Document mBgMn324q9kw5p19ZxbJ6adGB

ANALYTICAL METHOD VALIDATION FOR THE DETERMINATION OF PERFLUOROOCTANESULFONATE, POTASSIUM SALT (PFOS) IN PLANT TISSUES WILDLIFE INTERNATIONAL, LTD. PROJECT NUMBER: 454C-125 3M Environmental Lab Project No. U2723 AUTHORS: Raymond L. Van Hoven, Ph.D. Jon A. MacGregor, B.S. Willard B. Nixon, Ph.D. STUDY INITIATION DATE: August 28, 2001 STUDY COMPLETION DATE: October 22,2001 Submitted to: 3M Corporation Environmental Laboratory 935 Bush Avenue St. Paul, Minnesota 55106 Wildlife International, Ltd. 8598 Commerce Drive Easton, Maryland 21601 (410)822-8600 Page 1 of 51 Wildlife International. Ltd. 2- - Project Number 454C-125 GOOD LABORATORY PRACTICE COMPLIANCE STATEMENT SPONSOR: 3M Corporation TITLE: Analytical Method Validation for the Determination of Perfluorooctanesulfonate., Potassium Salt (PFOS) in Plant Tissues WILDLIFE INTERNATIONAL, LTD. PROJECT NUMBER: 454C-125 3M ENVIRONMENTAL LAB PROJECT NUMBER: U2723 STUDY COMPLETION: October 22,2001 This study was conducted in compliance with Good Laboratory Practice Standards as published by the U.S. Environmental Protection Agency in 40 CFR Part 160,17 August 1989 and OECD Principles of Good Laboratory Practice (ENV/MC/CHEM (98) 17) with the following exceptions: The stability of the test substance under storage conditions at the test site was not determined in accordance with Good Laboratory Practice Standards. STUDY DIRECTOR: Raympnd Z . Van Hoven, Ph.D. Scientist Wildlife International, Ltd. tO-Sa-Ol DATE SPONSOR APPROVAL: Wildlife International. Ltd. -3 - Project Number 454C-125 QUALITY ASSURANCE STATEMENT This study was examined for compliance with Good Laboratory Practice Standards as published by the U.S. Environmental Protection Agency in 40 CFR Part 160,17 August 1989 and OECD Principles of Good Laboratory Practice (ENV/MC/CHEM (98) 17). The dates of all inspections and audits and the dates that any findings were reported to the Study Director and Laboratory Management were as follows: ACTIVITY: Matrix Fortification Preparation Raw Data, Draft Report Final Report DATE CONDUCTED: DATE REPORTED TO: STUDY DIRECTOR: MANAGEMENT: August 30,2001 August 30,2001 September 4,2001 October 11,12 and 15,2001 October 15,2001 October 16,2001 October 22,2001 October 22,2001 October 22,2001 M ( j l ( L /yV James H. Coleman, B.S. Quality Assurance Representative DATE Wildlife International. Ltd._____ -4 - Project Number 454C-125 REPORT APPROVAL SPONSOR: 3M Corporation TITLE: Analytical Method Validation for the Determination of Perfluorooctanesulfonate, Potassium Salt (PFOS) in Plant Tissues WILDLIFE INTERNATIONAL, LTD. PROJECT NUMBER: 454C-125 3M ENVIRONMENTAL LAB PROJECT NUMBER: U2723 STUDY DIRECTOR: MANAGEMENT: Willard B. Nixon, Ph.D Director of Analytical Chemistry DATE DATE Wildlife International. Ltd.____________ Project Number 454C-125 -5- TABLE OF CONTENTS Title/Cover Page................................................................................................................................................1 Good Laboratory Practice Compliance Statement.......................................................................................... 2 Quality Assurance Statement...........................................................................................................................3 Report Approval............................................................................................................................................... 4 Table of Contents............................................................................................................................................. 5 Summary........................................................................................................................................................... 8 Introduction...................................................................................................................................................... 9 Objective......................................................................................................................... 9 Experimental Design.............................................:........................................................................ ................9 Materials and Methods................................................................................................. 9 Test Substance....................................................................................................................................... 9 Reagents and Solvents.........................................................................................................................10 Test Systems........................................................................................................................................ 10 Tissue Samples Onion........................................................................................................................................ 10 Alfalfa........................................................................................................ 10 Fruit Samples T o m a to ..................................................................................................................................... 10 Soybean....................................................................................................................................11 Analytical Validation Procedures........................................................................................................ 11 Primary and Secondary Stock Solutions............................................................................................. 12 Calibration Standards and Curves...................................................................................................... 12 Matrix Blanks and Matrix Fortifications............................................................................................ 12 Limit of Quantitation...........................................................................................................................12 Results ........................................................................................................................................................... 13 Reagent and Matrix Blank Samples................................................................................................... 13 Method Validation Samples.................................................................................................................13 Conclusions 13 Wildlife International. Ltd.____________ Project Number 454C-125 6- - TABLE OF CONTENTS - Continued - References...................................................................................................................................................... 15 TABLES Table 1 - Method Validation Recoveries for PFOS in Onion.............................................................. 16 Table 2 - Method Validation Recoveries for PFOS in Alfalfa.............................................................17 Table 3 - Method Validation Recoveries for PFOS in Tomato........................... 18 Table 4 - Method Validation Recoveries for PFOS in Soybean.......................................................... 19 APPENDICES Appendix 1 Appendix 2 - Protocol............................................................................................................................ 20 Certificate of Analysis..................................................................................................... 