EPA/600/R-18/056 | May 2021 www.epa.gov/emergency-response-research United States Environmental Protection Agency oEPA Direct Aqueous Injection of the Fluoroacetate Anion in Potable Water for Analysis by Liquid Chromatography/ Tandem Mass Spectrometry Office of Research and Development Homeland Security Research Program ------- EPA Report Number EPA/600/R-18/056 Date: May 2021 Direct Aqueous Injection of the Fluoroacetate Anion in Potable Water for Analysis by Liquid Chromatography/Tandem Mass Spectrometry Stuart Willi son and Emily Parry U.S. Environmental Protection Agency Office of Research and Development Homeland Security Research Program Cincinnati, OH 45268 ------- Disclaimer The U.S. Environmental Protection Agency (EPA) through its Office of Research and Development, National Homeland Security Research Center (NHSRC) funded and managed the research described herein under IA #DW-75-958671 in collaboration with the National Institute of Occupational Safety and Health (NIOSH), Centers for Disease Control and Prevention (CDC), a division of the U.S. Department of Health and Human Services (DHHS). It has been subjected to the Agency's review and has been approved for publication. Note that approval does not signify that the contents necessarily reflect the views of the Agency. Any mention of trade names, products, or services does not imply an endorsement by the U.S. Government or EPA. The EPA does not endorse any commercial products, services, or enterprises. ------- Table of Contents Disclaimer ii List of Tables iv List of Figures v Abbreviations and Acronyms vi Acknowledgments viii Executive Summary ix I.0 Introduction 1 2.0 Scope and Application 1 3.0 Summary of Method 2 4.0 Definitions 2 5.0 Interferences 4 6.0 Health and Safety 4 7.0 Equipment and Supplies 4 8.0 Reagents and Standards 6 9.0 Sample Collection, Preservation, and Storage 7 10.0 Quality Control 8 II.0 Instrument Calibration and Standardization 10 12.0 Analytical Procedure 11 13.0 Data Analysis and Calculations 12 14.0 Method Performance 13 15.0 Pollution Prevention 14 16.0 Waste Management 14 17.0 References 14 18.0 Tables and Validation Data 16 Appendix A: Methyl Fluoroacetate Analysis 24 iii ------- List of Tables Table 1: Summary of IDC with RW 16 Table 2: Parameters tested during method development 16 Table 3: Calibration Standards 17 Table 4: Water Quality Parameters for the four water samples used in this study 17 Table 5: LC Parameters 17 Table 6: LC Gradient 18 Table 7: ESI and MS Parameters 18 Table 8: Compound specific MRM parameters 18 Table 9: DL Calculation 20 Table 10: Half-Range Prediction Interval Calculation 20 Table 11: Initial Demonstration of Accuracy and Precision 21 Table 12: % FAA remaining compared to Day 0 22 Table 13: Sample Filtration 23 iv ------- List of Figures Figure 1 Formation of fluoroacetate anion (FAA) 2 Figure 2: Representative FAA Calibration Curve. Response is the area of the QT (57.26 m/z) divided by the internal standard quantification ion (36.13 m/z) 19 Figure 3: Blank and 1 |ig/L sample chromatogram. Extracted Ion Chromatogram (EIC) of 57.26 m/z 19 Figure 4:Low Standard Chromatograms for FAA 21 Figure 5: Hydrolysis of MFA to FAA in four different water treatments 25 v ------- Abbreviations and Acronyms ANOVA Analysis of Variance CAL Calibration Standard CCC Continuing Calibration Check CF Qualification Ion CI Confidence Interval DAI Direct Aqueous Injection DL Detection Limit EIC Extracted Ion Chromatogram EPA U.S. Environmental Protection Agency ESI Electrospray Ionization ESI- Electrospray Ionization, negative mode FAA Fluoroacetate anion FAA L Labeled fluoroacetate ion (13C2, 99%; 2-2D2, 98%) FAU Formazin Attenuation Units LC Liquid Chromatography HRpir Half Range for the Prediction Interval of Results IDA Initial Demonstration of Accuracy IDC Initial Demonstration of Capability IDP Initial Demonstration of Precision IRIS Integrated Risk Information System IS Internal Standard LC Liquid Chromatograph(y) LRB Laboratory Reagent Blank LFSM Laboratory Fortified Sample Matrix LFSMD Laboratory Fortified Sample Matrix Duplicate MeCN Acetonitrile MeOH Methanol MFA Methyl Fluoroacetate MRL Method Reporting Limit MRM Multiple Reaction Monitoring MS/MS Tandem Mass Spectrometry m/z Mass to Charge Ratio NTU Nephelometric Turbidity Unit PIR Prediction Interval of Results PPE Personal Protective Equipment PTFE Polytetrafluoroethylene PVDF Polyvinylidene fluoride QC Quality Control QT Quantification Ion % R % Recovery R2 Coefficient of Determination RE Relative Error RPD Relative Percent Difference RSD Relative Standard Deviation RT Retention Time vi ------- RW Reagent Water s Standard Deviation SAM Selected Analytical Methods for Environmental Remediation and Recovery SB Sequence Blank SDS Safety Data Sheet sss Stock Standard Solution TOC Total Organic Carbon VOA Volatile Organic Analysis WS Water Source vii ------- Acknowledgments We would like to acknowledge the following individuals and organizations for their contributions towards the development and/or review of this method. United States Environmental Protection Agency (EPA) Office of Research and Development, National Homeland Security Research Center Stuart Willison, Project Officer Emily Parry, Method Development Matthew Magnuson and Jim Starr, Reviewers Centers for Disease Control and Prevention National Institute for Occupational Safety and Health Jack Pretty, Laboratory Advisor Robert Streicher, Project Officer viii ------- Executive Summary Sodium fluoroacetate, also known as Compound 1080, is a pesticide registered for use in livestock protection collars, and it is acutely toxic to humans. In water, it dissociates into the fluoroacetate anion (FAA) and could greatly impact human health if it was released into a water system, either deliberately or accidently. The U.S. Environmental Protection Agency (EPA) is responsible for supporting water utilities in their response to water system contamination. In support of this objective, EPA's National Homeland Security Research Center publishes Selected Analytical Methods for Environmental Remediation and Recovery (SAM), which provides suggested methods for numerous hazardous chemicals in a variety of matrices including drinking water. Sodium fluoroacetate, fluoroacetic acid, and methyl fluoroacetate are all recommended in SAM to be analyzed as FAA. However, the current revision of SAM does not include a verified method for analysis in drinking water. The purpose of this report is to describe a quick, robust method to quantitate FAA in drinking water. In this method, samples are preserved with ascorbic acid (for dechlorination) and sodium omadine (an anti- microbial) and filtered prior to analysis. They are analyzed by liquid chromatography tandem mass spectrometry in negative ion mode (electrospray ionization, ESI-). Total analysis time, including column re-equilibration, is 32 minutes. An initial demonstration of capability (IDC) in reagent water (RW) showed linearity over two orders of magnitude (1 |ig/L to 100 jx/L), a detection limit (DL) of 0.4 |ig/L. and a minimum reporting level (MRL) of 2 |ig/L. Accuracy and precision were 2.5% (relative error, RE) and 3.4% (relative standard deviation, RSD), respectively. FAA stability was investigated in RW, as well as in four different drinking water samples with varying characteristics (e.g., organic content, hardness, etc.). FAA appeared stable in RW and the tested drinking water sources. Also included, is application of the method for the analysis of methyl fluoroacetate after its hydrolysis to FAA. Further testing would be required before implementation. ix ------- 1.0 Introduction The U.S. Environmental Protection Agency (EPA) is responsible for supporting the response to a deliberate/accidental chemical release or natural disaster damaging water and related infrastructure. In support of this objective, EPA's National Homeland Security Research Center publishes Selected Analytical Methods for Environmental Remediation and Recovery (SAM) [1] which provides suggested methods for numerous hazardous chemicals. SAM recommends that fluoroacetic acid, fluoroacetate salts, and methyl fluoroacetate all be analyzed as the fluoroacetate anion (FAA). However, SAM does not contain a verified method for performing this analysis in drinking water. The purpose of this report is to describe a quick, robust method to quantitate FAA in drinking water. One source of FAA is Compound 1080, registered for use in the United States (RN# 56228-22) for livestock protection collars. Predators attacking the livestock bite the collars, leading to ingestion of Compound 1080 and subsequent morbidity or mortality of the predator. Compound 1080 is the sodium fluoroacetate salt that dissociates in water to fluoroacetate anion (FAA). FAA is also present after pH-dependent deprotonation of fluoroacetic acid, as well as through the hydrolysis of methyl fluoroacetate, a precursor used in pharmaceutical synthesis. Human toxicity occurs at low levels 0.7-2.1 mg/kg [2], EPA's Integrated Risk Information System (IRIS) lists a reference dose of 0.00002 mg/kg/day [3], and 0.4 |ig/L in tap water is listed in the regional screening level tables [4], However, remediation goals would be site-specific. Reports of FAA analysis by liquid chromatography-tandem mass spectrometry (LC-MS/MS) are available; however, the reported analyses require multiple sample preparation steps [5-6], and a high-throughput method would be advantageous. This report describes a direct aqueous injection (DAI) method that minimizes sample preparation time. Drinking water samples are separated on a reverse phase octylsilane (C8) column. MS/MS fragmentation provides identification and quantitation. An isotopically labeled version of FAA, 13C2, 99%; 2-2D2, 98%, (FAA L) is used as an internal standard (IS) for quantitation and quality control purposes. The method has been tested for accuracy, precision, linearity, and detection limit in reagent grade water (RW). Four additional drinking water sources were evaluated to assess their effects on method performance and FAA stability. 2.0 Scope and Application Figure 1 shows several ways the fluoroacetate anion can form in water. The dissociation of sodium fluoroacetate, the deprotonation of fluoroacetic acid, and the hydrolysis of methyl fluoroacetate can all occur, depending on the environmental conditions. The scope of this study was determination of FAA by DAI-LC-MS/MS. The method was developed and evaluated in the same laboratory. Initial Demonstration of Capability (IDC) results are summarized in Table 1. Additional water samples were tested, representing a variety of drinking water types (chlorinated, chloraminated, etc.), to determine the effect on the method or stability of FAA. Quantitation and quality control includes the use of isotopically labeled FAA as an internal standard. The following analyte was tested: Chemical Name: fluoroacetate Abbreviation: FAA Empirical Formula: C2H2FO2 Molecular mass: 77.035 g/mol 1 ------- o 'OH Fluoroacetic acid CAS: 144-49-0 o o Na Sodium fluoroacetate CAS: 62-74-8 o o F^o- Fluoroacetate anion CAS: 513-62-2 'O Methyl fluoroacetate CAS: 453-18-9 Figure 1 Formation of fluoroacetate anion (FAA) 3.0 Summary of Method A 40-mL drinking water sample was collected in a bottle and preserved with a chlorine neutralizer (ascorbic acid) and an anti-microbial agent (sodium omadine). An aliquot of 990 |iL of sample was filtered with a 0.22-|im filter, placed into an autosampler vial, and the internal standard was added. A 20-|iL injection was made into an LC equipped with a C8 column interfaced to an MS/MS operated in negative electrospray ionization (ESI-) mode. FAA was identified by comparing the mass spectra, fragment ratios, and retention times to that of FAA calibration standards analyzed under identical conditions. FAA quantification was achieved by isotope dilution. 4.0 Definitions 4.1. ANALYSIS BATCH An Analysis Batch consists of a set of samples analyzed on the same instrument within a 24-hour period and included no more than 20 field samples, beginning and ending with the appropriate continuing calibration check (CCC) standards. Additional CCCs may be required depending on the number of samples (excluding quality control (QC) samples) in the analysis batch and/or the number of field samples. 4.2. CALIBRATION STANDARD The Calibration Standard (CAL) is a solution prepared from the analyte stock standard solution, the internal standard and diluted with water. The CAL solutions are used to calibrate the instrument response with respect to analyte concentration. 2 ------- 4.3. CONTINUING CALIBRATION CHECK The Continuing Calibration Check (CCC) is a calibration standard containing the method analyte and surrogate standard. The CCC is analyzed periodically to verify the accuracy of the existing calibration. 4.4. DETECTION LIMIT The Detection Limit (DL) is the minimum concentration of an analyte that can be identified, measured, and reported with 99% confidence that the analyte concentration is greater than zero. 4.5. INTERNAL STANDARD The Internal Standard (IS) is a pure chemical added to a standard solution in a known amount and is used to measure the relative response of other method analytes that are components of the same solution. The internal standard (IS) is a chemical that is structurally similar to the method analyte, has no potential to be present in the samples, and is not a method analyte. 4.6. LABORATORY FORTIFIED SAMPLE MATRIX The Laboratory Fortified Sample Matrix (LFSM) is a field sample to which known quantities of the method analytes are added in the laboratory. The LFSM is processed and analyzed exactly like a sample, and its purpose is to determine whether the sample matrix contributes bias to the analytical results. The background concentrations of the analytes in the sample matrix must be determined in a separate sample. 