Document Qg5ypXnrE2nMKR45KzdZYZp86

'. . '-. I_ -- ANALYTICAL METHOD VALIDATION FOR THE DETERMINA'TION 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 55 106 Wildlife International, Ltd. 8598 Commerce Drive Easton, Maryland 21601 (410) 822-8600 Page 1 of 51 WildlifeIntemational, Ltd. -2- -- Project Number 454C- 125 GOOD LABORATORY PRACTICE COMPLIANCE STATEMENT SPONSOR: 3M Corporation TITLE: Analytical Method Validation for the Determination of Perfluorooctanesulfonate!P, otassium Salt (PFOS) in Plant Tissues WILDLIFE INTERNATIONALyLTD. PROJECT NUMBER: 454C- 125 3M ENVIRONMENTAL LAB PROJECT NUMBER: U2723 STUDY COMPLETION: October 22,2001 This study was conducted in compliancewith Good Laboratory Practice Standards as published by the US. EnvironmentalProtection Agency in 40 CFR Part 16OY17August 1989and OECD PrinciplesofGood Laboratory Practice (ENVMCKHEM (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: Scientist Wildlife International, Ltd. c@-.m-o DATE SPONSOR APPROVAL: P Wildltfe InternationaI, Ltd. -- Project Number 454C- 125 QUALITY ASSURANCE STATEMENT This study was examined for compliancewith Good Laboratory Practice Standardsas publishedby the U.S. EnvironmentalProtection Agency in 40 CFR Part 160,17August 1989and OECD Principlesof 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: ACTMTY Matrix Fortification Preparation Raw Data, Draft Report Final Report DATE CONDUCTED: August 30,2001 October 11, 12 and 15,2001 October 22,2001 DATE REPORTED TO: STUDY DIRECTOR: MANAGEMENT: August 30,2001 September4,2001 October 15,2001 October 16,2001 October 22,2001 October 22,2001 Jam%sH. Coleman, B.S. Quality Assurance Representative io-32-01 DATE WildlifeInternational, Ltd. -4- Proiect Number 454C- 125 REPORT APPROVAL SPONSOR 3M Corporation TITLE: Analytical Method Validationfor the Determinationof Perfluorooctanesulfonate,Potassium Salt (PFOS)in Plant Tissues WILDLIFE INTERNATIONAL, LTD. PROJECT NUMBER: 454C- 125 3M ENVIRONMENTAL LAB PROJECT NUMBER: U2723 STUDY DIRECTOR: R a y d o d L . Van Hoven, Ph.D. Scientist MANAGEMENT: Willard B. Nixon, P h . d Director of Analytical Chemistry b WildlifeInternational. Ltd. -5- .. Project Number 454C- 125 TABLE OF CONTENT$ TitleKover 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 Tomato .................................................................................................................................. 10 Soybean ................................................................................................................................. 1 1 Analytical Validation Procedures...................................................................................................... 1 1 Primary and Secondary Stock Solutions ........................................................................... Calibration Standards and Curves ................ ................................................................ Matrix Blanks and Matrix Fortifications.......................................................................................... 12 Limit of Quantitation ........................................................................................................................ 12 Results .......................... ............................... ................................ Reagent and Matr ank Samples .................................................... .................................... 13 Method Validation Samples............ ........................... Conclusions ................................................................................................................................................. 13 WiIdltfe InternationaI, Ltd. -6- Proiect Number 454C- 125 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 Data ............................................................................... 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 PFOS ......................................................................... 35 Appendix 3.4 - Example Calculations for a Representative Sample ......................... Appendix 3.5 - Ion Chromatogram of a Low-level PFOS Standard....................................... Appendix 3.6 - Ion Chromatogram of a High-level PFOS Standard ........................ Appendix 3.7 - Ion Chromatogram of an Onion Matrix Blank .......................... Appendix 3.8 - Ion Chromatogram of a Low-level Onion Fortification Sample.................................... 40 Appendix 3.9 - Ion Chromatogram of a High-level Onion Fortification Sample .................................. 4 1 Appendix 3.10 - Ion Chromatogram of an Alfalfa Matrix Blank ............................................................ 