29 Appendix 3 - Analytical Method Validation D ata................................................................................32 Appendix 3.1 - Analytical Method Flowchart for the Analysis of PFOS in Plant Tissue and Fruit....33 Appendix 3.2 - Typical HPLC/MS/MS Operational Parameters........................................................... 34 Appendix 3.3 - A Typical Calibration Curve for PFO S......................................... 35 Appendix 3.4 - Example Calculations for a Representative Sample...................................................... 36 Appendix 3.5 - Ion Chromatogram of a Low-level PFOS Standard....................................................... 37 Appendix 3.6 - Ion Chromatogram of a High-level PFOS Standard..................... 38 Appendix 3.7 - Ion Chromatogram of an Onion Matrix Blank.............................................................. 39 Appendix 3.8 - Ion Chromatogram of a Low-level Onion Fortification Sample.................................... 40 Appendix 3.9 - Ion Chromatogram of a High-level Onion FortificationSample.................................... 41 Appendix 3.10 - Ion Chromatogram of an Alfalfa Matrix Blank............................................................. 42 Wildlife International. Lfri. -7- Project Number 454C-125 TABLE OF CONTENTS - Continued - Appendix 3.11 - Ion Chromatogram of a Low-level Alfalfa Fortification Sample...................................43 Appendix 3.12 - Ion Chromatogram of a High-level Alfalfa Fortification Sample...................................44 Appendix 3.13 - Ion Chromatogram of a Tomato Matrix Blank............................................................. 45 Appendix 3.14 - Ion Chromatogram of a Low-level Tomato Fortification Sample..................................46 Appendix 3.15 - Ion Chromatogram of a High-level Tomato Fortification Sample................................ 47 Appendix 3.16 - Ion Chromatogram of a Soybean Matrix Blank............................................................. 48 Appendix 3.17 - Ion Chromatogram of a Low-level Soybean Fortification Sample. Appendix 3.18 - Ion Chromatogram of a High-level Soybean Fortification Sample Appendix 4 - Personnel Involved in the Study...................................................... Wildlife International. Ltd. 8- - Project Number 454C-125 SUMMARY SPONSOR: 3M Corporation TITLE: Analytical Method Validation for the Determination of Perfluorooctanesulfonate, Potassium Salt (PFOS) in Plant Tissues WILDLIFE INTERNATIONAL, LTD. PROJECT NUMBER: 454C-125 3M ENVIRONMENTAL LAB PROJECT NUMBER: U2723 TEST DATES: Study Initiation: August 28,2001 Experimental Start (OECD): August 30, 2001 Experimental Start (EPA): August 30, 2001 Experimental Termination: October 4, 2001 TEST SYSTEM: Tissue Samples (Onion and Alfalfa) and Fruit Samples (Tomato and Soybean) FORTIFIED TEST CONCENTRATIONS: Onion and Alfalfa Tissue Samples (mg a.i./kg) Tomato and Soybean Fruit Samples (mg a.i./kg) 0.0500 0.500 5.00 50.0 0.0500 0.500 5.00 50.0 RESULTS: Samples of tissue matrix (onion and alfalfa) and fruit matrix (tomato and soybean) were each fortified in triplicate with PFOS to reflect nominal concentrations of 0.0500,0.500, 5.00 and 50.0 mg a.i./Kg. The samples were extracted with methanol, agitated, centrifuged, diluted into the instrumental calibration range with dilution solvent (50% methanol : 50% NANOpure water) and analyzed by high performance liquid chromatography triple quadrupole mass spectrometry (HPLC/MS/MS). Recoveries of PFOS in onion and alfalfa yielded overall mean percent recoveries of 96.7% (SD = 6.34; CV =6.56%; N = 12) and 94.6% (SD = 4.13; CV =4.37%; N = 12), respectively. Recoveries of PFOS in tomato and soybeans yielded overall mean percent recoveries of 90.5% (SD = 5.68; CV = 6.28%; N = 12) and 95.3% (SD = 4.92; CV = 5.16%; N = 12), respectively. Reagent blanks, consisting of solvents, and matrix blank samples, consisting of unfortified onion, alfalfa, tomato or soybean, were devoid of interfering components. The relative precision (CV) of triplicate fortifications of PFOS in plant tissue matrix taken through the methodology was typically 5 percent, or better for the 0.500, 5.00, and 50.0 mg a.i./Kg fortification levels. The relative precision at the 0.0500 mg a.i./Kg fortification level, ranged from 2.28 to 11.4 percent. Wildlife International. Ltd. -9- Project Number 454C-125 INTRODUCTION This study was conducted by Wildlife International, Ltd. for 3M Corporation at the Wildlife International, Ltd. analytical chemistry facility in Easton, Maryland. Validation samples were fortified with Perfluorooctanesulfonate, Potassium Salt (hereafter referred to as PFOS) and analyzed with high performance liquid chromatography mass spectrometry (HPLC/MS/MS) to evaluate the performance of a method for the determination of PFOS in plant tissues. Validation samples were prepared and analyzed between August 30, 2001 and October 4,2001. Raw data generated by Wildlife International, Ltd. and a copy of the final report are filed under Project Number 454C-125 in archives located on the Wildlife International, Ltd. site. OBJECTIVE The objective of this study was to verify the performance of a HPLC/MS/MS methodology for the analyses of PFOS in plant tissues. The validated method will be used in support of definitive plant ecotoxicology tests to be conducted with the test substance. EXPERIMENTAL DESIGN Tissue samples, defined herein as the entire plant system including leaves and stems, and fruit samples, defined herein as the seed-bearing structure of the flowering plant, were each fortified at four different concentrations in triplicate and analyzed based on HPLC/MS/MS methodology developed at Wildlife International, Ltd. The fortification levels bracketed the anticipated concentrations o f samples from anticipated plant toxicity tests. A reagent blank sample and matrix blank samples in each matrix were analyzed concurrently to evaluate potential analytical method interferences. A calibration curve was generated from analyses of standard solutions of PFOS with each series of samples analyzed. MATERIALS AND METHODS This study was conducted according to the procedures outlined in the protocol, "Analytical Method Validation for the Determination of Perfluorooctanesulfonate, Potassium Salt (PFOS) in Plant Tissues" (Appendix 1). Test Substance The test substance was received from 3M Corporation on October 29, 1998, assigned Wildlife International, Ltd. identification number 4675, and stored under ambient conditions. The test substance Wildlife International. Ltd. - 10- Project Number 454C-125 was described as a white powder. It was identified as FC-95 from lot number 217, with an expiration date of August 31, 2006. Information from the most recent Certificate of Analysis provided by the Sponsor indicated a purity of 86.9% (Appendix 2). Reagents and Solvents All solvents used in this study were HPLC grade or equivalent. All reagents were ACS reagent grade or equivalent. NANOpure water (equivalent to ASTM Type II Designation D1193-91) was used (1). Test Systems Tissue Samples Onion tissue was obtained from the Wildlife International, Ltd. Greenhouse in Easton, Maryland, on October 2, 200land identified as lot number 09-07-01. Upon receipt, the bulk onion tissue was initially placed in a -4C freezer until further processing. The bulk onion tissue was placed in a cyrogenic (-80C) freezer for approximately 30 minutes, transferred to a Waring blender and blended until homogenous. An appropriate number of 1.00 g aliquots were weighed from the onion homogenate. The remainder of the homogenized onion was then transferred to a zip-lock bag, and placed into the 4C freezer for use as control matrix in anticipated plant studies. Alfalfa