams

(AFFF), was reported to be biotransformed forming several unidentified volatile metabolites along with evidence of partial defluorination. Partial defluorination of trifluoroethane sulfonate (TES) was also evident from their studies using pure cultures of

Pseudomonas sp. strain D2 (235). This study clearly showed that defluorination can

occur on hydrogenated analogues of perfluoroalkyl sulfonates. Cleavage of C-F bonds

on hydrogenated analogues occurs only once it is activated and activation is aided by presence of surrounding functional groups such as carbonyl or sulfonyl groups. These

groups along with the perfluorinated chain result in the proton adjacent to the -CF2 group

to become highly acidic thus eliminated along with F".

Polyfluorinated Sulfonamides such as N-Ethyl perfluorooctane sulfonamide

(NEtFOSA) and N-ethyl perfluorooctane sulfoamidoethanol (NEtFOSE) shown in figure

1-12, belong to a class of perfluorinated chemicals (PFCs) thought to contribute to

perfluorosulfonate contamination in the environment. Analogous to the indirect source

hypothesis for PFCAs, these polyfluorinated sulfonamides are volatile compounds

thought to be precursors of perfluorinated sulfonates. These compounds have been

detected in indoor and outdoor air (247) and have been reported to partition to dust (248).

Also like the FTOHs, residual NEtFOSE was detected in a commercial product marketed

as a carpet protector and can be released to the environment from routine application

(201). Biotransformation studies of NEtFOSA and NEtFOSE in rainbow trout

(Onchorhynchus mykiss) (249) and rat liver microcosomes (250) showed PFOS formation 55 as a terminal metabolite. Biodegradation of NEtFOSE was observed in sludge microcosm experiments under aerobic conditions forming 2-(N- ethylperfluorooctanesulfonamido) acetic acid (N-EtFOSAA) and perfluorooctane sulfinate (PFOSI) as the only metabolites with 23% and 5% yields respectively (167). It was however found to be stable under anaerobic conditions (167). Recently, NEtFOSE was reported to biodegrade in activated sludge systems also forming N-EtFOSAA and

PFOSI as metabolites (251). Other metabolites reported were perfluorooctanesulfonylethylamide (NEtFOSA), 2 (perfluorooctanesulfonamido) acetic acid (FOSAA), perfluorooctane sulfonylamide (FOSA), perfluorooctanoic acid (PFOA), and perfluorooctane sulfonate (PFOS), again depicting degradation of NEtFOSE to only occur on the non-perfluorinated moiety of the compound. The proposed degradation pathway shows PFOSI to be the precursor of PFOS and also the precursor of PFOA

(251). N-EtFOSAA has also been previously measured in sediments and sludge at higher concentrations than PFOS (92). However, this was not considered entirely from possible

NEtFOSE degradation occurring in wastewater plants due to its presence in the influent flow or the primary sludge. Alternatively, it was suggested that presence of N-EtFOSAA in primary sludge could be from degradation along sewage transmission lines, metabolism from human exposure to NEtFOSE or also through direct input from

NEtFOSAA itself since it has been used as a surfactant in commercial applications (92). 56

F F F F F 0

NEtFOSE NEtFOSA

Figure 1-12 Chemical structures of polyfluorinated sulfonamides, N-ethyl perfluorooctane sulfoamidoethanol (NEtFOSE) and N-Ethyl perfluooctane sulfonamide (NEtFOSA).

Fluorotelomer alcohols (F(CF2)nCH2CH20H) as discussed in Section 1.4, were degraded forming the perfluorinated carboxylic acid PFOA in enrichment and wastewater sludge experiments (194, 197, 198). The initial step in the degradation was the sequential oxidation of the ethanol moiety, to the aldehyde and to the saturated telomer carboxylic acid. The following step was an apparent loss of-HF. The perfluorinated chain by virtue of its polarizing ability makes the hydrogen on the a-carbon more acidic and loss

of-HF likely leading to the formation of the unsaturated acid. A P-oxidation type of mechanism was proposed analogous to the metabolism of fatty acids, as a possible

mechanism of PFOA formation. A study by Wang et al. (197) had suggested that a p-

oxidation mechanism is unlikely due to the proton deficiency on the P-carbon that prevents the proton/electron shuffling that is essential for completion of the reaction.

However, a P-elimination type reaction has been observed in the biodegradation of

fluoropyruvate (Figure 1-13) (252). A similar mechanism may be involved in the

degradation of FTOH but mechanistic and enzymatic studies need to be pursued. Wang

et al. instead had proposed that the 7:3 FTCA metabolite that they have identified allows

for P-oxidation to occur easily. They proposed formation of the 7:3 acid from the 7:3 57 unsaturated acid which could have likely been a product of reductive defluorination of the 8:2 unsaturated acid.

H o o OH I II II H OH F C C C O- JLLL co2

H H \ / —^—»- r %t^* -R3

R\ R2 R, R2

H20

H3C C H2C=C H3C C OH

^\, ,N R3 \N R, .N R3

R1 Rl' R R2 R2 2

Figure 1-13 Proposed mechanism for the enzymatic defluorination of 3-fluoropyruvate catalyzed by a thiamin pyrophosphate (252).

Most of the studies on microbial metabolism of fluorinated organics have been

focused on fluorinated aromatic compounds that are beyond the scope of this review and

this thesis. The lack of evidence for defluorination of perfluorinated aliphatics is a likely

basis for the recalcitrance of these compounds in the environment.

1.7 GOALS AND HYPOTHESIS

In contrast to the numerous investigations describing the global distribution of

perfluorinated compounds in the environment, few studies have probed the sources of

these persistent and potentially toxic contaminants. The ultimate goal of this thesis was 58 to further examine this question of source by evaluating the contribution of a class of organofluorine compounds known as fluorotelomers to the environmental burden of

PFCAs. The focus of the research was to investigate whether the biodegradation of these compounds leads to formation of PFCAs.

In Chapter 2, the biodegradation of the 8:2 FTOH by a mixed culture and by wastewater sludge under aerobic conditions is presented. It was hypothesized that 8:2

FTOH will be degraded primarily at the non-fluorinated portion of the molecule leading to the production of PFOA. Hypothesized intermediates were synthesized to aid in identification of metabolites. Gas chromatography mass spectrometry (GCMS) as well as liquid chromatography tandem mass spectrometry (LCMSMS) was used to identify intermediate metabolites and a degradation pathway was proposed.

Despite detection of FTOHs in the atmosphere and their likely atmospheric and biotic degradation to PFCAs, sources of FTOH input to the environment were unknown.

Investigation into a potential source of FTOHs is described in Chapter 3 of this thesis. It was hypothesized that commercial and industrial fluorotelomer based polymers and surfactants would contain FTOHs of various chain lengths as residuals or impurities due to inefficient manufacturing or synthetic processes of these materials. A novel quantitative purge and trap method for removing FTOHs was developed that takes advantage of the inherent physical properties of FTOHs in order to detect and measure these compounds in several fluorinated materials.

Since FTOHs are primarily used as intermediates in the manufacture and synthesis of surfactants and polymers, one of the goals of this thesis project was to determine whether other fluorotelomer intermediates such monomers breakdown in the 59

environment to form PFCAs. Chapter 4 examines the biodegradation of telomer-based monomers with varying linkages - ester, ether and urethane - under aerobic conditions. It was hypothesized that linkage chemistry is critical in the stability of monomers that

incorporated FTOHs. Ester type monomers were found labile after addition to microcosms inoculated with wastewater treatment sludge. FTOHs were formed along with previously determined FTOH metabolites and PFOA.

The stability of fluorotelomer-based polymers is investigated in Chapter 5. It was

hypothesized that fluorotelomer based polymers when exposed to a biologically active

matrix, can degrade slowly forming FTOHs and ultimately PFCAs. A model

fluorotelomer acrylate based polymer was synthesized and characterized by Nuclear

Magnetic Resonance (NMR), Gel Permeation Chromatography (GPC) and Matrix

Assisted Laser Ionization Mass Spectrometry (MALDI-MS). Residual monomers and

FTOHs were removed prior to spiking into purge and trap microcosms to monitor for

FTOH production. Since the fluorotelomer industry is dominated by polymer-based

chemistry, the fate of these complex materials is of utmost concern to regulators.

Production of FTOHs and ultimately PFCAs from these compounds could mean a

significant source of contamination.

The work presented in this thesis is summarized in Chapter 6. The concluding

chapter also discusses the overall significance of the contribution of fluorotelomer

biodegradation to the burden of PFCAs in the environment. It presents some future

research perspectives including the need to probe whether degradation of other telomer

based materials such as surfactants are further sources of FTOHs to the environment. 60

Future studies investigating degradation of fluorotelomers under anaerobic conditions are also proposed.

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FLUOROTELOMER ALCOHOL BIODEGRADATION YIELDS POLY- AND PERFLUORINATED ACIDS

Mary Joyce A. Dinglasan, Yun Ye, Elizabeth A. Edwards and Scott A Mabury

Published in - Environ. Sci. Technol. 2004, 38, 2857-2864.

Reproduced with permission from Environmental Science and Technology Copyright ACS 2004

82 83

2.1 ABSTRACT

The widespread detection of environmentally persistent perfluorinated acids (PFCAs) such as perfluorooctanoic acid (PFOA) and its longer chained homologues (C9>C15) in biota has instigated a need to identify potential sources. It has recently been suggested that fluorinated telomer alcohols (FTOHs) are probable precursor compounds that may undergo transformation reactions in the environment leading to the formation of these potentially toxic and bioaccumulative PFCAs. This study examined the aerobic biodegradation of the 8:2 telomer alcohol (8:2 FTOH, CF3(CF2)7CH2CH20H) using a mixed microbial system. The initial measured half-life of the 8:2 FTOH was ~0.2 days mg"1 of initial biomass protein. The degradation of the telomer alcohol was monitored using a gas chromatograph equipped with an electron capture detector (GC/ECD). Volatile metabolites were identified using gas chromatography/mass spectrometry (GC/MS), and nonvolatile metabolites were identified and quantified using liquid chromatography/tandem mass spectrometry (LC/MS/MS). Telomer acids

(CF3(CF2)7CH2COOH; CF3(CF2)6CFCHCOOH) and PFOA were identified as metabolites during the degradation, the unsaturated telomer acid being the predominant metabolite measured. The overall mechanism involves the oxidation of the 8:2 FTOH to the telomer acid via the transient telomer aldehyde. The telomer acid via p-oxidation mechanism was further transformed, leading to the unsaturated acid and ultimately producing the highly stable PFOA. Telomer alcohols were demonstrated to be potential sources of PFCAs as a consequence of biotic degradation.

Biological transformation may be a major degradation pathway for fluorinated telomer alcohols in aquatic systems. 84

2.2 INTRODUCTION

The extensive use of perfluorinated organic compounds, in both commercial and industrial applications, has recently prompted research into the disposition, fate, persistence and overall environmental impact of this class of compounds. Their widespread application is attributed to the unique properties that the perfluoroalkyl chain imparts upon the compound.

Many of these compounds have been found to be highly stable in the environment due to the strength of the carbon-fluorine bond (1).

Extensive biological monitoring studies in recent years have revealed widespread global distribution of perfluorinated acids such as perfluoroalkane sulfonates, perfluorooctane sulfonate

(PFOS) and perfluorinated carboxylic acids (PFCAs) of which perfluorooctanoic acid (PFOA) and perfluorodecanoic acid (PFDA) are examples (2-5). Some long chained homologues of

PFCAs some of which were first reported in fish samples collected from a creek after a large spill of aqueous film forming foam (AFFF)(6); subsequent monitoring offish from this and nearby creeks suggests the PFCAs, other than PFOA, did not arise from the spill (6). More recently from biota samples collected from the Canadian artic (7) were shown to contain the full suite of PFCAs (C9-C15). PFOA have also been detected in trace concentrations from human serum samples worldwide (8). Long chain perfluorinated acids have been found to be environmentally persistent, bioaccumulative (9, 10) and potentially toxic (11, 12).

Perfluorinated acids are stronger acids as compared to their hydrocarbon counterparts and the correspondingly lower pKa, (i.e., PFOA is 2.80) (13), results in the dominance of the anionic form with little propensity to escape via volatilization. To explain the occurrence of PFCAs in remote regions we have postulated that another class of more volatile neutral compounds might 85 serve as atmospheric precursors. These would undergo environmental decomposition either biotically, or abiotically, to the more persistent acids (14).

Fluorotelomer alcohols (FTOHs) are polyfluorinated compounds typically characterized by even numbered perfluorinated carbons and two non-fluorinated carbons adjacent to a hydroxyl group. FTOHs are typically used as precursor compounds in the production of fluorinated polymers used in paper and carpet treatments and have similar applications as those of PFOS-based products (13). They are also used in the manufacture of a wide range of products such as paints, adhesives, waxes, polishes, metals, electronics and chaulks (13). During the years

2000-2002, an estimated 5 x 106kg yr"1 of these compounds were produced worldwide, 40% of which was in North America (14). Their name is derived from the telomerization process from which they are produced. FTOHs are given nomenclature based upon the number of perfluorinated carbons in relation to the number of hydrogenated carbons they possess (i.e. 8:2

FTOH; Table 2-1). Measured vapour pressures of FTOHs range from 140-990 Pa (75). The calculated dimensionless Henry's law constants for this class of compounds (ie. 270 for 8:2

FTOH) using the limited data available for water solubility and vapour pressure reveals the propensity of these compounds to partition into air. This is supported by a recent air sampling campaign in which FTOHs were detected at tropospheric concentrations typically ranging from

17-135 pg m"3 (16, 17) with urban locations apparently having higher concentrations than rural areas. A study by Ellis et al. shows that the atmospheric lifetime of short chain FTOHs as determined by its reaction with OH radicals is approximately 20 days (14). These results demonstrate that fluorotelomer alcohols are widely disseminated in the troposphere and are capable of long-range atmospheric transport. Sources of these compounds are currently unknown, although it is likely that they may be released from the decomposition of polymeric or 86 non-polymeric materials that incorporate FTOHs or from release of residual amounts of the fluorotelomer alcohols themselves that failed to be covalently linked to polymers during production (14). If polyfluorinated polymers are indeed a source for these compounds, then a potential fate of these materials is to end up in an aqueous environment such as sewage treatment plants as a result of routine activities such as carpet or upholstery cleaning. This type of an environment would subject these polymers to potential microbial degradation, possibly releasing

FTOHs to the aqueous systems where they too are subjected to biodegradation.

Table 2-1. Acronym, Structure and Molecular Weight of Perfluorinated Compounds of Interest

Compound Acronym Structure Molecular Weight (amu) 8:2 fluorotelomer alcohol 8:2 FTOH CF3(CF2)7CH2CH2OH 464 8:2 fluorotelomer aldehyde 8:2 FTAL CF3(CF2)7CH2CHO 462 8:2 fluorotelomer acid 8:2 FTCA CF3(CF2)7CH2C(0)0- 477 8:2 fluorotelomer 8:2 FTUCA CF3(CF2)6CF=CHC(0)0" 457 unsaturated acid ally lie 8:2 fluorotelomer ally lie 8:2 CF3(CF2)6CF=CHCH2OH 444 alcohol FTOH Perfluorooctanoic Acid PFOA CF3(CF2)6C(0)0" 413

An earlier study by Hagen et al. (18) showed compelling evidence for FTOH biotransformation. They identified 2H, 2H perfluorodecanoic acid (8:2 FTCA, Table 2-1) and

PFOA as metabolites in rats given a single dose of 8:2 FTOH using F NMR and a gas chromatograph equipped with a microwave plasma detector (GC/MPD) (18). They have also suggested the unsaturated form of the acid (8:2 FTUCA, Table 1) as another metabolite using retention time matching of a synthesized standard by gas chromatography using an electron capture detector (GC/ECD). A (3-oxidation mechanism was proposed to be involved in this biotransformation. This study suggests that FTOHs subjected to biotic reactions are sources of 87 the more stable perfluorinated acids. It has also been reported that a mixture of fluorochemical telomer alcohols were biodegraded when exposed to municipal wastewater treatment sludge

(19). This screening study has shown the production of both even and odd chained perfluorinated acids after a 16 day incubation period and proposes that P- and oc-oxidation mechanisms may be involved in the degradation pathway. Transient species detected by this study using LC/MS/MS included the unsaturated telomer acids of the corresponding alcohols (C8, C9, CIO, and C12).

In our investigation, we demonstrated that fluorotelomer alcohols could undergo biodegradation under aerobic conditions using 8:2 FTOH (Table 2-1) as a model telomer in a microbial enrichment culture known to degrade ethanol. We hypothesized that fluorotelomer alcohols can be oxidized to the corresponding aldehyde (Table 2-1), subsequently to the related acids and ultimately leading to the production of persistent PFCAs similar to what was initially shown by Hagen et al. in rats (18) and that of the screening study by Lange (19). To test this hypothesis, we developed a method to measure 8:2 FTOH via headspace using solid phase microextraction (SPME) coupled with GC/ECD. Methods were developed to identify volatile metabolites using gas chromatography/mass spectrometry (GC/MS) and liquid chromatography/tandem mass spectrometry (LC/MS/MS) for the non-volatile metabolites.

Unlike the use of gas chromatography for the analysis of PFCAs and telomer acids, applying the technique of LC/MS/MS involves little sampling preparation and eliminates the need for derivatization. It also provides enhanced confidence in chemical identification from MS/MS spectra. 88

2.3 EXPERIMENTAL SECTION

2.3.1 Media and Chemicals.

The enrichment culture used in the experiments was routinely grown in a defined mineral medium which contained the following constituents added to distilled and deionized water to

1 make one litre: 65mL of phosphate buffer (27.2 g KH2P04 and 38.4 g K2HP04 L" ), 10 mL of

1 salt solution (53.5 g NH4C1, 7.0 g CaCl2 • 6 H20, 2.0 g FeCl2 • 4 H20 I/ ), 2 mL of trace mineral solution (0.3 g H3BO3,0.1 g ZnCl2, 0.1 g Na2Mo04 • 2 H20, 0.75 g NiCl2 • 6 H20, 1.0 MnCl2 •

4 H20, 0.1 g CuCl2 • 2 H20, 1.5 g CoCl2 • 6 H20, 0.02 g Na2Se03, 0.1 g A12(S04)3 • 18 H20,

1 1 and 1 mL of concentrated H2S04 L" ), 2 mL of MgCl2 • 6 H20 solution (48.8 g L" ) and 10 mg of yeast extract. The mixture was autoclaved for 20 min at a temperature of 120°C and pressure of

18psi. The pH was subsequently adjusted to approximately 7 by the addition of IN HC1.

The 8:2 FTOH (97%) was purchased from Oakwood Research Chemicals (West Columbia, SC).

The 8:2 FTOH telomer aldehyde (8:2 FTAL) was synthesized as described by Napoli et al. (20) and the 8:2 FTOH telomer acid (8:2 FTCA) and the 8:2 FTOH unsaturated acid (8:2 FTUCA) were synthesized as described by Achilefu et al. (21). Characterization of these synthesized standards was done using 13C, 19F, and !H NMR along with high-resolution electron impact mass spectrometry, negative chemical ionization, and positive chemical ionization mass spectrometry.

Purity of the 8:2 FTAL, 8:2 FTCA, and 8:2 FTUCA was >95%. PFOA (96%) and mercuric chloride were purchased from Aldrich Chemical Co. (Milwaukee, WI).

2.3.2 Protein Analysis.

Protein measurements were carried out using the Bradford method, using a microassay kit (Bio-Rad Laboratories, Hercules CA) and bovine serum albumin (BSA) as a standard. 89

2.3.3 Growth Conditions and Culture Preparation for Degradation Experiments.

The enrichment culture was obtained from sediment and groundwater taken from a contaminated site and had been enriched with 1,2-dichloroethane and subsequently maintained using ethanol as the sole carbon source (22). This mixed culture was chosen because it was acclimated to degradation of chlorinated alkanes and alcohols and therefore may also be active on fluorinated alcohols. Cells were harvested by centrifugation, washed with defined mineral medium and resuspended in 2% of the total liquid volume used in the experiments. Degradation experiments were performed in triplicate using 1L glass vessels (Pyrex) filled with 950 mL of defined mineral medium and sealed with mininert® caps. The 8:2 FTOH was added to the culture vessels by adding 14 uL of a concentrated stock solution (50 ug uL"1) made up in ethanol to attain target aqueous concentration of 50 |o,g L" . The vessels were allowed to equilibrate for

24 hours and sampled for time zero concentrations prior to adding the inoculum. Sterile controls were prepared similarly except that 500 mg of mercuric chloride was added to inhibit microbial activity. All cultures were stored in the dark at room temperature on a shaker at 95 rpm to allow for continued mixing and to enhance mass transfer of oxygen from the headspace to the liquid phase. For degradation experiments using sewage treatment plant samples, activated sludge was obtained from Ashbridges Bay Treatment Plant (Toronto, ON) and was used as the inoculum and prepared as described previously without acclimation to ethanol.

2.3.4 GC/ECD and GC/MS Analysis of 8:2 FTOH and Volatile Transformation Products.

The degradation of the 8:2 FTOH was monitored using solid phase microextraction

(SPME). A 30um fibre with poly(dimethylsiloxane) (PDMS) coating (Supelco, Bellefonte, PA) 90 was exposed to the headspace of the sealed culture vessels and was allowed to equilibrate for 5 min. Headspace analysis was used to address the volatility of these fluoroalcohols. Stock et al. have measured the vapor pressure of the 8:2 FTOH at 212 Pa (75), and the water solubility was measured to be 148 ug L"1 (23) from which we calculated a Henry's law constant value of 270.

Aqueous concentrations of the 8:2 FTOH were then determined, to ensure that the system was below the water solubility, from the following relationship:

Mot = CL(FL + H XVg) (1) where Mtot is the total mass of the compound, CL is the aqueous concentration, H is the Henry's law constant, and Vg is the headspace volume; typical aqueous concentrations were -50 ug L"1.

Analysis was done using a Hewlett-Packard 5890 Series II gas chromatograph equipped with an electron capture detector (Agilent Technologies, Wilmington, DE) and a 30m x 0.5 mm x 250 urn DB-35 column (Phenomenex, Mississauga,ON). The injector temperature was 250 °C, and the detector temperature was set at 320 °C. The GC oven program was initially held at 45 °C for 2 min followed by a 10 °C min"1 ramp to 95 °C and held for 5 min and a final ramp of 30 °C min"1 to 250 °C. The carrier gas was hydrogen at a pressure of 5 psi, and the make-up gas was nitrogen. External standards were used for calibration. Standards used had aqueous

1 0 concentrations ranging from 2 to 55 ug L" , and response was linear with r typically >0.99.

A Hewlett-Packard 6890 gas chromatograph coupled to a mass selective detector

(Agilent Technologies, Wilmington, DE) was used under full scan positive chemical ionization mode to identify the volatile metabolites observed in the degradation. The carrier gas was helium, and methane was used as the ionizing gas at a flow rate of 1 mL min"1. The source temperature was 250 °C, and the electron energy was at 100 eV. Gas chromatographic separation was performed using a DB-Wax column (30 m x 0.25 mm x 250 urn) (J&W Scientific, Folsom, 91

CA). The initial oven temperature was 45 °C for 5 min and ramped at 15 °C min-1 to 210 °C.

Pulsed 1 uL splitless injections were performed at an initial pressure of 25 psi and 220 °C, returning to 10 psi at 1.2 min, and followed by an injector purge.

2.3.5 LC/MS/MS Analysis of Non-Volatile Metabolites.

Prior to analysis, 3mL samples were obtained from each experimental vessel and centrifuged to remove biomass. One mL of methanol and lmL of supernatant were then transferred to polypropylene autosampler vials for analysis. Measurement and identification of target nonvolatile metabolites were performed using a Waters 717 autosampler along with an

Alltech 426 isocratic pump equipped with an Alltech Econosil CI8 column (5 urn, 4.6 x 250 mm) at a flow rate of 400 uL min"1. Gradient elution was not applied because of contamination problems for PFOA with the available gradient pump. Isocratic elution proved to be an adequate and faster alternative for the analysis using a mobile phase comprised of 70% Optima grade methanol and 30% 18MQ deionized water. Samples were injected at a volume of 20 uL, and the

HPLC column eluate entered the mass spectrometer ion source without splitting. All analytes of interest eluted in less than lOmin.