42 b WildlifeInternational. Ltd. -7- .. Project Number 454C-125 TABLE OF CONTENTS .Continued . Appendix 3.1 1 .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................................ 49 Appendix 3.18 .Ion Chromatogram of a High-level Soybean Fortification Sample............................... 50 Appendix 4 . Personnel Involved in the Study ..................................................................................... 51 I I WildlifeInternational, Ltd. -8- -- Project Number 454C- 125 SUMMARY SPONSOR: 3M Corporation TITLE: AnalyticalMethod Validationfor the Determinationof 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 ExperimentalStart (OECD): August 30,2001 ExperimentalStart (EPA): August 30,2001 ExperimentalTermination: October 4,200 1 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 concentrationsof O.O:jOO, 0.500, 5.00 and 50.0 mg a.i./Kg. The samples were extracted with methanol, agitated, centrifuged, diluted into the instrumentalcalibration range with dilution solvent (50% methanol : 50% NANOpure@water) and analyzed by high performance liquid chromatography triple quadrupolemass spectrometry(HPLCMSMS). Recoveries of PFOS in onionand 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 interferingcomponents. The relativeprecision (CV) of triplicate fortificationsof 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./Kgfortificationlevel, ranged from 2.28 to 11.4 percent. WildlcfeInternational, 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 sampleswere fortifiedwith Perfluorooctanesulfonate,Potassium Salt (hereafterreferred to as PFOS) and analyzedwith high performance liquid chromatography mass spectrometry (HPLC/MS/MS) to evaluate the performance of a method for the determinationof PFOS in plant tissues. Validation samples were prepared and analyzed between August 30, 200 1and October4,200 1. Raw data generated by Wildlife International, Ltd. and a copy of the finalreport 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 Ieaves and stems,and h i t 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 of samples from anticipated plant toxicxty tests. A reagent blank sample and matrix blank sampks 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. MATERIALSAND 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 4 WiIdlife International., Ltd. - 10- -_ Proiect Number 454C-125 was described as a white powder. It was identified as FC-95 from Iot number 217,with an expirationdate of August 3 1, 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 I1 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, 2001and 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 appropriatenumber of 1.OO g aliquots were weighed from the onion hlomogenate. 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 2 1,2001, and identified as lot number 12-18-01. Upon receipt, &.ebulk aLfalfatissuewas 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 3 15 1 with an expiration date of 8/30/02. The tornatoes were quartered, WildlifeInternational, 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-OZF. rench-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.OO g aliquots were weighed from the soybeanhomogenate. Theremainderof the 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 h i t samples (tomato and soybean). A reagent blank (containing everything except PFOS and matrix) was carried through the entire procedure for each of the foiur matrices. The tissue sampleswere extractedwith methanol, agitated for a minimum of 30 minuteson a gyratoryshaker table at approximately 250 rpm and then centrifuged. The fruit samples were extracted with methanol, agitated for a minimumof one minute manually and then centrifuged. Dilutions into the calibration range of the HPLCIMSNS methodology were performed with a solution of 50% methanol (HPLC gradle, 99.9+%) and 50% NANOpure@water. Sampleswere then analyzed by direct injection. Concentrationsol'PFOS were determined by reverse-phase high performance liquid chromatography using a Hewlctt-Packard Model 1100 High Performance Liquid Chromatograph (HPLC) with a Perkin-Elmer API 30001,C Mass Spectrometer equipped with a Perkin-Elmer TurboIonSpray ion source. Chromatographic separationswere aclievedusing a Keystone Betasil Cl8column(50 mm x 2.0 mm, 3-pm particle size) fittedwith aKeystoneJavelin CISguard 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. I I WildlifeInternational, 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 bly 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 5 0 5 0 methanol: NANOpure@water by appropriatcdilutions of the 1.00 and 0.100 mg a.i./L stock solutions of PFOS in methanol. Calibration standards of PFOS, 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 simples. 