tissue was obtained from the Wildlife International, Ltd. Greenhouse in Easton, Maryland, on September 21,2001, and identified as lot number 12-18-01. Upon receipt, the bulk alfalfa tissue was placed in a -4C freezer until further processing. The alfalfa was removed from frozen storage and manually chopped using an Ekco 8" chefs knife and plastic cutting board and then returned to the -4C freezer. The frozen tissue was then further homogenized using a Waring blender. An appropriate number of 1.00 g aliquots were weighed from the alfalfa homogenate. The remainder of the homogenized alfalfa was then transferred to a zip-lock bag, and placed into the -4C freezer for use as control matrix in anticipated plant studies. Fruit Samples Tomatoes used were purchased on August 30,2001 from Wal-Mart Super-Center, Milford, Delaware, and identified as lot number 3151 with an expiration date of 8/30/02. The tomatoes were quartered, Wildlife International. Ltd. -11 - Project Number 454C-125 placed in a Waring blender and blended until homogenous. An appropriate number of 1.00 g aliquots were weighed from the tomato homogenate. The remainder of the homogenized tomato was then transferred to a 16-oz. French-square bottle and placed into the -4C freezer for use as control matrix in anticipated plant studies. Soybeans were obtained from the Wildlife International, Ltd. Greenhouse in Easton, Maryland on September 6, 2001, and identified as lot number 05-15-01, with an expiration date of 9/6/02. The soybeans were placed in a Waring blender and blended until homogenous. An appropriate number of 1.00 g aliquots were weighed from the soybean homogenate. The remainder ofthe homogenized soybean was then transferred to a zip-lock bag, and placed into the -4C freezer for use as control matrix in anticipated plant studies. Analytical Validation Procedures Wildlife International, Ltd. conducted a validation trial using procedures developed at Wildlife International, Ltd. (Appendix 3). Matrix blanks and matrix fortification samples were prepared in tissue samples (onion and alfalfa) and in fruit samples (tomato and soybean). A reagent blank (containing everything except PFOS and matrix) was carried through the entire procedure for each of the four matrices. The tissue samples were extracted with methanol, agitated for a minimum of 30 minutes on a gyratory shaker table at approximately 250 rpm and then centrifuged. The fruit samples were extracted with methanol, agitated for a minimum of one minute manually and then centrifuged. Dilutions into the calibration range of the HPLC/MS/MS methodology were performed with a solution of 50% methanol (HPLC grade, 99.9+%) and 50% NANOpure water. Samples were then analyzed by direct injection. Concentrations ofPFOS were determined by reverse-phase high performance liquid chromatography using a Hewlett-Packard Model 1100 High Performance Liquid Chromatograph (HPLC) with a Perkin-Elmer API 3000LC Mass Spectrometer equipped with a Perkin-Elmer TurboIonSpray ion source. Chromatographic separations were achieved using a Keystone Betasil Cis column (50 mm x 2.0 mm, 3-pm particle size) fitted with a Keystone Javelin Cis guard column (20 mm x 2.0 mm). A method flowchart is provided in Appendix 3.1 and the instrument parameters are summarized in Appendix 3.2. Wildlife International. Ltd. - 12- Project Number 454C-125 Primary and Secondary Stock Solutions All primary and secondary stock preparations were adjusted for the purity of the test substance (86.9%). A 10.0 mg a.i./mL primary stock solution of PFOS in methanol was prepared by weighing 1.1508 g of the test substance and bringing to a final volume of 100 milliliters with methanol. Secondary stock solutions (1000, 100, 10.0, 1.00, and 0.100 mg a.i./L) of PFOS in methanol were prepared by serial volumetric dilution from the primary stock. Calibration Standards and Curves Calibration standards were prepared in 50:50 methanol: NANOpure water by appropriate dilutions of the 1.00 and 0.100 mg a.i./L stock solutions of PFOS in methanol. Calibration standards ofPFOS, ranging in concentration from 0.400 to 5.00 pg a.i./L, were analyzed with each sample set. The same and most prominent peak response for PFOS was utilized to monitor PFOS in all calibration and study samples. No attempt was made to quantify PFOS on the basis of individual isomeric components. The calibration standard series was injected at the beginning and end of each run, and one standard was injected, at a minimum, after every five samples. Linear regression equations were generated using peak area responses versus the respective concentrations of the calibration standards. A typical calibration curve is presented in Appendix 3.3. The concentration of PFOS in the samples was determined by substituting the peak area responses into the applicable linear regression equation (Appendix 3.4). Representative ion chromatograms of low and high calibration standards are presented in Appendices 3.5 and 3.6, respectively. Matrix Blanks and Matrix Fortifications Selected 1.00-g aliquots of each matrix type were fortified with the appropriate secondary stock solutions of PFOS prepared in methanol using a gas-tight syringe. The matrix blanks for each matrix type were unfortified tissue or fruit. Limit of Quantitation The method limit of quantitation (LOQ) in each matrix was 0.0400 mg a.i./Kg calculated as the product of the lowest calibration standard (0.000400 mg a.i./L) and the overall dilution factor of the matrix blank samples (100 L/Kg). Wildlife International. Ltd. - 13 - Project Number 454C -125 RESULTS Reagent and Matrix Blank Samples A reagent blank and three matrix blank samples in each matrix were analyzed to determine possible interferences. No interferences were observed at or above the LOQ (Tables 1,2, 3 and 4). Representative ion chromatograms of onion, alfalfa, tomato and soybean matrix blank samples are presented in Appendices 3.7, 3.10, 3.13 and 3.16, respectively. Method Validation Samples Onion tissue was fortified in triplicate at 0.0500, 0.500, 5.00 and 50.0 mg a.:i./Kg resulting in mean recoveries of 100, 91.0,97.6, and 98.2, respectively (Table 1). Representative ion chromatograms of low and high-level fortifications in onion are presented in Appendices 3.8 and 3.9, respectively. Alfalfa tissue was fortified in triplicate at 0.0500, 0.500, 5.00 and 50.0 mg a.i./Kg resulting in mean recoveries of 96.4 94.4 92.8, and 94.8%, respectively (Table 2). Representative ion chromatograms of low and high-level fortifications in alfalfa are presented in Appendices 3.11 and 3.12, respectively. Tomato fruit was fortified in triplicate at 0.0500, 0.500, 5.00 and 50.0 mg a.i./Kg resulting in mean recoveries of 87.5, 90.6, 93.4 and 90.6%, respectively (Table 3). Representative ion chromatograms of low and high-level fortifications in tomato are presented in Appendices 3.14 and 3.15, respectively. Soybean fruit was fortified in triplicate at 0.0500, 0.500, 5.00 and 50.0 mg a.i./Kg resulting in mean recoveries of 87.9, 95.9, 98.5 and 99.2%, respectively (Table 4). Representative ion chromatograms o f low and high-level fortifications in soybean are presented in Appendices 3.17 and 3.18, respectively. The relative precision (CV) of the methodology, as demonstrated by the analysis of triplicate samples was 5 percent, or better for the 0.500, 5.00, and 50.0 mg a.i./Kg fortification levels in all testing matrices. The relative precision at the 0.0500 mg a.i./Kg fortification level, ranged from 2.28 to 11.4 percent in the four testing matrices. CONCLUSIONS Onion, alfalfa, tomato and soybean were fortified with PFOS to reflect nominal concentrations of 0.0500, 0.500, 5.00 and 50.0 mg a.i./Kg. The samples were extracted with methanol, agitated for the appropriate amount of time, and centrifuged. Dilutions into the calibration range of the HPLC/MS/MS methodology were performed with a solution of 50% methanol and 50% NANOpure water. Samples were Wildlife International. Ltd. - 14- Project Number 454C-125 then analyzed by high performance liquid chromatography mass spectrometry (HPLC/MS/MS). Recoveries of PFOS in onion yielded an overall mean percent recovery of 96.7% (SD = 6.34; CV = 6.56%; N = 12). Recoveries of PFOS in alfalfa yielded an overall mean percent recovery of 94.6% (SD = 4.13; CV = 4.37%; N = 12). Recoveries of PFOS in tomato yielded an overall mean percent recovery of 90.5% (SD = 5.68; CV = 6.28%; N = 12). Recoveries of PFOS in soybean yielded an overall mean percent recovery of 95.3% (SD = 4.92; CV = 5.16%; N = 12). Reagent blanks, consisting of solvents, and matrix blank samples, consisting of unfortified plant matrix, were devoid of interfering components. 