4.7. LABORATORY FORTIFIED SAMPLE MATRIX DUPLICATE The Laboratory Fortified Sample Matrix Duplicate (LFSMD) is fortified and analyzed identically to the LFSM. The LFSMD is used to assess method precision when the observed concentrations of the method analytes are low. 4.8. LABORATORY REAGENT BLANK The Laboratory Reagent Blank (LRB) is a blank matrix treated exactly the same as a sample including exposure to all glassware, equipment, solvents, reagents, preservatives, and surrogate standards used in the analysis batch. The LRB is used to determine if method analytes or other interferences are present in the laboratory environment, the reagents, or the apparatus. 4.9. MINIMUM REPORTING LEVEL The minimum reporting level (MRL) is the minimum concentration that can be reported as a quantitated value for a method analyte in a sample following analysis. This defined concentration can be no lower than the concentration of the lowest calibration standard for that analyte and can be used only if acceptable QC criteria for this standard are met. 4.10. PRECURSOR ION For the purpose of this report, the precursor ion is the deprotonated molecule ([M-H]") or adduct ion of the method analyte. In tandem mass spectrometry (MS/MS), the precursor ion is mass-selected and fragmented by collision induced dissociation to produce distinctive product ions of lower mass. 4.11. PRODUCT ION For the purpose of this report, a product ion is one of the fragment ions produced in MS/MS by collision inducted dissociation of the precursor ion. 3 ------- 4.12. SAFETY DATA SHEET The Safety Data Sheet (SDS) contains written information provided by vendors concerning a chemical's toxicity, health hazards, physical properties, fire, and reactivity data including storage, spill and handling precautions. 4.13. SEQUENCE BLANKS Sequence blanks (SBs) are prepared with the same dilutant and IS as the CAL standards. They are included in all injection sequences to ensure that no contamination or interferences have occurred in the preparation of the CAL standards. They also provide verification that there is no carryover between sample injections. 4.14. STOCK STANDARD SOLUTION The Stock Standard Solution (SSS) is a concentrated solution containing the method analyte prepared in the laboratory using assayed reference materials or purchased from a reputable commercial source. Interferences Method Interferences can be caused by contaminants in solvents, reagents (including reagent water (RW)), sample bottles and caps, and other laboratory supplies or hardware that lead to discrete artifacts and/or elevated baselines in the chromatograms. All items contacting the sample were routinely demonstrated to be free from interferences (less than 1/3 the DL) under the conditions of the analysis by analysis of a LRB. Subtracting blank values from sample results was not permitted. Relatively large quantities of the preservatives were added to added to sample bottles. Trace-level organic contaminants could be present in these reagents. Interferences were monitored by analysis of LRBs. Health and Safety FAA toxicity is documented, and other chemicals used in the method have known carcinogenicity. All were treated as a potential health hazards and exposure was minimized through the proper use of personal protective equipment (PPE) and engineering controls. PPE consisted of close-toed shoes, eye protection, lab coat, and nitrile gloves A reference file of SDSs was made available to all personnel involved in chemical analyses. Equipment and Supplies References to specific equipment brands and catalog numbers are provided as examples and do not constitute an endorsement of the use of such products or suppliers. Glassware, reagents, supplies, equipment, and settings other than those listed in this report may be employed, provided that method performance is demonstrated and documented. Analyses may be performed with any system capable of performing LC-MS/MS, as long as performance criteria are met. 7.1. LC-MS/MS INSTRUMENT 7.1.1. Liquid Chromatography (LC) System An Accela 1250 (Thermo, San Jose, CA) LC pump and autosampler with a CTO-IOAVP (Shimadzu, Columbia, MD) column oven were used for method development any equivalent is acceptable. 4 ------- 7.1.2. Analytical Column Sunfire™ -dC8, 150 mm x 4.6 mm, 5 |im particle size (Waters, Milford, MA, Catalog #186002737), or equivalent. Additional columns tested are listed in Table 2. 7.1.3. Tandem Mass Spectrometer (MS/MS) System A Thermo Quantum TSQ (San Jose, CA) was used for method development. Any equivalent system may be used. 7.1.4. Data System An interfaced data system is required to acquire, store, reduce, and output mass spectral data. Xcalibur 2.2 (Thermo, San Jose CA) software was used for method development any equivalent compatible with the LC-MS/MS instrument may be used. 7.2. GLASSWARE AND MISCELLANEOUS SUPPLIES 7.2.1. Autosampler Vials Amber 2-mL autosampler vials with pre-slit Teflon® - lined screw tops (Waters Corp., Milford, MA), or equivalent. 7.2.2. Disposable Sterile Syringes 10.0 mL ± 1% accuracy BD Safety-Lok™ syringes (Catalog No. 14-829-32, Fisher Scientific, Pittsburgh, PA), or equivalent. 7.2.3. Auto Pipettes 1000 (j,L, 100 (iL and 10 (iL ± 1% accuracy. 7.2.4. Desolvation Gas Nitrogen gas generator, or equivalent nitrogen gas supply that aids in the generation of an aerosol for the ESI liquid spray and should meet or exceed instrument manufacturer's specifications. 7.2.5. Collision Gas Argon gas used in the collision cell in the MS/MS instruments and should meet or exceed instrument manufacturer's specifications. 7.2.6. Analytical Balance Accurate to 0.1 mg; reference weights traceable to Class S or S-l weights. 7.2.7. Standard Solution Flasks Class A volumetric glassware. 7.2.8. Syringe Filter Millex® GV Syringe-driven polyvinylidene fluoride (PVDF) 13-mm filter unit, 0.22 |im (Millipore Corporation, Billerica, MA, Catalog # SLGV013NL), or equivalent. 7.2.9. Sample Collection Containers Twenty- or 40-mL volatile organic analysis (VOA) vials with polytetrafluoroethylene (PTFE) screw caps 5 ------- (Environmental Sampling Supply, product no. 0040-0310-PC, San Leandro, CA), or equivalent. 7.2.10. pHMeter Accumet ® pH Meter 50 with electrode (Fisher Scientific, Pittsburg, PA), or equivalent. 7.2.11. Vortex mixer Maxi Mix II (Thermo Scientific, San Jose, CA), or equivalent. 8.0 Reagents and Standards 8.1. STANDARDS, SOLVENTS AND REAGENTS All reagents used during the course of this study were analytical grade or equivalent. 8.1.1. Standards FAA and FAA_L were purchased from Cambridge Wasotopes (Tewskbury, MA). 8.1.2. Solvents and Chemicals Solvents utilized for this study were acetonitrile (MeCN), methanol (MeOH), and reagent grade water (RW) (Fwasher, Optima®, LC-MS grade) (Waltham, MA) and were demonstrated to be free of analytes and interferences. Chemicals included acetic acid (Fwasher, Optima™, >99.7%), sodium omadine (Sigma Aldrich, >96%) and L (+)-ascorbic acid (Acros Organics™, 99%). 