Acquisition of the mass spectra was carried out using a Micromass Quattro micro

Triple Quadrupole Mass Spectrometer (Micromass; Manchester, UK) operated under negative electrospray ionization mode. A standard with a concentration of 500 ug L"1 in methanol was infused through the equipped syringe pump at a flow rate of 30 uL min" for positioning of the ion sprayer and tuning of the mass spectrometer. The capillary voltage was 2.9 kV while the

cone voltage ranged from 14 to 15V. Specific operating parameters are listed in Table 2-2. The

source block and desolvation temperatures were 110 and 300°C, respectively and the dwell time 92 was 0.5s. The nebulizer and desolvation gas flow rate were 20 and 260 L hr"1 respectively. For tandem mass spectrometric analysis, argon was used as the collision gas (2.86x10"3 mbar) and collision energies (Table 2-2) were varied to optimize for the sensitivity of each compound.

Table 2-2 Optimized MS/MS Conditions for Metabolite Confirmation

„ , Parent Ion Cone Vol. Daughter Collision Compound (m/Z) (V) Ion (m/Z) Energy (eV) 393 15 8:2FTCA 477 15 63 8 39 10 8:2 FTUCA 457 15 393 15 369 10 PFOA 413 15 219 15 169 18

Quantification was achieved under multiple reaction monitoring (MRM) mode and by calibrating the primary daughter ion peak area versus the concentration. Four-point matrix- matched calibration curves were generated daily by using freshly prepared standards. Standard concentrations ranged from 100 jag L"1 to 1000 (j,g L"1, and curve r2 was typically>0.99. Cone voltages and collision energies were optimized by standards made up in methanol for each individual compound to ensure the sensitivity of MS/MS analysis. Late in the study, when 8:2

FTUCA concentrations were declining and correspondingly PFOA concentrations were rising, some suppression of the PFOA signal was revealed. Standard additions were performed for

PFOA at the final time point of the experiment (day 81) so that an accurate mass balance was determined; no suppression was observed for the other analytes of interest during the investigation. Along with the hypothesized metabolites, other prospective products such as perfluorononanoic acid (PFNA) and perfluoroheptanoic acid (PFHpA) were also monitored

although neither were detected in any samples. 93

2.3.6 QA/QC.

To guarantee data quality, a reagent blank (methanol) was injected after each time-point sample group (four samples/ group) to reveal any problems of carryover. A typical chromatogram is shown in Figure. 2-1. Three parent-daughter transitions were monitored for 8:2

FTC A and PFOA to confirm their identity and only one for 8:2 FTUCA since no further fragmentation was observed for this compound. Each ion was monitored under its own optimal condition listed in Table 2-2. The ratio of the peak areas of the two less-intense product ions to the strongest ion was calculated and compared between samples and standards. As outlined by the European Analytical Guidelines, the repeatability of the product-ion ratios obtained in the confirmation procedure is the average ratio of three replicate injections of both the standards and the samples (Figure 2-2); the difference between samples and standards in this study were within the recommended tolerance set by the guidelines (24). 94

24-

20- 8:2 FTOH

16- \ altylic 8:2 FTOH

12-

10-

10(h 6.72

% 8:2FTCA 477>393

T•^ftT*•!"*pn • 'I '•' i i l ' i i i | i i i i | '

100n 6.33

% 8:2FTUCA 457>393

T,TTnFr}t-TT-nrt| .,11,1, r) , , | , ,

100^ 5.8S PFOA % 413>369

' l ' ' ' ' l ' ' ' ' l ' 1 i i ' i i l ' ' ' ' l ' ' ' ' l

100 B.72 Total Ion Chromatogram

.-nTlme 0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00

Figure 2-1 (a) Typical GC/ECD chromatogram showing the 8:2 FTOH and the impurity ally lie 8:2 FTOH. (b) Typical LC/MS/MS chromatogram of the acid metabolites. 95

m/z m/z m/z m/z • standard

Compounds

Figure 2-2 Confirmation of metabolites observed from the degradation of 8:2 FTOH. Comparison of transition parent-daughter m/z ratios in samples and standards. Base peak set at 100%.

2.4 RESULTS AND DISCUSSION

Despite the rising number of published studies looking at the detection of perfluorinated acids in the environment, only a small number have examined their potential sources. Ellis et al.

(25) have identified thermolysis of fluoropolymers as an abiotic mechanism that can potentially lead to the production of these persistent compounds while Hagen et al. have been the first to observe the production of PFOA from the 8:2 telomer alcohol in a biotic system (18)and more recently, the production of perfluorocarboxylic acids were observed from a telomer alcohol biodegradation screening study by Lange (19). The study presented here not only provides 96 further evidence that telomer alcohols are a potential source of PFCAs through biotransformation reactions, but it also presents a plausible biotic mechanism in a microbial system.

In this laboratory study, an initial mass of 750 (j,g (1.5umol) of 8:2 FTOH was added to microcosms (1 Litre) inoculated with microorganisms. As seen in Figure 2-3, the 8:2 FTOH spiked was 85% degraded as of day 7, and was below detection limit levels (2 ug L"1) by day 16.

Triplicate vessels showed similar trends, although rates differed by as much as 35%. The initial half-life of the 8:2 FTOH was calculated to be ~0.2 days mg"1 of initial biomass protein followed by a second half-life of ~0.8 days mg"1. The complex kinetics observed in the degradation of 8:2

FTOH maybe attributed to a couple of probable explanations. First, there may have been a change in the activity of specific microbial populations comprising the mixed inoculum during the experiment that could explain the observed change in the rate of the degradation after day 3.

Second, more than one mechanism of degradation may be operative, resulting in varying rates of reaction involved; this study however could only provide evidence for one dominant mechanism. 0 10 20 30 40 50 60 70 80 90 Time (Days)

Figure 2.4-3 Typical transformation kinetics and mass balance of metabolites observed in degradation experiments; degradation of 8:2 FTOH in active microcosm (vessel B). Loss of 8:2 FTOH, production of 8:2 FTCA, PFOA, and overall mass balance. No observable loss of 8:2 FTOH in sterile control (n=l) (inset). PFOA values were obtained using standard additions at day 81.

The degradation of the 8:2 FTOH was presumably due to microbial activity since the sterile control showed little to no transformation during the experimental period as shown in

Figure 2-3 (inset). The small decrease in the concentration observed in the sterile control is attributed to losses of volatiles from the vessel during sampling. The sterile control was not routinely sampled for 8:2 FTOH past day 16 of the study. Further evidence that the degradation of the telomer alcohol was driven by microbial activity in active bottles was the observed and quantified acids in the active bottles and their absence in the sterile control (Figure 2-3). The degradation of the 8:2 FTOH occurred concurrently with the production of the telomer acid, 8:2

FTCA and the telomer unsaturated acid, 8:2 FTUCA. This transformation step occurred via the formation of the telomer aldehyde, 8:2 FTAL. This volatile metabolite was detected and 98

confirmed in sewage treatment plant samples by matching gas chromatograph peak retention times with synthesized standards as well as with mass spectral data (Figure 2-4a). The

synthesized standard of the 8:2 FTAL showed a distinctive double peak for its molecular ion m/z

463 (M +1), the presence of which was also confirmed in the samples. The 8:2 FTOH aldehyde

appeared to be a transient intermediate and confident quantification was not possible with our

current method. The 8:2 FTCA and 8:2 FTUCA were more stable in the system. 99

8:2 FTAL Abundance Abundanc 40 Sarrmle 3500 300 m/z 463 30 ] m/z 463 250 200 20 150 100 10 50 -fVf>.^l.-t.af*1ttrM,»1W|..^f.H^"T'V'Vi'^ *|Mf :M*UiW Mi °19 n 12.5 13.0 13.5 14.0 14.5 15.0 15.5 9.£6 ' ii.5 'lib' 13^ 'i4.b' i4.5 'iS.b i5.5 Abundanc Abundanc 3500 3Rnn m/z 379 m/z 3000 | 3000 2500^ 2500 2000 r 2000 1500 | 1500 1000 1000 500 ; 500 Time- - 12.0 12.5 13.0 13.5 14.0 14.5 15.0 15.5 Time-12.0 12.5 13.0 13.5 14.0 14.5 15.0 15.5

Media Blank Sterile Control Sample •too. 1(Xh 100

8:2 FTCA 8:2 FTCA °*A 477>393 477>393

P^'t'i'n'^'i'n'i'ih'i'i'i'i'i'iii'ii'H'ii'ii^Trri'i'i'i'ii'tiiiiTr OUT i|i..i[iiii|iii.|ini[iiii|ini|iii.|HM|ii

10(h 100i i«h

8:2 FTUCA 8:2 FTUCA % 457>393 % 457>393

100 100i 100- PFOA PFOA % 413>369 413>369

Figure 2-4 (a) GC/MS (PCI) extracted chromatograms of synthesized 8:2 FTAL standard and 8:2 FTAL detected in samples inoculated with sludge obtained from a sewage treatment plant. Distinctive double peak for m/z 463 (M+l) in standard was also detected in samples, (b) LC/MS/MS chromatogram of sample taken from a blank, sterile control, and an active bottle. 8:2 FTCA, 8:2 FTUCA, and PFOA were detected in the active sample and were absent in the blank and sterile control. 100

Upon the depletion of the 8:2 FTOH in the system, the loss of the 8:2 FTCA was observed coincident with an increase in the production of 8:2 FTUCA. There are potentially two mechanisms by which the observed 8:2 FTUCA was produced in the system, via abiotic hydrolysis where the 8:2 FTCA loses an -HF, or biotically, where perhaps an acyl-coA dehydrogenase type of enzyme oxidizes the Ca-Cp bond. This reaction proceeds via the removal of the a-proton, followed by hydride transfer of the P-proton presumably to a cofactor such as flavin adenine dinucleotide (FAD). Other experiments in our lab indicate that the abiotic elimination of HF from FTCAs are slower (half-lives > lweek) than observed in these biological systems, although it is likely that both pathways were involved as the experiment progressed.

The production of the unsaturated acid can also be attributed to the degradation of the allylic 8:2 FTOH present as an impurity in the 8:2 FTOH alcohol (Figure 2-la). Mass spectral data from previous studies within the group have identified the allylic form of the 8:2 FTOH as the only impurity present since the 8:2 FTOH used in this study was of 97% purity (26), and we can assume that the allylic 8:2 FTOH comprised at most 3% of the total mass of FTOH initially.

This impurity would likely be metabolized in analogous fashion as the saturated alcohol, presumably forming the unsaturated acid via the unsaturated aldehyde. The detection of the 8:2

FTUCA early in the experiment (day 2-5) at approximately the same time as the detection of 8:2

FTCA may be a consequence of the oxidation of the allylic form of the 8:2 FTOH.

PFOA was detected in the system at very low concentrations beginning at day 16. A sample chromatogram is presented in Figure 2-4 of all non-volatile metabolites detected in samples and their absence in the blank and in the sterile control. It appears that the highest concentrations of PFOA detected in the system within the duration of the experiment occurred at the peak concentration of the 8:2 FTUCA. By day 81, PFOA was detected at approximately 3% 101 of the total mass. This production of PFOA may be attributed to the degradation of the earlier produced 8:2FTUCA,and we suggest that further degradation of the 8:2 FTUCA in the system may lead to an increase in the production of this perfluorinated acid. Current studies are looking at degradation products when only the 8:2 FTUCA is spiked in a microbial system. Hagen et al.

(18) in their earlier study identified 8:2 FTUCA in their system but were unable to definitively show that this was the initial step in the biotransformation process prior to forming PFOA.

Lange et al. (19) reported the detection of 6-7% of PFOA at the conclusion of their biodegradation study of telomer alcohol mixtures and have also identified the unsaturated acid as an intermediate in the degradation process.

An assessment of the mass balance between the parent compound and the nonvolatile metabolites resulted in approximately 55% of products accounted for at the conclusion of the study (Figure 2-3), with 8:2 FTUCA being the most abundant metabolite along with PFOA.

During the time interval, day 1-5, there was also a noticeable loss in the total mass measured

(Figure 2-3), which suggests that metabolite(s) produced early on in the pathway was unaccounted. This apparent loss in mass may be due to our inability to quantify the 8:2 FTAL, along with other observed but unidentified volatile metabolites. Analyses via GC/ECD have shown other unknown chromatographic peaks produced in the early stage of the degradation in experimental bottles and their absence in the sterile control. By day 81, the observed 45% loss of the products may be due to a number of reasons. As previously alluded to, other volatile metabolites observed in the degradation that were left unidentified may account for partial loss in measured products, as well as that volatile metabolites may have been lost during routine sampling. It is also possible the unaccounted mass could arise from the unsaturated metabolites

(i.e., 8:2FTUCAand 8:2 unsaturated aldehyde) being covalently bound by biological macromolecules such as extra polymeric substances (EPS) produced extensively by most bacteria leading to its perceived loss. The unsaturated metabolites are presumably quite electrophilic and hence susceptible to attack by endogenous nucleophiles present in biological systems. The observation that the unsaturated acid was the dominant metabolite produced from telomer alcohol biodegradation may be of significance since it may very well be a toxic metabolite for organisms.

Ion chromatography was used in an attempt to quantify inorganic fluorine (F") in the system that may account for a small part of the observed loss in mass. If the telomer acids and

PFOA were the only primary metabolites of telomer alcohol degradation as observed in this study, then it is suggested that these compounds fail to undergo extensive defluorination; hence, expected fluoride concentrations would be low. The high amounts of chloride present in the matrix (mineral medium) prevented the detection limits for fluoride to be any lower than 1 mg L

\ The use of a fluoride specific electrode was also considered, although a similar type of interference was expected from the matrix. This limit of detection for fluoride was too high for the telomer alcohol concentration used in this study; the initial spike of 750 ug of 8:2 FTOH would have produced approximately 60 (j,g L"1 of fluoride (1:2 molar ratio) from the hypothesized pathway where 1 equiv is produced upon generation of the unsaturated acid. The concentration used in this study for dosing the FTOH was chosen to be well below its saturated water solubility, which made it impossible to determine the evolved fluoride. It should be noted that if the FTOH underwent complete defluorination, the resulting fluoride concentration

(>1 mg L"1) would have been observable.

A proposed biodegradation scheme (Figure 2-5) is based upon results of this laboratory study and built on earlier results presented by Hagen et al. (18). Under this proposed pathway, 103

8:2 FTOH can be oxidized by an alcohol dehydrogenase enzyme, fairly common in bacteria (27-

29), to form the 8:2 FTAL. Subsequent oxidation of the terminal carbon leads to the formation of the 8:2FTCAperhaps via an aldehyde dehydrogenase type of enzyme. Murphy et al. reported the isolation of an aldehyde dehydrogenase enzyme capable of converting fluoroacetaldehyde to fluoroacetate in Streptomyces cattleya (30). Although this study was looking at the probable enzymes involved in the biosynthesis of fluorinated compounds, it confirms that bacteria indeed possess enzymes capable of mediating such metabolic transformations. This proposed degradation pathway for the fluorotelomer alcohols involves reactions similar to dehydrogenation reactions seen in the conversion of ethanol to acetic acid in the absence of molecular oxygen (31). Dehydrogenation reactions also require the coenzyme nicotinamide adenine dinucleotide (NAD+), which serves as hydrogen carriers. However, in the presence of molecular oxygen, typical oxidation reactions producing similar products as stated previously may be aided by mixed function oxidases (MFO) or monooxygenase type of enzymes such as cytochrome P450, also widespread in microorganisms, animals, and humans (31).

Despite performing the biodegradation experiments under aerobic conditions, the oxygen concentration was not measured; hence, we are unable to definitively determine which of these two types of reactions is involved. The transformation of the 8:2 FTCA leading to its unsaturated form and ultimately to PFOA is an example of a P-oxidation mechanism as previously proposed by Hagen et al. (18). Several critical enzymes are possibly involved in such a mechanism. We suggest that enzymes such as acyl-CoA synthases and crotonases may be required. The oxidation step from the unsaturated acid to PFOA is thermodynamically costly and hence is expected to be slow. This hypothesis was consistent with our observation that

PFOA was first detected in the system in the latter phase of the experiment (day 16). This 104

suggests that oxidation of the FTUCA, a-carbon is difficult given its high electrophilicity,

although it appears to be transformed to the highly persistent perfluorocarboxylic acid (e.g.,

PFOA). Corollary experiments using the activated sludge from a sewage treatment plant showed

that a similar pathway was operable with all the identified degradation products indicated in

Figure 2-5 observed (data not shown). The previous observations suggest that other telomer

alcohols may degrade analogously producing their corresponding even perfluorocarboxylic acid

under the proposed mechanism (i.e., 10:2 FTOH may biodegrade producing the

perfluorodecanoic acid, and the 6:2 FTOH would form the perfiuorohexanoic acid).

F F., F F, F FE, F , FF ft F*,F \f\f\f 9 c . / v "ClV'S-CoA "CH/ OH I? F" F F~ F F* V WXfr 8:2 FTCA Hydrolysis

F* f F* F F Q V "OH F. F F. F F„ F \ ,F F F •W&F* 'V F* FF&' 'F H F \/ \/ \/ 1 ° ,CH F w 8:2 FTUCA Tf A A A F 8:2 FTAL l F F FVF FVF OH O FVVVS4CH^S-COA F* F FF F F Fh P-oxidation

F. F F. F F. F t F F„ F F. F 0 O CH CH OH F-^T AAA 2 2 CHf "S-CoA # F F F F F F F i F.FF.FF. F O S-CoA

Figure 2-5 Proposed biodegradation pathway and products of 8:2 FTOH based upon laboratory experiments. Structures in brackets are proposed transitional intermediates and were not determined in this study. 105

The current study showed that telomer alcohols readily biodegrade, producing telomer acids and perfluorinated acids, with the unsaturated telomer acid being the predominant metabolite; we are currently investigating whether the FTUCAs are commonly observed in environmental samples. Microbial transformation reactions such as demonstrated by these experiments have strong implications for other biological transformations since microorganisms can be seen as surrogates for metabolic reactions of higher organisms. Thus, these reactions may serve as probable sources of PFOA and other carboxylic fluorinated acids detected widely in biota and as previously demonstrated by Hagen et al. (18). There are likely several pathways under which telomer alcohols can biodegrade when released into the environment, although it appears that the (3-oxidation pathway described previously is a principal fate for these compounds. There exists the potential for R-oxidation of the FTOHs to yield the odd numbered

FTCAs recently detected in biota (7); our investigation, however, indicated no evidence for this pathway being operable under these microbial conditions. Further studies are underway to determine the identity of other volatile metabolites observed in these experiments. Hagen et al.

(18) were also unable to identify a major metabolite in their experiments. The identity of these unknown volatile metabolites may provide further clues to the existence of alternative degradation pathways. Microbial degradation of telomer alcohols may very well be a primary fate for these compounds since the potential sources for these compounds (i.e., polymers) often end up in sewage treatment plants.

2.5 ACKNOWLEDGEMENTS

The authors would like to acknowledge Dr. David Ellis for his input to this manuscript, along with Dan Mathers, of the ANALEST facility at the University of Toronto and Naomi 106

Stock for their technical support. We would like to thank Ivan Lee for assistance in experimental sampling. The Natural Science and Engineering Research Council of Canada through a strategic grant generously provided funding.

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(16) Martin, J. W., Muir, D. C. G., Moody, C. A., Ellis, D. A., Kwan, W. C, Solomon, K. R. and Mabury, S. A. Collection of Airborne Fluorinated Organics and Analysis by Gas Chromatography/Chemical Ionization Mass Spectrometry. Anal. Chem. 2002, 74, 584- 590.

(17) Stock, N. L., Lau, F. K., Ellis, D. A., Martin, J. W., Muir, D. C. G. and Mabury, S. A. Polyfluorinated Telomer Alcohols and Sulfonamides in the North American Troposphere. Environ. Sci. Technol. 2004, 38, 991-996.

(18) Hagen, D. F., Belisle, J., Johnson, J. D. and Venkateswarlu, P. Characterization of fluorinated metabolites by a gas chromatographic-helium microwave plasma detector - the biotransformation of 1H,1H,2H,2H - perfluorodecanol to perfluorooctanoate. Anal. Biochem. 1981,118, 336-343.

(19) Lange, C. C. Biodegradation screen study for telomer-type alcohols, 3M Environmental Laboratory; November 6, 2002

(20) Napoli, M., Scipioni, A., Legnaro, C. E. and Krotz, L. N. A modified procedure for the preparing perfluoroalkyl acetic acids from perfluoroalkyl iodides. J. Fluor. Chem. 1994, 66, 249-252.

(21) Achilefu, S., Mansuy, L., Selve, C. and Thiebault, S. Synthesis of 2H,2H-perfluoroalkyl and 2H-perfluoroalkenyl carboxylic acids and amides. J. Fluor. Chem. 1995, 70, 19-26. 108

(22) Cox, E. E., Major, D. and Edwards, E. A. In Remediation of Chlorinated and Recalcitrant Compounds; Wickramanayake, G. B. and Hinchee, R. E., Eds.; Battelle Press: Columbus, OH, 2000; Vol. I.

(23) Program, T. R. Telomer research program update - Presented to the USEPA OPPT, Washington, D.C., November 5, 2002

(24) Communities, C. o. t. E. Commission Decision 2002/657/EC implementing Council Directive 96/23/ EC concerning the performance of analytical methods and the interpretation of results, Off. J. Eur. Communities; August 17, 2002

(25) Ellis, D. A., Mabury, S. A., Martin, J. W. and Muir, D. C. G. Thermolysis of fluoropolymers a a potential source of halogenated organic acids in the environment. Nature 2001, 412, 321-324.

(26) Information provided by Oakwood Products Inc. , West Columbia , SC.

(27) Ludwig, B., Akundi, A. and Kendall, K. A long-chain secondary alcohol dehydrogenase from Rhodococcus erythropolis ATCC-4277. Appl. Environ. Microbiol. 1995, 61, 3729- 3733.

(28) Van Iersel, M. F. M., Eppink, M. H. M., VanBerkel, W. J. H., Rombouts, F. M. and Abee, T. Purification and characterization of a novel NADP-dependent branched-chain alcohol dehydrogenase from Saccharomyces cerevisiae. Appl. Environ. Microbiol. 1997, 63, 4079-4082.

(29) Malone, V. F., Chastain, A. J., Ohlsson, J. T., Poneleit, L. S., Nemecek-Marshall, M. and Fall, R. Characterization of a Pseudomonas putida allylic dehydrogenase induced by growth on 2-methyl-3buten-2-ol. Appl. Environ. Microbiol. 1999, 65, 2622-2630.

(30) Murphy, C. D., Moss, S. J. and O'Hagan, D. Isolation of an aldehyde dehydrogenase involved in the oxidation of fluoroacetaldehyde to fluoroacetate in Streptomyces cattleya. Appl. Environ. Microbiol. 2001, 67.

(31) Crosby, D. G. Environmental Toxicology and Chemistry; Oxford University Press: New York, 1998. 109

2.7 ADDENDUM TO CHAPTER II

Since the publication of this chapter in Environmental Science and Technology in 2004, several studies have emerged in the scientific literature that both question and provide support to the results presented here. These studies along with how it relates to our work will be briefly discussed.

The observation of 8:2 FTOH biodegradation under aerobic conditions was also demonstrated by Wang et al. (1). Microcosms used in their study were amended with 14C- labelled 8:2 FTOH as the substrate and diluted activated sludge taken from a domestic wastewater treatment plant was added as the inoculum. In addition to detection of previously observed metabolites such as 8:2 FTCA, 8:2 FTUCA and PFOA (2), Wang et al. reported the

14 detection of a novel metabolite, CF3(CF2)6 CH2CH2COOH (2H,2H,3H,3H-perfluorodecanoic acid or 7:3 Acid). In contrast to what was previously observed, the dominant metabolite detected by Wang et al. was the 8:2 FTCA making up 27% of the total mass balance at the conclusion of their experiment that ran for 28 days. Our study had measured the 8:2 FTUCA as the dominant metabolite making up approximately 40% of the total mass balance after 81 days of degradation.

8:2 FTUCA made up only 6% of the initial mass of 14C-labelled FTOH at the conclusion of their study and the 7:3 FTCA accounted for 2.3%. 7:3 FTCA was not analyzed for in our earlier study, however it is likely that this metabolite makes up part of the 45% of unaccounted mass balance that we reported at the conclusion of our study. PFOA concentrations detected in both studies are comparable with reported yields of 2.1% by Wang et al. and 3% by our investigation.