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 seco:ndary 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 LKg). I 1 Wildlcfe 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 analyzedto determinepossible interferences. No interferenceswere 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 presentedin 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). Representativeion 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 3.1 1 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). Representativeion 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). Representativeion chromatograms of low and high-level forlificationsin soybeanare presented in Appendices 3.17 and 3.18, respectively. The relative precision (CV) of the methodology, as demonstrated by the analysisof triplicatesamples 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 2.25 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 HP:LC/MS/MS methodology were performed with a solutionof 50% methanol and 50% NANOPUX@water. Samples were , L WildlifeInternational, Ltd. - 14- -... Proiect Number 454C- 125 then analyzed by high performance liquid chromatographymass spectrometry(HPLCMSMS). 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. WiIdlife Int e m utionul?Ltd. -15- Project Number 454C-125 REFERENCES 1. American Society for Testing and Materials. 1991. Standard Specificationfor Reagent Water. D1193-91, ASTM Section I1 Water and Environmental Technology, Vol. I. 1.01:45-47. 6 WildlcfeInternational, Ltd. -16- -- Project Number 454C- 125 Table 1 Method Validation Recoveries for PFOS in Onion PFOS Concentration Sample (mg a.i./Kg) Mean Number Percent Measured > 454C-1_ 25- __FT ortified My easured' p Recoverede ' (mga.i./Kg) Mean Percent Recovery VREB-4 Reagent Blank 0 c LOQ' -- VMAB- 10 VMAB-11 VMAB -12 Matrix Blank Matrix Blank Matrix Blank __ 0 c LOQ 0 c LOQ -- 0 c LOQ -- VMAS - 37 VMAS - 38 VMAS - 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= 1: 0.00567 89.2 cv=:11.4% VMAS - 40 VMAS-41 VMAS - 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= :t0.0161 93.6 cv=.3.54% VMAS - 43 Matrix Fortlfication 5.00 4.84 VMAS - 44 Matrix Fortification 5.00 4.86 VMAS - 45 Matrix Fortification 5.00 4.93 96.9 4.88 97.6 97.2 SD= :f:0.0473 98.7 CV=o.969% VMAS - 46 Matrix Fortification 50.0 47.4 VMAS - 47 Matrix Fortification 50.0 50.1 VMAS - 48 Matrix Fortification 50.0 49.7 94.7 49.1 98.2 100 SD==f 1.46 99.4 CV==2.97% Overall Mean = 96.7 Standard Deviation = 6.34 CV= 6.56% ' N= 12 Measured and PercentRecovered values were calculated using MacQuan, version 1.6 software. Manual calculationsmay vary slightly. b ) 'The limit of quantitation(LOQ)was 0.0400 mg a.i./Kg based upon the product of the lowest calibration standard -_._ WildlifeInternational, Ltd. - 17- Project Number 454C-125 Table 2 Method Validation Recoveries for PFOS in Alfalfa Number (454C-125) Sample Type WEB-3 Reagent Blank VMAB-7 VMAB-8 VMAB-9 Matrix Blank Matrix Blank Matrix Blank VMAS - 49 VMAS - 50 VMAS-51 Matrix Fortification Matrix Fortification Matrix Fortification VMAS - 28 VMAS - 29 VMAS - 30 Matrix Fortification Matrix Fortification Matrix Fortification VMAS-31 VMAS - 32 VMAS - 33 Matrix Fortification Matrix Fortification Matrix Fortification VMAS - 34 VMAS - 35 VMAS - 36 Matrix Fortification Matrix Fortification Matrix Fortification PFOS Concentration (mg a.i./Kg) Fortified Measured' 0 <LOQ~ 0 c LOQ 0 < LOQ 0 e LOQ Percent Recovered' __ -_-_ -- Mean Measured (mg a.i./Kg) Mean Percent Recovery -- -_ 0.0500 0.0500 0.0500 0.0521 0.0477 0.0449 104 0.0482 96.4 95.3 SD= f0.00363 89.7 CV= 7.52% 0.500 0.500 0.500 0.458 0.467 0.490 91.6 0.472 94.4 93.4 SD= f 0.0165 98.0 CV= 3.50% 5.00 4.40 5.00 4.74 5.00 4.77 87.9 4.64 92.8 94.9 SD==f0.206 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- f0.902 96.6 cv= 1.90% Overall Mean = 94.6 - Standard Deviation = cv= N= 4.13 4.37% 12 l i t i o n s may vary slightly. 'The limit of quantitation(LOQ) was 0.0400 mg a.i./Kgbased upon the product of the lowest calibration standard ~ 0 L/Kg). WildlifeInternational, Ltd. - 18- Project Number 454C-125 Table 3 Method Validation Recoveries for PFOS in Tomato Number 454C-125- Sample Type VREB-1 Reagent Blank VMAB-1 VMAB-2 VMAB-3 Matrix Blank Matrix Blank Matrix Blank VMAS-1 VMAS-2 VMAS-3 Matrix Fodication Matrix Fortlfication Matrix Fortification VMAS-4 VMAS-5 VMAS-6 Matrix Fortification Matrix Fortification Matrix Fortification VMAS-7 VMAS-8 VMAS-9 Matrix Fortification Matrix Fortifcation Matrix Fortification VMAS-10 VMAS-11 VMAS-12 Matrix Fortlfication Matrix Fortification Matrix Fortification PFOS Concentration (mg a.i./Kg) Fortified Measuredl 0 < LOQ* 0 < LOQ 0 < LOQ 0 < LOQ 0.0500 0.0500 0.0500 0.0493 0.0409 0.041 1 0.500 0.500 0.500 0.430 0.480 0.449 5.00 5.00 5.00 4.52 4.99 4.50 50.0 50.0 50.0 45.9 44.3 45.7 Percent Recovered' -- -_-_ -- 98.6 81.9 82.1 85.9 95.9 89.8 90.3 99.9 90.0 91.8 88.7 91.4 Mean Mean -- Measured (mg a.i./Kg) Percent Recovery 0.0438 SD= f0.00479 cv= 11.0% 0.453 SD= f 0.0252 Cv=5.57% 4.67 SD- f0.277 cv= 5.94% 45.3 SD==f 0.872 Cv= 1.92% 87.5 90.6 93.4 90.6 Overall Mean = 90.5 Standard Deviation = 5.68 - CV= 6.28% N= 12 ~ , t i o nmay s vary slightly. 