8.1.3. Mobile Phase A Prepared by adding 50 |iL of acetic acid to a mobile phase bottle containing 1 L of RW. Mixed and stored at room temperature. The solution expired in five days. 8.1.4. Mobile Phase B Prepared by adding 50 |iL of acetic acid to a mobile phase bottle containing 1 L of LC/MS grade MeCN. Mixed and stored at room temperature. The solution expired in 1 month. 8.1.5. Sodium Omadine Solution Prepared by transferring 0.8 g (±0.1g) of sodium omadine, accurately weighed, into a 25 mL, Class A volumetric flask. Diluted with RW and mixed by vortex. Resulting nominal concentration was 32 g/L. Prepared a lOx dilution of the stock solution (3.2 g/L). Stored solutions at 4°C (± 3°C) 8.1.6. Ascorbic Acid Solution Prepared by transferring 0.0625 g (±0.001 g) ascorbic acid, accurately weighed, into a 25 mL, Class A volumetric flask. Dissolved and diluted with RW. Mixed by vortex. Resulting nominal concentration was 2.5 g/L. Stored solutions at 4 °C (±3 °C) and prepared fresh daily. 8.1.7. Needle Wash 1 Combined 190 mL of RW, 10 mL of MeCN and 10 (iL of acetic acid. Mixed well and stored at room temperature for up to 30 days. 8.1.8. Needle Wash 2 Combined 100 mL of RW and 100 mL of MeOH. Mixed and stored at room temperature for up to 30 6 ------- days. 8.2. STANDARD SOLUTIONS 8.2.1. FAA Standard Solutions Prepared 10 mg/L FAA stock solution by diluting 100 |iL of the purchased 1000 mg/L FAA solution (> 98% purity) into 10 mL of RW. Transferred the remaining purchased stock to a 4-mL vial. Prepared 1- mg/L by diluting 1 mL of the 10-mg/L FAA solution into 10 mL of RW. Prepared 50-(ig/L by diluting of 500 |iL of 10 mg/L solution into 10 mL of RW. Peak areas and calibration slopes were monitored for consistency to ensure integrity of the solutions. Stored all solutions at 4 °C (± 3°C) for a maximum of six months. 8.2.2. IS Solutions Prepared 10 mg/L FAA_L stock solution by diluting 100 |iL of the purchased 1000-mg/L FAA_L solution into 10 mL of RW. Transferred the remaining purchased stock to a 4-mL vial. Prepared 2.5-mg/L standard by diluting 2.5 mL of the 10 mg/L FAA solution into 10 mL of RW. Stored all solution at 4 °C (± 3 °C) for a maximum of six months. 8.2.3. Calibration Standards Prepared the calibration (CAL) standards by transferring a set amount of FAA dilution stocks into 1-mL vials. Added the 3.2-g/L sodium omadine solution (20 jxL) and 2.5-/L ascorbic acid solution (100 |iL) to each CAL standard. Added the 2.5-mg/L FAA L IS solution (10 jxL) to each vial, and diluted to volume with RW and mixed by vortex. Table 3, Calibration Standards, details the dilution series. These samples were also used as the CCC. 9.0 Sample Collection, Preservation, and Storage 9.1. SAMPLE COLLECTION FROM TREATMENT UTILITIES Treated water samples were obtained from four drinking water utilities across the U.S. Utilities were selected to provide samples that had varying water quality parameters (Total Organic Carbon (TOC), conductivity, etc.) and residual disinfectant (chlorine or chloramine) to determine whether these parameters had any effect on FAA stability or the method. Tested water quality parameters for the four water sources (WS) are listed in Table 4. 9.2. SAMPLE PRESERVATION Drinking water samples were preserved to prevent any compound degradation from residual chlorine and microbial degradation. Dechlorination was achieved with ascorbic acid (250 mg/L, final concentration) and sodium omadine (64 mg/L, final concentration) served as an anti-microbial. Additional additives that were tested, but did not achieve optimal performance are listed in Table 2. 9.3. WATER FILTRATION STUDY High and low FAA concentrations in RW were prepared to assess loss during filtration. An aliquot of each sample was analyzed, filtered and unfiltered. Samples were prepared by adding sodium omadine (80 |iL) and ascorbic acid (10 mg) to two separate 40-mL VOA vials filled with RW. Either 16 or 160 jxL of 10 mg/L FAA stock was added to create a low (4 |ig/L) or high (40 |ig/L) concentration sample. Four aliquots were removed, filtered, and placed in LC vials. Four additional replicates were removed from the VOA vial (unfiltered). All replicates were fortified with FAA_L, mixed by vortex, and analyzed. 7 ------- 9.4. HOLDING TIME STUDY IN REAGENT WATER A holding time study was performed to determine the stability of RW samples spiked with FAA for up to 28 days. The following preparation scheme was used: 1. Added 40 mL of RW to a VOA vial. Preserved by adding 80 jxL of 32 g/L of sodium omadine and 10 mg of ascorbic acid with final concentrations of 64 mg/L and 250 mg/L, respectively. Added 40 mL of RW and mixed by vortex. 2. Added either 16 (iL or 160 (iL of 10 mg/L FAA solution to obtain concentrations of either 4 |ig/L or 40 |ig/L. respectively. Mixed by vortex. 3. Withdrew ~ 4 mL of sample solution with a syringe, fixed a 0.22-|im filter onto the syringe, and dispensed the solution into a clean container. Aliquoted 990 jxL of filtered solution into each LC vial (n=3). 4. Added 10 jxL of 2.5 mg/L FAA L solution to each LC vial. Capped and mixed by vortex. 5. Placed the remaining sample solution into 4 °C (± 3 °C) storage conditions. 6. Analyzed samples and appropriate QCs. 7. Repeated steps 3-6 for each subsequent time point (1, 3, 7, 14, 21, 28 days). 9.5. WATER STABILITY STUDY IN TAP WATERS Water from four different sources representing a variety of water types (chlorinated, chloraminated, hard water, organic content) was prepared and tested with the procedure described in Section 9.4. 10.0 Quality Control Quality control (QC) requirements included the Initial Demonstration of Capability (IDC) and ongoing QC requirements to be met when preparing and analyzing samples. This section describes the QC parameters, their required frequency, and the performance criteria required to meet quality control objectives. 10.1. INITIAL DEMONSTRATION OF CAPABILITY The Initial Demonstration of Capability (IDC) was successfully performed prior to analyzing any field samples. Prior to conducting the IDC, an acceptable initial calibration following the procedure outlined in Section 11.3 was generated. The following subsections describe how the IDC was generated for this report. 