4 Wildlife International. Lfrl - 15 - Project Number 454C-125 REFERENCES 1. American Society for Testing and Materials. 1991. Standard Specification for Reagent Water. D 1193-91, ASTM Section II Water and Environmental Technology, Vol. 11.01:45-47. Wildlife International. Ltd. -16- Project Number 454C-125 Table 1 Method Validation Recoveries for PFOS in Onion Sample Number (454C-125-) Type PFOS Concentration (mg ai./Kg) Fortified Measured1 Percent Recovered1 Mean Measured (mg a.i./Kg) Mean Percent Recovery V R E B -4 Reagent Blank 0 <LOQ2 - -- VM AB-10 V M A B -11 V M A B -12 Matrix Blank Matrix Blank Matrix Blank 0 <LOQ 0 <LOQ 0 <LOQ ___ -- - ___ ___ VM AS-37 VM AS-38 VM AS-39 Matrix Fortification Matrix Fortification Matrix Fortification 0.0500 0.0500 0.0500 0.0559 0.0494 0.0446 112 0.0500 100 98.9 SD = 0.00567 89.2 CV== 11.4% VM AS-40 V M A S-41 VM AS-42 Matrix Fortification Matrix Fortification Matrix Fortification 0.500 0.500 0.500 0.437 0.460 0.468 87.3 0.455 91.0 92.0 SD = 0.0161 93.6 CV== 3.54% VM AS-43 VM AS-44 VM AS-45 Matrix Fortification Matrix Fortification Matrix Fortification 5.00 5.00 5.00 4.84 4.86 4.93 96.9 4.88 97.6 97.2 SD= 0.0473 98.7 CV= 0.969% VM AS-46 VM AS-47 V M A S-48 Matrix Fortification Matrix Fortification M atrix Fortification 50.0 50.0 50.0 47.4 50.1 49.7 94.7 49.1 98.2 100 SD = 1.46 99.4 CV= 2.97% Overall Mean = 96.7 Standard Deviation = 6.34 CV = 6.56% ___________________________________________________ N = 12____________________________ 1Measured and Percent Recovered values were calculated using MacQuan, version 1.6 software. Manual calculations may vary slightly. 3The limit of quantitation (LOQ) was 0.0400 mg a.i./Kg based upon the product of the lowest calibration standard (0.000400 mg a.i./L) and the overall dilution factor of the matrix blank samples (100 L/Kg). Wildlife International. Ltd. - 17- Project Number 454C-125 Table 2 Method Validation Recoveries for PFOS in Alfalfa Sample Number (454C-125-) Type V R E B -3 Reagent Blank VM AB-7 VM AB-8 VM AB-9 Matrix Blank Matrix Blank Matrix Blank VM AS-49 VM AS-50 V M A S-51 Matrix Fortification Matrix Fortification Matrix Fortification VM AS-28 VM AS-29 VM AS-30 Matrix Fortification Matrix Fortification Matrix Fortification V M A S-31 VM AS-32 VM AS-33 Matrix Fortification Matrix Fortification Matrix Fortification VM AS-34 VM AS-35 VM AS-36 Matrix Fortification Matrix Fortification M atrix Fortification PFOS Concentration (mg a.i./Kg) Fortified Measured1 Percent Recovered1 Mean Measured (mg a.i./Kg) Mean Percent Recovery 0 <LOQ2 - -- -- 0 <LOQ 0 <LOQ 0 <LOQ -- -- -- -- 0.0500 0.0500 0.0500 0.0521 0.0477 0.0449 104 0.0482 96.4 95.3 SD= 0.00363 89.7 CV= 7.52% 0.500 0.500 0.500 5.00 5.00 5.00 0.458 0.467 0.490 4.40 4.74 4.77 91.6 0.472 94.4 93.4 SD= 0.0165 98.0 CV= 3.50% 87.9 4.64 92.8 94.9 SD== 0.206 95.3 CV= 4.43% 50.0 47.5 50.0 46.5 50.0 48.3 95.1 47.4 94.8 92.9 SD = 0.902 96.6 CV= 1.90% Overall Mean = 94.6 Standard Deviation = 4.13 CV = 4.37% N = 12 1Measured and Percent Recovered values were calculated using MacQuan, version 1.6 software. Manual calculations may vary slightly. 2The limit of quantitation (LOQ) was 0.0400 mg a.i./Kg based upon the product of the lowest calibration standard (0.000400 mg a.i./L) and the overall dilution factor of the matrix blank samples (100 L/Kg). Wildlife International. Ltd. - 18- Project Number 454C-125 Table 3 Method Validation Recoveries for PFOS in Tomato Sample Number (454C-125-) Type V R E B -1 Reagent Blank VM AB-1 VM AB-2 VM AB-3 Matrix Blank Matrix Blank Matrix Blank VM AS-1 VM AS-2 VM AS-3 Matrix Fortification Matrix Fortification Matrix Fortification VM AS-4 VM AS-5 VM AS-6 Matrix Fortification Matrix Fortification Matrix Fortification VM AS-7 VM AS-8 VM AS-9 Matrix Fortification Matrix Fortification Matrix Fortification VM AS-10 VMAS - 11 V M A S - 12 Matrix Fortification Matrix Fortification M atrix Fortification PFOS Concentration (mg a.i./Kg) Fortified Measured1 Percent Recovered1 Mean Measured (mg a.i./Kg) Mean Percent Recovery 0 <LOQ2 ~ -- 0 <LOQ -- ---- 0 <LOQ -- 0 <LOQ -- 0.0500 0.0500 0.0500 0.0493 0.0409 0.0411 98.6 0.0438 87.5 81.9 SD = 0.00479 82.1 CV= 11.0% 0.500 0.500 0.500 5.00 5.00 5.00 0.430 0.480 0.449 4.52 4.99 4.50 85.9 0.453 90.6 95.9 SD= 0.0252 89.8 CV= 5.57% 90.3 4.67 93.4 99.9 SD = 0.277 90.0 CV= 5.94% 50.0 45.9 50.0 44.3 50.0 45.7 91.8 45.3 90.6 88.7 SD== 0.872 91.4 CV= 1.92% Overall Mean = 90.5 Standard Deviation = 5.68 CV = 6.28% N = 12 1Measured and Percent Recovered values were calculated using MacQuan, version 1.6 software. Manual calculations may vary slightly. 2The limit o f quantitation (LOQ) was 0.0400 mg a.i./Kg based upon the product of the lowest calibration standard (0.000400 mg a.i./L) and the overall dilution factor of the matrix blank samples (100 L/Kg). Wildlife International. Ltd. - 19- Project Number 454C-125 Table 4 Method Validation Recoveries for PFOS in Soybean Sample Number (454C-125-) Type V R E B -2 Reagent Blank VM AB-4 VM AB-5 VM AB-6 Matrix Blank Matrix Blank Matrix Blank VM AS-13 VM AS-14 VM AS-15 Matrix Fortification Matrix Fortification Matrix Fortification VM AS-16 V M A S - 17 V M A S - 18 Matrix Fortification Matrix Fortification Matrix Fortification V M A S -1 9 3 Matrix Fortification VMAS- 2 0 Matrix Fortification VMAS-2 1 Matrix Fortification VMAS-2 2 VMAS-2 3 VMAS-2 4 Matrix Fortification Matrix Fortification M atrix Fortification PFOS Concentration (mg a.i./Kg) Fortified Measured1 Percent Recovered1 Mean Measured (mg a.i./Kg) Mean Percent Recovery 0 <LOQ2 - -- 0 <LOQ -- -- -- 0 <LOQ - 0 <LOQ -- 0.0500 0.0500 0.0500 0.0440 0.0449 0.0429 87.9 0.0439 87.9 89.9 SD= 0.00100 85.7 CV= 2.28% 0.500 0.500 0.500 0.487 0.473 0.478 97.3 0.479 95.9 94.7 SD= 0.00709 95.6 CV= 1.48% 5.00 5.01 5.00 4.83 5.00 4.94 100 4.93 98.5 96.6 SD= 0.0907 98.9 CV= 1.84% 50.0 50.0 50.0 48.7 50.0 50.1 100 49.6 99.2 97.4 SD = 0.781 100 CV= 1.57% Overall Mean = 95.3 Standard Deviation = 4.92 CV= 5.16% _________ _________________________________________ N = 1 2 ________ ______________ 1Measured and Percent Recovered values were calculated using MacQuan, version 1.6 software. Manual calculations may vary slightly. 2The limit o f quantitation (LOQ) was 0.0400 mg a.i./Kg based upon the product of the lowest calibration standard (0.000400 mg a.i./L) and the overall dilution factor of the matrix blank samples (100 L/Kg). 3Measured value is the mean of one re-dilution, duplicate injection, 454C-125-19R1, and 19R2.