'The limit of quantitation(LOQ)was 0.0400mg a.i./Kg based upon the product of the lowest calibration standard l g ) . WildlifeInternational, Ltd. - 19- Project Number 454C- 125 Table 4 Method Validation Recoveries for PFOS in Soybean Number (454C-125-) Sample TYPe WEB-2 Reagent Blank VMAB-4 VMAB-5 VMAB-6 Matrix Blank Matrix Blank Matrix Blank VMAS - 13 VMAS - 14 VMAS - 15 Matrix Fodication Matrix Fortification Matrix Fortification VMAS - 16 VMAS - 17 VMAS - 18 Matrix Fortification Matrix Fortification Matrix Fortification VMAS - 193 VMAS - 20 VMAS - 2 1 Matrix Fortification Matrix Fortifcation Matrix Fortification VMAS - 22 VMAS - 23 VMAS - 24 Matrix Fortification Matrix Fortification Matrix Fortiiication PFOS Concentration (mg a.i./Kg) Fortified Measured' 0 LOQ~ 0 < LOQ 0 < LOQ 0 < LOQ Percent Recovered' -_ __ -- -- 0.0500 0.0440 87.9 0.0500 0.0449 89.9 0.0500 0.0429 85.7 0.500 0.487 97.3 0.500 0.473 94.7 0.500 0.478 95.6 5.00 5.01 100 5.00 4.83 96.6 5.00 4.94 98.9 50.0 50.0 100 50.0 48.7 97.4 50.0 50.1 100 Mean Mean Measured Percent (mga.i./Kg) Recovery P P -- __ 0.0439 87.9 SD= M.OO1OO CV=2.28% 0.479 95.9 SD= k 0.00709 CV= 1.48% 4.93 98.5 SD= k 0.0907 CV= 1.84% 49.6 99.2 SD- f 0.781 CV= 1.57% Overall Mean = 95.3 Standard Deviation = 4.92 CV = 5.16% N= 12 _ I _ s d a n u a l calculat'ions may vary slightly. The limit of quantitation(LOQ) was 0.0400 mg a.i.Kg based upon the product of the lowest calibration standard (0.000400mg a.i./L) and the overall dilution factor of the matrix blank samples (100 LKg). Measured value is the mean of one re-dilution, duplicateinjection, 454C-125-19R1, and 19R2. WildlifeInternational?Ltd. - 20 - Appendix 1 Protocol Project Number 454C- 125 -- WildlifeInternational, Ltd. Project Number 454C- 125 -21 - PROTOCOL ANALYTICAL h4EXHOD VALIDATION FOR THE DFXERh4INATION OF PERFLUOROOCT~SULFONATE,POTASSIUM SALT (PFOS)IN PLANT TISSUES En- Laboratory Request Number U2723 Submitted to 3M Corporation Environmental -wry 935 Bush Avtrme st. Paul,Minntsata 55106 WildlijieInternational Ltd. 8598 Commerce Drive Easton,MatyM 21601 (410) 822-8600 May 2,2001 WildlgfeIntemational, Ltd. - 22 - Project Number 454C-125 wildlife International, Ltd. -2- ANALYTICAL METHOD VALIDATION FORTHE DETERMINATION OF PERFLUOROOC"ANESULF0NATE.POTASSIUM SALT (PFOS)IN PLANT TISSUES SPONSOR 3M Corporation Environmental Laboratory P.O. Box 33331 St. Paul Minnesata 55133 SPONSOR'S REPRESENTATIW Ms.Rochelle Robideau TESTING FACILITY: w i l a JnbmatiwLtd. 8598 commerceDrive Easton, Maryland21601 STUDY DIRECTOR Raymond L. VanHoven, Ph.D. Scientist Wddliie International, Ltd. LABORATORY MANAGEMENT: Willard B. Nixon, Ph.D. Manager of Analytical Chemistry Wildlife International, Ltd. FOR LABORATORY USE ONLY ProjcctNo.: 454C- 125 TestSubstamxNo.: 4675 - PROTOCOL NO.: 454/05020l/MV-PT/SUB454 EnvironmentalLaboratory Request Number U2723 b -- WildlifeInternational, Ltd. Proiect Number 454C- 125 - 23 - wildlife International, Ltd. -3- INTRODUCTION Wildlie International, Ltd. will condud analyses to validate the performance of a liquid chromatography mass spectromet~~ (LCIMS) method for the determination of Perfluorooctanesulfonate,Potassium Salt (hereafterreferred to as PFOS)inplanttissues and h i t . The study will be p e r f o d at the Wildlife h-onal, Ltd. analytical chemistry faciity in Easton, Maryland. The method de~~lopeatdWildlife Manational, Ltd. will be validated by fortifying plant tissues and quantifyingthe rccOvcrieS of PFOS. Raw data for all work performeda! Wildlife Intema- tional, ud and a copy of the final report will be filed by project number in archives located on the Wildlife International,Ltd. site, or at an alternativelocationto be specified in the final repoa. OBJECTIVE The objective of this study is to validate the performance of LUMS methodology for determinationof PFOS residues in plant tissues and h i t s used by Wildlife International. Ltd. EXPERIMENTAL DESIGN Plant tissues andor hits f?om several species of plants will be fortified with at least four d i f f m t c ~ ~ c e n t r a tainod~a~n~alyzed using LUMS methodology developed at Wildlife International, Ltd. 