10.1.1. Initial demonstration of low system background A Laboratory Reagent Blank (LRB) was prepared by analyzing blank RW prepared with the same additives as a standard (i.e., ascorbic acid and sodium omadine) and internal standards. To be acceptable, method analytes were not detected in the LRB at concentration > 1/3 of the DL. 10.1.2. Minimum Reporting Level Confirmation Seven replicates of the minimum reporting level were prepared at the CAL 2 level (see Table 3) and 8 ------- analyzed. The mean measured concentration and standard deviation for the method analytes in the seven replicates were calculated, and the Half Range for the Prediction Interval of Results (HRPir) was determined using the following formula: HRpir =3.963s where: s = standard deviation 1.963 = a constant value for seven replicates The upper and lower limits for the Prediction Interval of Result (PIR) were required to meet the following upper and lower recovery limits based on the following formulas: The upper PIR limit requirement was < 150% recovery Mean + HRpir Fortified Concentration The lower PIR limit requirement was > 50% recovery Mean — HR x 100% < 150% PIR x 100% > 50% Fortified Concentration 10.1.3. Detection Limit Determination The Detection Limit (DL) was verified by the preparation and analysis of seven (7) replicates of a standard concentration 1 Ox the noise limit, and 7 LRB replicates were prepared in tandem over the course of three (3) days. The DL was calculated using the following formula: DL — S * t(n-l, l-a=0.99) where: s = standard deviation of replicate analyses t(n-l, l-a=0.99) =3.143 n = number of replicates 10.1.4. Initial Demonstration of Precision Seven (7) replicates of CAL 5 (Table 3) were prepared for the Initial Demonstration of Precision (IDP) study and analyzed. To pass acceptability criteria, the calculated relative standard deviation from the replicate analyses was required to be < 20%. 10.1.5. Initial Demonstration of Accuracy The same seven (7) replicates of CAL 5 that were generated for the IDP study were used for the Initial Demonstration of Accuracy (IDA) study. To pass acceptability criteria, the calculated mean recovery from the replicate analyses was required to be ± 30%. 10.2 ONGOING QC REQUIREMENTS 10.2.1. Laboratory Reagent Blanks An LRB was required with each analysis batch (Sect. 4.8) to confirm that background contaminants were not interfering with the identification or quantitation of FAA. If the LRB showed a peak within the RT window of FAA, the source of the contamination was determined and eliminated before processing samples. Background from FAA or other contaminants that interfere with the measurement must be below 1/3 of the MRL. Blank contamination was estimated by extrapolation, if the concentration was below the lowest CAL standard. This extrapolation procedure was not allowed for sample results as it may not meet data quality objectives. If FAA was detected in the LRB concentrations equal to or greater 9 ------- than 1/3 the MRL, then all data must be considered invalid for all samples in the analysis batch. 10.2.2. Sequence Blanks Sequence Blanks (SBs) were run in tandem with the CCCs, bracketing every five samples, to periodically verify a background free of interferences and FAA contamination/carryover. 10.2.3. FAA verification FAA identification was confirmed by elution of the sample peak within ± 1.5 % of the average RT observed in the current calibration. Additional confirmation was provided by the presence of the CF/QT ratio comparable (± 30 %) to the CF/QT ratio in the most recent calibration. 10.2.4. Continuing Calibration Check The calibration was confirmed by analysis of a CCC at the beginning and end of a sample analysis batch. CCCs were then injected after every five samples alternating between a high level (CAL 8) and a low level (CAL 2) to ensure instrument sensitivity and stability. The following requirements were required to be met for a batch to meet acceptability criteria: 1. The absolute area counts of the IS had to be within 50-150% of the average area measured in the most recent calibration. 2. The calculated amount for FAA in the high level CCCs had to be within ± 30% of the true value. 3. The calculated amount for FAA in the low level CCCs had to be within ± 50% of the true value. 10.2.5. Internal Standard The analyst monitored internal standard (IS) areas in all injections during the analysis. The IS peak areas in any chromatographic run were within 50-150% of the average IS area in the most recent calibration curve. If the IS areas did meet this criterion, inject a second aliquot from the same autosampler vial was injected. 10.2.6. Laboratory Fortified Sample Matrix Analysis of a Laboratory Fortified Sample Matrix (LFSM) was required in each analysis batch and was used to determine that the sample matrix did not adversely affect method accuracy. Assessment of method precision was accomplished by analysis of a Laboratory Fortified Sample Matrix Duplicate (Sect. 4.7). 10.5. STABILITY STUDIES All stability batches must meet CCC requirements to be acceptable. The concentrations of the stored (stability) samples were compared to the concentrations analyzed at time zero and % R calculated. To generate a % R for the reference value, Day 0 replicates were compared to their mean value and a % R generated. One-way analysis of variance (ANOVA) across RW and drinking water samples relating % R and time as a factor must show no significance (a =0.05) for FAA to be considered stable. 10.6. WATER FILTRATION STUDY The average concentration of the filtered samples was compared to the average concentration of the non- filtered samples at both concentrations. To be reported as comparable, the sample sets should show no significance when compared via a one-sided t-test with a =0.05. 11.0 Instrument Calibration and 10 ------- Standardization 11.1. LC INSTRUMENT AND PARAMETERS The LC method parameters are in Table 5 and the gradient is described in Table 6. 11.2. ESI-MS/MS The [M-H]" signal was optimized for FAA and FAAL by infusing approximately 1 |ig/m L of each analyte directly into the MS. The MS parameters were optimized for the precursor m/z, the product m/z value and the collision energies were determined. See Table 7 for the optimized ESI-MS/MS conditions and Table 8 for the Multiple Reaction Monitoring (MRM) Transitions. 11.3. INITIAL CALIBRATION Nine CAL standards were used in this report. The lowest CAL was required to be at or below the MRL. The curve was created by normalizing FAA peak area by the IS response. The LC-MS/MS data system software was used to generate a linear regression calibration curve with 1/x weighting. 11.4. CALIBRATION CURVE Linearity was studied over two orders of magnitude. Calibration curves consisted of nine nonzero samples (each at different concentrations) covering the nominal concentration range of 1 |ig/L - 100 |ig/L. An SB was analyzed at the beginning and end of the series of CAL injections. Plots of the peak area divided by internal standard area versus nominal standard concentration were constructed using a best-fit line determined by regression analysis. A curve-weighting factor of 1/x with linear regression was utilized. Linearity was acceptable at a coefficient of determination (R2) > 0.99 11.5. CALIBRATION ACCEPTANCE CRITERIA Each calibration point (except the lowest point) should calculate to be within 70-130% of its true value. The lowest CAL point should calculate to be within 50-150% of its true value. 