Wang et al. had suggested that (3-oxidation cannot occur on the 8:2 FTUCA as our study had proposed due to the lack of protons on the [3-carbon. They suggested that this proton deficiency prevents the proton/electron shuffling that is essential for completion of one cycle of 110

P-oxidation similar to what has been observed with fatty acids. Instead, they proposed that p- oxidation of the 7:3 FTC A is more likely to occur leading to PFOA production. The authors however fail to provide a possible explanation as to how this new metabolite is formed from the

8:2 FTOH. We contend that production of PFOA from p-oxidation of 8:2 FTUCA is likely occurring due to observed formation of PFOA from degradation experiments where 8:2 FTCA and 8:2 FTUCA were spiked as substrates under similar conditions as the 8:2 FTOH degradation

(unpublished data). Additionally, a P-elimination mechanism similar to what had been proposed for the degradation of 3-fluoropyruvate involving catalysis by a thiamin pyrophosphate (J) may provide some insight into the formation of PFOA from 8:2 FTUCA. Enzymatic studies are clearly warranted to provide clarity on the mechanism of PFOA formation. The absence of perfluorononanoic acid (PFNA) production from both studies (1, 2) is indicative that cc-oxidation is not a significant mechanism of degradation for the 8:2 FTOH in microbial systems.

A follow up study by Wang et al. (4) reported additional new metabolites from the

14 degradation of C-labelled 8:2 FTOH. The newly identified metabolites were: CF3(CF2)6-

14 14 CHOHCH3 (7-2 sFTOH), CF3(CF2)6 CH=CHCOOH (7-3 unsaturated acid or 7-3 FTUCA

14 acid) and CF3(CF2)6 CH=CHCONH2 (7-3 u amide). The 7:3 FTUCA was first observed by

Martin et al. in a rat metabolism study (5). Inoculum used in this second study by Wang et al. include diluted activated sludge obtained from several wastewater treatment facilities as well as mixed bacterial culture also from a wastewater treatment plant but acclimated on 5 to 10 mg L"1 of 8:2 FTOH. It is likely that different consortia of microorganisms are capable of degradation via alternative degradation pathways. A critical finding in this study is the observed production

14 of C02 from 8:2 FTOH degradation amounting to 12% of the initial 8:2 FTOH added. This

C02 production was observed along with equivalent amounts of perfluorohexanoic acid Ill

(PFHxA). Authors of the study suggest the evolution of 14C02 in the microcosms is indicative of possible mineralization of telomer alcohols. CO2 formation increased with addition of ethanol in microcosms indicating that addition of organic nutrients to microbial systems may accelerate degradation. Overall yield of PFOA at 6% from this study is comparable to previous measurements (1, 2, 6). This study proposed several alternative degradation pathways for the newly identified metabolites but did not propose a pathway for the formation of CO2. Wang et al. had suggested that formation of CO2 and PFHxA would involve defluorination of-CF2- groups from the intermediate compounds such as the 8:2 FTUCA and or the 7:3 FTUCA and not from PFOA based on the lack of observed C02 production in vessels spiked only with PFOA.

Microbial defluorination of -CF2- groups however is uncommon due to the strength of the C-F bond (7) and is rarely reported (8).

Significance of PFOA production demonstrated in laboratory investigations of 8:2 FTOH biodegradation has been highly relevant with studies that followed that examined presence of perfluorinated chemicals in sludge and wastewater samples. Several studies have been published that quantified perfluorinated chemicals (PFCs) in wastewater influent and effluent from various wastewater facilities (9-14). These studies suggest that high concentrations of PFCAs and PFCs in general from sludge and wastewater samples may be due to possible degradation of precursors that are present in the wastewater system. Concentrations of PFOA in wastewater effluent from six wastewater treatment plants (WWTPs) range from 58 up to 1050 ng L'l(14). Concentrations reported by Schultz et al. (12) were lower ranging from 0.7 to 97 ng LI. Total PFCs measured by Higgins et al. (10) from sludge amounted to 176 to 3390 ng g"1. These values indicated that concentrations of PFCAs and PFCs in general vary amongst different WWTPs and are likely influenced by the input of the surrounding area (ie. residential, commercial or industrial) of the 112 facility. The presence of these contaminants in wastewater effluent in significant amounts makes them potential point sources to natural waters (14).

A closer examination of mass flows in wastewater treatment plants were carried out by

Sinclair et al.(/4)and Schultz et al.(ii). Both studies reported elevated concentrations of PFCs detected in effluent versus influent wastewater samples. Schultz et al. reported an observed increase in PFOA concentrations of up to 352% while Sinclair et al. measured an increase in

PFOA as well as the C9, CIO and CI 1 homologue concentrations in the effluent samples ranging from 211 ± 87%. to as high as 556 ± 182%. Further more, Sinclair et al. reported a general trend observed where greater amounts of even chained PFCAs were measured in comparison to odd chained PFCAs. Authors of these studies suggest that their observations all point to possible presence of precursor compounds in the wastewater streams such as FTOHs or FTOH related compounds that are transformed by microorganisms leading to increase in PFCA production.

The pattern even >odd was suggested to be indicative of PFCA formation from microbial (3- oxidation of these telomer based compounds (10, 14). An even more significant evidence of 8:2

FTOH degradation occurring in the environment is the detection of intermediate compounds, 8:2

FTCA and 8:2 FTUCA from wastewater samples at concentrations ranging from 2.5 ng L" to 29 ng L"1. These compounds are not commercially produced and have no known industrial application. Despite the evidences that allude to presence of PFCA precursors in wastewater systems, there has been no study to date that has detected these compounds in these samples.

Clearly a method is warranted for detection of possible precursor compounds in various environmental matrices such as wastewater, sludge and sediment. 113

2.8 LITERATURE CITED IN ADDENDUM

(1) Wang, N., Szostek, B., Folsom, P. W., Sulecki, L. M., Capka, V., Buck, R. C, Berti, W. R. and Gannon, J. T. Aerobic Biotransformation of 14C-Labeled 8-2 Telomer B Alcohol by Activated Sludge from a Domestic Sewage Treatment Plant. Environ. Sci. Technol. 2005, 39, 531-538.

(2) Dinglasan, M. J. A., Ye, Y., Edwards, E. A. and Mabury, S. A. Fluorotelomer Alcohol Biodegradation Yields Poly- and Perfluorinated Acids. Environ. Sci. Technol. 2004, 38, 2857-2864.

(3) Leung, L. S. and Frey, P. A. Fluoropyruvate: An unusual substrate for escherichia coli pyruvate dehydrogenase. Biochem. Biophys. Res. Commun. 1978, 81, 274-279.

(4) Wang, N. S., Bogdan; Buck, Robert C; Folsom, Patrick W.; Sulecki, Lisa M.; Capka, Vladimir; Berti, William R.; Gannon, John T. Fluorotelomer Alcohol Biodegradation - Direct Evidence that Perfluorinated Carbon Chains Breakdown. Environ. Sci. Technol. 2005,59,7516-7528.

(5) Martin, J. W., Mabury, S. A. and O'Brien, P. J. Metabolic products and pathways of fluorotelomer alcohols in isolated rat hepatocytes. Chem. Biol. Interac. 2005,155, 165- 180.

(6) Lange, C. C. Biodegradation screen study for telomer-type alcohols, 3M Environmental Laboratory; November 6, 2002

(7) Kissa, E. Fluorinated surfactants and repellents; Marcel Dekker: New York, 2001.

(8) Key, B. D., Howell, R. D. and Criddle, C. S. Fluorinated organics in the biosphere. Environ. Sci. Technol. 1997, 31, 2445-2454.

(9) Boulanger, B. V., J. D.; Schnorr, J. L.; Hornbuckle, K. C. Evaluation of Perfluorooctane surfactants in a Wastewater treatment system and in a commercial surface protection product. Environ. Sci. Technol. 2005, 39, 5524-5530.

(10) Higgins, C. P., Field, J. A., Criddle, C. S. and Luthy, R. G. Quantitative determination of perfluorochemicals in sediments and domestic sludge. Environ. Sci. Technol. 2005, 39, 3946-3956.

(11) Furdui, V. I., Crozier, P. W., Reiner, E. J. and Mabury, S. A. Rapid trace levels of perfluorinated chemicals in Great Lakeswater. Chemosphere 2007, Accepted.

(12) Schultz, M. M., Barofsky, D. F. and Field, J. A. Quantitative Determination of Fluorinated Alkyl Substances by Large-Volume-Injection Liquid Chromatography Tandem Mass Spectrometry-Characterization of Municipal Wastewaters. Environ. Sci. Technol. 2006, 40, 289-295. 114

(13) Schultz, M. M., Higgins, C. P., Huset, C. A., Luthy, R. G., Barofsky, D. F. and Field, J. A. Fluorochemical mass flows in a municipal wastewater treatment facility. Environ. Sci. Technol. 2006, 40, 7350-7357.

(14) Sinclair, E. K., Kurunthachalam Mass Loading and Fate of Perfluoroalkyl Surfactants in Wastewater Treatment Plants. Environ. Sci. Technol. 2006, 40, 1408-1414. CHAPTER III

SIGNIFICANT RESIDUAL FLUORINATED ALCOHOLS PRESENT IN VARIOUS FLUORINATED MATERIALS

Mary Joyce A. Dinglasan-Panlilio and Scott A. Mabury

Published in -Environ. Sci. Technol. 2006, 40, 1447-1453.

Contributions - Mary Joyce A. Dinglasan-Panlilio designed and conducted the experiments. All data analysis, interpretation and manuscript preparation were also carried out by Mary Joyce A. Dinglasan-Panlilio. Intellectual input and critical comments were provided by Scott A.Mabury.

Reproduced with permission from Environmental Science and Technology Copyright ACS 2006

115 116

3.1 ABSTRACT

Polyfluorinated telomer alcohols and sulfonamides are classes of compounds recently identified as precursor molecules to the perfluorinated acids detected in the environment. Despite the detection and quantification of these volatile compounds in the atmosphere, their sources remain unknown. Both classes of compounds are used in the synthesis of various fluorosurfactants and incorporated in polymeric material used extensively in the carpet, textile and paper industry. This study has identified the presence of residual unbound fluoro telomer alcohols (FTOHs) in varying chain lengths (C6-C14) in several commercially available and industrially applied polymeric and surfactant materials. NMeFOSE, a perfluoroalkyl sulfonamido alcohol, was also detected in a commercially available carpet protector product. A method was developed to remove these residual compounds from polymeric and surfactant materials by dispersion in water and stripping of the volatiles using a constant flow of air and trapping on

XAD resin. Using gas chromatography mass spectrometry analysis, it was determined that the fluorinated materials examined consist of 0.04-3.8% residual alcohols on a fluoroalcohol to dry mass basis. These values indicate that residual alcohols, left unreacted and unbound from the manufacturing process of fluorinated polymers and surfactants, could be a significant source of the polyfluorinated telomer alcohols and sulfonamides released into the environment. This study suggests that elimination or reduction of these residual alcohols from all marketed fluorinated polymers and fluorosurfactants is key in reducing the prevalence of perfluorinated acids formed in the environment. 117

3.2 INTRODUCTION

Compounding evidence has been reported recently that supports the hypothesis that fluorinated telomer alcohols (FTOHs) can act as precursors to the perfluorinated acids (PFCAs) that have been detected widely in the environment. FTOHs were found pervasive (11-165 pg m"3) in the North American atmosphere (1,2) and have been identified as a source of a suite of perfluorinated acids through atmospheric degradation (3). Extensive kinetic and mechanistic studies of the FTOHs with CI atoms and OH radicals have demonstrated the formation of PFCAs as degradation products (4,5). It has been determined, in a number of experiments, that biotic degradation of telomer alcohols under aerobic conditions also generates these persistent environmental contaminants (6,7). Perfluorinated acids such as PFOA have been a concern to environmental scientists and regulators in recent years due to their ubiquity in various environmental matrices, particularly in blood samples of occupationally exposed workers but most specially the general human population (8). Its carcinogenic effects (9) and developmental toxicity to rats (10) have also instigated the demand for further study of these compounds and their sources by regulators to determine potential risk if any to humans. It has been shown that metabolism of the 8:2 telomer alcohol in rats produce PFOA (11,12) which has further emphasized that PFOA and other PFCAs found in human blood may be partially due to exposure to these volatile compounds (12). In addition, isomeric profiles of PFCAs detected in polar bears suggest they originated primarily from a linear isomer source (13).

Similarly, the perfluoroalkyl sulfonamido alcohols have been suggested to be precursors of perfluoroalkane sulfonates, another class of perfluorinated acids (2); they have also been detected at appreciable amounts in both outdoor (1,2) and indoor air (14). Biotransformation studies of JV-ethyl perfluorooctane sulfonamidoethanol (NEtFOSE) have confirmed the 118 production of perfluorooctane sulfonate (PFOS) as a terminal metabolite in rats (15). PFOS like

PFOA, has been detected in various environmental matrices and found resistant to degradation

(16). Research in our laboratory is currently investigating whether sulfonamido alcohols undergo atmospheric degradation to produce PFOS. In response to the widespread detection of PFOS in the environment, 3M, a major manufacturer of perfluorinated sulfonamides voluntarily phased out their production of compounds with perfluorooctane chemistry in 2000-2002. These compounds include the sulfonamido alcohols, TV-methyl (NMeFOSE) as well as N-ethyl (NEtFOSE) perfluorooctane sulfonamidoethanol.

The sources of both fluorotelomer and sulfonamido alcohols have yet to be clearly identified since they are not understood to be directly used in industrial or commercial applications. They are however largely used in the synthesis of various fluorosurfactants as well as incorporated into a wide array of fluorinated polymers.

Fluorinated polymers and surfactants have been in use for over half a century and have been incorporated into a vast array of products. These polymers are used widely in various industries such as paints, carpet, and paper coatings (17) and recently, have found their way into other commercial products such as household cleaning agents (18). The widespread application of these polymers can be directly linked to their unique ability as effective surface-active agents, delivering both water and stain repellency. Fluorotelomer based polymers are characterized by a hydrocarbon backbone, from which telomer alcohols (FTOHs) of various chain lengths (C6-18, or

4:2 up to 16:2 FTOHs) are appended through ester, amide, urethane and ether linkages (19).

Polymers make up approximately 80% of the reported production in the fluorotelomer market

(20). A critical question is whether the alcohols observed in the atmosphere are a consequence of 119 the scission at the polymer linkage releasing the fluoro alcohols, or merely caused by sufficient residual material escaping directly from the fluorinated polymer or surfactants.

This investigation has specifically sought to quantify unbound FTOHs as well as the sulfonamido alcohols, hereafter referred to as "residuals", from various fluorinated materials, prior to or following application to consumer or industrial products to assess the potential contribution to the flux of these compounds to the environment. These residual alcohols, presumably a function of incomplete synthesis or lack of purification prior to marketing of materials (Figure 3-1), may be a significant source of release to the environment. Quantification and identification of residuals from seven different commercially and industrially available fluorinated materials were performed using a purge and trap method coupled with gas chromatography-mass spectrometry analysis. Commercially available materials are those that are available to retail consumers while industrially applied materials are those that are not generally available to regular retail consumers but instead are recommended for the use of manufacturers and industry. All fluorinated materials studied may or may not contain other non-fluorinated components in their formulation. This study identifies a source of these precursor compounds, which may ultimately comprise a significant fraction of the contribution to the perfluorinated acids in the environment. 120

'Offgassing" "Offgassing" u E Ft Ft FE F 'Offgassing"

F H2FFFFFF

F free-radical F ^F esterification F polymerization F-

OH TelomerAlcohol 0 CV=^H "Residual"

"Monomer" "Polymer' o=c o=c o=c o=c + o H,C H II H RO' Nc=c(

H-3VC H-y Carpet Treatment

Figure 3-1 Stylized schematic of postulated steps leading to the production of telomer-based polymers using 8:2 FTOH as an example. The potential source of unreacted telomer alcohols is depicted from the reaction producing the monomer and leading to its point of release to the environment from polymeric materials if left unpurified. Polymeric materials produced will have a mixture of varying chain lengths of perfluorinated and hydrocarbon chains as well as varying carbon backbone depending on reactants used.

3.3 MATERIALS AND METHODS

3.3.1 Chemicals and Standards.

Zonyl® FSO-100 and Zonyl® FSE were obtained from DuPont chemicals (Wilmington,

DE). Teflon® Advance Carpet Protector was purchased from Kleen Kuip Supply Mart Inc.

(Toronto, ON) and Polyfox-L-diol was provided by Omnova Solutions Inc. (Fairlawn, OH).

Motomaster® Windshield fluid with Teflon® was purchased from Canadian Tire (Toronto, ON);

Scotchgard® Rug and Carpet Protector was purchased from Home Depot (Toronto, ON) and

3,3,4,4,5,5,6,6,7,7,8,8,9,9,10,10,10-Heptadecafluorodecyl methacrylate (97%) was purchased

from Aldrich (Oakville, ON). Fluorotelomer alcohols (FTOHs) 4:2, 6:2, 8:2 and 10:2 standards

(all 97%) were purchased from Oakwood Products, Inc. (West Columbia, S.C.) and 3M Corp. (St. 121

Paul, MN) generously provided the standard for iV-methyl perfluorooctane sulfonamidoethanol

(NMeFOSE).

3.3.2 Preparation of Experimental Vessels.

Purge and trap vessels were made using 1L media bottles (Pyrex) with caps drilled to accommodate Orbo™ Amberlite™ XAD-2 cartridges (lOOmg) (Supelco, Bellefonte, PA) and gas diffuser tubes (Pyrex, VWR International Ltd., Mississauga, ON). Distilled deionized water (500-

750 mL) was added to the vessels along with 10-100 mg of the fluorinated materials of interest.

These aqueous suspensions were manually shaken thoroughly and extra care was taken to ensure that the transfer of fluorinated materials to the vessels was done quickly to ensure minimal loss of volatiles. Materials that caused excessive foaming such as Zonyl® FSO-100 and Zonyl® FSE, were put on a shaker at 140 rpm for continuous mixing. The vessels were tightly sealed and carbon filtered in-house air was sparged through the vessels at a flow rate of ~40mL min"1.

Vessels were stored at room temperature. A schematic of the experimental set-up can be found in the supporting information. Each compound was analyzed, generally in triplicate or N=6, and a blank vessel was included for each batch of experiments. Blank vessels consisted of deionized water without addition of any fluorinated material and treated the same as the spiked vessels.

Vessels were purged continuously for duration of up to 3 weeks with intermittent sampling of the

XAD cartridges. The XAD resin and the glass wool from the cartridges were extracted using two

5 mL aliquots of ethyl acetate and both fractions were subsequently combined. Samples of 2 mL each were then transferred to autosampler vials for gas chromatographic analysis. Scotchgard™ spiked samples were blown down to a final volume of 200 uL using a gentle stream of purified nitrogen prior to analysis due to higher detection limits for NMeFOSE but were otherwise 122 prepared analogous to the other materials studied. New cartridges were installed in the vessels after each sampling period.

3.3.3 Instrumental Analysis and Quantification.

Sample analyses were carried out using a Hewlett Packard 6890 gas chromatograph equipped with a 5973 inert mass spectrometer detector. Optimal separation of the FTOHs and

NMeFOSE was done using a 30m Rtx®-35MS with Intra Guard column (0.25mm i.d., 0.5[mi film thickness, Chromatographic Specialties, Brockville, ON). The following GC oven program was employed: initial temperature of 45°C held for 2 minutes; 10°C min"1 ramp to 95°C; 30°C min"1 ramp to 150°C min"1; 7°C min"1 ramp to 180°C and a final ramp of 50°C min"1 to 240°C and held for 2 minutes. Helium was used as the carrier gas at a flow rate of lmL min"1 with pulsed splitless injection at an initial pressure of 20 psi at 220°C for 1.2 min followed by an injector purge at 40.0mL min"1 for 0.8 min. Definitive identification of fluoro alcohols was performed under both electron ionization (EI) and positive chemical ionization (PCI) modes. Quantification proceeded under PCI in single ion monitoring mode, and the molecular ion (M+l) was monitored for all target analytes (4:2, 6:2, 8:2, 10:2, 12:2 and NMEFOSE). Calibration was performed using external standards prepared in ethyl acetate and ranging in concentration from 25 to 500 pg juL~ for the telomer alcohols and 2.5 to 500 ng juV1 for the NMeFOSE. Standards were run in between each sample set (ie. 4-6 samples) and the mean response was used to plot a calibration curve.

Good linearity was observed with typical r2 > 0.98 and retention times for all analytes of interest were well conserved (± 0.04 min). Limit of quantitation was defined as the lowest standard to give a signal-to-noise ratio >10, corresponding to 25 and 2.5 ng fiL'1 for the telomer alcohols and

NMeFOSE, respectively. 123

3.3.4 Dry Mass Determination.

To normalize the residual fluoro alcohols measured from the various fluorinated polymers and surfactants, values presented in this investigation are expressed as mass of fluoro alcohols to dry mass of material examined. Dry masses were obtained by measuring the mass of an aliquot of the polymer or surfactant before and after storage in a desiccator at room temperature for several days. It is assumed that the fluorinated polymeric or surfactant material was not volatile and was not lost during the drying process. Some of the fluorinated materials examined contained other compounds in their formulations such as ethylene glycol along with varying amounts of water. Expressing the quantity of fluoro alcohols in this fashion was deemed as the most appropriate method for direct comparison between different materials. This expression should provide a conservative estimate of the amounts of residuals measured and a value that allows comparison across materials and further studies. It is an attempt to prevent confusion in the reporting of residual amounts. It would be preferable to report percent residual FTOHs, for example based on total FTOHs on the polymer, but this requires data that to date are not generally available.

3.3.5 Quality Control and Assurance.

Spike and recovery experiments were performed to validate the applicability of the purge and trap vessels. Fluorinated material was spiked into the vessels and stripped of residuals.

Known amounts of telomer alcohols and NMeFOSE were then added to the same vessels, purged, trapped and quantified. Recovery of target analytes was >80% ± 20 for all compounds except the

4:2 FTOH where recoveries were <50%. Breakthrough portions of the XAD cartridges were analyzed separately to ensure breakthrough of analyte did not compromise the analysis. The 4:2 124

FTOH was readily found in these breakthrough analyses, which suggests poor recoveries were due to inefficient trapping. Values expressed were not corrected for recovery values.

3.4 RESULTS AND DISCUSSION

Recent investigations into the environmental fate of FTOHs and perfluorinated sulfonamides have provided further evidence that they undergo degradation to pervasive and recalcitrant perfluorinated acids. In earlier studies, we have suggested that fluorinated polymers and fluorosurfactants, known to incorporate these volatile compounds, may be a source of these alcohols through fugitive emission of residuals or as they degrade post application (2,6). In this study, 7 different fluorinated materials, ranging from industrially applied to direct consumer products, were all found to consist of free or unbound fluorinated alcohols. These compounds were quantified to determine their overall contribution to the accumulation of fluoro alcohols in the atmosphere and ultimately of PFCAs to the environment.

The materials analyzed included two commercially available products known as Teflon®

Advance Carpet Protector and Scotchgard® Rug and Carpet Protector. These products are marketed as a post application treatment for carpets to infer water and soil repellency and are readily available at hardware and carpet care retail stores. These products are relevant in the study because of their similarity to industrial scale products. Fluorinated polymers and surfactants formulated over the past decade for industrial or commercial applications are not expected to differ significantly with respect to their basic chemistry or to the presence of fluorochemical residuals (21). Teflon® Advance Carpet Protector is a mixture containing both urethane and acrylic FTOH based copolymers (22). Scotchgard™ Rug and Carpet protector is reported to contain a mixture of fluoroalkyl copolymers as well as other acrylic based polymers (23). 125

Fluorotelomer based surfactants such as Zonyl® FSO-100 and Zonyl® FSE were also studied.

These compounds are industrial scale products obtained directly from the producer and not generally available to regular retail consumers. FSO-100 is described as an ethoxylated non-ionic fluorosurfactant able to impart low aqueous surface tensions at low concentrations. It has a generic formula Rf-CH2CH20(CH2CH2)xH where Rf = F(CF2CF2)y; x can be 0 to -15 and y = 1 to ~7. Its recommended applications include incorporation into caulks, paints, coatings and adhesives (24). Zonyl" FSE is a water-soluble, anionic phosphate fluorosurfactant recommended for use in floor waxes and coatings. Its structure consists of (RfCH2CH20)xP(0)(ONH4)y where x and y = 1 or 2 and again the Rf- is a perfluorinated chain containing 1-7 carbons (25). The synthesis of fluorotelomer based polymeric compounds is also known to proceed via the polymerization of telomer-based monomers (Figure 3-1), hence a monomer

3,3,4,4,5,5,6,6,7,7,8,8,9,9,10,10,10-heptadecafluorodecyl methacrylate (8:2 methacrylate) was also investigated to compare the amounts of residuals obtained from this unpolymerized compound to those of polymeric and surfactant type materials. Polyfox-L-diol, a fluorinated polyoxetane that is not commercially available nor industrially applied, was also a subject of this study. Polyfluorooxetanes are synthesized using the polymerization of fluorinated oxetane monomers (26). This product is an example of a well-characterized polymeric material obtained directly from industry containing an ether-based polymer linkage. Finally, a windshield washer fluid available at common hardware and retail stores in Ontario, Canada called Motomaster

Windshield Washer with Teflon® was also examined to determine whether new products that have recently incorporated fluorinated polymers in their formulation would also be sources of volatile fluorinated alcohols. 126

Unbound fluorinated alcohols were detected in all seven materials analyzed in this study.