____________ Wildlife International. Ltd. -20- Appendix 1 Protocol Project Number 454C-125 Wildlife international. Ltd. -21 - Project Number 454C-125 PROTOCOL ANALYTICAL METHOD VALIDATION FOR THE DETERMINATION OF PERFLUOROOCTANESULFONATE, POTASSIUM SALT (PFOS) IN PLANT TISSUES Environmental Laboratory Request Number U2723 Submitted to 3M Corporation Environmental Laboratory 935 Bush Avenue St. Paul, M innesota 55106 Wildlife International Ltd. 8598 Commerce Drive Easton, Maryland 21601 (410) 822-8600 May 2,2001 Wildlife International. Ltd. -22- Projet;! Number 454C-125 W ildlife International, Ltd. - 2- ANALYTICAL METHOD VALIDATION FOR THE DETERMINATION OF PERFLUOROOCTANESULFONATE, POTASSIUM SALT (PFOS) IN PLANT TISSUES SPONSOR: 3M Corporation Environmental Laboratory P .O .B ox 33331 St. Paul, Minnesota 55133 SPONSOR'S REPRESENTATIVE: M s. Rochelle Robideau TESTING FACILITY: W ildlife International, Ltd. 8598 Commerce Drive Easton, Maryland 21601 STUDY DIRECTOR Raymond L. VanHoven, Ph.D Scientist W ildlife International, Ltd. LABORATORY MANAGEMENT: W illard B. N ixon, Ph.D. Manager o f Analytical Chemistry W ildlife International, Ltd. FOR LABORATORY USE ONLY Proposed Dates: Experimental Start Date: X-'l-Ol Experimental Termination Date: - 3 O 'O ' Project No.: 454C- I 2 . S T est Substance N o. 4675 T est Concentrations: e.oSoe; o.SoOj 5.00 w J 80*0 tnj ./fcfr- DATE PROTOCOL NO.: 454/050201/MV-PT/SUB454 Environmental Laboratory Request Number U2723 Wildlife International. Ltd. -23 - Project Number 454C-125 W ildlife International, Ltd. -3 - INTRODUCTION W ildlife International, Ltd. w ill conduct analyses to validate the performance o f a liquid chromatography m ass spectrometry (LC/M S) method for the determination o f Perfluorooctanesulfonate, Potassium Salt (hereafter referred to as PFOS) in plant tissues and fruit. The study w ill be performed at the W ildlife International, Ltd. analytical chemistry facility in Easton, Maryland. The method developed at W ildlife International, Ltd. w ill be validated by fortifying plant tissues and quantifying the recoveries o f PFOS. Raw data for all work performed at W ildlife Interna tional, Ltd. and a copy o f the final report w ill be filed by project number in archives located on the W ildlife International, Ltd. site, or at an alternative location to be specified in the final report. OBJECTIVE The objective o f this study is to validate the performance o f LC/MS methodology for determination o f PFOS residues in plant tissues and fruits used by W ildlife International, Ltd. EXPERIMENTAL DESIGN Plant tissues and/or fruits from several species o f plants w ill be fortified with at least four different concentrations and analyzed using LC/MS methodology developed at W ildlife International, Ltd. The method w ill be based on procedures developed at W ildlife International, Ltd. Reagent and matrix blanks w ill be analyzed to evaluate potential analytical interferences. One calibration curve w ill be prepared and analyzed with each series o f matrix fortification sam ples. The accuracy, precision, and lim it o f quantitation o f the method w ill be determined. M ATERIALS AND M ETHODS Test Substance The test substance w ill be Perfluorooctanesulfonate, Potassium Salt. Information on the characterization o f test, control or reference substances is required by Good Laboratory Practice Standards (GLP). The Sponsor is responsible for providing W ildlife International, Ltd. written verification that the test substance has been characterized according to GLPs prior to initiation o f the study. The reference material in this study w ill be the test substance. If written verification o f GLP test substance characterization is not provided to W ildlife International, Ltd., it w ill be noted in the compliance statement o f the final report. PROTOCOL NO.: 454/050201/MV-PT/SUB454 Environmental Laboratory Request Number U2723 Wildlife International, Ltd. -24- Project Number 454C-125 W ildlife International, Ltd. -4 - Thc Sponsor is responsible for all information related to the test substance and agrees to accept any unused test substance and/or test substance containers remaining at the end o f the study. R eagents and Solvents All solvents used in the method or procedure w ill be o f reagent grade or better. All reagents w ill be o f ACS grade or better. Plant Tissues Plant tissues w ill be obtained from onion and alfalfa plants that have no known exposure to the test substance. There are no levels o f contaminants reasonably expected to be present in the plant tissues that are considered to interfere with the purpose or conduct o f the study. Fruits Fruits w ill be obtained from two species o f plants (tom atoes and soybeans) that have no known exposure to the test substance. There are no levels o f contaminants reasonably expected to be present in the plant tissues that are considered to interfere with the purpose or conduct o f the study. Analytical V erification Procedures The analytical method to be used w ill be LC/MS based upon procedures developed at W ildlife International, Ltd. The method used w ill be documented in the raw data and summarized in the final report. Prior to the analysis o f analytical samples, primary stock solution s) w ill be prepared directly from the test substance. Calibration standards w ill be prepared by appropriate dilution o f die primary stock solution s). Primary Stock Solution(s), Calibration Standards and C urves A minimum o f five calibration standards (o f different concentrations) w ill be prepared and analyzed along with each analysis set. The calibration standard series w ill be injected at the beginning and one standard injected, at a minimum, after every five samples. A minimum o f two injections o f each standard in the calibration standard series w ill be injected with the analysis set. One calibration curve and regression equation w ill be prepared from the series o f calibration standards. PROTOCOL NO.: 454/050201/MV-PT/SUB454 Environmental Laboratory Request Number U2723 Wildlife International. Ltd. -25 - Project Number 454C-125 W ildlife International, Ltd. -5 - Fortification Stock Solutions Plant tissue and fruit homogenates w ill be fortified with a stock solution(s) o f PFOS at levels anticipated to bracket levels in the bioconcentration study. Evaluation o f Interferences One reagent blank and three matrix blanks w ill be analyzed to detect possible interferences. The reagent blank w ill contain everything except the matrix and the test substance. The matrix blanks w ill contain everything except the test substance. V erification A nalyses - M ethod Perform ance Matrix fortifications (spiked plant tissues and fruits or seeds), prepared at known concentrations o f the test substance, w ill be analyzed to determine the recovery o f the analyte and to evaluate method performance. The anticipated verification series w ill consist o f the following analyses: SUMMARY OF A TYPICAL VERIFICATION ANALYSIS SCHEME A nalysis Type C o n cen tra tio n Reagent Blank (Solvent) O(Control) M atrix Blank (Plant tissues) 0 (Control) Matrix Fortifications Level 1 - Low1 Level 2 Number o f T issue Sam ples Onion A lfalfa 1l 33 33 33 Level 3 Level 4 -H igh 33 33 Total Number o f Analyses 16 16 'M atrix fortifications w ill be prepared at four concentrations. Num ber of F ru it S am p les Tomato Soybean 11 33 33 33 33 33 16 16 . Matrix fortifications w ill represent the range o f bioconcentration that is anticipated to be found in plant tissue in subsequent effects tests and w ill be selected in consultation with the Sponsor. PROTOCOL NO. : 454/050201/MV-PT/SUB454 Environmental Laboratory Request Number U2723 Wildlife International. Ltd. -26- Project Number 454C-12.5 W ildlife International, Ltd.