'zhc method will be based on prooeduns developed at Wildlife htemaiional, Ltd. Reagent and matrix blanks will be analyzed to evaluate potensial analytical interferemxs. One calit~rationcurve will be pnpared and analyzed with each series of matrix fortification samples. ne acarracy. pncision, and limitof quaositationof the methodwillbedetermined. MATERIALSANDMETHODS Test Substance Ihc test substance will be P e d u o r m & m e d f ~ Potassium Salt. Information on the cbar;lderitatioa of tcst, COntFol or rcferaKx substances is mquired by Good Laboratory Practice Standards (GLP). Tlst Sponsor is nsponsible for providing Wildlife htematid. Ltd. written verification thatthe test substancehas been characttn'zedaccording to GLPs prior to initiation ofthe study. Tbt rcfcrence mated in this study will be thetest substance. If written verificationof GLP test substance charactcritation is not provided to Wildlife International, Ltd., it will be noted in the compliance statanent of the final report. PROTOCOLNO.: 454/05020l/MV-PT/SUB454 Environmental Laboratory Request Number U2723 WildltfeInternational?Ltd. Project Number 454C- 125 w i l d l i f e International, Ltd. - -4- The Sponsor is responsible for all information related to the test substance and agrees to accept any unused test substanceand/or test substance containers remainingat the end ofthe study. Reagents and Solvents All solvents used in the methcd or procedure will be of reagent grade or better. All reagents will be ofACS grade or better. PlMt Tissues Plant tissues will be obtained !?om onion and alfalfa plants that have no known cxposure to the test substance. There are no levels ofcoatamioantsreasonably cxpeded to be present in the plant tissues that are considered to interfere with the purpose or conduct of the study. Fruits Fruits will be obtained from two species of plants (tomatoes and soybeans) that have no known exposure to the test substance. There L e no levels of contaminants reasonably expected to be present in the plant tissues that are considered to interfere with the purpose or conduct of the study. Analytical Verification Procedures 'Ihe analytical method to be used will be Lc/MS based upon procedures developed at Wildlife International, Ltd. 'Ihemetbodusedwill bedoaunentadinthe raw dataand summanz. ed inthe.final report. Prior to the analysis of analytical samples, primary stock solution(s) will be prepared directly from the test substance. Calibration standards will be pnparcd by appropriate dilution ofthe primary stock solution(s). Primary Stock Solution(s), Calibration Standards and Curves A minimum of five calibration standards (of different conCentratons) will be prepared and analyzed along with each analysis set. The calibration standard series will be injected at the b e g i i and one standard injected, at a minimum, after every five samples. A minimum of two injections of each standard in the calibration standard series will be injected with the analysis set. One calibration curve and regression equation will be prepared from the series of calibration standards. PROTOCOL NO.: 454/0502OI/MV-PT/SuB454 Environmental Laboratory Request Number U2723 -- WildlifeInternational, Ltd. Project Number 454C- 125 - 25 - 'wildlveInternational, Ltd. -5 - Fortification Stock Solutions Plant tissue and h i t homogenates will be fortified with a stock solution(s) of PFOS at levels anticipatedto bracket levels in thebiownmtration study. Evaluation of Interferences One reageat blank and threc matrix blanks will be analyzed to detect possible intcrferasces. Thc mgeat blank will contain cvtrythiog except the matrix and the test substance. The matrix blanks will containcvgrthing except the test substance. Verification Analyses - Method Performance Matrix fortifications (spiked plant tissues and fruits or seeds), prepared at known conCentrationsof the test substance, will be anaIyzedto determine the ncovery of the analyte and to evaluate method performance. The anticipated verification series will consist of the following analyses: SUMMARY OF A TYPICAL VERIFICATION ANALYSIS SCHEME onion Alfalfa Tomato IRmgeatBlank (Sol-) Matrix Blank (Plant tissues) Matrix Fottificatioas 0 (Control) 0 (Coatrol) Lcvd 1 -Low' Level2 Lcvcl3 1 1 3 3 3 3 3 3 3 3 I 1 3 3 3 3 3 3 3 3 Mat& fortifications will represent the range of bimnmtration that is anticipated to be found in plant tissue in subsequent ef' tests and will be selected in consultation with the Sponsor. PROTOCOLNO.: 454/050201~-PT/SuB454 Environmental Laboratory Request Number U2723 WiId1ife IntemationaI , Ltd. Proiect Number 454C- 12j wildlifeInternational, Ltd. -6- Individual samples (identified by project number and a unique sample