Scotchgard® carpet and rug protector, the only nontelomer based material analyzed, had free

NMeFOSE detected using the purge and trap method. It is important to note that this product was of the initial formulation prior to the withdrawal of perfluoroctane sulfonyl fluoride (POSF) based materials and other related sulfonamides in 2000 to 2002 by 3M and is no longer produced. The detection of NMeFOSE as residuals clearly reflects the presence of perfluorooctyl-based compounds. All other materials consisted of free FTOHs of varying chain length.

Purging of the Teflon Advance product resulted in detection of unbound telomer alcohols with chain lengths ranging from 8-14 carbons (6:2 up to 12:2 FTOHs). The 8:2 fluorotelomer alcohol (8:2 FTOH) was found in greatest abundance, followed by the 10:2 then the 6:2. The

12:2 FTOH was also identified in an earlier range finding study using solid phase microextraction

(SPME) headspace analysis but was below the limit of quantitation when purge and trap analysis was utilized. The lack of authentic 12:2 FTOH standard prevented spike and recovery analysis and the low levels measured by purge and trap analysis could have been due to poor recovery of this compound. A typical GCMS chromatogram is shown in Figure 3-2. The identities of the telomer alcohols were confirmed using both their EI and PCI spectra (inset). Analysis applying positive chemical ionization gave a strong signal for the molecular ions m/z 365, 465, and 565 for

6:2, 8:2, and 10:2 respectively where each telomer alcohol differs by 100 molecular mass units.

The characteristic fragments 327, 427 and 527 were evident and were attributed to the loss of -HF

+ H20 from the M+l molecular ion as previously reported (27). In materials that contained 4:2

FTOH, the molecular ion m/z 265 and characteristic fragment 227 were observed, although in materials that contained 12:2 FTOH, molecular ion m/z 665 was the only familiar fragment observed and the expected 627 was not evident. Analysis of telomer alcohols using electron impact ionization (EI) often fails to produce a strong signal for the molecular ion, typical fragments observed however for these compounds are m/z 69, 95 and 131 (28). This was evident when EI was used to verify the presence of FTOHs from the samples. 128

8:2 FTOH 10:2 FTOH

Abundance Abundance 465 PCI 1200 PCI 28000

24000 1000 565

20000 800

16000 600 427 12000 400 527

8000 200 593 493 4000 1 1 ' ' ' ' 1 ' m/z~> 450 500 550 600 0 I J. m/z-> 400 450 500

6:2 FTOH

Abundance 36 5 PCI 95000

75000

55000 327

35000

15000 393

0 1 ! 1 m/z-> 300 320 340 360 380 400

_j ! r r 4.50 5.00 Time (Minutes)

Figure 3-2 GCMS chromatogram of sample obtained from purging of Teflon™ Advance Rug and Carpet Protector, a commercially available product designed for post application carpet treatment. The telomer alcohols detected were 6:2, 8:2, 10:2 and 12:2 as identified by their mass spectra under positive chemical ionization (PCI) (inset) mode. 129

Profiles of FTOHs removed from the other fluorinated materials investigated differed, although 8:2 FTOH appears to be the predominant material identified in the Teflon™ Advance

(Fig. 3) and Polyfox-L-Diol, the two known telomer-based polymeric materials used in the study.

This is expected since it has been reported that telomer compounds with carbon chain length equaling ten (8 perfluorinated carbons) is the key surface-active ingredient in polymeric materials (20). Contact angle is a physical property critical in imparting repellency, where higher contact angles decrease adhesion. It was reported that the contact angle achieved by polymers with a general structure of F(CF)2)n(CH2)202C[C(CH3)-CH2]2 where the perfluorinated chain measures 8 carbons is optimal for water and soil repellency (17). This may explain the abundant use of the 8:2 FTOH in various fluorinated polymeric materials. The monomer or the

8:2 methacrylate (3,3,4,4,5,5,6,6,7,7,8,8,9,9,10,10,10-heptadecafluorodecyl methacrylate), tested consisted mainly of the 8:2 FTOH as expected but it also contained small amounts of the shorter chain 6:2 FTOH. The relatively low percent residual (Table 1) is presumably due to the extensive separation steps required to isolate the 8:2 methacrylate from the other chain length methacrylates likely generated in the synthesis. Zonyl® FSO-100 and Zonyl® FSE contained unbound 6:2 FTOH as the primary component of telomer alcohols purged; it has been reported that 6:2 FTOH is primarily incorporated into non-polymeric, surfactant materials (20). The

Motomaster® Windshield Washer was shown to contain mostly the 6:2 FTOH; it may be that the surface-active component of this consumer product is surfactant based rather than polymeric. 130

• 4:2 FTOH -a 100 CD H6:2FTOH 3 10 (O • 8:2 FTOH CD E 80 • 10:2 FTOH • NMeFOSE •g 'in CD

Polyfox-L-diol Teflon Zonyl FSO Zonyl FSE Motomaster 8:2 Scotchgard Advance 100 Windshield Methacrylate Washer Figure 3-3 Profiles of residual unbound polyfluorinated alcohols detected in the seven fluorinated materials tested. Values are expressed as percent of total residuals measured.

This study demonstrated the effective removal of the volatile compounds from

fluorinated polymeric and surfactant materials by air stripping. Figure 3-4 shows the relationship

between accumulated telomer alcohols extracted versus days purged in vessels containing

Zonyl® FSO-100. The majority of the volatile compounds investigated were stripped in less than

2 days and no significant increase in accumulation was observed in subsequent days. For the

other materials studied (results given in the supporting information) accumulation of residuals

ceased at various times from 1 day for the 8:2 methacrylate, to 38 days for the Teflon™ Advance.

The method of continuous purging of the material while dispersed in water takes advantage of

the inherent physical properties of the fluorinated alcohols. Telomer and sulfonamido alcohols 131 are considered to be hydrophobic compounds that are expected to preferentially partition into air due to their high vapour pressures and low water solubility (29). Hence, the technique applied in this study is expected to be superior to more traditional solvent extraction methods of purification though it may not be applicable on an industrial scale.

~ 300 o E 6:2 FTOH S 250 > A A •oo >* I 200 x LU 150

Figure 3-4. Cumulative residual alcohols extracted versus days purged in Zonyl™ FSO 100, an ethoxylated nonionic fluorosurfactant. 4:2 FTOH is not adequately retained on the XAD cartridges used and mass shown is a significant underestimation.

Total masses of fluoro alcohols purged from headspace of fluorinated polymers and surfactants studied ranged from 0.04-3.8% on the basis of mass per dry mass of initial material

(Table 3-1). In this investigation, variation was observed in three of the measured materials,

Zonyl® FSE, Zonyl® FSO-100, and Teflon™ Advance. This may be attributed to non- homogenous consistency of materials tested. Both Zonyl products were viscous aqueous dispersions that were resistant to mixing, while the Teflon advance product was milk-like in 132 consistency. These values are in agreement with those reported by 3M of residuals in concentration of being 1-2% or less in their final products (30). A study by Boulanger et al. (31), published after the initial submission of this manuscript, reported the measurement of residual fluorinated monomers in a single electrochemical based surface protector product. The % residuals reported are extremely low which is not surprising since the dominant fluorinated material used in surface protection in this product line was the N-MethylFose alcohol (30); this dominant material was apparently not analyzed in their study.

Table 3-1 Total residual polyfluorinated telomer alcohols and sulfonamides removed from various fluorinated polymeric and surfactant materials.

% of dry weight of initial Fluorinated Material fluorinated material Polyfox-L-Diol 0.1 V (0.03) Teflon™ Advance 0.34a (0.20) Zonyl®FSO100 1.03 (0.61) Zonyl®FSE 3.80(1.09) 8:2 Methacrylate Monomer 0.04 (0.01) lotomaster® Windshield Washer with 0.36 (0.01) Teflon™ Scotchgard™ Rug and Carpet Protector 0.39 (0.06) Data are means (standard deviations). a average percentage of 6 experimental vessels. All other materials tested were measured in triplicate.

To assess the potential contribution of residuals to the overall flux of the telomer alcohols to the environment, key assumptions need to be made. First, values obtained from this study are conservative estimates since information is lacking concerning postproduction processing that the compounds may have undergone prior to handling in our lab. It is probable that additional release of the volatile compounds occurred prior to packaging and shipping of the various materials investigated in this study. On the basis of manufacturing data in 2000-2002, telomer 133 alcohols had an estimated global production of 5 to 6.5 x 106 kg yr"1 (20). This has increased to

11 to 14 x 106 kg yr"1 (32) perhaps related to the withdrawal by 3M of their POSF based products in 2000-2002 (2).

The method applied in this study was designed to efficiently force out the residual compounds from the materials tested. Actual rates of residual release to the environment are difficult to determine due the diverse applications of these fluorinated products, yet undoubtedly the potential exists for a significant proportion of the residual fluoro alcohols to be released.

Ellis et al. in their study of the atmospheric lifetime of telomer alcohols, have reported that based on crude calculations, a flux on the order of 100,000 - 1,000,000 kg yr_1is needed to maintain the concentrations currently observed in the atmosphere (33). The data presented in this paper suggests the residual fluoro alcohol contribution to the atmospheric load of these chemicals is significant and may be the dominant source. Evidence supporting this is the reported measurement of the highest concentrations of these fluoro alcohols in Griffin, Georgia (US), a known hub for the carpet manufacturing industry. An average of 148 pg m"3 of telomer alcohols and 403 pg m"3 of sulfonamido alcohols were detected at this sampling location (2) although widespread detection of these compounds was also observed throughout the North American atmosphere. This observation indicates that release of residual fluoroalcohols may occur all along the supply chain from production, through application, into actual consumer use. These suppositions need verification by additional measurements inside and outside the manufacturing plant as well as within areas containing the coated materials. Supportive of these data is the recent paper by Shoeib et al. reporting indoor air concentrations of fluorinated sulfonamidoethanol related chemicals (34). 134

Removal of these volatile compounds from various fluorinated materials is critical to reduce the occurrence of fluoro alcohols in the atmosphere and ultimately minimize the contributions to perfluorinated acid accumulation in the environment. The detection of the unbound alcohols from the monomer "stage" of the process, as demonstrated by the presence of residuals from the methacrylate monomer studied here, show that perhaps these residuals are simply a function of incomplete synthesis or poor purification at the monomer step of the manufacturing process. A typical synthetic reaction where fluorinated monomeric acrylates are produced using transesterification reaction of the 8:2 FTOH along with ethyl pyruvate was reported to have an over all yield of 70% (35) indicating that unreacted material may still be present if left unpurified. Elimination of unreacted alcohols at this stage in the manufacturing process will reduce the amounts present at the polymerization step, assuming that the polymerization reaction itself does not cleave the linkage between the fluorinated alcohols and the polymeric backbone.

This study indicates the potential for a significant amount of fluorinated alcohols to be released as residuals from a suite of fluorinated materials that are industrially applied and commercially available and hence contributes substantially to the atmospheric burden of FTOHs.

The ability of these compounds to undergo long-range atmospheric transport provides a means to the production of PFCAs in the environment as they degrade. This study also suggests that direct exposure of the general population to these compounds is plausible if these materials are applied in homes and are outgassing after treatment of surfaces, including carpet, textiles, or paper products. Metabolism of these volatile precursors would then lead to the perfluorinated acids detected in human blood samples worldwide. The elimination of these precursor compounds from marketed fluorinated materials would appear to significantly reduce the likely production of perfluorinated acids in the environment; and in a recent presentation to the

Environmental Protection Agency, a leading manufacturer of telomer based-materials, has announced the removal of residuals from their products by 2006 (32). Studies are underway in our laboratory to determine whether abiotic and biotic degradation of polymeric materials and surfactants, post-application, can release additional fluoro alcohols to the environment.

3.5 ACKNOWLEDGEMENTS

We acknowledge NSERC for a strategic grant and Hira Syed for her assistance in sample preparation.

Supporting Information Available

Further information regarding the experimental set-up, blank samples, spike and recovery data, amounts of fluorinated alcohols measured, average dry weights of fluorinated materials, and plots of accumulated alcohols removed versus days for the other fluorinated materials investigated can be found in Appendix B. 136

3.6 LITERATURE CITED

(1) Martin, J. W.; Muir, D. C. G.; Moody, C. A.; Ellis, D. A.; Kwan, W. C; Solomon, K.R.; Mabury, S. A. Collection of airborne fluorinated organics and analysis by gas chromatography/chemical ionization mass spectrometry. Anal. Chem. 2002. 74, 584-590.

(2) Stock, N. L.; Lau, F. K.; Ellis, D. A.; Martin, J. W.; Muir, D. C. G.; Mabury, S. A. Polyfluorinated telomer alcohols and sulfonamides in the North American trophosphere. Environ. Sci. Technol. 2004. 38, 991-996.

(3) Ellis, D. A.; Martin, J. W.; De Silva, A. O.; Mabury, S. A.; Hurley, M. D.; Andersen, M. P. S.; Wallington, T. J. Degradation of fluorotelomer alcohols: A likely atmospheric source of perfluorinated carboxylic acids. Environ. Sci. Technol. 2004. 38, 3316-3321.

(4) Hurley, M. D.; Wallington, T.J.; Andersen, M.P.S.; Ellis, D.A.; Martin, J.W., Mabury S.A. Atmospheric chemistry of fluorinated alcohols: Reactions with CI atoms and OH radicals and atmospheric lifetimes. J. Phys. Chem. A. 2004.108, 1973-1979.

(5) Hurley, M. D.; Wallington, T.J.; Andersen, M.P.S.; Ellis, D.A.; Martin, J.W., Mabury S.A. Atmospheric chemistry of 4:2 fluorotelomer alcohol: Products and mechanism of CI atom initiated oxidation. J. Phys. Chem. A. 2004.108, 5635-5642.

(6) Dinglasan, M. J.; Ye, Y.; Edwards, E. A., Mabury, S. A. Fluorotelomer alcohol biodegradation yields poly- and perfluorinated acids. Environ. Sci. Technol. 2004. 38, 2857-2864.

(7) Wang, N.; Szostek, B.; Folsom, P. W.; Sulecki, L. M.; Capka, V.; Buck, R.; Berti, W.; Gagnon, J. Aerobic biotransformation of 14C-labelled 8-2 telomer B alcohol by activated sludge from a domestic sewage treatment plant. Environ. Sci. Technol. 2005. 39, 531- 538.

(8) Kennedy Jr., G. L.; Butenhoff, J. L.; Olsen, G. W.; C'Connor, J. C; Seacat, A. M.; Perkins, R. G.; Biegel, L. B.; Murphy, S. R.; Farrar, D. G. The toxicology of perfluorooctanoate. Crit. Rev. Toxicol. 2004. 34, 351-384.

(9) Biegel, L. B.; Hurtt, M. E.; Frame, S. R.; O'Connor, J.; Cook, J. C. Mechanism of extrahepatic tumor induction by peroxisome proliferators in male rats. Toxicol. Sci. 2001. 60, 44-55.

(10) Lau, C; Butenhoff, J. L.; Rogers, J. M. The developmental toxicity of perfluoroalkyl acids and their derivatives. Toxicol. App. Pharm. 2004.198, 231-241.

(11) Hagen, D. F.; Belisle, J.; Johnson, J. D.; Venkateswarlu, P. Characterization of fluorinated metabolites by a gas chromatographic-helium microwave plasma detector - the biotransformation of 1H,1H,2H,2H - perfluorodecanol to perfluorooctanoate. Anal. Biochem. 1981. 118, 336-343.

(12) Martin, J. W.; Mabury, S. A.; O'Brien, P. J. Metabolic products and pathways of fluorotelomer alcohols in isolated rat hepatocytes. Chem. Biol. Interact. 2005.155, 165- 180.

(13) Desilva, A.O.; Mabury, S.A. Isolating isomers of perfluorocarboxylates in polar bears (ursus maritimus) from two geographical locations. Environ. Sci Technol. 2004. 38, 6538-6545.

(14) Shoeib, M.; Harner, T.; Ikonomou, M.; Kannan, K. Indoor and outdoor air concentrations and phase partitioning of perfluoroalkyl sulfonamides and polybrominated diphenyl ethers. Environ. Sci. Technol. 2004. 38, 1313, 1320.

(15) Xu, L.; Krenitsky, D. M.; Seacat, A. M.; Buttenhoff, J. L.; Anders, M. W. Biotransformation of N-ethyl-N-(2-hydroxyethyl)perfluorooctanesulfonamide by rat liver microcosomes, cytosol, and slices and by expressed rat and human cytochromes P450. Chem. Res. Toxicol. 2004.17,161-115.

(16) Giesy, J. P.; Kannan, K. Global distribution of perfluorooctane sulfonate in wildlife. Environ. Sci. Toxicol. 2001. 35, 1339-1342.

(17) Kissa, E. Fluorinated surfactants and repellents; Surfactant Science Series 97; Marcel Dekker: New York, NY, 2001.

(18) Giacobbi, E.; Scialla, S.; Stiros, P. (Procter and Gamble) Composition and Methods for Treating Surfaces. US Patent 20020142097A1 (2002).

(19) Moody, R. After-treatment method for imparting oil-and-water-repellency to fabric. United States Patent. Patent No. 6165545. 2000.

(20) Telomer Research Program "Telomer Research Program Update - Presented to the USEPA OPPT," November 25, 2002; U.S. Environmental Protection Agency public docket AR226-1141.

(21) Falco, M. 3M Corp., Personal Communication, July 19, 2005.

(22) Material Safety Data Sheet for Teflon® Advance Carpet Protector, DuPont Canada, Inc. Mississauga, ON, 2001. (23) Material Safety Data Sheet for Scotchgard™ Carpet and Rug Protector, 3M Corp., London, ON, Id. 09-3245-9. 2003.

(24) DuPont™ Zonyl® FSO-100. Product Information sheet.

(25) DuPont™ Zonyl® FSE. Product Information sheet. 138

(26) Kausch, C; Leising, J. E.; Medsker, R.; Russell, V.; Thomas, R. R. Synthesis, characterization, and unusual surface activity of a series of novel architecture, water- dispersible poly(fluorooxetane)s. Langmuir. 2002. 18, 5933-5938.

(27) Ellis, D.; Mabury, S. A. Chemical ionization pathways of polyfluorinated chemicals - A connection to environmental atmospheric processes. J. Am. Soc. Mass. Spectrom. 2003. 14, 1177-1191.

(28) Napoli, M.; Krotz, L. Scipioni, A. Mass spectrometry of some CeFu-compounds and their CeHu-analogs. Rapid Commun Mass Spectrom. 1993. 7, 789-794.

(29) Lei, Y. D.; Wania, F.; Mathers, D.; Mabury, S. A. Determination of vapour pressures, octanol-air, and water-air partition coefficients for polyfluorinated sulfonamide, sulfonamidoethanols, and telomer alcohols. J. Chem. Eng. Data. 2004. 49, 1013-1022.

(30) Fluorochemical use, distribution and release overview. Prepared by the 3M Company for USEPA OPPT, May 26, 1999. U.S. Environmental Pretection Agency public docket AR226-0550.

(31) Boulanger, B.; Vargo, J. D.; Schnorr, J. L.; Hornbuckle, K. C. Evaluation of Perfluorooctane surfactants in a Wastewater treatment system and in a commercial surface protection product. Environ. Sci. Technol. 2005. 39, 5524-5530.

(32) "DuPont Global PFOA Strategy - Comprehensive Source Reduction". Presented to the USEPA OPPT, January 31, 2005" US Environmental Protection Agency public docket AR226-1914.

(33) Ellis, D.; Martin, J.W.; Mabury, S. A.; Hurley, M. D.; Sulbaek Andersen, M. P.; Wallington, T. J. Atmospheric lifetimes of fluorotelomer alcohols. Environ Sci Technol. 2003.37,3816-3820.

(34) Shoeib, M.;Harner, T.; Wilford, B.H.; Jones, K. C; Zhu, J. Perfluorinated Sulfonamides in indoor and outdoor air and indoor dust: occurrence, partitioning, and human exposure. Environ. Sci. Technol. 2005. 39, 6599-6606.

(35) Guyot, B.; Ameduri, B.; Boutevin, B.; Sideris, A. Synthesis and polymerization of fluorinated acrylic monomers substituted in a-position. 4. Applications to 2- perfluorooctylethyl a -acetoxyacrylate and a -propionyloxy aery late. Macromol Chem Phys. 1995. 196, 1875-1886. CHAPTER IV

BLODEGRADATION OF FLUOROTELOMER BASED MONOMERS AS A SOURCE OF FLUOROTELOMER ALCOHOLS

Mary Joyce Dinglasan-Panlilio, Elizabeth A. Edwards and Scott A. Mabury

To be submitted to - Environ. Sci. Technol.

Contributions - Mary Joyce A. Dinglasan-Panlilio designed and conducted all experiments. Data analysis, interpretation and all versions of the manuscript were prepared by Mary Joyce A. Dinglasan-Panlilio. Scott A. Mabury and Elizabeth A. Edwards provided critical comments.

139 140

4.1 ABSTRACT

Fluorotelomer alcohols (FTOHs) are known precursors of the perfiuorinated carboxylic acids (PFCAs) that are ubiquitous in the environment. The detection of FTOHs in both urban and remote atmospheres has been partially attributed to release from the use of fluorinated materials that contain these volatile compounds as residuals or impurities. The current study demonstrates that biodegradation of fluorotelomer monomers can add to the burden of FTOHs to the environment that may ultimately contribute to PFCA contamination. These monomers are building blocks for fluorotelomer-based polymers that comprise 80% of the fluorotelomer chemical industry. Various chemical linkages were tested to determine whether ester, ether, or urethane-based monomers vary in their stability. Using solid phase microextraction (SPME) coupled with gas chromatography mass spectrometry (GC/MS), ester linked monomers 2

(Perfluorooctyl) ethyl acrylate and 2 (Perfluorooctyl) ethyl methacrylate were observed to degrade to 8:2 FTOH in batch microcosm experiments inoculated with activated sludge. The telomer alcohol was then observed to further degrade forming previously determined metabolites including the telomer acids, 8:2 FTCA and 8:2 FTUCA along with PFOA via liquid chromatography tandem mass spectrometry (LC/MS/MS). An ether-linked monomer, known as

8:2 oxetane, in contrast was not observed to produce the fluorinated alcohol, although loss of the monomer was observed. No loss was observed in the urethane monomer tested. These experiments provide evidence that fluorotelomer acrylate ester monomers are susceptible to microbially mediated hydrolysis forming FTOHs as metabolites. The observed degradation of the ester telomer monomers could be another important indirect source of PFCAs in the environment and may be indicative of the susceptibility of ester fluorotelomer polymers to similar degradation. 141

4.2 INTRODUCTION

Fluorotelomer alcohols (FTOHs) have been identified as precursors to the environmentally ubiquitous perfluorinated carboxylic acids. This volatile class of polyfluorinated compounds has been detected in both the North American troposphere (1, 2) and in remote regions such as the Arctic (3). The detection of FTOHs in the Arctic further supports the predictions that these compounds are capable of long-range atmospheric transport (4). FTOHs undergo atmospheric degradation (5-7) and biodegradation to produce PFCAs (8-12). Detection of fluorotelomer unsaturated acids (FTUCAs) - known intermediates in the degradation pathway

- in precipitation (13, 14), arctic sediment and surface waters (15, 16), as well as in wastewater treatment plant effluent (17), is likely direct evidence of these transformation processes occurring in the environment. FTOHs are primarily used as intermediates in the synthesis of commercial and industrial fluorinated materials including surfactants and monomers. FTOH monomers are then ultimately incorporated into polymers. It has been suggested that FTOH impurities or residuals contained in surfactant and polymeric materials are a significant source of these PFCA precursors observed widely in the atmosphere (9).