*1234567890 - 6- Individual sam ples (identified by project number and a unique sample identification number) w ill be prepared and analyzed for verification o f the analytical methodology. If difficulties arise in the verification process (e.g. low recoveries or interferences), the Sponsor w ill be notified and the need for additional verification or method development w ill be deter mined through discussion with the Sponsor. Upon com pletion o f die method verification, the Sponsor w ill review the results and authorize use o f the methodology for analysis o f sam ples, or w ill authorize further method development trials. The acceptable range o f recoveries is 70 to 120% o f the nominal concentrations. D ata A nalysis One calibration curve w ill be established for each analytical run. A regression equation o f the concentration versus peak area for the calibration standards w ill be generated. The concentration o f the sam ples w ill be determined by substituting the respective peak area into the regression equation. Statistics to be determined and reported w ill include the mean and standard deviation for each level o f fortification. RECORDS TO BE M AINTAINED Records to be maintained for data generated by W ildlife International, Ltd. w ill include: 1. A copy o f die signed protocol. 2. Identification and characterization o f the test substance and/or analytical standard, if provided by the Sponsor. 3. Dates o f initiation and termination o f the test. 4. Storage conditions for test substance, analytical standards, and/or samples. 5. T est substance and/or analytical standard use log. . 6. Concentration calculations and records o f solution preparation. 7. Instrument operating conditions and chromatograms. 8. Statistical calculations. 9. A copy o f the final report. 10. Documentation that the steps in the method were followed. PROTOCOL NO.: 454/050201/MV-PT/SUB454 Environmental Laboratory Request Number U2723 Wildlife International, Ltd. -27- Project Number 454C-125 W ildlife International, Ltd. -7 - FINAL REPORT The report w ill summarize the findings o f the verification, the procedural recoveries obtained, and the methods and instrumentation employed. Upon receipt o f these findings, the Sponsor w ill review the methods and results and evaluate the results for acceptability prior to initiating any fate and/or effects studies. The final report w ill include, but not be lim ited to, the following: 1. Name and address o f the facility performing foe study. 2. Dates on which foe study w as initiated and completed. 3. Objectives and procedures stated in foe approved protocol, including any changes in foe original protocol or deviations from the protocol. 4. The test substance and/or analytical standard identification, including name, chemical abstract number or code number, strength, purity, com position, date o f receipt, lot number, storage conditions, physical characteristics, stability and method o f preparation o f test concentrations, if provided by foe Sponsor. 5. A detailed summary o f foe analytical methodology: A description o f foe experimental measurements, example calculations, sample preparation (sam ple weights and dilutions), instrumentation employed, reagents and solvents used, class o f water used, and any major modifications to foe method. 6. A description o f circumstances that may have affected the quality or integrity o f the data. 7. The name o f foe Study Director, foe names o f other scientists or professionals, and foe names o f all supervisory personnel involved in foe study. 8. A description o f foe transformations, calculations, or operations performed on foe data. 9. The signed and dated reports o f each o f the individual scientists or other professionals involved in foe study, i f applicable. . 10. The location where raw data and final report are stored at foe conclusion o f the study. 11. A statement prepared by foe Quality Assurance Unit listing foe dates that study inspections were made and foe dates o f any findings reported to the Study Director/Management. PROTOCOL NO.: 454/050201/MV-PT/SUB454 Environmental Laboratory Request Number U2723 Wildlife International. Ltd. -28- Project Number 454C-125 W ildlife International, Ltd. - 8- CHANGING OF PROTOCOL Planned changes to the protocol w ill be in the form o f written amendments signed by the Study Director and the Sponsor's Representative. Amendments w ill be considered as part o f the protocol and w ill be attached to the final protocol. Any other changes w ill be in the form o f written deviations signed by the Study Director and filed with the raw data. A ll changes to the protocol w ill be indicated in the final report GOOD LABORATORY PRACTICES This study w ill be conducted in accordance with Good Laboratory Practice Standards for EPA (40 CFR Part 160 and/or Part 792); and OECD Principles o f Good Laboratory Practice (ENV/M C/CHEM (98) 17). Each study conducted by W ildlife International, Ltd. is routinely examined by the W ildlife International, Ltd. Quality Assurance Unit (QAU) for compliance with Good Laboratory Practices, Standard Operating Procedures and the specified protocol. The final report w ill also be reviewed by the QAU. A statement o f compliance with Good Laboratory Practices w ill be prepared for all portions o f the study conducted by W ildlife International, Ltd. The Sponsor w ill be responsible for certification o f compliance with Good Laboratory Practices for procedures performed by other laboratories. Raw data for all work performed at W ildlife International, Ltd. and a copy o f die final report w ill be filed by project number in archives located on the W ildlife International, Ltd. site, or at an alternative location to be specified in the final report. PROTOCOL NO.: 454/050201/MV-PT/SUB454 Environmental Laboratory Request Number U2723 Wildlife International. Ltd. -29- Project Number 454C-125 Appendix 2 Certificate of Analysis INTERIM CERTIFICATE OF ANALYSIS Revision 1(9/7/00) Centre Analytical Laboratories COA Reference #: 023-018A 3M Product: PFOS,Lot217 Reference#: SD-018 __________ _________ Purity: 86.9%______________ Test Name Specifications P urity1 Result 86.9% Appearance Identification. NMR M etals (ICP/M S) 1. Calcium 2 . M agnesium 3. Sodium 4. Potassium 2 5. Nickel 6. Iron 7. M anganese T otal % Impurity (NM R) T otal % Impurity (LC/M S) Total % Impurity (G C/M S) Related Compounds POAA Residual Solvents (TGA) Purity by DSC Inorganic Anions (IC) 1. Chloride 2 . Fluoride 3 . Bromide 4 . Nitrate 5. Nitrite 6. Phosphate 7 . Sulfate1 Organic A cids 5 (IC) 1. TFA 2. PFPA 3. HFBA 4. NFPA Elem ental Analysis11: 1. Carbon 2. Hydrogen 3. Nitrogen 4. Sulfur 5. Fluorine W hite Crystalline Powder ' > i / :1 . ::'?.i!v:1;.:?t:! V i >> < n< `r:>:;i ^: i >:! r:; X i! >i j t .....*.......... ]. ' I'. ...A....... J .., . " ,ytj 1 y r " > " > ; > u ' v 1''1'""........ 'ii,,mi > ,> ,'' **! '* ,,V i , ' ' ' / w sfir ik i ! " ' > !* ,< >> , , ` . . '` -v . h , ,, ................................ r ....... ,------- ; '------------ i'- ' ; > " * >< : `` ^> 1. Theoretical Value = 17.8% 2. Theoretical Value = 0% 3. Theoretical Value = 0% 4. Theoretical Value = 5.95% 5. Theoretical Value = 60% C onform s P o sitiv e 1. 0.005 wt./wt.% 2. 0.001 wt./wt.% 3. 1.439 wt./wt.% 4. 6.849 wt./wt.% 5. <0.001 wtVwt.% 6. 0.005 wt./wt.% 7. <0.001 wt./wt.% 1.93 wt./wt.% 8.41 wt./wt.% None Detected 0.33 wt./wt.% None Detected N ot Applicable3 1. <0.015 wt./wt.% 2. 0 .59 wtAvt.% 3. <0.040 wtVwt.% 4 . <0.009 wtVwt.% 5. <0.006 wt./wt.% 6. <0.007 wt./wt.% 7. 8.76 wt./wt.% 1. <0.1 wt./wt.% 2. <0.1 w tfw t.% 3. 0.10 wt./wt.% 4. 0.28 wt./wt.% 1. 12.48 wt./wt.% 2. 0.244 wt*/wt.% 3. 1.74 wt./wt.% 4. 8.84 wt./wt.% 5. 54.1 wt./wt.