identScation number) will be prepared and analyzed for verification of the analyticalmethodology. If diEcuIties arise in the veriiication process (e.g. low recovtfies or interferences), the Sponsor will be n o t 3 4 and the need for additional veri6cationor method development d be determined through discussion with the Sponsor. Upon completion of the method verification, the Sponsor will review the results and a u t h o h use of the methodology for analysis of samples, or will authorizC further method developmenttrials. The acceptable range of Cecoveries is 70 to 120%of the nominal concQltratons. Data Analysis One calibration curve will be established for each analytical run. A regression equation of the concentration versus peak area for the calibration standards will be generated. The concentration of the samples will be determined by substituting the respective peak area into the regression equation. Statistics to be determined and reported will include the mean and standard deviation for each level of fortification. RECORDS TO BE MAINTAINED Recordstobcmaintainedfor data gentrated by Wildlife International, ud.will include: 1. A copy of the signed pmtocol. 2. Identificationandchamcten.zat`lonof the test substance and/or analytical standard, if bythespasor. 3. Dates of initiationdtcmrioatiOn ofthe test. 4. Storage conditions for test substame, analytical standards, and/or samples. 5. Tcst substance ador analytical standarduse log. 6. concentrationcalculationsand recordsof solutionpreparation. 7. Instnunentaperatingmditionsaadchromatognuns. 8. statisticalcalculations. 9. A copy ofthe finalreport. 10. Documentation that the steps in the method were followed. PROTOCOLNO.: 454/05020l/MV-PT/SuB454 EnvironmentaJ LaboratoryRequest Number U2723 WildltfeInternational, Ltd. - 27 - Project Number 454C-125 wildlife International, Ltd. -7- FINAL REPORT The report will Summarize the findings of the verification,the procedural recoveries obtained, and the methods and instrumentation employed. Upon receipt of these hdings, the Sponsor will review the methods and results and evaluate the results,foracceptability prior to initiating any fate d o r &e& studies. The fiaalreport will include, but not be limitedto, the following: 1. Name and address ofthe facility performing the study. 2. Dates on whichthe study was initiatedandcompleted. 3. Objectives and procedures stated in the approved protocol, including any changes in the originalprotocol or deviations from the protocol. 4. The test substance andor analytical standard identification, including name,chemical abstract number or code number, streng& purity, composition, date of receipt, lot number, storage .. conditions, physical &m#emh cs, stability and method of preparation of test concendons, if provided by the Sponsor. 5. A detailed summary of the analytical methodology: A description of the experimental measurements. example calculations, sample preparation (sample weights and dilutions), instrumentation employed, reagents and solvents used, class of water usad, and any major modificationsto the method. 6. A descriptionof ciraunstanwsthat may have *the quality or integrityofthc data 7. The name of the Study Dinctor, the names of other scientists or professionals, and the names ofall supervisorypmonnelinvolvedin the study. 8. Ademiptionofthe- 'om,calculatims,or operations performedon thedata. 9. The signed and dated reports of each of the individual scientists or other professionals involved in the study, ifapplicable. 10. The location when raw data and final report are stored at theconclusion ofthe study. 11. A statement prepared by the Quality Assurance Unit listing the dates that study inspectionS were made and the dates of any findings reported to the Study Dimtorh4anagemcnt. PROTOCOL NO.: 454/050201/MV-PT/SUB454 Environmental Laboratory Request Number U2723 WildlifeInternational, Ltd. -28- Project Number 454C- 125 w i l d l y e International, Ltd. - -8- CHANGING OF PROTOCOL Planned changes to the protocol will be in the form of written amendments signed by the Study Director and the Sponsor's Representative. Amendments will be considered as part of the protocol and will be attached to the final protocol. Any other changes will be in the forni of written deviations signed by the Study Director and filed with the raw data. AU changes to the protocol will beindicatedinthefinalrepolt. GOOD LABORATORY PRACTICES This study will be conducted io accotdancewith Good Laboratory Practice Standards for P A (40 CFR Part 160 andlor Part 792); and OECD Principles of Good Laboratory Practice (ENV/MUCHEM (98) 17). Each study d u c t e d by Wildlife Merdonal, Ltd. is routinely examined by the Wildlife