11.6. CONTINUING CALIBRATION CHECK The initial calibration must be verified at the beginning and end of each group of analyses, and after every fifth sample. Continuing Calibration Checks (CCCs) alternated between a low and medium concentration CAL standard. The absolute areas of the quantitation ions of the IS must be within 50% - 150% of the average areas measured in the most recent calibration. Additionally, the calculated amount for each analyte for medium level CCCs must be within ± 30% of the true value and ± 50% at the lowest calibration level. 12.0 Analytical Procedure The following procedure was used for preparation of samples for analysis (i.e., Stability Samples, LFSM and LFSMD, etc.) 1. Filter > 1 mL of sample water into a clean container 2. Transfer 990 |iL of filtered sample into an autosampler vial. 3. Add 10 fxL of FAA L to each samples. 11 ------- 4. Mix by vortex and cap for analysis. 13.0 Data Analysis and Calculations 13.1. DESCRIPTIVE STATISTICS Descriptive statistics [mean, standard deviation (s), relative standard deviation (% RSD), relative error RE) and percent difference] were calculated for this method. 13.1.1. Sample concentration Results were expressed as a concentration based on the calibration curve. The concentration was calculated as follows: , ¦ / rr \ ((response-v int)\ Sample concentration (|ig/L) = ^ ) where: response = Peak area of the analyte versus IS in the sample y int = y-intercept obtained from the calibration curve slope = slope obtained from the calibration curve 13.1.2. Method Accuracy Method accuracy was expressed as percent relative error (% RE) which was calculated based on the nominal concentration as follows: % Relative error = N^ x 100 N where: D= determined concentration N= nominal concentration 13.1.3. Method Precision Method precision was expressed as percent relative standard deviation (% RSD) when the number of samples (n) > 3 and was calculated as follows: % Relative Standard Deviation = x 100 where: s= standard deviation X= mean 13.1.4. Confidence Interval To provide estimation of probability of sample mean, the 95% confidence interval was calculated as follows: 95% Confidence Interval = X ± 1.96 -7= \]n where: X= mean s= standard deviation n= number of samples 13.1.5. Stability To evaluate stability, the mean concentration after the storage time was compared to the mean concentration at Time 0 as follows: % of Time 0= -xl00 Y where: X= mean concentration after storage time Y= mean determined concentration at Time 0. 12 ------- 14.0 Method Performance 14.1. LINEARITY The coefficient of determination (R2) for the calibration standards of a particular batch was required to be 0.99 or greater for the batch to be acceptable. Calculated concentrations for each point should be within 70-130 % of the nominal value and 50-150% for the lowest CAL point. An example calibration curve is shown in Figure 2. In the displayed instance, R2 = 0.9983, and the greatest deviation from the nominal concentration was 116% (16% RE) at the lowest calibration level, 1 (.ig/L. 14.2. CONTINUING CALIBRATION CHECKS CCC primarily performed within parameters. If a CCC failed, the samples bracketed by the failing check were re-run. 14.3. INITIAL DEMONSTRATION OF LOW SYSTEM BACKGROUND FAA was not detected in a sample spiked with preservatives and internal standards at concentrations that were > 1/3 of the DL. A representative chromatogram of a blank sample is shown in Figure 3. 14.4. MINIMUM REPORTING LEVEL CONFIRMATION The MRL was determined from seven replicates of samples at the CAL 2 level. The HRpir was determined to be 0.65 |ig/L. Based on this result, the lower PIR was calculated to be 57.0% and the upper PIR to be 122%. These values meet both the upper and lower PIR limit requirements of < 150% for the upper PIR and > 50% for the lower PIR. The MRL confirmation is shown in Table 10. Representative chromatograms of both 1 |ig/L and 2 |ig/L. the MRL, are shown in Figure 4. 14.5. DETECTION LIMIT DETERMINATION FAA detection limit was determined from seven replicates of samples at the CAL 3 level, with batches prepared over three days. The DL was calculated to be 0.4 |ig/L. using at value of 3.143 for n=7. The DL calculation is presented in Table 9. 14.6. INITIAL DEMONSTRATION OF PRECISION The IDP was determined from seven replicates at the CAL 5 concentration level, calculated versus a calibration curve. The precision (% RSD) was 3.4%. This value was within the % RSD acceptability criteria of < 20 %. The IDP results are summarized in Table 11. 14.7. INITIAL DEMONSTRATION OF ACCURACY The IDA was determined from the same seven CAL 5 replicates that were used for the IDP study, calculated vs. a calibration curve. The IDA (% RE) was 2.46%. This value was within the % RE acceptability criteria of ± 30%. The nominal value was within the confidence interval. The IDA results are summarized in Table 11. 14.8. HOLDING TIME STUDY IN REAGENT WATER Both concentration levels (40 and 4 |ig/L) of FAA in RW were compared across all time points. One-way ANOVA analysis showed no significant difference (p=0.34) in the recovery during the 28-day test period. The data set is available in Table 12. 13 ------- 14.9. HOLDING TIME STUDY IN DRINKING WATER Stability was tested at two concentrations in four different drinking waters. The data set is available in Table 11. FAA met the stability criteria (Section 10.5) for all drinking water samples (WS 1-4) across the 28-day time frame with a one-way ANOVA showing no statistical difference (p=0.08). 14.10. WATER FILTRATION One-sided t-tests (a=0.05) showed no difference in the means of the unfiltered and filtered water samples for both concentration levels. The water filtration study results are summarized in Table 13. Filtering the samples at either high or low concentrations did not affect the recovery of the target analyte. 15.0 Pollution Prevention This method utilized ESI-LC/MS/MS for the analysis of FAA in water. The method required the use of small volumes of organic solvent and small quantities of pure analytes, thereby minimizing the potential hazards to both the analyst and the environment. All waste was collected and disposed of in accordance with regulations. 16.0 Waste Management The analytical procedures described in this method generated relatively small amounts of waste since only small amounts of reagents and solvents were used. The matrices of concern were finished drinking water. Laboratory waste management practices were in adherence with all applicable rules and regulations. The laboratory protected the air, water and land by minimizing and controlling all releases from fume hood and bench operations. Compliance with any sewage discharge permits and regulations, particularly the hazardous waste identification rules and land disposal restrictions were followed. 