% COA023-018A Page 1 of 3 Wildlife International. Ltd. -30- Project Number 454C-125 INTERIM CERTIFICATE OF ANALYSIS Centre Analytical Laboratories COA Reference #: 023-018A Date o f Last Analysis: 08/31/00 Expiration Date: 08/31/01 Storage Conditions: Frozen <-10C Re-assessment Date: 08/31/01 'Purity = 100% - (sum of metal impurities, 1.45% +LC/MS impurities, 8.41%+Inorganic Fluoride, 0.59%+NMR impurities, 1.93%+organic acid impurities, 0.38%+POAA, 0.33%) Total impurity from all tests = 13.09% Purity = 100% - 13.09% = 86.9% 2Potassium is expected in this salt form and is therefore not considered an impurity. 3Purity by DSC is generally not applicable to materials o f low purity. No endotherm was observed for this sample. 4Sulfur in the sample appears to be converted to SO4 and hence detected using the inorganic anion method conditions. The anion result agrees well with the sulfur determination in the elemental analysis, lending confidence to this interpretation. Based on the results, the SO4 is not considered an impurity. 5TFA HFBA NFPA PFPA Trifluoroacetic acid Heptafluorobutyric arid Nonofluoropentanoic acid Pentafluoropropanoic acid 'Theoretical value calculations based on the empirical formula, CsFnS0 3 ~K+(MW=53 8) This work was conducted under EPA Good Laboratory Practice Standards (40 CFR 160). / COA023-018A Page 2 of 3 Wildlife International. Ltd. -31 - Project Number 454C-125 INTERIM CERTIFICATE OF ANALYSIS Centre Analytical Laboratories COA Reference t: 023-018A LC/MS Purity Profile: Impurity C4 C5 C6 C7 Total w t/w t % 1.22 1.33 4.72 1.14 8.41 Note: The C4 and C6 values were calculated using the C4 and C6 standard calibration curves, respectively. The C5 value was calculated using the average response factors from the C4 and C6 standard curves. Likewise, the C7 value was calculated using the average response factors from the C6 and C8 standard curves. Prepared By: ____________________________ ____ ! David S. Bell Date Scientist, Centre Analytical Laboratories Reviewed B y :_____________________ '______ ____ John Flaherty Date Laboratory Manager, Centre Analytical Laboratories COA023-018A Page 3 of 3 Wildlife International. Ltd._ -32- Appendix 3 Analytical Method Validation Data Project Number 454C-125 Wildlife International, Ltd. -33 - Project Number 454C-12.5 Appendix 3.1 Analytical Method Flowchart for the Analysis of PFOS in Plant Tissue and Fruit Weigh approximately 1.00 gram of homogenized plant matrix per sample into a tared vial. Record weights. For matrix fortification samples, fortify each sample with the appropriate volume of PFOS stock solution in methanol with a gas-tight syringe. The matrix blank is unfortified plant matrix. For each sample, measure 10.0 milliliters of methanol with a Class A volumetric pipette and transfer into the vial. Add 10.0 milliliters of methanol into an empty vial and process as for the plant seimples. The additional sample is the reagent blank. i For plant tissue samples, cap vials and place on a shaker table. Allow the samples to shake for a minimum of 30 minutes at approximately 250 rpm. For the fruit samples, cap vials and shake manually for a minimum of one minute. I Centrifuge samples for approximately ten minutes at 2000 ipm. 4 Dilute samples into the calibration range of the PFOS LC/MS methodology: Partially fill Class A volumetric flasks with dilution solvent (50% methanol: 50% NANOpure water). Add appropriate volume of sample with a gas-tight syringe and bring to volume with dilution solvent. Process matrix blank samples using the same dilution and aliquot volumes as for the lowest fortification level. 4 Ampulate samples and submit for HPLC/MS/MS analysis. Wildlife International. Ltd. -34- Project. Number 454C-125 INSTRUMENT: Appendix 3.2 Typical HPLC/MS/MS Operational Parameters Hewlett-Packard Model 1100 High Performance Liquid Chromatograph with a Perkin-Elmer API 3000 Mass Spectrometer equipped with a PerkinElmer TurboIonSpray ion source. Operated in multiple ion reaction monitoring (MRM) mode. ANALYTICAL COLUMN: GUARD COLUMN: OVEN TEMPERATURE: STOP TIME: FLOW RATE: MOBILE PHASE: Keystone Betasil Cu column (50 mm x 2 mm I.D., 3-pm particle size) Keystone Javelin Cis column (20 mm x 2 mm I.D.) 40C 5.00 minutes 250 pL/minute 70.0% Methanol: 30.0% NANOpure Water containing 0.1% Formic Acid INJECTION VOLUME: PFOS RETENTION TIME: 10.0 pL Approximately 4.0 minutes PFOS MONITORED MASS: 499.0 amu - 99.1 amu Wildlife International. Ltd. -35- Appendix 3.3 A Typical Calibration Curve for PFOS PROS 49 9 .0 -> 9 9 .1 No Internal Standard Weighted (1/x) Intercept = 352.9491 Slope = 9618.9219 Correlation Coeff. = 0.99906 Area Project Number 454C-125 Wildlife International. Ltd, -36- Project Number 454C-125 Appendix 3.4 Example Calculations for a Representative Sample Sample number 454C-125-VMAS-1, nominal concentration of 0.0500 mg a.i./Kg in Tomato Peak Area 8465 Y-intercept = 2383.6709 Slope = 12340.8760 Primary Dilution: Initial Weight (W ) = 1.00 g Original Final Volume (V0= 10.0 mL Secondary Dilution: Initial Volume (Vi) = 1.00 mL Final Volume (Vf) = 10.0 mL 100 mL 100L Overall Dilution Factor (Vi/Wj x V/Vi) = ~ = V a (peak area - y-intercept) PFOS at instrument (pg a.i./L) = slope (8465 - 2383,6709) 12340.8760 0,49278 pg a.i. L PFOS in sample (mg a.i./Kg) = PFOS at instrument (pg a.i./L) x overall dilution factor (L/Kg) x conversion factor (0.001 mg/pg) 0.49278 pg a.i. 100L 0.001 mg PFOS in sample (mg a.i./Kg) = L Kg pg 0.049278 mg a.i. Kg PFOS (mg a.i./L) in sample Percent of Nominal Concentration = PFOS (mg a.i./L) nominal x 0,049278 0.0500 x 100 = 98.6% Instrument Software: MacQuan, version 1.6. Wildlife International. Ltd. -37- Project Number 454C-125 Appendix 3.5 Ion Chromatogram of a Low-level PFOS Standard PFOS_1 STD 0.400 ug a.L/L 4 6 7 5 A -0 1 1D-26 4.98 in 1 period PROS No Internal Standard Use Area 1: 4.97 MRM, 298 scans 499.0->99.1 Noise Thres. Quant Thres. Min. Width Mult. Width Base. Width RT Win. (secs) Smooth Expected RT 2.0 1.0 12 10 30 10 2 4.37 Area 4336 Height 471 Start Time End Time Integration Width 3.58 4.04 0.45 Retention Time 3.77 Integration Type A* - BB Tue, Oct 2, 2001 09:49 10Oi 9080706050 40 30 20 10 o-l intensity: 6000 cps 28 41 0.69 76 81 1.36 123 155 121 161 2.03 2.70 225 198 261 201 241 281 Scan 3.37 4.04 4.71 Time Nominal concentration: 0.000400 mg a.i./L Wildlife International. Ltd -38- Project Number 454C-125 Appendix 3.6 Ion Chromatogram of a High-level PFOS Standard PF0S_6 STD 5.00 ug a .IA 4675A-011D-31 4.98 in 1 period PFOS No Internal Standard Use Area 1: 4.97 MRM, 298 scans 499.0->99.1 Noise Thres. 2.0 Quant Thres. 1.0 Min. Width 12 Mult. Width 10 Base. Width 50 RT Win. (secs) Smooth 10 2 Expected RT 4.37 Area 48627 Height 5149 Start Time 3.53 End Time 4.14 integration Width Retention Time 0.60 3.77 Integration Type A-VB Tue, Oct 2, 2001 10:19 intensity: 0000 cps Nominal concentration: 0.00500 mg a.i./L Wildlife International, Ltd. 39- - Appendix 3.7 Ion Chromatogram of an Onion Matrix Blank PFOSjlO 454C-125- VMAB-11 4.98 in 1 period PROS No Internal Standard Use Area 1: 4.97 MRM, 298 scans 499.0->99.1 Noise Thres. 2.0 Quant Thres. 0.5 Min. Width 12 Mult. Width 10 Base. Width 40 RT Win. (secs) 1 0 Smooth 2 Expected RT 3.72 Area 0 Height 0 Start Time End Time 0.00 0.00 Integration Width 0.00 Retention Time 0.00 Integration Type Wed. Oct 3. 2001 12:55 Project Number 454C-125 Intensity: 4000 cps Sample number 454C-125-VMAB-11. The arrow indicates the approximate retention time ofPFOS. Wildlife International. Ltd. -40- Project Number 454C-125 Appendix 3.8 Ion Chromatogram of a Low-level Onion Fortification Sample PFOS 14 454C-125- VMAS-38 4.98 in 1 period FFOS No Internal Standard Use Area 1: 4.97 MRM, 298 scans 499.0->99.1 Noise Thres. 