Identification of sources of PFCAs has become a priority for scientists and regulators due to their persistence, potential toxicity (18) and widespread detection in environmental matrices

(19). PFCA contamination has been attributed to both direct and indirect sources. Direct sources to the environment are a result of the manufacture and widespread use of this class of compounds as agents in the synthesis of fluorinated polymers (20). Indirect sources are those where PFCAs are formed from abiotic or biotic degradation of precursor compounds such as

FTOHs (7, 12). Further study is required to determine the extent of FTOH contribution to PFCA formation in the environment. Identification of other sources of telomer alcohol input to the environment is critical in this evaluation. It has previously been hypothesized that these compounds may also be released from the decomposition of polymeric and surfactant materials that incorporate FTOHs (12). A recent rat metabolism study of a telomer phosphate surfactant showed evidence of FTOH release and production of FTOH metabolites (21). The current study examined the biodegradation of different fluorotelomer-based monomers under aerobic conditions to determine whether they could be sources of FTOH release to the environment.

These monomers are building blocks of the fluorotelomer polymers that make up 80% of the telomer industry (20) and they are likely to be found as residuals or impurities in the polymers themselves. A large scale effort by industry is underway to determine the stability of telomer based polymers since these are the leading materials marketed as surface active agents to make carpets and textiles water and soil resistant (22). We believe, however, that the environmental fate of monomers is also critical in evaluating sources of PFCAs. FTOHs are incorporated into monomers via different linkages and are then polymerized using traditional radical polymerization or emulsion polymerization methods to form polymers with various characteristics (23-25). Since different chemical bonds have different strengths, linkage chemistry may be important in the stability of FTOH monomers and ultimately of the polymers that incorporate them. This study aims to interrogate different 8:2 FTOH based monomers with ester, ether and urethane linkages (Table 1) through biodegradation experiments using inocula taken from an urban wastewater treatment plant. Biodegradation is a critical environmental fate of these compounds because they are likely released to wastewater treatment plants resulting from routine carpet and upholstery cleaning. A number of studies have reported higher 143 concentrations of PFCAs in WWTP effluent than in the influent, suggesting formation of these persistent compounds from unknown precursors (17, 26), thus the purpose of our investigation is to probe one possible class of precursors that may be in WWTP influent, which also may be suggestive of the fate of larger polymeric based materials.

Table 4-1 Structures and molecular ions of analytes telomer monomers investigated.

Chemical Name Structure m/z (M+l)

^ F3CF2CF2CF2CF2CF2CF2CF2CF2CH2CH20^ \c=c' / \ H H

8:2 Methacrylate ~^cv^ /H 533 F3Cr2Cr2CF2Cr2Cr2CF2^' 2Gr2Crl2Cn20 ^^p—p H,C H

CH3 CH2OCH2CH2CF2CF2CF2CF2CF2CF2CF2CF3 8:2 Oxetane ^C 549

CF .CF3 , » F C F2C 2 \ CF yCF2 / 2

F2C^ **\ CF CF2 r 2 1 F c 8:2Urethane F2c * x 897 F F2C\F2 2C CF2

CH2 CH2 / H C O H Ho 2 I O—C—N—CH2CH2CH2CH2CH2CH2-N-C—O 4.3 MATERIALS AND METHODS

4.3.1 Media and Chemicals.

Components and preparation of the mineral medium used in this study was described previously (12). 8:2 fluorotelomer alcohol (8:2 FTOH, 97%), perfluorohexanoic acid (PFHxA,

95%) and 2(Perfluorooctyl)ethyl acrylate (8:2 acrylate, 97%) were purchased from Oakwood

Research Chemicals (West Columbia, SC). 3,3,4,4,5,5,6,6,7,7,8,8,9,9,10,10,10-

Heptadecafluorodecyl methacrylate (8:2 methacrylate, 97%), perfluorononanoic acid (PFNA,

97%), perfluorooctanoic acid (PFOA, 96%), perfluoroheptanoic acid (PFHPa, 99%), mercuric chloride and sodium azide were purchased from Aldrich Chemical Co. (Milwaukee, WI).

Standard for the 2H,2H,3H,3H-Perfluorodecanoic acid (7:3 telomer acid, 97%) was obtained from Synquest Co. (Alachua, FL). The 8:2 oxetane monomer was donated by Omnova Solutions

(Akron, OH) and the 8:2 urethane monomer was provided by Interface Biologies (Toronto, ON).

The 8:2 saturated telomer acid (8:2 FTC A) and the 8:2 unsaturated telomer acid (8:2 FTUCA) were synthesized as previously described (27).

4.3.2 Growth Conditions and Microcosm Preparation for Degradation Experiments.

Degradation experiments for ester and ether monomers were performed using 250mL glass vessels filled with 125mL of defined mineral medium prepared as described previously

(12). Experiments with the 8:2 urethane monomer were conducted in 250mL Nalgene® vessels due to loss of compound to the glass walls. Vessels were sealed using Mininert® caps (Alltech

Associates Inc., Deerfield, IL) for ease of headspace sampling. Fluorinated monomers were added at a target concentration ranging from 100|ng/L-1000ug/L (aq) from a concentrated stock made up in ethanol. Concentrations of these stock solutions were 2-10ug/uL and typical volume 145 of ethanol added to the vessels was approximately 5-10fiL. Spiked vessels were allowed to equilibrate for at least 24 hours prior to sampling for time zero concentrations and inoculum addition. Inoculum was obtained from the Ashbridges Bay sewage treatment plant (STP) in

Toronto, ON. Prior to start of the experiment, STP samples were shaken and allowed to settle for 15 minutes. Four milliliters were then taken from the supernatant and centrifuged for

15mins. at 2000 rpm. The supernatant was removed and 4mL of fresh mineral media was added to resuspend the pellet as a wash step. The sample was centrifuged again and the supernatant removed. The pellet was resuspended in 2mL of mineral medium before addition to experimental vessels. Active vessels were prepared in triplicate and sterile control in duplicate for each experiment. A "medium control" that was spiked with monomers but without the addition of inoculum was also included. Mercuric chloride and sodium azide (150mg of each in the 125 ml of medium) were added to the "sterile" and "medium controls" to inhibit microbial activity.

Experimental vessels were stored statically in the dark, at room temperature with an initial oxygen headspace concentration equal to that of ambient air. The vessels were manually shaken daily and there was no further addition of air or oxygen during the incubation.

4.3.3 GC/MS Analysis of Volatile Monomers and FTOHs.

The inherent volatility of the ester (8:2 acrylate and 8:2 methacrylate) and ether (8:2 oxetane) monomers used in this study allowed for the application of solid phase microextraction

(SPME) coupled with GC/MS analysis. A 100p.m fibre with polydimethylsiloxane (PDMS) coating (Supelco, Bellefonte, PA) was exposed to the headspace of the sealed culture vessels via the mininert cap and allowed to equilibrate for 1 minute. The fibre was then desorbed for 30 seconds at a GC injection temperature of 220°C. Sample analyses were carried out using a 146

Hewlett Packard 6890 gas chromatograph equipped with a 5973 inert mass spectrometer detector and a 30m Rtx®-35MS with Intra Guard column (0.25mm i.d., 0.5um film thickness,

Chromatographic Specialties, Brockville, ON). The following GC oven program was employed: initial temperature of 45°C held for 2 minutes; 10°C/min ramp to 95°C; 30°C min"1 ramp to

150°C/min; 7°C min"1 ramp to 180°C and a final ramp of 50°C to 240°C held for 2 minutes.

Helium was used as the carrier gas at a flow rate of lmL min"1 with pulsed splitless injection at an initial pressure of 20psi for 1.2 min followed by an injector purge at 40.0mL min"1 for 0.8 min. Quantification proceeded under positive chemical ionization (PCI) using methane as an ionizing gas, in single ion monitoring mode, and the molecular ion (M+l) was monitored for all target analytes. A summary of the molecular ions analyzed for each target analyte is shown in

Table 4-1. Calibration was performed using external standards and retention times for all analytes of interest were well conserved (± 0.04 min). There are currently no data available for

Henry's law constants of all of the volatile monomers investigated to allow determination of headspace concentration, thus aqueous concentrations were used in the calibration. The volume ratio of headspace to aqueous phase in standards and experimental vessels were kept constant throughout the experiment to allow for headspace analysis.

4.3.4 LC/MS/MS Analysis of Non-Volatiles.

Vessels were manually shaken prior to taking 2mL aqueous samples for analysis of non volatile metabolites. Samples were extracted using a previously described method for extracting fluorinated chemicals from biological samples (28). Extracted samples were reconstituted in 500

|j.L of 50:50 methanol:water and transferred to 0.7mL polypropylene autosampler vials for

LCMSMS analysis. Quantitative analysis was performed using a Waters 717 autosampler along with a

Waters 300 series pump equipped with a Gemini CI8 column (2 urn, 4.6*250mm, Phenomenex,

CA). Isocratic elution was used with an eluent composed of 80:20 methanohwater with 20mM ammonium acetate at a flow rate of 150 uL/min. A Micromass Quattro micro™ Triple

Quadrupole Mass Spectrometer (Micromass; Manchester, UK) under negative electrospray ionization and multiple reaction monitoring (MRM) mode was used to monitor the following compounds: PFOA, 8:2 FTUCA, 8:2 FTCA, 7:3 FTCA, PFNA, PFHPa and PFHxA.

Quantification was performed using internal standard calibration for compounds where labeled standards were available namely, PFOA, 8:2 FTUCA, PFNA and PFHPa. External calibration was used for 8:2 FTCA, 7:3 FTCA and PFHxA.

The urethane monomer was not volatile and was analyzed via LCMSMS. Aqueous samples from the urethane experiment were extracted using the same protocol as above except that ethyl acetate was used as the extraction solvent instead of MTBE as described by Hansen et al (28). Samples were also reconstituted in 500 uL of 50:50 methanol:water after evaporation of ethyl acetate. Separation was performed on the same system but equipped with a Gemini C8 column (2 urn, 4.3><50mm, Phenomenex, CA) with gradient elution using initial conditions of

75:25 methanol:water buffered with 20mM ammonium acetate at a flow rate of 200 (xL/min for

30 seconds followed by a 1.5 minute ramp to 100% methanol and held for 4 minutes before reverting back to initial conditions at 8 minutes. This chromatographic method allowed for simultaneous detection of the urethane monomer and the hypothesized FTOH metabolites.

Positive electrospray ionization was used to measure the urethane monomer. 148

4.4 RESULTS AND DISCUSSION

PFCA production from abiotic and biotic transformation of FTOHs is now widely reported in the published literature. The point of discussion has now shifted to the relative importance of FTOHs as an indirect source of PFCA contamination. Therefore, all potential sources of FTOHs to the environment need to be investigated. FTOHs are known intermediates in the manufacture of fluorotelomer polymers. They are first made into monomers with different linkages then polymerized via traditional polymerization techniques (22-24). This study examines FTOH production from the biodegradation of three different types of monomers that vary according to their linkage chemistry.

1200 12000 Active Vessel Sterile Control 1000 10000 T=0 days T=0 days

800 8000

600 8:2 Methacrylate 6000 8:2 Methacrylate

4000 400

2000 200 8:2 FTOH 8:2 FTOH ^ 0 L.

12000 12000 T=3 days T=3 days 10000 10000

8000 8000

6000 8:2 Methacrylate 8:2 Methacrylate

8:2 FTOH 8:2 FTOH L .A L 5.00 6.00 7.00 8.00 9.00 10.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00

Figure 4-1 Sample chromatograms of an active vessel vs sterile control in 8:2 methacrylate biodegradation experiment at T=0 and T=3 days. Presence of residual 8:2 FTOH evident in both vessels at T-0. 149

4.4.1 Experiments with an Ester Monomer

Two ester-linked monomers were investigated. Both compounds were volatile and amenable to analysis via SPME GC/MS. A spike concentration of 100 ug L"1 (aq) of the 8:2 methacrylate degraded rapidly forming the 8:2 FTOH in active vessels. Figure 4-1 shows sample chromatograms of the 8:2 methacrylate and 8:2 FTOH at T=0 days and 3 days after addition of the inoculum in an active vessel and sterile control. Small amounts of residual 8:2

FTOH were present in the vessels at T=0 days (Figure 4-1) prior to addition of the inoculum.

This was expected as we previously determined that at least 0.04% residual FTOH (mass per dry weight of monomer) is present in this telomer based monomer (9). This residual FTOH was degraded by day 1 in active vessels. A significant increase in 8:2 FTOH concentration was then observed after 3 days (Figure 4-2). This increase in 8:2 FTOH occurred concurrently with a decrease in 8:2 methacrylate and is clearly attributed to microbial activity due to the lack of similar production in the sterile controls. Sterile controls showed some loss of the 8:2 methacrylate without evidence of 8:2 FTOH production (Figure 4-2 inset). This decrease in concentration may be attributed to loss during sampling or compound adhesion to the walls of the vessels and the PTFE lining of the Mininert caps (29). 8:2 FTOH concentration in the active vessels peaked at day 4 as the 8:2 methacrylate was further depleted in the vessels. 8:2 methacrylate was 80% degraded by day 4 of the experiment (Figure 4-2). The detection of previously observed metabolites from 8:2 FTOH degradation (12) in the active vessels and their absence in the sterile control (Figure 4-2) is further evidence that the degradation was microbially-mediated. The saturated (8:2 FTC A) and unsaturated telomer (8:2 FTUCA) acids were both produced then further transformed to PFOA. PFOA was the terminal metabolite observed m these experiments and there was no evidence of shorter chain acids produced or carbon fluorine bond breakdown as reported by Wang et al. (27). Their observation of shorter chain acids could have been unique to degradation pathways of microorganisms present in their inoculum (27). It could also be due to the acclimation of the microorganisms used in their study where an alternate degradation pathway had developed. The inoculum used in their study was exposed to high concentrations of 8:2 FTOH (5-10mg L"1) and was periodically transferred into fresh medium prior to the initiation of their experiment. These sequential transfers would have promoted enrichment of microorganisms that thrive on 8:2 FTOH and its metabolites, thus the inoculum used may not represent a typical consortium of microorganisms present in the environment. The inoculum used in our study was not exposed to any known telomer based compounds prior to the experiment. Wang et al. (15, 27) had also observed the 7:3 telomer acid as an intermediate in their study. This compound was monitored throughout our experiment but was not detected in any of the samples. This may be due to high limits of detection for this compound (LOD 5ppb) in our analytical system. The products measured at the end of the experiment accounted for -65% of the initial monomer added to the vessels. It is reasonable to assume that some loss of the parent material occurred due to adsorption to glass walls and PTFE cap lining as was observed in the controls where about 20% of the monomer was lost (Figure 4-

2). Assuming similar losses occurred in the active vessels, the total recovery would increase to

-85%. 151

0 5 10 15 20 25 30 35 40 45 Time (Days)

Figure 4-2: Degradation of ester monomer, 8:2 methacrylate, over time in active vessels (n=3) and sterile control (inset, n=2).

The 8:2 acrylate monomer was also investigated to determine whether FTOH production occurs with other ester-linked monomer degradation. The 8:2 acrylate is structurally similar to the methacrylate except for the absence of a methyl group (Table 4-1). This monomer behaved analogously to the 8:2 methacrylate, forming the 8:2 FTOH and its metabolites (Figure 4-3) as it was degraded. Some loss in the sterile control was also observed but without simultaneous production of the 8:2 FTOH (Figure 4-3 inset). Peak production of the 8:2 FTOH in the active vessels was seen at day 3 of the experiment and was subsequently depleted by day 15. This loss of the 8:2 FTOH was followed by the production of the saturated and the unsaturated telomer acids as previously observed. PFOA production was also observed as the terminal metabolite but was not produced to the same extent as seen in the methacrylate experiment. The source of the inoculum used in both experiments were the same, however the consortium of microorganisms present cannot be assumed to be identical given the time that elapsed between the two experiments. Even though the overall degradation kinetics of the two acrylate ester 152 monomers differed, it was evident that both were susceptible to degradation forming the 8:2

FTOH as a major metabolite. During the degradation of both ester monomers, approximately molar equivalents of the 8:2 FTOH were produced. This degradation likely proceeded through enzymatic hydrolytic attack of the ester bond forming an alcohol and carboxylic acid as products

(30). Phthalate esters, industrial compounds added to polymers as plasticizers, are transformed by microorganisms in the same manner (11, 31). Esterase enzymes common in various microorganisms are known to catalyze these hydrolytic reactions. Our results suggest that it is highly probable that these ester type telomer monomers make up some of the unknown precursors hypothesized as sources of PFCAs generated from WWTPs (17, 26).

Sterile Control

10 1E

o o

Time (Days) Figure 4-3: Degradation of ester monomer, 8:2 acrylate, over time in active vessels (n=3) and sterile control (inset, n=2) 153

4.4.2 Experiments with an Ether Monomer.

The 8:2 oxetane is the monomer used in this investigation that contains an ether linkage

(Table 4-1). This particular monomer is not used industrially or commercially but can be polymerized to form polyfluorooxetane polymers through a cationic ring-opening mechanism

(32). They were of interest in this study because it allowed for direct comparison of 8:2 FTOH production with the ester monomers and because of access to the material. The monomer however contained the 6:2, 10:2, 12:2 and 16:2 oxetanes as impurities. Thus, while observing for any 8:2 FTOH production, the 6:2, 10:2 and 12:2 FTOHs were also monitored during the biodegradation experiment. All chain lengths of the oxetane monomers decreased in concentration over time, however no corresponding formation of 8:2 FTOH or any of the other

FTOHs of interest were observed in the active vessels (Figure 4-4). Sterile controls had some

Time (Days)

Figure 4-4: Ether monomer degradation in active vessels (n=3) and sterile control (inset, n=2). No production of 8:2 FTOH observed. 154 loss but also also showed no significant FTOH production. The loss of this ether monomer in the active vessels could be attributed to possible transformation via a different pathway not forming the FTOH as an immediate metabolite from the scission of the ether linkage. Ether linkages are known to be resistant to biodegradation due to the strength of the C-0 bond energy (360 kJ/mol)

(33). An alternate degradation pathway for this monomer could likely be an enzymatic attack on the oxetane ring forming a hydroxylated product. To determine if FTOH was formed later on as a metabolite further down the degradation pathway, vessels were incubated for more than two months. Periodic sampling over this time for evidence of FTOH and FTOH metabolite formation was performed and none were detected. It cannot be ruled out that PFCAs would be the ultimate product of the breakdown of this monomer hence further study is required to elucidate its degradation pathway.

4.4.3 Experiments with a Urethane Monomer.

The physical properties of the urethane linked monomer tested were significantly different from the ester or ether monomers investigated in this study. The urethane monomer was a white solid at room temperature, non-volatile and was analyzed using the LCMSMS. The structure of this monomer consisted of two FTOH chains linked by an isocyanate bridge via urethane bonds (Table 4-1) making it significantly larger than the other monomers investigated.

The urethane monomer was a mixture made up of 90% of two 6:2 FTOH chains and 10% of a

6:2 FTOH and 8:2 FTOH chained version (proportions measured by peak area). Urethane monomers with a single FTOH pendant, similar to the ether and ester-linked monomers, were not available. No significant loss of the urethane monomers was observed in sterile or active vessels during the degradation. The concentration over time of the two 6:2 FTOH chained monomer is 155 shown in Figure 4-5. There was an increase in the measured concentration of the monomer between sampling at T=0 and T=l possibly due to the vessels not being in equilibrium at time of the initial sampling. Unlike the ester and ether monomers that were sampled for T=0 after 24 hours of equilibration due to their volatility, the urethane monomer was sampled for T=0 only several hours after the addition of the compound having assumed that equilibration period was shorter as it was non-volatile. 6:2 FTOH, 8:2 FTOH or their metabolites were not detected in the samples indicating that the microorganisms were unable to transform the urethane monomers.

The microorganisms appeared to be viable during the experiment based on an observed increase in optical density. It is likely that this growth was promoted by the addition of ethanol to the vessels, as the carrier solvent for the monomer, which may have served as the carbon source for the microorganisms. Low molecular mass compounds with urethane bonds are susceptible to hydrolysis by microorganisms catalyzed by their esterase enzymes (34). Little is known however of the degradability of higher molecular weight compounds although some urethane polymers are now typically developed and marketed as a biodegradable alternative to other polymers (35). The lack of degradation observed with this monomer can be attributed to its high molecular weight or steric hindrance caused by the two FTOH pendants. This steric hindrance may render the molecule inaccessible to attack by microbial enzymes. The use of a two-pendant monomer perhaps was not the best probe to assess the stability of urethane monomers. 156

2.50 -, » 2.00 - Sterile Controls o essels ) 3.00 itio n (nmo l Urethane Monomer ±j " 0.50 4 o> c 0.00 ' 1 • • •-»—- • • —• 8:2 FTOH o O 5 10 15 20 2.50 -0.50 - Time (Days)

2.00 tn "5 E 1.50 c o 2 1.00 c u0> c 0.50 oo

0.00 * 8 10 12 14 16 18 20 Time (Days) Figure 4-5 No significant loss observed in urethane monomer biodegradation in both active vessels (n=3) and sterile controls (inset, n=2)

FTOH input to the environment attributed to residuals from various materials was estimated to be within the amounts required to maintain observed concentrations in the atmosphere (9). It was suggested that about 200 tonnes of the estimated flux calculated by Ellis et al. (100-1000 tonnes) could be coming from residuals (9). From the current study, degradation of ester monomers such as the 8:2 acrylate and methacrylate is also a likely contributor to that

FTOH input. These monomers are sold as chemical intermediates to the fluorinated polymer industry and can be emitted to the environment at manufacturing centers. Alternatively, analogous to FTOH residuals, these monomers can be present in significant amounts from 157 unpurified polymeric materials and released through their routine use and application. The resulting environmental effect of the voluntary action by leading manufacturers of telomer compounds to remove residual FTOHs from their products by 2015 (36) is yet to be determined.

An objective of an ongoing long-term air sampling study in our laboratory is to evaluate the changes in atmospheric concentrations of these compounds as a result of this voluntary action.

In addition to direct releases from residuals due to their volatility, FTOH ester monomers may be another source of FTOHs ultimately contributing to the overall burden of PFCA precursors to the environment.

Monomers exist with various linkage chemistries, used to create diverse polymeric structures with different characteristics. Ester, ether and urethane monomers were tested to determine whether linkage chemistry affects stability in a biologically active system. Acrylate ester monomers were found to be readily transformed to 8:2 FTOH and its known metabolites, while the urethane monomer was recalcitrant under similar conditions. However, based on known reactivity of urethanes, this class of monomers is also expected to break down. The ether monomer tested was observed to be degraded without subsequent FTOH or PFCA production.

Further studies are required to determine the degradation pathway of this monomer. Based on these results, emission of FTOH ester monomers to the environment, as residuals from fluorinated materials or from point sources in manufacturing centres, are expected to contribute to the environmental burden of FTOHs and ultimately PFCAs. The stability of telomer-based polymers that incorporate labile monomers is of immediate interest. Ester based telomer polymers are likely to make up a significant portion of the fluorinated polymer market based on numerous patents that make use of fluorinated alkyl esters as monomers (37). Results from our investigation suggest that design of monomers with more stable linkages such as ether bonds 158 could result in materials resistant to degradation and formation of FTOHs. Studies are underway in our laboratory to determine whether an ester type telomer based polymer biodegrades to form

FTOHs, which can ultimately produce PFCAs in the environment. This study is critical in the evaluation of potential risks of these compounds and in future regulations. Environment Canada had recently issued a permanent ban on the import and manufacture of four telomer based polymers in the country to further protect the health of Canadians and the environment from exposure to perfluorinated substances (38).

4.5 ACKNOWLEDGEMENTS

The authors would like to thank Interface Biologies for donation of the urethane monomer and to OMNOVA Solutions for the ether monomer. We would also like to acknowledge the Natural Science and Engineering Research Council as well as the Omnova

Foundation for financial support of this work.

4.6 LITERATURE CITED

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(2) Stock, N. L., Lau, F. K., Ellis, D. A., Martin, J. W., Muir, D. C. G. and Mabury, S. A. Polyfluorinated Telomer Alcohols and Sulfonamides in the North American Troposphere. Environ. Sci. Technol. 2004, 38, 991-996.

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(5) Hurley, M. D., Wallington, T. J., Andersen, M. P. S., Ellis, D. A., Martin, J. W. and Mabury, S. A. Atmospheric chemistry of fluorinated alcohols: Reactions with CI atoms and OH radicals and atmospheric lifetimes. J. Phys. Chem. 2004,108, 1973-1979.

(6) Hurley, M. D., Wallington, T. J., Andersen, M. P. S., Ellis, D. A., Martin, J. W. and Mabury, S. A. Atmospheric chemistry of 4:2 fluorotelomer alcohol: Products and mechanism of CI atom initiated oxidation. J. Phys. Chem. 2004,108, 5635-5642.

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(8) Hagen, D. F., Belisle, J., Johnson, J. D. and Venkateswarlu, P. Characterization of fluorinated metabolites by a gas chromatographic-helium microwave plasma detector - the biotransformation of 1H,1H,2H,2H - perfluorodecanol to perfluorooctanoate. Anal. Biochem. 1981,118, 336-343.