17.0 References 1. U.S. EPA. Selected Analytical Methods for Environmental Remediation and Recovery (SAM) 2012. U.S. Environmental Protection Agency, Washington, DC, EPA/600/R-12/555, 2012 2. Connolly, G. Technical Bulletin for the Sodium Fluoroacetate (Compound 1080) Livestock Protection Collar EPA Registration Number: 56228-22; U.S. Department of Agriculture, Animal and Plant Health Inspection Service, Wildlife Services: Riverdale, MD, February 2017. 3. U.S. EPA. 1991. Integrated Risk Information System. Retrieved from US EPA website: http://www.epa.gov/iris/subst/0469.htm [11/01/2017], 4. U.S. EPA. Regional Screening Levels (RSLs) - Generic Tables (June 2017). Retrieved from US EPA website: https://www.epa.gov/risk/regional-screening-levels-rsls-generic-tables-iune-2017 5. Bessaire, T., Tarres, A., Goyon, A., Mottier, P., Dubois, M., Tan, W.P., Delatour, T. Quanitative determination of sodium monofluoroacetate "1080" in infant formulas and dairy products by isotope dilution LC-MS/MS. Food Additives & Contaminants: Part A, 2015, 32 (11) 1885-1892. 6. Hamelin, E.I., Mawhinney, D.B., Parry, R., Kobeleski, R.J. Quantification of monofluoroacetate and monochloroacetate in human urine by isotope dilution liquid chromatography tandem mass spectrometry. Journal of Chromatography B, 2010, 878, 1045-1050. 14 ------- 7. CSID:5046, http://www.chemspider.com/Chemical-Structure.5046.html (accessed 15:06, Nov 30, 2017) 15 ------- 18.0 Tables and Validation Data Table 1: Summary of IDC with RW Test Parameter Fluoroacetate Anion Matrix RW Quantitation LC-MS/MS Regression Type Linear (1/x) Linear Range 1 |jg/L to 100 |jg/L IDC Tests Acceptance Criteria Results Coefficient of Determination NA >0.99 (R2) Minimum Reporting Level <150% Upper PIR Limit > 50% Lower PIR Limit Upper limit = 122 % Lower Limit = 56.9% Detection Limit Determination NA 0.41 (jg/L Initial Demonstration of Low Background < 1/3 of minimum Non-detect System Background reporting Level Initial Demonstration of < 20 % RSD 3.4 % Precision Initial Demonstration of ± 30 % Mean Recovery (RE) 2.5 % Accuracy FAA Holding Time Study3 Test p-valueb Reagent Water (RW) One-way ANOVA 0.34 Drinking Water (WS 1-4) One-way ANOVA 0.09 statistical results for both RW and drinking water samples across all time points bResults considered significant if p < 0.05 Table 2: Parameters tested during method development Test Parameters Test System Performance Ammonium acetate (2 Added as a dechlorinating Caused FAA peak .. M) - Final agent with sodium broadening Preservatives concentration in omadine as an anti- sample 1.5 g/L microbial Columns Waters BEH Amide column SIELC Primesep B2 Water + 0.01 % formic acid + 5mM ammonium formate, gradient elution Direct aqueous injection is incompatible with hydrophilic interaction chromatography 60:40 MeCN: Water + 0.05% Acetic acid FAA RT not stable among waters with different pH values. 16 ------- Table 3: Calibration Standards Calibration Source Source 2.5 g/L 3.2 g/L 2.5 mg/L Final Nominal Solution Solution Solution Ascorbic Sodium FAA L Volume Solution Name Volume Acid Omadine Standard of Con- (ML) Solution Solution Solution Solution centration Volume (ML) (ML) (mL) (mq/l) (ML) CAL 1 0.05 mg/L FAA 20 100 20 10 1 1 CAL 2 0.05 mg/L FAA 40 100 20 10 1 2 CAL 3 0.05 mg/L FAA 80 100 20 10 1 4 CAL 4 0.05 mg/L FAA 160 100 20 10 1 8 CAL 5 1 mg/L FAA 12 100 20 10 1 12 CAL 6 1 mg/L FAA 20 100 20 10 1 20 CAL 7 1 mg/L FAA 40 100 20 10 1 40 CAL 8 1 mg/L FAA 60 100 20 10 1 60 CAL 9 1 mg/L FAA 100 100 20 10 1 100 Table 4: Water Quality Parameters for the four water samples used in this study WS 1 WS 2 WS 3 WS 4 Date Collected 2/22/2017 2/23/2017 8/01/2017 8/10/2017 PH 9.35 8.8 7.14 7.69 Turbidity3 0.27 NTU 0.05 NTU 0.28 NTU 0 FAU Conductivity (MS/cm) 348 287 1124 388 Alkalinity (mg/L) 76 63 346 65 Hardness (mg/L) 76 114 436 127 Free chlorine (mg/L 0.03 1.3 0.4 0.07 Chloramine (mg/L) NA NA NA 3.11 (mono) Total Organic Carbon (mg/L) 7.85 1.13 NA NR aNTU=Nephelometric Turbidity Unit, FAU=Formazin Attenuation Units Table 5: LC Parameters Parameter Setting Mobile Phase A 0.005% Acetic Acid in water Mobile Phase B 0.005% Acetic Acid in MeCN Column Sunfire™ -dC8, 150 mm x 4.6 mm, 5 |jm particle size or equivalent Column 37 °C Temperature Injection Volume 20 (jL 17 ------- Table 6: LC Gradient TIME %A %B FLOW LC FLOW (MIN) RATE (mL/min) 0 95 5 0.5 Waste 1 95 5 0.5 11 60 40 0.5 Instrument (10 to 21 14 0 100 0.5 min) 19 0 100 0.5 20 95 5 0.5 21 80 20 1 Waste 24 0 100 1 30 95 5 1 30.5 95 5 0.5 32 95 5 0.5 Die 7: ESI and MS Parameters Parameter Value Mass Spectrometer Thermo Quantum TSQ Software Thermo Xcalibur 2.2 SP1.48 Ionization Mode Heated Electrospray, negative Scan Mode Multiple reaction monitoring (MRM) Spray Voltage 3300 V Vaporizer Temperature 250 °C Sheath Gas Pressure 50 Ion Sweep Gas 0 Auxiliary Gas Pressure 0 Capillary Temperature 275 °C Capillary Offset -85 Tube Lens Offset -90 Collision Gas Pressure 0.7 Table 8: Compound specific MRM parameters COMPOUND NAME FLUOROACETATE 13C2,2-2D2 FLUOROACETATE aQT=Quantification transition, CF=Confirmation transition MONITORED DWELL COLLISION RETENTION TYPE TRANSITION TIME (MS) ENERGY (EV) TIME (MIN) 77.1 > 57.26 100 14 19.4 QT 77.1 > 33.04 100 14 19.4 CF 80.95 >36.13 100 14 19.4 QT 80.95 > 59.9 100 14 19.4 CF 18 ------- y=0.01736-0.0154x R2=0.9983 w:1/x 40 60 80 Concentration (jjg/L) 100 120 Figure 2: Representative FAA Calibration Curve. Response is the area of the QT (57.26 m/z) divided by the internal standard quantification ion (36.13 m/z). 4000 1 ppb FAA blank £>• £>' -T 4?' ^ Time Figure 3: Blank and 1 pg/L sample chromatogram. Extracted Ion Chromatogram (EIC) of 57.26 m/z. 19 ------- Table 9: DL Calculation Nominal Determined Sample Concentration Concentration (Mg/L) (Mg/L) DL CAL 3 Day 1 4 4.2 DL CAL 3 Day 2 4.1 DL CAL 3 Day 2 3.8 DL CAL 3 Day 2 4.2 DL CAL 3 Day 3 4.0 DL CAL 3 Day 3 4.1 DL CAL 3 Day 3 4.0 Average 4.0 s 0.13 Calculated DL 0.4 Table 10: Half-Range Prediction Interval Calculation Sample Name Nominal Determined Concentration Concentration Level (Mg/L) (Mg/L) DL CAL 2 1 1.7 DL CAL 2 2 2.0 DL CAL 2 3 2.0 DL CAL 2 4 2 1.7 DL CAL 2 5 1.6 DL CAL 2 6 1.9 DL CAL 2 7 1.6 Average 1.8 s 0.2 HRpir 0.65 20 ------- Table 11: Initial Demonstration of Accuracy and Precision Sample Nominal Concentration (mq/l) Determined Concentration 1 DP/I DA Cal 5_1 12.1 I DP/I DA Cal 5_2 11.5 I DP/I DA Cal 5_3 11.3 I DP/I DA Cal 5_4 12 11.2 I DP/I DA Cal 5_5 12.2 I DP/I DA Cal 5_6 11.7 I DP/I DA Cal 5_7 11.9 Average 11.7 s 0.397 Precision (% RSD) 3.4% Accuracy (% RE) 2.5% 95% Confidence 11.7 ±0 .29 interval 3000 2500 2000 g 1500 1000 500 1 |jg/L CF 1 |jg/L QT 0 16.0 16.8 17.7 18.6 19.5 20.4 Time (min) 17.7 18.6 Time (min) 20.4 Figure 4:Low Standard Chromatograms for FAA 21 ------- Table 12: % FAA remaining compared to Day 0. Day 0 Day 1 Day 3 Day 7 Day 14 Day 21 Day28/31a RW Low3 Average (Mg/L) 3.3 3.7 3.9 3.7 3.7 3.5 3.5 % RSD 5 9 6 8 11 5 12 % of day 0 ref 112 120 114 111 107 108 RW High Average (Mg/L) 44.9 39.0 43.4 39.0 40.0 41.9 38.2 % RSD 8 3 6 13 8 4 13 % of day 0 ref 87 97 87 89 93 85 WS 1 Low3 