Intedonal. Ltd. Quality Assurance Unit (QAU) for compliance with Good Labratory Practices, Standard Operating Procedures and the specified protocol. n e final report will also be reviewed by the QAU. A statement of compliance with Good Laboratory Practices will be prepared for all portions of the study conducted by Wddlife International,Ltd. The Sponsor will be responsible for certi6cation of c o m p l i c e with Good Laboratory Practices for procedures performed by other laboratories Raw data for all work performed at Wildlife International, Ltd. and a copy of the final report will be filed by project number in archives located on the Wildlife International,Ltd.site, or at an altemativelocation to be p i t i e din the finalreport. PROTOCOL NO.:454/05020lIMV-PT/SUB454 EnvironmentalLaboratory Request Niumber U2723 WildlifeInternational, Ltd. - 29 - -- Project Number 454C-125 Appendix 2 Certificate of Analysis INTERIM CERTIFICATE OF ANALYSIS Reviswn ipnmo) Centre Analytical Laboratories COA Reference #: 023-018A 3M Product: PFOS,Lot 217 Reference #: SD-018 1. Carbon COA023418A 1. 4.015 wt./wt.% 2. 0.59wt.hvt.% 3. 4 . 0 4 0 wtJwt.0,~ 4. 4.009Wt./wt.% 5 . -4.006 wt./wt.a/o 6. 4.007wt./wt.a/o 1 Theoretical Value = 17.8% 2 TheoreticalValue = 0% 3 ?heoreticalvaluc=00% 1. 12.48 Wt./wt.% 2. 0.244wta/wt.% 3. 1.74 wt./wt.% Page 1 of3 WildltfeInternational, Ltd. - 30 - -- Project Number 454C-125 INTERIM CERTIFICATE OF ANALYSIS Centre Analytical Laboratories COA Reference #: 023-018A Date of Last Analysis: 0813 1/00 ExpirationDate: 08/31/01 Storage Conditions: Frozen S-IO'C Re-assessment Date: 08131/01 'Purity = 100% - (sum of metal impurities, 1.45% + L W S impurities, 8.41%+Inorganic Fluoride, 0.59%+Nh4R impurities, 1.93%+organicacid impurities,0.38%+PO,@ 0.33%) - Total impurity fromall tests = 13.09% Purity = 100% 13.09% = 86.9% 2Potassiumis expected in this salt form and is therefore not considered an impurity. 'Purity by DSC is generally not applicable to materials of low purity. No endotherm was observed for this sample. 4Su&r in the sample appearsto be converted to SO4 and hence detectedusing the inorganicanion method conditions. The anion result agrees well with the sulfiir determinationin the elemental analysis, lending confidenceto this interpretation. Based on the results, the SO4 is not consideredan impurity. %A HFBA NFPA PFPA Trifluoroaceticacid Heptafluorobutyric acid Nonofluoropentanoic acid Pentatluoropropanoicacid 6Theoretical value calculationsbased on the empiricalformula, CsF1,SO~~+~vZW-538) This work was conducted under EPA Good Laboratory Practice Standards (40 CFR 160). i COA023-018A Page 2 of 3 WildlifeInternational, Ltd. -31 - Project Number 454C-125 INTERI..CERTIFICATE OF ANALYSIS Centre Analytical Laboratories COA Reference #: 023-018A LClMS Purity Profile: Impurity c4 cs C6 wtfwt. Yo 1.22 1.33 4.72 , Note: The C4 and C6 values were calculated using the C4 and C6 standard calibration curves,respectively. The CS value was calculatedusing the average response factors from the C4 and C6 standard curves. Likewise, the C7 value was calculatedusing the average response factors from the C6 and C8 standard curves. Prepared By: David S.Bell - Date Scientist, Centre AnalyticalLaboratories Reviewed By: I John Flaherty - Date Laboratory Manager, Centre Analytical Laboratories COA023-0 18A Page 3 of 3 I -_ WildlifeInternational, Ltd. - 32 - Appendix 3 Analytical Method Validation Data Project Number 454C- 125 * WildlifeInternational, Ltd. - 33 - .Project Number 454C-125 Appendix 3.1 Analytical Method Flowchart for the Analysis of PFOS in Plant Tissue and Fruit Weigh approximately 1.OO 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. J 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 szunples. The additional sample is the reagent blank. 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 h i t samples, cap vials and shake manually for a minimum of one minute. Centrihge samples for approximately ten minutes at 2000 rpm. Dilute samples into the calibration range of the PFOS LCMS methodology: Partially fill Class A volumetric flasks with dilution solvent (50% methanol: 50% NANOpure@water). Add appropriate volume of samplewith 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. Ampulate samples and submit for HPLC/MS/MS analysis. WildlifeInternational, Ltd. - 34 - Project Number 454C-125 INSTRUMENT: Appendix 3.2 Typical HPLC/MS/MS Operational Parameters Hewlett-Packard Model 1100 High Performance Liquid IChromatogaph with a Perkin-Elmer API 3000 Mass Spectrometer equippd With aPerkin- Elmer TurboIonSpray ion source. Operated in multiple ion reaction monitoring (MRM) mode. ANALYTICAL COLUMN: GUARD COLUMN: OVEN