(9) Dinglasan-Panlilio, M. J. A. and Mabury, S. A. Significant Residual Fluorinated Telomer Alcohols Present in Various Fluorinated Materials. Environ. Sci. Technol. 2006, 40, 1447-1453.

(10) Wang, N. S., Bogdan; Buck, Robert C; Folsom, Patrick W.; Sulecki, Lisa M.; Capka, Vladimir; Berti, William R.; Gannon, John T. Fluorotelomer Alcohol Biodegradation - Direct Evidence that Perfluorinated Carbon Chains Breakdown. Environ. Sci. Technol. 2005, 39, 7516-7528.

(11) Wang, Y., Fan, Y. and Gu, J. D. Microbial Degradation of the Endocrine-Disrupting Chemicals Phthalic Acid and Dimethyl Phthalate Ester Under Aerobic Conditions. Bull. Environ. Contam. Toxicol. 2003, 71, 810-818.

(12) Dinglasan, M. J. A., Ye, Y., Edwards, E. A. and Mabury, S. A. Fluorotelomer Alcohol Biodegradation Yields Poly- and Perfluorinated Acids. Environ. Sci. Technol. 2004, 38, 2857-2864.

(13) Loewen, M., Halldorson, T., Wang, F. and Tomy, G. Fluorotelomer Carboxylic Acids and PFOS in Rainwater from an Urban Center in Canada. Environ. Sci. Technol. 2005, 39,2944-2951.

(14) Scott, B. F., Spencer, C, Mabury, S. A. and Muir, D. C. G. Poly and Perfluorinated Carboxylates in North American Precipitation. Environ. Sci. Technol. 2006, 40, 7167- 7174.

(15) Stock, N. L., Marvin, C. H., Mabury, S. A. and Muir, D. C. G. Perfluoroalkyl Contaminants in Lake Ontario Sediment: Contribution to Mass Balance. Environ. Sci. Technol. 2007, In Prep. 160

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(17) Sinclair, E. K., Kurunthachalam Mass Loading and Fate of Perfluoroalkyl Surfactants in Wastewater Treatment Plants. Environ. Sci. Technol. 2006, 40, 1408-1414.

(18) Kennedy Jr., G. L. B., J. L.; Olsen, G. W.; 0"Connor, J. C; Seacat, A. M.; Perkins, R. G.; Biegel, L. B.; Murphy, S. R.; Farrar, D. G. The toxicology of perfluorooctanoate. Critical Reviews in Toxicology 2004, 39.

(19) Houde, M., Martin, J. W., Letcher, R. J., Solomon, K. R. and Muir, D. C. G. Biological Monitoring of Polyfluoroalkyl Substances: A Review. Environ. Sci. Technol. 2006, 40, 3463-3473.

(20) Prevedouros, K., Cousins, I. T., Buck, R. C. and Korzeniowski, S. H. Sources, Fate and Transport of Perfluorocarboxylates. Environ. Sci. Technol. 2006, 40, 32-44.

(21) D'eon, J. C. and Mabury, S. A. Production of perfluorinated carboxylic acids (PFCAs) from the biotransformation of polyfluoroalkyl phosphate surfactants (PAPS): Exploring routes of human contamination. Environ. Sci. Technol. 2007, 41, 4799-4805.

(22) Consent Agreement and Final Order - PFOA Settlement, E. I. duPont de Nemours and Company; December 14, 2005

(23) Kissa, E. Fluorinated surfactants and repellents; Marcel Dekker: New York, 2001.

(24) Dupont ™ Zonyl Fluorochemical Intermediate Product Information Sheet. 2002

(25) Greenwood, E. J., Lore, A. L. and Rao, N. S. Oil- and water-repellent copolymers. E. I. Du Pont de Nemours and Company (Wilmington, DE), US Patent 4742140, 1988

(26) Schultz, M. M., Barofsky, D. F. and Field, J. A. Quantitative Determination of Fluorinated Alkyl Substances by Large-Volume-Injection Liquid Chromatography Tandem Mass Spectrometry-Characterization of Municipal Wastewaters. Environ. Sci. Technol. 2006, 40, 289-295.

(27) Achilefu, S., Mansuy, L., Selve, C. and Thiebault, S. Synthesis of 2H,2H-perfluoroalkyl and 2H-perfluoroalkenyl carboxylic acids and amides. J. Fluor. Chem. 1995, 70, 19-26.

(28) Hansen, K. J., Clemen, L. A., Ellefson, M. E. and Johnson, H. O. Compound-Specific, Quantitative Characterization of Organic Fluorochemicals in Biological Matrices. Environ. Sci. Technol. 2001, 35, 766-770.

(29) Wang, N., Szostek, B., Folsom, P. W., Sulecki, L. M., Capka, V., Buck, R. C, Berti, W. R. and Gannon, J. T. Aerobic Biotransformation of 14C-Labeled 8-2 Telomer B Alcohol by Activated Sludge from a Domestic Sewage Treatment Plant. Environ. Sci. Technol. 2005,39,531-538. 161

(30) Muller, R. J., Kleeberg, I. and Deckwer, W. D. Biodegradation of polyesters containing aromatic constituents. Journal of Biotechnology 2001, 86, 87-95.

(31) Nalli, S. C, D.G.; Nicell, J.A. Biodegradation of Plasticizers by Rhodoccocus rhodochrous. Biodegradation 2002,13, 343-352.

(32) Kausch, C. L., J. E.; Medsker, R.; Russell, V.; Thomas, R. R. Synthesis, characterization, and unusual surface activity of a series of novel architecture, water-dispersible poly(fluorooxetane)s. Langmuir 2002,18, 5933-5938.

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(34) Nakajima-Kambe, T., Shigeno-Akutsu, Y., Nomura, N., Onuma, F. and Nakahara, T. Microbial degradation of polyurethane, polyester polyurethanes and poly ether polyurethanes. App. Microbiol. Biotech. 1999, 51, 134-140.

(35) Rutkowska, M., Krasowska, K., Heimowska, A., Steinka, I. and Janik, H. Degradation of polyurethanes in sea water. Polymer Degradation and Stability 2002, 76, 233-239.

(36) DuPont 2010/15 PFOA Stewardship Program, U.S. Environmental Protection Agency;

(37) Audenaert, F., van der Elst, P. J. and Roily, D. G. Fluoropolymer of fluorinated short chain acrylates or methacrylates and oil- and water repellent compositions based theron. 3M Innovative Properties Company, EP 1493761 Al, 2005

(38) Canada Gazette Part I. Notice of Action Plan for the Assessment and Management of Perfluorinated Carboxylic Acids and their Precursors. June, 17, 2006. CHAPTER V

INVESTIGATION INTO THE BIODEGRADATION OF FLUOROTELOMER- BASED POLYMERS 5.1 ABSTRACT

Fluorinated telomer based polymers are complex synthetic materials widely used in the textile and carpet industries as stain and water repellants. These materials make up 80% of the fluorotelomer chemical industry. Our earlier studies have found the building blocks of these polymers, the telomer alcohols (FTOHs) and ester linked telomer monomers, susceptible to biodegradation under aerobic conditions forming perfluorinated carboxylic acids (PFCAs).

PFCAs are, persistent, potentially toxic, moderately bioaccumulative and ubiquitous in the environment. The current study investigates the stability of a model telomer based polymer to determine whether these materials also biodegrade to produce FTOHs which can ultimately form

PFCAs in the environment. Ester monomers, 2(Perfluorooctyl) ethyl acrylate (8:2 acrylate) and the hexadecyl acrylate, were polymerized using a radical polymerization technique. Two size batches were synthesized to determine whether low molecular weight polymers are more labile to biodegradation than higher molecular weight polymers; size was determined using Gel

Permeation Chromatography (GPC) and Matrix Assisted Laser Desorption Ionization Mass

Spectrometry (MALDI-MS). Residual FTOHs and monomers were removed prior to the initiation of the degradation experiment. Experiments were performed in purge and trap vessels designed to trap volatile metabolites and inoculated with dilute activated sludge taken from a local wastewater treatment plant. Undiluted activated sludge experiments were also conducted to monitor for non-volatile metabolites. No significant amount of FTOH or PFOA were observed after 34 days of aerobic degradation suggests that the synthesized telomer acrylate polymer is more stable than FTOHs and the ester telomer monomer. Future experiments were proposed to better study the stability of these fluorinated macromolecules. 164

5.2 INTRODUCTION

Fluorotelomer polymers are the dominant products of the telomer chemical industry with a reported 80% share of the market (1). These compounds are widely used in the textile, upholstery and carpet industries to render them soil and water repellant. They are also frequently employed as components of varnish and paint formulations as well as protective coatings for stone materials (2). Telomer alcohols (FTOHs), intermediates used in the production of these macromolecules, have been identified as precursors to PFCAs. PFCAs such as perfluorooctanoic acid (PFOA) are widespread environmental contaminants (3) and are a concern due to their recalcitrance and potential toxicity (4). Fluorotelomer based polymers' widespread application and estimated mass production have garnered attention from regulators as to their potential contribution as a source of perfluorinated acid (PFCA) contamination in the environment.

PFCA contamination has been attributed to direct sources where PFCAs themselves are released directly to the environment (5, 6). They are released from manufacturing emissions as well as from emissions of the fiuoropolymer industries that routinely apply PFCAs as a processing agent. The fluorotelomer chemical industry has been linked to the global contamination of PFCA as an indirect source. FTOHs have been found to form PFCAs from oxidation reactions in the atmosphere (7-10) as well as from biotic transformation reactions in rats (11-13) and microorganisms (14-16). FTOHs are found as residuals, in various telomer- based materials such as polymers and surfactants in significant amounts and are likely released to indoor and outdoor environments from the application of these materials (17-19). It is believed that FTOH released to the environment will ultimately lead to PFCA production; hence sources of FTOHs are considered to be sources of PFCAs. This was further emphasized in a recent study where a biodegradation of 8:2 FTOH based monomer with an ester linkage was observed to form 165

FTOHs as metabolites, that were then further transformed to PFOA (20). These monomers are common building blocks in the synthesis of telomer based acrylate polymers (2, 21-26) and are probable residuals left in commercial formulations of these polymers. Production of PFCAs was also demonstrated from a rat metabolism study of a telomer phosphate based surfactant material commonly used in paper packaging for food products (27).

This investigation examined the biodegradation potential of a model telomer based polymer. Like other telomer-based materials, fiuorotelomer-based polymers are of interest due to their potential to degrade leading to FTOH release in the environment. Environmental regulators in Canada have precluded the importation of four different telomer-based polymers on the basis of their being potential sources of PFCAs to the environment (28). Polymers, in spite of their high molecular weights, have been known to biodegrade. Microorganisms are capable of using extracellular enzymes to break down polymers into smaller components that can then be transported inside the cell for further metabolism (29). Perhaps the most well known example of this process occurring in the environment is the breakdown of cellulose, the most abundant polymer found on earth, by bacteria.

The goal of this investigation was two-fold, to synthesize a well-characterized fluorinated telomer-based polymer, virtually free of residual monomers or FTOHs, and to subject the polymer to biodegradation conditions to determine whether PFCAs are produced. In this study, we synthesized a copolymer using, 2(perfluorooctyl)ethyl acrylate (8:2 FTOH acrylate) and heptadecyl acrylate as monomers via a conventional radical polymerization technique. Non- fluorinated monomers such as heptadecyl acrylate are typically used in industrial and commercial synthesis to create hybrid fluorinated polymers with improved solubility (30). The copolymerization of two monomers, one fluorinated and another non fluorinated has drawn interest due to their varied and useful properties (31). Unlike the fluorotelomer polymer synthesized in this study, varying chain lengths of telomer acrylate monomers up to 14 carbons

(4:2 to 14:2) are routinely used in commercial formulations (32). Biodegradation experiments were performed in open, purge and trap systems using dilute wastewater samples as inoculum as well as in open systems using undiluted activated sludge, both under aerobic conditions.

The use of sludge as inoculate in this study was highly relevant as these compounds are likely to end up in wastewater treatment plants as part of their life cycles. In addition, wastewater treatment plants have been shown to be point sources of PFCA release to the environment (33, 34). PFCAs can be further disseminated through discharge of wastewater effluent into natural waters as well as through the agricultural application of sludge where these contaminants may sorb to soil. Effluent samples taken from wastewater treatment facilities have repeatedly described higher amounts of these contaminants when compared to influent wastewaters. Schultz et al. had reported an increase in PFOA concentrations of from 9 to 352% in 7 of 10 WWTPs examined (35). PFOA, PFNA and PFUnDA all increased in concentrations from 211 to 345% in effluent wastewater samples taken from six WWTPs in New York State

(34).

It has been suggested that PFCAs detected in influent wastewaters may be due to direct input of PFCAs though it cannot be ruled out that they are generated somewhere along the waste stream. It was proposed that an increase in the effluent PFCA levels, was due to microbially mediated degradation of precursors such as telomer compounds present within the wastewater stream (35). Based on these observations, it appears that biodegradation may be an important process in the overall environmental fate of telomer-based compounds including polymeric materials. Fluorinated polymers are potential precursors of PFCAs that could be present in wastewater systems. Telomer based polymers are likely to be released from treated products (ie. carpet and textile) as a result of routine activities such as cleaning or washing and thus ultimately ending up in wastewater influent.

5.3 MATERIALS AND METHODS

5.3.1 Chemicals

The fluorinated telomer monomer used in the polymer synthesis was

2(Perfluorooctyl)ethyl acrylate (8:2 FTOH acrylate, 97%), purchased from Oakwood Research

Chemicals (West Columbia, SC). The hydrocarbon monomer, hexadecyl acrylate (97%) was obtained from Monomer-Polymer & Dajac Laboratories (Feasterville, PA). Preparation and components of the mineral medium used in the biodegradation experiments are described elsewhere (14). Standards for the 8:2 fluorotelomer alcohol (8:2 FTOH, 97%) and perfluorohexanoic acid (PFHxA, 95%) were purchased from Oakwood Research Chemicals

(West Columbia, SC). Perfluorononanoic acid (PFNA, 97%), perfluorooctanoic acid (PFOA,

96%>), perfluoroheptanoic acid (PFHPa, 99%>), mercuric chloride and sodium azide were purchased from Aldrich Chemical Co. (Milwaukee, WI). The standard for the 2H,2H,3H,3H-

Perfluorodecanoic acid (7:3 telomer acid, 97%) was obtained from Synquest Co. (Alachua, FL) and the 8:2 saturated telomer acid (8:2 FTC A) and the 8:2 unsaturated telomer acid (8:2

FTUCA) were synthesized as previously described (36).

5.3.2 Polymer Synthesis

The copolymerization of equimolar mixtures of 2(perfluorooctyl)ethyl acrylate (8:2

FTOH acrylate) and hexadecyl acrylate was performed in ethyl acetate using a,a-azobis- 168 isobutyronitrile (AIBN) as the initiator. A schematic of the reaction is presented in figure 1.

Reaction composition was typically a 90-10 ratio of solvent to solids with good product yields of

70-80%. Synthesis was performed under a nitrogen atmosphere in a three-necked round bottom flask equipped with a condenser; AIBN used in reactions ranged from 0.1% to 1% in order to vary the size distribution of polymers synthesized. Reaction times were also altered from 8 hours to 19 hours. Temperature was maintained at 70°C throughout the duration of the reaction.

The product was precipitated using methanol, vacuum filtered and air-dried in the fumehood; product appeared as white crystals after drying. Residual unreacted monomers were removed from the product by means of multiple wash steps using 75:25 methanol: water solution. Each wash cycle involves vigorous mixing of product in solution followed by air-drying in the fumehood.

,CH, .CH, H,C=

AIBN +

H,C

/ / o=cx H H,Q c=cv P P H H 8:2 Acrylate Monomer o=c o=\ o=/ o=/ c C CH C- H H H

H3C Hexadecyl Acrylate Monomer Model Fluorotelomer Polymer

Figure 5-1 Schematic representation of fluorotelomer based polymer synthesis. 169

5.3.3 Polymer Characterization

F and !H NMR spectroscopies were used to confirm synthesis of polymers on a Varian

400 system operating at 376.14 MHz for 19F analysis and at 400MHz for lH analysis. The system was equipped with an ATB8123-400 auto switchable probe. Deuterated chloroform was utilized as the solvent with tetramethylsilane (TMS) as the internal standard. Samples were run at 25°C typically with 256 repetitions, relaxation delay of 2 seconds with 30 degrees pulse.

Chemical shifts were recorded at a range of-70 to -130 ppm and 1 to 8 ppm for 19F and lH respectively.

Gel permeation chromatography (GPC) was used to measure average molecular weight

(Mn) and size average molecular weight (Mw) of polymers. Samples were dissolved in tetrahydrofuran (THF) and analyzed using a Viscotek Model 100 system equipped with a refractive index detector and a phenogel linear X3 column at a flow rate of 1.0 mL min"1.

Calibration was performed using polystyrene standards.

Matrix Assisted Laser Desorption Ionization Mass Spectrometry (MALDI-MS) was employed to gain information regarding the chemical structure of the synthesized polymer.

Samples along with, Dithranol and Litium trifluoroacetate (LiTFA) were dissolved in tetrahydrofuran (THF) at a concentration of 20mg/ml, lOmg/ml and 10 mg/ml respectively. The ratio of polymer samples, Dithranol, and LiTFA is 2:10:1. The mixture was spotted on sample target at approximately 0.5 AuL. The mass spectra were acquired by using a Bruker REFLEX

III time-of-flight (TOF) mass spectrometer (Bruker Daltonics, Billerica, MA) equipped with a nitrogen laser (337nm), a single-stage pulsed ion extraction ion source, a two-stage grid-less reflector, and two dual microchannel plate detectors for detection in linear and reflectron modes. 170

MS spectra were measured under reflectron mode, with the ion source and reflector lens potentials set at 20keV and 22.5keV, respectively. The attenuation of the nitrogen laser was adjusted to get the optimal sensitivity without causing fragmentation of the polymer. The mass scale was calibrated by using 6 peaks of a polymethylmethacrylate standard (PMMA) that has an approximate molecular weight of 2000 amu.

5.3.4 Biodegradation Experiments

5.3.4a Purge and Trap Experiment

Modified purge and trap vessels were used to allow for continuous flow of air through the microcosms and to trap potential FTOHs produced during the experiment. A description of the experimental set-up used is described in detail elsewhere (18) (Chapter 3) and only briefly summarized here. Polymers were added as solids at target concentrations of 200 mg L"1 in Pyrex media bottles (1L) filled with 800mL sterile mineral media. Vessels contained approximately

160 mg of polymers and were covered with caps modified to fit Orbo™ Amberlite™ XAD-2 cartridges (lOOmg) (Supelco, Bellefonte, PA) and gas diffuser tubes (Pyrex, VWR International

Ltd., Mississauga, ON). Vessels were purged continuously with carbon-filtered air for approximately two weeks prior to addition of inoculum. Intermittent sampling of the XAD cartridges was done during this duration to check for presence and amount of residual FTOHs and 8:2 acrylate monomer. Inoculum was only added once residual amounts were at a minimum.

Inoculum was obtained from the Ashbridges Bay wastewater treatment plant (WWTP) in

Toronto, ON. Prior to the start of the experiment, STP samples were shaken and allowed to settle for 15 minutes. Sixteen milliliters were then taken from the supernatant and centrifuged for 15 minutes at 3400 rpm. The supernatant was removed and 16 mL of fresh mineral media 171 was added to resuspend the pellet as a wash step. The sample was centrifiiged again and the supernatant removed. The pellet was resuspended in 5 mL of mineral medium before addition to experimental vessels. Active vessels were prepared in quadruple and sterile controls were prepared in duplicate. Sterile controls consisted of 800 mL autoclaved distilled deionized water without addition of inoculum.

XAD cartridges were sampled intermittently during the degradation experiment. XAD resin and glass wool from the cartridges were extracted using two 2mL aliquots of ethyl acetate and both fractions were subsequently combined. Four hundred microliters were then transferred to autosampler vials for gas chromatographic analysis.

5.3.4b Activated Sludge Experiment

Experiments were also carried out using 125mL Nalgene® bottles filled with 50mL of undiluted activated sludge taken from the Ashbridges Bay sewage treatment plant (Toronto, ON) and capped with rubber septa. Polymers were added as solids at 1 to 1.5 mg to get a target concentration of approximately 30 mg L"1 in active and sterile control vessels. Active vessels were prepared in quadruplicate and sterile controls in duplicate, which were autoclaved to cease microbial activity. Two vessels were prepared as above without the addition of polymers (no spike vessels) to measure any background production of the analytes of interest. Aerobic conditions were maintained by flowing carbon-filtered air in the active and no spike microcosms using a stainless steel needle punctured through the rubber septa. Another needle was punctured to act as a vent to prevent pressurizing of vessels. Sterile controls received no further amendment of air. 172

5.3.5 Instrumental Analysis and Quantification

5.3.5a GCMS Analysis of Purge and Trap Experiment Samples

Analysis of XAD extracts from the purge and trap experiment were carried out using a

Hewlett Packard 6890 gas chromatograph equipped with a 5973 inert mass spectrometer detector. Optimal separation of the FTOHs and the 8:2 aery late monomer were done using a

30m DB®-Wax column (0.25mm i.d., 250um film thickness, J &W Scientific, Folsom, CA). The following GC oven program was employed: initial temperature of 55°C held for 1 minute; 5°C min1 ramp to 75°C; 15°C min1 ramp to 130°C min1 and a final ramp of 30°C min"1 to 240°C and held for 1 minute. Helium was used as the carrier gas at a flow rate of lmL min"1 with pulsed splitless injection at an initial pressure of 25 psi at 220°C for 1.2 min followed by an injector purge at 40.0mL min"1 for 0.8 min. Quantification proceeded under positive chemical ionization (PCI) in single ion monitoring mode, and the molecular ion (M+l) was monitored for the target analytes. Calibration was performed using external standards prepared in ethyl acetate and ranging in concentration from 5 pg uL"1 to 500 pg uL"1 for the 8:2 acrylate and internal standard calibration were used for 8:2 FTOH quantification using a 13C labeled standard. Good linearity was observed and retention times for all analytes of interest were well conserved (± 0.04 min). The limit of quantitation was defined as the lowest standard to give a signal to noise ratio

>10, corresponding to 5 pg uL"1.

5.3.5b LCMSMS Analysis of Non-Volatile Metabolites

To monitor for any non-volatile metabolites produced in the purge and trap vessels, 2mL aqueous samples were taken at 90 days after the start of the experiment. Samples were 173 processed using a previously described method for extracting fluorinated chemicals from biological samples (37). Extracts were reconstituted in 200 uL of 75:25 methanol:water and transferred to 300uL polypropylene autosampler vials for LCMSMS analysis.

The undiluted sludge vessels were sampled weekly by taking 1 mL samples and extracted in a similar manner with an added clean-up step using the addition of a hexafluoropropanol

(HFP) previously used in the analysis of biological samples (38).

Quantitative analysis was performed using a Waters Acquity UltraPerformance Liquid

Chromatographic system (UPLC®) equipped with a Gemini C18 column (2 um, 4.6><250mm,

Phenomenex, CA). Isocratic elution was used with an eluent composition of 85:15 methanokwater, both with lOmM ammonium acetate at a flow rate of 150 uL/min. A Micromass

Quattro micro™ Triple Quadrupole Mass Spectrometer (Micromass; Manchester, UK) under negative electrospray ionization and multiple reaction monitoring (MRM) mode was used to monitor the following compounds: PFOA, 8:2 FTUCA, 8:2 FTCA, 7:3 FTCA, PFNA, PFHPa and PFHxA. Quantification was performed using internal standard calibration for compounds where labeled standards were available namely: PFOA, 8:2 FTUCA, PFNA and PFHPa.

External calibration was used for 8:2 FTCA, 7:3 FTCA and PFHxA.

5.4 RESULTS AND DISCUSSION

Limited studies have been done to determine the environmental fate of fluorotelomer based polymers. Intermediates in the synthesis of these compounds including the fluorotelomer alcohols, as well as the telomer acrylate and methacrylate monomers have all been shown to be labile, forming PFCAs as a metabolite of microbial degradation under aerobic conditions (14-16,

20). This suggests the linkage is potentially labile to biologically driven scission with the 174

corresponding release of a FTOH. In this study, a model fluorinated telomer acrylate polymer

was synthesized and subjected to biodegradation under aerobic conditions to determine its

potential degradability and whether PFCAs are formed as metabolites.