Average (Mg/L) 3.9 3.9 3.3 4.3 3.7 3.5 3.5 % RSD 13 8 10 5 9 7 8 % of day 0 ref 101 86 110 94 89 91 WS1 High3 Average (Mg/L) 41.1 39.5 36.9 39.0 41.8 38.0 44.2 % RSD 4 5 7 2 3 2 11 % of day 0 ref 96 90 95 102 92 107 WS 2 Low3 4.0 3.8 3.7 3.8 3.2 3.3 3.0 Average (Mg/L) % RSD 15 7 2 10 15 10 26 % of day 0 ref 94 92 95 80 81 76 WS 2 High3 41.5 39.2 37.6 40.2 38.3 36.8 45.3 Average (Mg/L) % RSD 2 4 1 4 1 4 8 % of day 0 ref 94 91 97 92 89 109 WS 3 Low Average (Mg/L) 4.0 3.6 2.5 3.1 3.0 3.1 2.4 % RSD 5 13 13 6 33 14 15 % of day 0 ref 89 63 76 74 78 59 WS 3 High Average (Mg/L) 40.3 38.5 36.2 40.0 38.4 44.4 44.2 % RSD 11 3 3 9 8 9 3 % of day 0 ref 96 90 99 95 110 110 WS 4 Low Average (Mg/L) 4.0 4.0 3.9 4.0 4.0 3.5 3.6 % RSD 1 10 21 11 20 9 29 % of day 0 ref 100 99 101 99 88 90 WS 4 High Average (Mg/L) 40.6 37.3 36.6 41.3 37.4 39.8 37.4 % RSD 3 2 5 6 3 2 5 % of day 0 ref 92 90 102 92 98 92 aFinal measurement was conducted after 31 days. All values are calculated from three replicates. RSD = relative standard deviation. Ref = Day 0 was the comparison value so recovery is considered 100%. 22 ------- Table 13: Sample Filtration Condition Low Concentration3 High Concentration13 Average0 3.4 37.3 Unfiltered s 0.84 2.22 RSD 25% 6% Average0 3.0 38.5 Filtered s 0.61 3.33 RSD 20% 9% p-valued 0.25 0.29 aLow concentration = 4 |ig/L bHigh concentration = 40 |ig/L cn=4 done-tailed t-test, a=0.05 23 ------- Appendix A: Methyl Fluoroacetate Analysis The FAA method was evaluated to determine if it could be used to measure Methyl fluoroacetate (MFA), a hydrolysis product of FAA. MFA spiking experiments verified that the transformed product could be measured using the FAA method. Preliminary experiments, results and important considerations are discussed below. A. 1. INTRODUCTION Methyl fluoroacetate (MFA) is a methylated analog of FAA, which has similar toxicity (3-4 mg/kg, orally, rat) [1]. MFA is available from U.S. vendors and is used as a pharmaceutical precursor or as a starting material during consumer product formulation. MFA is vulnerable to both acid- and base-catalyzed hydrolysis in water, transforming into the free acid, FAA. Base-catalyzed hydrolysis is purported to be faster. Price and Jackson [2] reported a minimum rate of hydrolysis between pH of 2-3.5 and a hydrolysis half-life of less than an hour at pH=7. Structure-based calculations (estimated by EPI Suite and accessed on ChemSpider) estimate a half-life of 9.67 h at pH =8 and a half-life of 4.03 days at pH=7 [3] at 25°C. MFA is also more volatile than FAA with Henry's constant of 3.49 xlO"4 atm m3/mol [3], making the loss to volatilization from water more likely than with the free acid. Preliminary experiments were conducted to verify hydrolysis of MFA in tap water, examine the effect of water pH, and whether FAA measurements would be a feasible way to characterize contamination levels. Accordingly, tests were evaluated in RW and WS 2 utilizing the procedures described for FAA analysis. A.2. MATERIALS AND METHODS MFA was purchased from TCI America (Portland, OR) and diluted to 10 mg/L in MeCN. Four water samples were prepared, two with RW and two with WS 2. One of each water type was preserved as described in the FAA method (Section 9.0). A method blank was also prepared with preservatives. After mixing, the pH was measured. Samples were fortified to 0.54 (j,M (50 |ig/L) of MFA, and the time was recorded. Filtered samples were aliquoted into LC vials. Sample handling, analytical and calculation procedures were as described for FAA. Samples were injected in sets of five (i.e., one injection of each treatment) interspersed with CCC and blanks. Each set (~ 3.5 h) was marked as one time point and the total covered approximately 24 h. While awaiting injection, samples were stored in the sample tray at 10°C. A 3. RESULTS Water pH ranged from 3.71 to 8.5, and values are listed associated with the treatment in Figure 5. Water utilities typically try to maintain a basic pH to minimize corrosion, but the pH range can span 6-8.5. Addition of the ascorbic acid preservative lowered water pH to outside that range, so it is not strictly representative of tap water other than to show the effect of preservatives/pH. The MFA stock in MeCN was also analyzed and showed a consistent level of -0.078 (iM (7 |ig/L) FAA, demonstrating the transformation of MFA without the presence of water. The method blanks showed a consistent level of FAA. During the FAA method development, no other method blank samples showed FAA contamination. Given the position of the LRB in sample injection sequence, two contamination scenarios are plausible. First, the LRB was contaminated during preparation due to the more volatile nature of MFA. Second, the LRB was run at the end of the five injection series. It is possible that the untransformed MFA was retained and transformed into FAA on the column and eluted during subsequent injections. The second explanation seems slightly more likely since the LRB was the same preparation as the RW plus preservatives (RW + P) and had a relatively low pH. 24 ------- Only the MFA in the tap water source (no preservatives) (Tap) displayed complete transformation to FAA over the course of 24 h. No transformation was observed with MFA in tap water source plus preservatives (Tap + P) This result is consistent with previous literature on hydrolysis rates and the estimated physicochemical properties of MFA. In all other water treatments, the pH was too low (<6.5) to facilitate hydrolysis in the studied time-frame. Time point Figure 5: Hydrolysis of MFA to FAA in four different water treatments. A.4. CONCLUSIONS 1. The alkaline pH maintained by many water utilities will likely begin MFA conversion to FAA upon its addition. However, it may take many half-lives (depending on amount added) to achieve complete transformation. 2. Due to MFA volatility, sample collection may need to be headspace-free to reduce losses. 3. If using FAA to estimate MFA contamination, exhaustive hydrolysis of samples may need to be conducted to ensure complete conversion prior to analysis. Otherwise, toxicity may be underestimated due to presence of MFA that is not converted. Likely this will include pH adjustment to pH >8 and vigorous shaking for longer than 24 h. A.5. REFERENCES 1. Floss, G.L., The Toxicology and Pharmacology of Methyl Fluoroacetate (MFA) in Animals with Some Notes on Experimental Therapy. Brit. L. Pharmacol. (1948), 3, 118. 2. Price, C.C.; Jackson, W.G., Some Properties of Methyl Fluoroacetate and Fluoroethanol, Journal of the American Chemical Society (1947), 69(5) 1065-1068. 3. CSID:9565, http://www.chemspider.com/Chemical-Structure.9565.html (accessed 14:57, Nov 30, 2017) 25 ------- vvEPA United States Environmental Protection Agency PRESORTED STANDARD POSTAGE & FEES PAID EPA PERMIT NO. G-35 Office of Research and Development (8101R) Washington, DC 20460 Official Business Penalty for Private Use $300 ------- |