2.0 Quant Thres. 0.5 Min. Width 12 Mult. Width 10 Base. Width 40 RT Win. (secs) Smooth Expected RT 10 2 3.72 Area 4605 Height 472 Start Time End Time 3.57 4.24 Integration Width 0.67 Retention Time 3.80 Integration Type A-BB Wed, Oct 3, 2001 13:19 100-1 908070605040302010 o4 3752 41 0.69 Intensi!/: 4000 cps 1--011--2r0 150 81 121 161 1.36 2.03 2.70 227 199 ^l,. 201 241 3.37 4.04 281 Scan 4.71 Time Sample number 454C-125-VMAS-38, 0.0500 mg a.i./Kg nominal concentration, overall dilution factor = 100 L/Kg. Wildlife International. Ltd. -41 - Project; Number 454C-125 Appendix 3.9 Ion Chromatogram of a High-level Onion Fortification Sample PFOS_24 454C-125- VMAS-47 4.98 in 1 period pros No Internal Standard Use Area 1: 4.97 MRM, 298 scans 499.0->99.1 Noise Thres. Quant Thres. Min. Width Mult. Width Base. Width RT Win. (secs) Smooth Expected RT 2.0 0.5 12 10 40 10 2 3.72 Area 16775 Height 1869 Start Time End Time Integration Width Retention Time Integration Type 3.55 4.22 0.67 3.80 A-B8 Wed. Oct 3. 2001 14:20 intensity: 4000 cps Sample number 454C-125-VMAS-47, 50.0 mg a.i./Kg nominal concentration, overall dilution factor 25000 L/Kg. Wildlife International. Ltd. -42- Appendix 3.10 Ion Chromatogram of an Alfalfa Matrix Blank PFOS_10 454C-125- VMAB-8 Tue, Oct 2, 2001 10:43 4.98 in 1 period pros No Internal Standard Use Area 1: 4.97 MRM, 298 scans 499.0->99.1 Noise Thres. Quant Thres. 2.0 1.0 Min. Width Mult. Width Base. Width RT Win. (secs) Smooth Expected RT 12 10 50 10 2 4.37 Area 0 Height 0 Start Time End Time Integration Width Retention Time Integration Type 0.00 0.00 0.00 0.00 Project; Number 454C-125 intensity: 6000 cps Sample number 454C-125-VMAB-8. The arrow indicates the approximate retention time of PFOS. Wildlife International. Ltd. -43 - Project Number 454C-125 Appendix 3.11 Ion Chromatogram of a Low-level Alfalfa Fortification Sample PFOS_9 454C-125- VMAS-49 Thu, Oct 4, 2001 12:39 4.98 in 1 period pros No Internal Standard Use Area 1: 4.97 MRM, 298 scans 499.0->99.1 Noise Thres. Quant Thres. Min. Width Mult. Width Base. Width RT Win. (secs) Smooth Expected RT 2.0 0.5 12 10 40 10 2 3.72 Area 5449 Height 537 Start Time 3.55 End Time 4.17 Integration Width 0.62 Retention Time 3.80 Integration Type A-VB 10On 90- 80- 70- 60- 50- 40- 30- 2050 10o-l 2 2 41 0.69 95 81 121 1.36 2.03 intensity: 5000 cps 227 168 i-IS.i*8~. 161 201 2.70 3.37 265 241 281 Scan 4.04 4.71 Time Sample number 454C-125-VMAS-49, 0.0500 mg a.i./Kg nominal concentration, overall dilution factor = 100 L/Kg. Wildlife International. Ltd. -44- Project Number 454C-125 Appendix 3.12 Ion Chromatogram of a High-level Alfalfa Fortification Sample PFOS.23 454C-125- VMAS-34 4.98 fn 1 period pros No Internal Standard Use Area 1: 4.97 MRM, 298 scans 499.0->99.1 Noise Thres. 2.0 Quant Thres. 1.0 Min. Width 12 Mult. Width 10 Base. Width SO RT Win. (secs) 10 Smooth 2 Expected RT 4.37 Area 18647 Height 1901 Start Time End Time Integration Width Retention Time Integration Type 3.60 4.25 0.65 3.89 A-VB Tue, Oct 2. 2001 12:01 100i 90807060sa 4a 3a 2a 1a o4 41 0.69 81 121 1.36 2.03 intensity: 6000 cps 232 281 Scan 4.71 Time Sample number 454C-125-VMAS-34,50.0 mg a.i./Kg nominal concentration, overall dilution factor = 25000 L/Kg. Wildlife International. Ltd. -45- Appendix 3.13 Ion Chromatogram of a Tomato Matrix Blank PFOS 10 454C-125- VMAB-2 Sat, Sep 1, 2001 12:04 4.98 in 1 period PFOS No internal Standard Use Area 1: 4.97 MRM, 298 scans 499.0->99.1 Noise Thres. 2.0 Quant Thres. 1.0 Min. Width 12 Mult. Width 10 Base. Width 40 RT Win. (secs) Smooth Expected RT 10 2 3.82 Area 816 Height 79 Start Time 3.69 End Time 4.04 Integration Width 0.35 Retention Time 3.80 Integration Type A -BB Project Number 454C-125 intensity: 8000 cps Sample number 454C-125-VMAB-2. The arrow indicates the approximate retention time of PFOS. Wildlife International, Ltd. -46- Project Number 454C-125 Appendix 3.14 Ion Chromatogram of a Low-level Tomato Fortification Sample PFOS_12 454C-125- VMAS-1 Sat, Sep 1, 2001 12:16 4.98 in 1 period pros No Internal Standard Use Area 1: 4.97 MRM, 298 scans 499.0->99.1 Noise Thres. 2.0 Quant Thres. 1.0 Min. Width Mult. Width 12 10 Base. Width 25 RT Win. (secs) Smooth Expected RT 10 2 3.82 Area 8465 Height 972 Start Time 3.57 End Time Integration Width 3.99 0.42 Retention Time Integration Type 3.77 A'-BB intensity: 8000 cps Sample number 454C-125-VMAS-1, 0.0500 mg a.i./Kg nominal concentration, overall dilution factor 100 L/Kg. Wildlife International. Ltd -47- Project Number 454C-125 Appendix 3.15 Ion Chromatogram of a High-level Tomato Fortification Sample PFOS_24 454C-125- VMAS-11 4.98 in 1 period pros No Internal Standard Use Area 1: 4.97 MRM, 298 scans 499.0->99.1 Noise Thres. 2.0 Quant Thres. 1.0 Min. Width 12 Mult. Width 10 Base. Width 40 RT Win. (secs) 10 Smooth 2 Expected RT 3.82 Area 24270 Height 2598 Start Time End Time Integration Width Retention Time Integration Type 3.55 4.09 0.54 3.79 A-VB Sat, Sep 1, 2001 13:28 intensity: 8000 cps Sample number 454C-125-VMAS-11, 50.0 mg a.i./Kg nominal concentration, overall dilution factor = 25000 L/Kg. Wildlife International. Ltd. -48- Project Number 454C-125 Appendix3.16 Ion Chromatogram of a Soybean Matrix Blank PFOS_9 454C-125- VMAB-4 Fri, Sep 7, 2001 12:38 4.98 In 1 period PFOS No Internal Standard Use Area 1: 4.97 MRM, 298 scans 499.0->99.1 Noise Thres. Quant Thres. Min. Width 2.0 1.0 12 Mult. Width 10 Base. Width 40 RT Win. (secs) 10 Smooth 2 Expected RT 3.82 Area 0 Height 0 Start Time 0.00 End Time 0.00 Integration Width 0.00 Retention Time 0.00 Integration Type 100i 9n 8070605040302010o-l intensity: 4000 cps i 66 89 109 41 81 121 0.69 1.36 2.03 167 161 2.70 199 232 275 201 241 281 Scan 3.37 4.04 4.71 Time Sample number 454C-125-VMAB-4. The arrow indicates the approximate retention time of PFOS. Wildlife International. Ltd. -49- Project Number 454C-125 Appendix 3.17 Ion Chromatogram of a Low-level Soybean Fortification Sample PFOS_12 454C-125- VMAS-13 Fri, Sep 7, 2001 12:56 4.98 in 1 period pros No Internal Standard Use Area 1: 4.97 MRM, 298 scans 499.0->99.1 Noise Thres. Quant Hires. 2.0 0.5 Min. Width 12 Mult. Width Base. Width 10 60 RT Win. (secs) 10 Smooth 2 Expected RT 3.82 Area 4065 Height 416 Start Time 3.50 End Time 4.19 Integration Width 0.69 Retention Time 3.87 Integration Type A* - BB intensity: 4000 cps Sample number 454C-125-VMAS-13, 0.0500 mg a.i./Kg nominal concentration, overall dilution factor = 100L/Kg. Wildlife International. Ltd. -50- Project: Number 454C-125 Appendix 3.18 Ion Chromatogram of a High-level Soybean Fortification Sample PFOS.26 454C-125- VMAS-24 4.98 in 1 period pros No Internal Standard Use Area 1: 4.97 MRM, 298 scans 499.0->99.1 Noise Thres. Quant Thres. Min. Width 2.0 1.0 12 Mult. Width Base. Width RT Win. (secs) Smooth Expected RT 10 40 10 2 3.82 Area 15377 Height 1645 Start Time End Time Integration Width Retention Time Integration Type 3.60 4.27 0.67 3.84 A-BB Fri, Sep 7, 2001 14:20 100-1 90- 8070605040302010- 20 39 o-J T" 41 0.69 intensity: 4000 cps 229 75 1-0r 2 1 2 4 81 121 1.36 2.03 200 154 1 7 5 X V -, 161 201 2.70 3.37 241 4.04 281 Scan 4.71 Time Sample number 454C-125-VMAS-24, 50.0 mg a.i./Kg nominal concentration, overall dilution factor 25000 L/Kg. ** I* Wildlife International. Ltd. -51 - Project Number 454C-125 Appendix 4 Personnel Involved in the Study The following key Wildlife International, Ltd. personnel were involved in the conduct or management of this study: 1. Willard B. Nixon, Ph.D., Director, Analytical Chemistry 2. Raymond L. Van Hoven, Ph.D., Scientist 3. Jon MacGregor, B.S., Scientist 4. Frank J. Lezotte, B.S., Chemist