5.4.1 Polymer Characterization

The synthesis of a telomer-based polymer was confirmed using 19F and lH NMR

spectroscopy. A comparison of the NMR spectra of the fluorinated and non-fluorinated

monomers used along with the synthesized polymer is shown in Figure 5-2 and Figure 5-3. The

NMR spectra of the synthesized polymer show presence of broad peaks characteristic of

polymeric materials. Another defining characteristic as shown by the 'H NMR spectra of the

two monomers is the loss of the vinylic protons, indicative of the reaction on the double bond of

the monomer during polymerization.

F3C(F2Cj)7CH2CH20^ \C = C'

H H H H

I pps* 1 ppra

Figure 5-2 lH NMR spectra of monomers, (a) 'H NMR of hexadecyl acrylate, (b) lH NMR of 8:2 FTOH acrylate. 175

Figure 5-2 (c) !H NMR of synthesized Polymer I with box showing loss of vinylic protons. 176

F3C(F2C)7CH2CH2(r \c / H

-60 -SS -AttO -J.il' 0 -140 j»p*

Figure 5-3 F NMR of 8:2 acrylate monomer (a) and synthesized Polymer I (b). 177

Two different sized distributions of polymers were synthesized to examine if susceptibility to degradation was affected by molecular weight. Synthesis of different sized polymers was achieved by varying the amount of initiator used in the reaction and/or altering the length of time the reaction was run. Average number molecular weight (Mn) and weight average molecular weight (Mw) determined by GPC analysis of the two polymers synthesized is summarized in Table 5-1. Polymer I was considered to be the high molecular weight polymer and Polymer II was the low molecular weight polymer. Molecular weights up to 55,000amu have been reported in commercial patents for similar types of polymers (32).

Table 5-1 Molecular weights of synthesized polymers based on GPC analysis.

Average Number Weight Average Polymer Name Molecular Weight Molecular Weight (Mn) (Mw) Polymer I 11,100 amu 25,900 amu

Polymer II 6,380 amu 8,290 amu

Key structural information of the synthesized polymers was provided by MALDI-MS analysis. Figure 5-4 shows a sample spectrum of the 4,000 - 6,000 amu region of polymer I along with ratios of the monomers that correspond to each respective mass of polymeric chains.

Ratios of the two monomers were variable but were within desired target ratios of 1:1. The size distribution of Polymer I and Polymer II according to MALDI-MS analysis was significantly lower than determined by GPC. Polymer I was measured to have an Mn of-2000 while

Polymer II was measured to have an Mn of- 1200. MALDI-MS is reported to provide good estimates of Mn (39). GPC however is prone to solvent effects, whereby solvent can often lead 178 to polymer micellization or dimerization resulting in higher than true molecular weights values

(40). The Mark-Houwink constant measured for the synthesized polymers were low, determined at 0.099 for Polymer I and 0.534 for Polymer II. These low values are indicative of poor solvent-polymer compatibility that may explain the discrepancy with the MALDI-MS measurements (40). Further GPC analysis of the polymers using a different solvent is warranted to reconcile the molecular mass measurements of the two different modes of analysis.

Irrespective of the discrepancy, two different sized polymers were synthesized that can be used to test the effect of size on potential degradability.

Figure 5-4 Expanded MALDI-MS spectra from mass range 4,000 to 6,000 of Polymer I 5.4.2 Biodegradation of Model Telomer Polymer

Purge and trap vessels were used in a biodegradation experiment that focused primarily on monitoring for FTOH production from potential polymer degradation. In addition to several wash steps in the synthesis reaction that aided in the removal of residuals, polymers were added to vessels with sterile mineral media and purged for 18 days prior to addition of inoculum. This method of residual removal has been reported effective on a laboratory scale for several commercial and industrial telomer-based materials (18). As shown in Figure 5-5, residual 8:2 acrylate monomer was removed from the vessels by the purge and trap method after approximately 18 days of continuous purging. XAD cartridges were sampled every 2 days during the residual removal phase of the experiment. Residual 8:2 FTOH present was significantly less than the 8:2 acrylate monomer as expected, since 8:2 FTOH is only an impurity of the 8:2 FTOH acrylate monomer used in the synthesis. Total mass of residual 8:2 FTOH and

8:2 acrylate removed at the end of day 18 was equal to 11.68 ± 5.90 ug and 133.72 ± 30.91ug respectively which corresponds to 0.007% and 0.08% of the initial mass of polymer added.

These values are significantly lower than those expected for commercially available fluorinated polymers that were shown to be as high as 1-3% (18). Inoculum was only added to vessels after day 18 when most of the residual FTOH and 8:2 FTOH acrylate had been depleted from the vessels. 1

300 I8:2FT0H > • Active Vessels 18:2 Aery late O o • Sterile Water Controls Inoculum

Day 2 Day 4 Day 6 Day 8 Day 10 Day 12 Day 14 Day 16 Day 18

Day of Purging Figure 5-5 Removal of residual 8:2 FTOH and 8:2 acrylate monomer from Polymer II spiked vessels. 8:2 FTOH measured before and after inoculum addition in Polymer II spiked vessels (inset). Error bars represent standard deviation (active vessels n=3, sterile controls n=2)

Figure 5-6 is a summary of the cumulative FTOH produced in vessels spiked with

Polymer I and Polymer II. No significant production of FTOHs was observed in either set of vessels after 77 days and production of FTOHs appeared to plateau by day 40 of the study.

Cumulative amounts of FTOHs observed were small considering the amount of polymers added to the vessels were in the milligram quantities. More importantly, no significant difference was observed in amounts of FTOHs produced between control and active vessels. The 8:2 FTOH observed was likely due to residuals that continued to be released from the polymeric material during the duration of the experiment. This is also shown in the Figure 5-5 (inset) where amounts of 8:2 FTOH measured did not differ between the residual removal phase of the experiment and after addition of the inoculum (degradation phase). It is currently unclear as to 181 why this compound remains in the purge and trap system even after 77 days of continued purging. In comparison, the 8:2 acrylate was depleted from the system after approximately 10 days. It cannot be ruled out that slow hydrolysis of the polymer is occurring leading to some

FTOH production, further studies are required to test this hypothesis. Aqueous samples were taken at day 90 of the experiment to determine whether non-volatile metabolites were present in the system. The lack of significant detection of FTOHs could have been due to further metabolism of this intermediate to other non-volatile metabolites in the degradation pathway, however, analysis of aqueous samples showed no evidence that this occurred. 8:2 FTOH metabolites of interest such as 8:2 FTCA, 8:2 FTUCA, 7:3 FTCA and PFOA were not detected in amounts above blanks from the analysis of day 90 aqueous samples. Concentrations of these compounds in blank samples ranged from 2 to 5 ug L"1.

•Active Vessels • Active Vessels • • Water Controls • 20.00 • • Water Controls m - • 35.00 • • 30.00 . • • • • • • • • | 25.00 - • • J 20.00 • • Mas s (ug ) • • f 15.00 • • Polymer I 10.00 Polymer II (High MW) 5.00 (Low MW) I 1

0 10 20 30 40 50 60 70 80 9 0 0 10 20 30 40 50 60 70 80 90 Days Days

Figure 5-6 Cumulative FTOH produced from active and control vessels spiked with Polymer I and Polymer II.

The apparent lack of degradation suggests that the synthesized telomer-based acrylate polymer is substantially more stable than FTOHs and the ester telomer monomer. Alternatively, it was hypothesized that the lack of degradation could be due to poor solubility of polymer in the aqueous media and/or poor contact between microorganisms and polymer as a result of the 182 diluted inoculum used. To test the latter hypothesis, a degradation experiment was conducted in undiluted activated sludge spiked with lower concentrations of both polymers. Unlike the purge and trap experiment, this study did not attempt to measure 8:2 FTOH production, alternatively its focus was on quantification of the non-volatile metabolites of 8:2 FTOH particularly PFOA.

There are inherent challenges that are associated with biodegradation experiments involving perfluorinated compounds and undiluted activated sludge. Several studies that examined samples from wastewater treatment facilities receiving industrial and residential inputs have indicated that sludge is highly contaminated with PFCAs of varying chain lengths (33-35,

41, 42), hence they are likely present in the sludge prior to any addition of the telomer-based polymer. An additional challenge to this experimental set-up is the inability to remove residual

FTOHs and monomer via the purge and trap method before exposure of the polymer to the microbes. An accurate quantification of residuals is required to determine the amount of metabolites that can be attributed to degradation of residuals alone. The total amount of residuals measured from the purge and trap experiment were applied in this study.

Spike and recovery experiments were performed to assess the applicability of the extraction method to sludge samples. Sludge samples used for the spike and recovery experiments were autoclaved to cease microbial activity. Recoveries for 8:2 FTUCA and PFOA were measured at 72.3% ±7.1 and 83.3% ± 14.3 (n=8) respectively but recoveries for the 8:2

FTCA and 7:3 FTCA were 179.7 + 52 and 162.0% ± 43.8 respectively. The excellent recoveries for 8:2 FTUCA and PFOA were likely aided by the use of labeled internal standards. Labeled standards for 7:3 FTCA are not commercially available and labeled standards for the 8:2 FTCA were not accessible at the beginning of this study. External calibration was used for the 7:2

FTCA and the 8:2 FTCA. The enhancement of the signal for 7:3 acid and 8:2 FTCA may likely 183 be due to matrix effects from sludge samples during the LCMSMS analysis, a common problem encountered when analyzing complicated samples using electrospray ionization(43).

PFOA along with other PFCAs, PFHxA, PFHpA and PFNA were present in sludge samples at concentrations ranging from 0.07 to 2.2 ug L"1 prior to the addition of polymer, with

PFOA being the most abundant. PFHxA was also detected in samples but were below the limits of quantitation. The 8:2 FTOH intermediates 8:2, FTCA, 8:2 FTUCA and 7:3 FTCA were also detected at concentrations ranging from 1.2 to 2.2 ug L"1, 0.2 to 0.8 ug L"1 and 2.6 to 4.4 ug L"1 respectively. PFCA concentrations measured in this study are in agreement with those previously reported in sludge and wastewater (34, 35, 41). This is the first report of 7:3 FTCA detection from environmental samples. Measured concentrations in this study were not corrected for recoveries and thus values for the 8:2 FTCA and 7:3 FTCA may be inflated. Standard additions will be performed in future analyses to add confidence to measured concentrations.

Preliminary data from the degradation of the synthesized telomer-based polymers in undiluted sludge, indicate a small increase in the detected amounts of PFOA after 34 days

(Figure 5.7). However, this increase was also observed in killed controls and in the no spike vessels. No spike vessels demonstrated higher PFOA production than any other experimental vessels in the study. These vessels appeared to contain a higher amount of solids at the start of the experiment which may have resulted in higher biomass. More importantly PFOA production in these controls suggests that other precursors may already be present in sludge. Hence, it is quite uncertain whether the observed increase of PFOA in polymer-spiked vessels is a consequence of degradation of the spiked polymer or other precursors present in the sludge samples. 8:2 FTUCA and 8:2 FTCA were not consistently detected in the samples and no clear trends can be observed. 7:3 FTCA increased in vessels spiked with Polymer I but decreased in 184 concentration over time in vessels spiked with Polymer II which may be indicative of further degradation of this intermediate. The total amount of 8:2 FTOH metabolites detected by day 34 of the study was less than the theoretical amount of metabolites that can be attributed to residual degradation (Table 5-2) and hence cannot be related to potential polymer degradation. The experiment is currently ongoing and further samples will be taken to determine whether amounts of PFOA will continue to increase. Results presented here will be used to design more robust investigations in the future using undiluted sludge as a matrix.

4.50

4.00 • Poly I Active 0) U Poly II Active (A (A 3.50 a> "~A—,Poly I Killed > V. O 3.00 —•—Poly II Killed a. v> 2.50 * No Spike o 2.00 c o 1.50 c o o 1.00 c o o 0.50

0.00

Figure 5-7 PFOA production in undiluted activated sludge experiment. 185

Table 5-2 Summary of measured 8:2 FTOH metabolites from undiluted sludge experiment

Total Residuals Total PFOA at start Total PFOA Vessels present in Polymers of experiment detected after day (nmols) (nmols) 34 (nmols) Polymer I spiked 0.26 ± 0.03 0.86 + 0.07 vessels 1.89+ 0.3 Polymer I spiked 1.94+ 0.00 0.24 ± 0.03 0.91 ±0.26 vessels (Killed) Polymer II spiked 2.02+ 0.34 0.28 + 0.05 0.87 ±0.08 Vessels Polymer II spiked 2.38 ± 1.94 0.26 ± 0.004 0.68 ± 0.06 Vessels (Killed) No Spike Vessels - 0.22 ± 0.00 3.96 ±1.04

In a soil biodegradation study of a commercial telomer-based polymer, amounts of PFCA observed as metabolites were suggested to be attributed only to the degradation of large amounts of residuals present in their study (44). This suggestion was based on a proposed conversion rate of 100% of all residuals to PFOA. Reported production rates of PFOA from biodegradation of

FTOHs have only ranged from 2-6% in experiments some lasting up to 18 months (14-16). It is thus probable that the measured amounts of PFCAs from this soil investigation are a combination of degradation products from residuals as well as from degradation of the polymer. A reliable method of measuring actual loss of polymer from microcosms is clearly required to directly relate PFOA production from breakdown of polymer.

Synthesis of two different sized distributions of fluorotelomer-based polymer was achieved in this study. Biodegradation experiments using a purge and trap system with diluted inoculum showed little FTOH production which indicates that polymers are substantially more stable than the ester monomer or FTOH itself. To further assess the stability of the synthesized polymer, an undiluted sludge experiment was performed in order to promote association of 186 microorganisms to polymers to ensure that limited microbial population is not the limiting factor in the degradation. Preliminary data from this experiment show metabolites produced are minimal. Further samples need to be taken and analyzed to determine any extent of degradation of these polymeric compounds. This study is ongoing and further experiments need to be performed before stability of telomer-based polymers is assessed. Ultimately, development of reliable methods of quantifying polymers in aqueous or sludge type matrix is essential in providing solid evidence of degradation of these materials.

5.5 ACKNOWLEDGEMENTS

The authors would like to thank Dr. Bill Coggio of Dyneon and Dr. Rick Thomas for valuable discussion on polymer synthesis; Dr. Rick Thomas also for assistance with GPC analysis; and Dr. Chris Wesdimiotis and Kittisak Chaicharoen of University of Akron for

MALDI-MS analysis of the synthesized polymer. Assistance of Derek Jackson with NMR analysis is greatly appreciated. We are grateful to Wellington Laboratories (Guelph, ON) for the donation of labeled standards. Environment Canada is also acknowledged for its financial support.

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(10) Hurley, M. D., Wallington, T. J., Andersen, M. P. S., Ellis, D. A., Martin, J. W. and Mabury, S. A. Atmospheric chemistry of 4:2 fluorotelomer alcohol: Products and mechanism of CI atom initiated oxidation. J. Phys. Chem. 2004,108, 5635-5642.

(11) Hagen, D. F., Belisle, J., Johnson, J. D. and Venkateswarlu, P. Characterization of fluorinated metabolites by a gas chromatographic-helium microwave plasma detector - the biotransformation of 1H,1H,2H,2H - perfluorodecanol to perfluorooctanoate. Anal. Biochem. 1981,118, 336-343.

(12) Martin, J. W., Mabury, S. A. and O'Brien, P. J. Metabolic products and pathways of fluorotelomer alcohols in isolated rat hepatocytes. Chem. Biol. Interac. 2005,155, 165- 180.

(13) Fasano, W. J., Carpenter, S. C, Ganno, S. A., Snow, T. A., Stadler, J. C, Kennedy, G. L., Buck, R. C, Korzeniowski, S. H., Hinderliter, P. M. and Kemper, R. A. Absorption, distribution, metabolism and elimination of 8:2 fluorotelomer alcohol in the rat. Toxicol. Sci. 2006, 91, 341-355.

(14) Dinglasan, M. J. A., Ye, Y., Edwards, E. A. and Mabury, S. A. Fluorotelomer Alcohol Biodegradation Yields Poly- and Perfluorinated Acids. Environ. Sci. Technol. 2004, 38, 2857-2864.

(15) Wang, N., Szostek, B., Folsom, P. W., Sulecki, L. M., Capka, V., Buck, R. C, Berti, W. R. and Gannon, J. T. Aerobic Biotransformation of 14C-Labeled 8-2 Telomer B Alcohol 188

by Activated Sludge from a Domestic Sewage Treatment Plant. Environ. Sci. Technol. 2005,39,531-538.

(16) Wang, N. S., Bogdan; Buck, Robert C; Folsom, Patrick W.; Sulecki, Lisa M.; Capka, Vladimir; Berti, William R.; Gannon, John T. Fluorotelomer Alcohol Biodegradation - Direct Evidence that Perfluorinated Carbon Chains Breakdown. Environ. Sci. Technol. 2005,59,7516-7528.

(17) Larsen, B. S., Stchur, P., Szostek, B., Bachmura, S. F., Rowand, R. C, Prickett, K. B., Korzeniowski, S. H. and Buck, R. C. Method development for the determination of residual fluorotelomer raw materials and perflurooctanoate in fluorotelomer-based products by gas chromatography and liquid chromatography mass spectrometry. J. Chromatogr. A 2006,1110, 117-124.

(18) Dinglasan-Panlilio, M. J. A. and Mabury, S. A. Significant Residual Fluorinated Telomer Alcohols Present in Various Fluorinated Materials. Environ. Sci. Technol. 2006, 40, 1447-1453.

(19) Sinclair, E., Kim, S. K., Akinleye, H. B. and Kannan, K. Quantitation of gas-phase perfluoroalkyl surfactatns and fluorotelomer alcohols released from nonstick cookware and microwave popcorn bags. Environ. Sci. Technol. 2007, 41, 1180-1185.

(20) Dinglasan-Panlilio, M. J. A., Edwards, E. A. and Mabury, S. A. Biodegradation of fluorotelomer based monomers as a source of fluorotelomer alcohols. Environ. Sci. Technol. 2007, To be submitted.

(21) Greenwood, E. J., Lore, A. L. and Rao, N. S. Oil- and water-repellent copolymers. E. I. Du Pont de Nemours and Company (Wilmington, DE), US Patent 4742140, 1988

(22) Castelvetro, V., Aglietto, M., Ciardelli, F., Chiantorre, O. and Lazzari, M. Structure control, coating properties, and durability of fluorinated acrylic-based polymers. J. Coatings Technol. 2002, 74, 57-66.

(23) Saidi, S., Guittard, F., Guimon, C. and Geribaldi, S. Fluorinated acrylic polymers: surface properties and XPS investigations. J. App. Poly. Sci. 2005, 99.

(24) Zuev, V. V., Bertini, F. and Audisio, G. Investigation on the thermal degradation of acrylic polymers with fluorinated side chains. Poly. Deg. Stabl. 2006, 91, 512-516.

(25) Huber, C. D., Grant, R. J. and Smith, R. J. Fabric repellent treatment from hydrocarbon solvent system. Minnesota Mining and Manufacturing Co. (St. Paul, Mn), US5284902, 1994

(26) Yamamoto, K. and Yuzuru, Y. Aqueous emulsion component for a water- and oil- repellent agent. Nippon Mektron Limited (Tokyo, JP), US6121372, 2000 189

D'eon, J. C. and Mabury, S. A. Production of perfluorinated carboxylic acids (PFCAs) from the biotransformation of polyfluoroalkyl phosphate surfactants (PAPS): Exploring routes of human contamination. Environ. Sci. Technol. 2007, 41, 4799-4805.

Portugais, J. Notice of Action Plan for the Assessment and Management of Perfluorinated Carboxylic Acids and their Precursors. Canada Gazette Part I, June, 17, 2006.

Maier, R. M., Pepper, I. L. and Gerba, C. P. Environmental Microbiology; Academic Press: San Diego, California, 2000.

Imae, T. Fluorinated Polymers. Curr. Opin. Colloid Interface Sci. 8 2003, 5, 307-314.

Ame'duri, B., Boutevin, B., Guida-Pietrasanta, F. and Rousseau, A. Fluorinated oligomers, telomer and (co)polymers: synthesis and applications. J. Fluor. Chem. 2001, 707, 397-409.

Audenaert, F., van der Elst, P. J. and Roily, D. G. Fluoropolymer of fluorinated short chain acrylates or methacrylates and oil- and water repellent compositions based theron. 3M Innovative Properties Company, EP 1493761 Al, 2005

Boulanger, B. V., J. D.; Schnorr, J. L.; Hornbuckle, K. C. Evaluation of Perfluorooctane surfactants in a Wastewater treatment system and in a commercial surface protection product. Environ. Sci. Technol. 2005, 39, 5524-5530.

Sinclair, E. K., Kurunthachalam Mass Loading and Fate of Perfluoroalkyl Surfactants in Wastewater Treatment Plants. Environ. Sci. Technol. 2006, 40, 1408-1414.

Schultz, M. M., Barofsky, D. F. and Field, J. A. Quantitative Determination of Fluorinated Alkyl Substances by Large-Volume-Injection Liquid Chromatography Tandem Mass Spectrometry-Characterization of Municipal Wastewaters. Environ. Sci. Technol. 2006, 40, 289-295.

Achilefu, S., Mansuy, L., Selve, C. and Thiebault, S. Synthesis of 2H,2H-perfluoroalkyl and 2H-perfluoroalkenyl carboxylic acids and amides. J. Fluor. Chem. 1995, 70, 19-26.

Hansen, K. J., Clemen, L. A., Ellefson, M. E. and Johnson, H. O. Compound-Specific, Quantitative Characterization of Organic Fluorochemicals in Biological Matrices. Environ. Sci. Technol. 2001, 35, 766-770.

Butt, C. M., Muir, D. C. G., Stirling, I., Kwan, M. and Mabury, S. A. Rapid Response of Arctic Ringed Seals to Changes in Perfluoroalkyl Production. Environ. Sci. Technol. 2007, 41, 42-49.

Latourte, L., Blais, J. and Tabet, J. Desorption behavior and distribution of fluorinated polymers in MALDI and electrospray ionization mass spectrometry. Anal. Chem. 1997, 69, 2742-2750.

Thomas, R. R. OMNOVA Solutions Inc., Personal Communication, 190

(41) Higgins, C. P., Field, J. A., Criddle, C. S. and Luthy, R. G. Quantitative determination of perfluorochemicals in sediments and domestic sludge. Environ. Sci. Technol. 2005, 39, 3946-3956.

(42) Schultz, M. M., Higgins, C. P., Huset, C. A., Luthy, R. G., Barofsky, D. F. and Field, J. A. Fluorochemical mass flows in a municipal wastewater treatment facility. Environ. Sci. Technol. 2006, 40, 7350-7357.

(43) Mallet, C. R., Lu, Z. and Masseo, J. R. A study of ion suppression effects in electrospray ionization from mobile phase additives and solid phase extracts. Rapid Commun. Mass Spectrom. 2004,18, 48-58.

(44) Russell, M., Berti, W. R., Szostek, B. and Buck, R. C. Biodegradation potential and environmental impact of fluorotelomer-based acrylate polymer. Society of Environmental Toxicology and Chemistry Europe 17th Annual Meeting, Porto, Portugal, May 20-24, 2007. CHAPTER VI

SUMMARY, CONCLUSIONS AND FUTURE RESEARCH PERSPECTIVES 192

6.1 SUMMARY AND CONCLUSIONS

Large research efforts have been committed to investigating potential sources of perfluorocarboxylic acids (PFCAs) in the environment primarily due to their prevalence (7), persistence (2)and potential toxicity (3). Identification of major sources may lead to possible solutions to this current environmental problem. The primary objective of this thesis project was to investigate potential indirect sources of PFCAs to the environment from biological reactions.

Fluorotelomer alcohols (FTOHs), a class of perfluoroalkyl chemicals, were hypothesized to be precursors of PFCAs due to their widespread detection in the atmosphere and production of

PFCAs from atmospheric oxidation reactions (4). This research aimed to determine the potential for PFCA formation from the biodegradation of FTOHs under aerobic conditions and to investigate other sources of FTOHs that may ultimately lead to PFCA production in the environment.

FTOHs are intermediates in the production of telomer based surfactants and polymers used to render textiles, carpet and upholstery dirt and water repellant (2). Routine application of these materials may release these compounds to wastewater treatment plants where they are likely to be degraded by microbial organisms. Hence, microbial degradation may be an important environmental fate for these organofluorine contaminants.

The biodegradation potential of FTOHs under aerobic conditions was investigated in

Chapter 2 (5) using an enrichment culture acclimated on ethanol as well as dilute wastewater sludge. 8:2 FTOH rapidly degraded in active vessels forming PFOA as a terminal metabolite.

No significant degradation was observed in sterile controls, a confirmation of microbially mediated degradation. Along with the detection of PFOA, the 8:2 fluorotelomer aldehyde (8:2

FTAL) was first identified as a transient intermediate. The telomer carboxylic acids, 8:2 193 fluorotelomer acid (8:2 FTCA) and 8:2 fluorotelomer unsaturated acid (8:2 FTUCA), were the more stable intermediates detected and quantified. Based on these detected metabolites, a P- oxidation mechanism was proposed as a probable degradation mechanism for these compounds if exposed to a microbially active matrix such as wastewater sludge or sediment. The production of the odd acid (PFNA) was not observed in this system indicating that a-oxidation is not a significant degradation pathway for these compounds in microorganisms. This study provided further evidence that FTOHs are precursors to PFCAs under biodegradation conditions.

Since FTOHs are intermediates in the production of more complex fluorinated materials and not directly used by consumers and industry, their widespread detection in the atmosphere led to the question of their origin or release (6-11). Chapter 3 (12) examined a potential source for these volatile precursors to the environment. Several fluorinated polymers and surfactants were analyzed and found to contain residual FTOHs of varying chain lengths. These residuals or impurities were hypothesized to be the consequence of inefficient synthesis or purification of telomer based materials. FTOH flux to the environment has been proposed to be in the order of

100 - 1000 t yr"1 to maintain current observed (13) atmospheric concentrations. Using measured data from this study and combining it with current available global production estimates of

FTOHs, it was determined that residuals make up a significant proportion of this estimated

FTOH input to the environment. A method of removing these unbound volatile compounds was also developed involving a purge and trap apparatus that takes advantage of FTOHs' volatility and low water solubility. In addition, NEtFOSE, a polyfluoro sulfonamidoethanol, was also detected and quantified in a commercial upholstery protector product. This compound is hypothesized to be a precursor to PFOS. Presence of these residuals suggests potential for their release to the environment from routine application and use of the materials that contain them. It was also suggested that these residuals are likely to offgas in indoor atmospheres from surfaces treated with telomer-based products, making it a possible route for human exposure (14).

To further evaluate the contribution of FTOHs to the burden of PFCA contamination in the environment, it was important to resolve other potential sources of FTOHs in addition to that of residuals. The biodegradation potential of various telomer-based monomers was investigated in Chapter 4 to determine whether these compounds degrade forming FTOHs. 8:2 FTOH based monomers with ester, ether and urethane linkages were interrogated to determine whether

FTOHs are produced from the breaking of these linkages. This study aimed to provide insight on whether the type of linkage is critical in the stability of these telomer based monomers. The environmental fate of these monomers is important because they are the building blocks of the telomer-based polymers and they are likely found as residuals and impurities in the polymer formulations as well. Using microcosms inoculated with wastewater sludge samples, two types of ester telomer monomers were degraded forming FTOHs and ultimately PFOA under aerobic conditions. Previously reported metabolites of FTOHs were also measured in the experiments.

In contrast, the urethane monomer tested showed no degradation and depletion of the ether monomer was observed without immediate FTOH formation. The degradation of the ether monomer likely proceeded via an alternate pathway that does not form FTOHs though may form

PFOA further down the degradation pathway. There was nevertheless no evidence of the latter from the study. The urethane monomer used in the experiment was not deemed to be the best probe to test urethane linkages due to its significantly higher molecular weight and structural difference when compared to the ester and ether analogues. The lack of degradation could have been due to the inability of microbial enzymes to attack the bond as a result of steric effects.

This study showed evidence that if emitted, ester telomer monomers, through its biodegradation 195 adds to the burden of FTOHs in the environment and ultimately of PFCAs. These ester monomers are compounds used extensively in the synthesis of telomer aery late polymers (15,

16).

Telomer based polymers make up 80% of the telomer chemical industry. Chapter 5 attempts to provide much needed insight to the environmental fate of these organofluorine macromolecules by probing the biodegradation potential of a model telomer-based acrylate polymer. A telomer-based copolymer was synthesized using the 8:2 FTOH acrylate monomer along with the heptadecyl acrylate, a non-fluorinated monomer, via a radical polymerization reaction. Polymers with two different size distributions, high and low molecular weights, were investigated. Sizes of polymers were measured using Gel Permeation Chromatography (GPC) and polymer structure was confirmed using Matrix Assisted Laser Desorption Ionization - Mass

Spectrometry (MALDI-MS). Polymers were purified of residuals using solvent extractions as well as purging of air while dispersed in mineral media prior to the addition of microorganisms in the vessels. After 90 days of degradation, no significant FTOH production was observed in purged vessels inoculated with diluted sludge taken from a wastewater treatment facility. Data suggests that the synthesized telomer-based polymer is more stable than the intermediate FTOHs and ester telomer monomers. The possibility that the lack of polymer degradation was due to poor contact between microbes and substrate was also explored. Absence of contact between polymer and microbes prevents induction of hydrolytic enzymes that may be required for degradation to occur. To investigate this hypothesis, experiments using pure activated sludge were performed that resulted in some PFOA production after ~ 30 days of degradation. Amount of PFOA measured however, was not significant which further points to the potential stability of 196 fluorinated telomer aery late polymers. This study is ongoing to determine whether PFOA concentrations will continue to increase over time.

From these investigations, it was apparent that fluorotelomer compounds such as telomer alcohols and telomer ester monomers could add to the burden of PFCAs to the environment.

These compounds were degraded forming the recalcitrant PFCAs by microorganisms enriched from a contaminated site as well as those present from a typical wastewater facility, receiving inputs from residential and commercial sources. Substantial FTOH emissions to the environment are attributed to release of alcohol residuals from various telomer-based materials in addition to degradation from telomer based monomers. In contrast, preliminary studies demonstrate increased stability of telomer-based polymers when compared to FTOHs and ester telomer monomers. Stability of these fluorinated macromolecules may indicate that they are not a significant source of PFCAs to environment in comparison to FTOHs and telomer ester monomers.

High global production rates of telomer based compounds which includes FTOHs and telomer ester monomers, in conjunction with the observed formation of PFCA ensuing from their biodegradation is indicative of an important contribution of telomer compounds to the observed

PFCA contamination in non-remote regions. Voluntary actions of industry to remove residuals from their products may have a promising impact on preventing further input of PFCAs to the environment. There is a possibility as well that use of more stable monomers in the synthesis of polymers could create telomer-based materials resistant to biodegradation. Further investigation into the biodegradation of telomer polymers is warranted to fully assess its contribution to the widespread PFCA contamination. 197

6.2 FUTURE RESEARCH PERSPECTIVES

Research presented in this thesis has made a significant impact in the understanding of microbial metabolism of fluorinated telomer compounds and its relation to the environmental contamination of PFCAs. Several potential research avenues have arisen from this work and are discussed below. It is apparent that further study is required in these areas to fully elucidate the extent of fluorotelomer contribution to the PFCA problem.

6.2.1 Additional Polymer Degradation Experiments

An additional polymer degradation experiment using pure activated sludge is warranted to determine whether observed lack of PFOA production, indicative of polymer stability is reproducible. Future studies require use of an improved clean up step for sludge samples in order to ensure accurate quantitation of metabolites in the complex sludge matrix. A sample preparation method similar to what was utilized by Higgins et al. (1 7) is recommended.

Removal of residuals still present in the synthesized polymer using more rigorous methods such as distillation may be valuable in obtaining pure polymeric material that can be used in the study.

Further more, the effect of adding solubilized polymer in sludge systems should be investigated.

The synthesized polymer is soluble in solvents such as tetrahydrofuran (THF) and ethyl acetate.

A study on the overall fate of the polymer in a small scale agricultural soil system amended with sludge and plants would add to the knowledge of how these compounds partition and behave in a multi component environmental system. 198

6.2.2 Degradation of other telomer based compounds

Various other types of fluorinated telomer compounds are manufactured by industry or produced as byproducts of the manufacture of telomer alcohols or monomers. These compounds namely telomer olefins and ethers similar to FTOHs are likely present as residuals in commercial products or formulations that have the potential to enter the environment. Investigation into their presence in the environment, as well as studies on their environmental fate is clearly warranted.

Little is also known of the environmental fate of telomer based surfactants. There are various types of these compounds including phosphate, phosphonates and sulfate analogues.

Phosphate type of telomer surfactants have been metabolized by rats forming PFCAs (18) and transformation of sulfate type of telomer surfactants have also been reported (19). Clearly, these compounds are somewhat labile under environmental conditions and their environmental fate should be further probed.

6.2.3 Degradation under various conditions and matrices

Most studies on the biodegradation of telomer compounds have been performed under aerobic conditions. There are no currently published studies in the scientific literature that examine biodegradation of FTOH and FTOH related materials under anaerobic conditions.

Anaerobic conditions prevail at landfill sites where many products that incorporate these telomer based materials are likely to end up; hence investigation of possible degradation under these conditions is highly relevant to the complete assessment of telomer contribution to PFCA formation.

The detection of FTOH intermediates in various sediment samples alludes to biodegradation of FTOH and FTOH related materials occurring in this environmental 199 compartment. Research into the biodegradation of these compounds in sediment and soil would provide insight as to whether this compartment is a source or sink of PFCAs in the environment.

6.2.4 Mechanistic and Enzymatic Studies on FTOH biodegradation

Despite acceptance of PFCA formation from FTOH biodegradation, there are still questions unanswered with regards to the mechanism. Enzymatic and mechanistic studies are needed to add clarity to the degradation mechanism of FTOHs. Two studies have emerged since the first publication of FTOH biodegradation (Chapter 2). These studies used labeled substrates and report production of several novel metabolites (20, 21). The mechanism of formation of these metabolites is unclear and needs to be further explored.

6.2.5 Reactivity of FTOH Intermediates

The investigation into the biodegradation of FTOHs has led to the identification of intermediate compounds that could potentially have more interesting chemistry than the PFCAs themselves. The unsaturated fluorotelomer carboxylic acid for example was found to be the dominant metabolite detected in the biodegradation of the 8:2 FTOH (5) and more importantly were found to be more toxic than the PFCAs (22) in aquatic organisms from a recent study.

MacDonald et al. had also reported that the saturated telomer acids are more toxic than their unsaturated counterparts. The telomer aldehyde was a transient intermediate in the biodegradation of FTOHs. Its transient nature was thought to be due to its high electrophilicity that may contribute to its reactivity. Little is known about potential reactivity of these compounds. It is likely that these intermediates have more potential harmful effects to biota than the PFCAs and hence should be investigated. 200

6.2.6 Method Development for Measuring Precursors in Environmental Samples

Studies that examined mass flows in wastewater treatment plants have suggested that increase in the concentrations of PFCAs in effluent vs influent flows may be due to the degradation of telomer precursors present in the wastewater (i 7, 23, 24). This research has provided evidence of that these precursors could likely be FTOHs, telomer based monomers or polymers. These compounds, however, have not yet been detected in environmental matrices with the exception of FTOHs in air. Method development is required in order to analyze wastewater effluents for presence of precursor compounds whether FTOHs, monomers, or polymers. These studies are essential in confirming the hypothesis that the observed PFCA increase in effluent wastewaters are due to degradation of FTOH based compounds. Other matrices that these precursors should be investigated are in: air for volatile monomers such as the esters, and sediment for presence of telomer based polymers.

6.3 LITERATURE CITED

(1) Houde, M., Martin, J. W., Letcher, R. J., Solomon, K. R. and Muir, D. C. G. Biological Monitoring of Polyfluoroalkyl Substances: A Review. Environ. Sci. Technol. 2006, 40, 3463-3473.

(2) Kissa, E. Fluorinated surfactants and repellents; Marcel Dekker: New York, 2001.

(3) Lau, C. B., J. L.; Rogers, J. M. The developmental toxicity of perfluoroalkyl acids and their derivatives. Toxicological Applied Pharmacology 2004,198, 231-241.

(4) Ellis, D. A., Martin, J. W., De Silva, A. O., Mabury, S. A., Hurley, M. D., Andersen, M. P. S. and Wellington, T. J. Degradation of fluorotelomer alcohols: A likely atmospheric source of perfluorinated carboxylic acids. Environ. Sci. Technol. 2004, 38, 3316-3321.

(5) Dinglasan, M. J. A., Ye, Y., Edwards, E. A. and Mabury, S. A. Fluorotelomer Alcohol Biodegradation Yields Poly- and Perfluorinated Acids. Environ. Sci. Technol. 2004, 38, 2857-2864. 201

(6) Martin, J. W., Muir, D. C. G., Moody, C. A., Ellis, D. A., Kwan, W. C, Solomon, K. R. and Mabury, S. A. Collection of Airborne Fluorinated Organics and Analysis by Gas Chromatography/Chemical Ionization Mass Spectrometry. Anal. Chem. 2002, 74, 584- 590.

(7) Stock, N. L., Lau, F. K., Ellis, D. A., Martin, J. W., Muir, D. C. G. and Mabury, S. A. Polyfluorinated Telomer Alcohols and Sulfonamides in the North American Troposphere. Environ. Sci. Technol. 2004, 38, 991-996.

(8) Jahnke, A., Ahrens, L., Ebinghaus, R., Berger, U., Barber, J. L. and Temme, C. An improved method for the analysis of volatile polyfluorinated alkyl substances in environmental air samples. Anal. Bio. Chem. 2007, 387, 965-975.

(9) Jahnke, A., Ahrens, L., Ebinghaus, R. and Temme, C. Urban versus Remote Air Concentrations of Fluorotelomer Alcohols and Other Polyfluorinated Alkyl Substances in Germany. Environ. Sci. Technol. 2006.

(10) Jahnke, A., Berger, U., Ebinghaus, R. and Temme, C. Latitudinal gradient of airborned polyfluorinated alkyl substances in the marine atmosphere between Germany and South Africa. Environ. Sci. Technol. 2007, 41, 3055-3061.

(11) Shoeib, M., Harner, T. and Vlahos, P. Perfluorinated Chemicals in the Arctic Atmosphere. Environ. Sci. Technol. 2006, 40, 7577-7583.

(12) Dinglasan-Panlilio, M. J. A. and Mabury, S. A. Significant Residual Fluorinated Telomer Alcohols Present in Various Fluorinated Materials. Environ. Sci. Technol. 2006, 40, 1447-1453.

(13) Ellis, D. A., Martin, J. W., Mabury, S. A., Hurley, M. D., Andersen, M. P. S. and Wallington, T. J. Atmospheric lifetime of fluorotelomer alcohols. Environ. Sci. Technol. 2003, 37, 3816-3820.

(14) Shoeib, M. H., T.; Ikonomou, M.; Kannan, K. Indoor and outdoor air concentrations and phase partitioning of perfluoroalkyl sulfonamides and polybrominated diphenyl ethers. Environ. Sci. Technol. 2004, 38, 1313-1320.

(15) Audenaert, F., van der Elst, P. J. and Roily, D. G. Fluoropolymer of fluorinated short chain acrylates or methacrylates and oil- and water repellent compositions based theron. 3M Innovative Properties Company, EP 1493761 Al, 2005

(16) Greenwood, E. J., Lore, A. L. and Rao, N. S. Oil- and water-repellent copolymers. E. I. Du Pont de Nemours and Company (Wilmington, DE), US Patent 4742140, 1988

(17) Higgins, C. P., Field, J. A., Criddle, C. S. and Luthy, R. G. Quantitative determination of perfluorochemicals in sediments and domestic sludge. Environ. Sci. Technol. 2005, 39, 3946-3956. (18) D'eon, J. C. and Mabury, S. A. Production of perfluorinated carboxyhc acids (PFCAs) from the biotransformation of polyfluoroalkyl phosphate surfactants (PAPS): Exploring routes of human contamination. Environ. Sci. Techno!. 2007, 41, 4799-4805. (19) Key, B. D., Howell, R. D. and Criddle, C. S. Defluorination of organofluorine sulfur compounds by Pseudomonas sp. strain D2. Environ. Sci. Technol. 1998, 32, 2283-2287.

(20) Wang, N., Szostek, B., Folsom, P. W., Sulecki, L. M., Capka, V., Buck, R. C, Berti, W. R. and Gannon, J. T. Aerobic Biotransformation of 14C-Labeled 8-2 Telomer B Alcohol by Activated Sludge from a Domestic Sewage Treatment Plant. Environ. Sci. Technol. 2005,39,531-538.

(21) Wang, N. S., Bogdan; Buck, Robert C; Folsom, Patrick W.; Sulecki, Lisa M.; Capka, Vladimir; Berti, William R.; Gannon, John T. Fluorotelomer Alcohol Biodegradation - Direct Evidence that Perfluorinated Carbon Chains Breakdown. Environ. Sci. Technol. 2005,39,7516-7528.

(22) MacDonald, M. M., Dinglasan-Panlilio, M. J. A., Mabury, S. A., Solomon, K. and Sibley, P. K. Fluorinated telomer acids are more toxic than perfluorinated acids. Environ. Sci. Technol. 2007, Accepted.

(23) Schultz, M. M., Barofsky, D. F. and Field, J. A. Quantitative Determination of Fluorinated Alkyl Substances by Large-Volume-Injection Liquid Chromatography Tandem Mass Spectrometry-Characterization of Municipal Wastewaters. Environ. Sci. Technol. 2006, 40, 289-295.

(24) Sinclair, E. K., Kurunthachalam Mass Loading and Fate of Perfluoroalkyl Surfactants in Wastewater Treatment Plants. Environ. Sci. Technol. 2006, 40, 1408-1414. APPENDIX A

SUPPORTING INFORMATION FOR CHAPTER II:

Fluorotelomer Alcohol Biodegradation Yields Poly- and Perfluorinated Acids 204

Table A-l Calibration data for protein measurements of inoculum used in 8:2 FTOH biodegradation experiment. Inoculum taken from 1,2-DCA enrichment culture known to degrade ethanol.

Protein (ug) Absorbance Protein Calibration (595nm) 0 0 y = 0.0467X + 0.0308 ? 1 R2 = 0.9846 3 0.175 8 6 0.29 a °- o 0.6 9 0.54 ra *^*^ € 0.4 15 0.7 0 in 21 1 5 0.2

0 5 10 15 20 25 Protein (ug)

Table A-2 Protein measurements at start of 8:2 FTOH biodegradation experiment

Vessel Absorbance Protein Total inoculum Protein Cone. (595nm) (ug/mL) added(ug) (ug/mL) Active A 0.36 7.05 704.93 0.74 Active B 0.45 8.98 897.64 0.94 Active C 0.61 12.40 1240.26 1.31 Sterile Control 0.60 12.19 1218.84 1.28 205

+ 1/12 CH3CH2OH + 1/4 H20 = 1/6 C02 + H + e" + l VA 02 + H + e" = A H20

1/12 CH3CH2OH + VA 02 = % H20 + 1/6 C02 or

CH3CH2OH + 3 02 = 3H20 + 2 C02

Figure A-l Energy equations of oxygen consumption linked to ethanol oxidation

Volume of ethanol added in microcosms as carrier solvent for 8:2 FTOH = 14uL or 0.014mL

Density of ethanol = 0.789 g mL"1

Thus, mass of ethanol added to experimental vessels = 0.014 mL x 0.789 g mL"1 = 0.011 g

moles of ethanol added = 0.011 g / 46.068 g mol"1 = 0.00024 mol

If oxygen to ethanol ratio is 1 to 3 then to consume 0.00024 mol of ethanol requires 0.00072 mol of oxygen (0.00024 x 3).

How much oxygen is available in the headspace of the vessels? Volume of vessels = 1 L Aqueous phase = 0.8 L Headspace = 0.2 L

How much oxygen is in 0.2 L of headspace?

Knowing that air is composed of 20.95% of air then: 20.95% of 0.2 L = 0.0419 L

Using the ideal gas law, we can calculate moles of oxygen present in the vessels: PV = nRT (1 atm) (0.0419 L) = n 0.0821 (273K + 23) 0.0419 = 24.30n n = 0.0017 mol of oxygen

Therefore, there are 0.0017mol of oxygen available in the vessels and enough to consume the ethanol typically added (0.00024 mol). Even if microorganisms were to consume all of the added ethanol first in the active vessels, oxygen was not fully consumed and is still available for aerobic degradation of 8:2 FTOH.

Figure A-2 Calculation of oxygen requirement for ethanol degradation in batch microcosms. APPENDIX B

SUPPORTING INFORMATION FOR CHAPTER III:

Significant Residual Fluorinated Alcohols Present in Various Fluorinated Materials XAD Cartridge

Fluorinated material dispersed in distilled deionized water

Figure B-l Schematic of purge and trap vessels used in the study.

^HA-vMwiAjAA^t^^vv^^'^^^wviA^,

20 19 18 17 16 15 14 13 12 11 10 90

5.0 6.0 7.0 8.0 9.0 10. Figure B-2 Chromatogram of a typical blank sample. (Vessels not spiked with fluorinated material).

Table B-l Recovery of FTOHs and NMeFOSE from purge and trap vessels Compound Percent Recovery 4:2 FTOH 29.24(68.12) 6:2 FTOH 89.77 (26.07) 8:2 FTOH 92.07(18.27) 10:2 FTOH 79.34 (9.23) NMeFOSE 97.50 (5.62) Data are means (n=3) and values in brackets are relative standard deviations (%). 208

Table B-2 Summary of fluorinated alcohols measured and average dry weights of fluorinated materials Average mass (ug) Avg Dry Polymer 4:2 FTOH 6:2 FTOH 8:2 FTOH 10:2 FTOH NMeFOSE Total Wgt (mg) Polyfox-L-diol 1.00 1.42 35.62 17.91 55.95 52.07 (0.25) Teflon Advance 1.90 6.65 3.56 12.11 3.45(0.87) ZonylFSOlOO 88.19 44.86 23.59 156.64 16.09(2.40) Zonyl FSE 81.59 72.33 13.12 167.04 4.43(4.50) Motomaster Windshield Washer 0.12 5.32 3.68 1.00 10.12 4.88(3.60) 8:2 Methacrylate 0.13 12.64 0.00 12.77 31.34(2.20) Scotchgard 93.74 93.74 23.60 (2.20) Dry weight data are means (n=3) and values in brackets are relative standard deviations

•o 90 4-1

1o_ 80 4-CO1 X -4:2 FTOH 0) 70 S2 60 ""^jjs" -6:2 FTOH

0IU O ) -8:2 FTOH ol ) 50

te l 40 -10:2 FTOH (n m 30 "+*8 flj 3 20 p 3 11) o -# < 0 8 10 12 14 16 18 Days Purged O td Accumulated Mass Extracted Accumulated Telomers Extracted (nmol) (nmol) _i. _* l\> M Oi Ul o Ul o Ul o OM^ffiOOOM^OJQO ) O o o o O o 1—l„„,„:U ,- ....;.,!,., •• ,„ , I,,.,,:,,:;: 1 1 .... ~_1

O a 0) 03 >< >< (/) w TJ Tl O C (Q c (D Q. <3 Ol CD Q.

GO O O

NO Ul O

to O W Accumulated Mass Extracted (nmol) Accumulated Telomers (nmol)

OM^OCOOM-ti.C5>

M D D 0) Q) >< >< w (/> "0 TJ c— * 0 CD Q. Q. CO

Ol e»; com O) :*# M M NJ 00 CO o Kb -n ~n Tl -n H H H H O O O Xo X X o o X X X Ol O

O 211

Figure B-3 Cumulative residual alcohols extracted versus days purged in (a) Polyfox-L- Diol (b) Zonyl™ FSE (c) Teflon™ Advance Rug and Carpet Protector (d) Motomaster® Windshield Washer with Teflon™ (e) 8:2 Methacrylate (f) Scotchgard™ Rug and Carpet Protector. APPENDIX C

SUPPORTING INFORMATION FOR CHAPTER V:

Investigation into the Biodegradation of Fluorotelomer-based Polymers

212 213

0.70

0.60

0.50

0.40

0.30 •7:3 Acid •8:2 FTCA 8:2 FTUCA

10 15 20 25 30 35 40 Days

Figure C-l Other FTOH metabolites observed in active (n=3) and killed control vessels (n=2) (inset) of undiluted sludge experiment spiked with Polymer I.

•7:3 Acid •8:2 FTCA 8:2 FTUCA

Figure C-2 Other FTOH metabolites observed in active (n=3) and killed control vessels (n=2) (inset) of undiluted sludge experiment spiked with Polymer II. 214

» 7 7:3 Acid CD 03 8:2 FTC A | 6 8:2 FTUCA £ 5 m £ 4 c ~c 3 o 2 2 c o 1 3 0 10 15 20 25 30 35 4) Days

Figure C-3 Other FTOH metabolites observed over time in No Spike Vessels of undiluted sludge experiment (n=3).