TEMPERATURE: STOP TIME: FLOW RATE: MOBILE PHASE: INJECTION VOLUME: PFOS RETENTION TIME: PFOS MONITORED MASS: Keystone Betasil C18column (50 mm x 2 mm I.D., 3-pm particle size) Keystone Javelin CIScolumn (20 mm x 2 mi I.D.) 40C 5.00 minutes 250 pWminute 70.0% Methanol: 30.0%NANOpure@Water containing 0.1% Formic Acid 10.0 pL Approximately 4.0 minutes 499.0 amu +99.1 m u W i l d l i f e I nterna tional? L t d . - 35 - -- Proiect Number 454C-125 Appendix 3.3 ' A Typical Calibration Curve for PFOS PFOS 499.0->99.1 No Internal Standard Weighted (llx) Intercept = 352.9491 Slope = 9618.9219 Correlation Coeff. = 0.99906 Area 50000 40000 30000 2000c 1 oooc C I I I 0.60 , , , 1.20 , , , 1.80 I I I 2.40 1 I I 3.00 Conc. ug a.i./L I I I - -n 3.60 4.20 4.80 WildltfeInternational, Ltd. -36- Project Number 454C-125 Appendix 3.4 ' Example Calculationsfor a Representative Sample Sample number 454C-125-VMAS-1,nominal concentrationof 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 (VI)= 10.0mL Secondary Dilution: Initial Volume (Vi) = 1.OO mL Final Volume (Vf)= 10.0 mL Overall Dilution Factor (VJW,x Vfli) 100mL =- - - lOOL - g Kg eak area - y-intercept) PFOS at instrument(pg a.i./L) = (P 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 (LKg) x conversionfactor (0.001mg/pg) 0.49278 pg a.i. lOOL PFOS in sample (mg a.i./Kg) = x-x- L Kg 0.0013 PI; -- 0.049278 mg a i . Kg PFOS m a.i./L in sam le Percent of Nominal Concentration = p,os((~g nomin:l x 100 -- 0.049278 x 100=98.6% 0.0500 Instrument Software: MacQuan, version 1.6. Wild1ife Intemational, Ltd. - 37 - -- Project Number 454C-125 Appendix 3.5 ' Ion Chromatogram of a Low-level PFOS Standard PFOS-1 STD 0.400 ug a.lL 4675A-011 D-26 4.98 in 1 period pR3s No Internal Standard Use Area 1: 4.97 MRM, 298 499.0-r99.1 Noise Thres. Quant Thres. Min. Width Mutt. Width Ease. Width RT Win. (sea) Smooth Expected RT Area 4336 Height 471 Start Time End Time Integration Width Retention Time Integration Type scans 2.0 1.0 12 10 30 10 2 4.37 3.58 4.04 0.45 -3.77 A' EE Tue, Oct 2, 2001 09:49 1 intensity: 6000 cps Nominal concentration: 0.000400 mg a.i./L WildlifeIntemationa1, Ltd. - 38 - -Project Number 454C-125 Appendix3.6 . . Ion Chromatogram of a High-level PFOS Standard PFOS-6 STD 5.00 ug a.lJL 4675A-0110-31 4.98 in 1 period PFc6 No Internal Standard U s e Area 1: 4.97 MRM,298 scans 499.0->99.1 Noise Thres. 2.0 Quant Thres. 1 .O Min. Width 12 Mult. Width 10 Base.m 5 0 RTWin.(secs) 1 0 Smooth 2 Expected RT 4.37 Area 48627 HeiQht 5149 Start Time 3.53 End Time 4.14 lntegratlon Wldth 0.60 Retention Time Integration Type 3.77 A - VB Tue, Oct 2, 2001 10:19 inlensily: 6000 cps 9O 3 225 8 7 6 5 4 3 2 1 I1 scan 0.69 1.36 2.03 2.70 3.37 4.04 4.71 Time Nominal concentration: 0.00500 mg a.i./L WildlifeInternational, Ltd. -39- -- Proiect Number 454C- 125 Appendix 3.7 . Ion Chromatogram of an Onion Matrix Blank PFOS-10 454C-125- VMAB-11 m4.98 in 1 period No Internal Standard Use Area 1: 4.97 MRM, 298 scans 499.0->99.1 Ndse Thres. 2.0 Quant Thres. 0.5 Min. Width 12 Mull. Widlh 10 Base. Width 40 RTWin.(secs) 1 0 Smooth 2 Expected RT 3.72 Area 0 Height 0 Start Time 0.00 End Time 0.00 integration Width 0.00 Retention Time 0.00 Integration Type ,;Wed, Oct 3, 2001 12:55 10 543 2 1 intensity 4000 cps 1 Sample number 454C-125-VMAB-11. The arrow indicates the approximate retention time of PFOS. WiId1ijfe Intemationa1, 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 pFo6 No Internal Standard Use Area 1: 4.97 MRM, 298 scans 499.0->99.1 Noise Thm. 2.0 auantnres. 0.5 Mln. Width 12 Mult. Width 10 Base. Width 40 RTWin.(secs) 1 0 Smooth 2 ExpededRT 3.72 Area 4605 Height 472 Start Time 3.57 End Time 4.24 lnlegratlon Width 0.67 Retention Time Integration Type -3.80 A BE Wed, Oct 3, 2001 13:19 intensity: 4000 cps :I'1 78J 3 2 227 1 3752 101120 150 xg-. ' 4'1 ' 8'1 ' lil 161 ' 261 0.69 1.36 2.03 2.70 3.37 4.04 4.71 Time Sample number 454C-I25-VMAS-38,0.0500 mg a.i./Kg nominal concentration,overall dilution factor = 100 LKg. WildlifeInternational? Ltd. -41 - Proiect 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 Fms No Internal Standard U s e 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 RTWin.(secs) 10 Smooth 2 Expected RT 3.72 Area 16775 Height 1869 Start Time 3.55 End Time 4.22 lntwration Width Retention lime Integration Type 0.67 -3.80 A B8 Wed, Ocl 3, 2001 14:20 10 4 3 2 1 41 0.69 81 1.36 121 2.03 161 2.70 intensity: 4000 cps 227 - 273 , l ' 261 seal 3.37 4..04 4.71 Time Sample number 454C-125-VMAS-47,50.m0g a.i./Kgnominal concentration, overall cidution factor = 25000 LKg. WiIdltfe InternationaI? Ltd. - 42 - -_ Proied. Number 454C-125 Appendix 3.10 ' Ion Chromatogram of an Alfalfa Matrix Blank PFOS-10 454C-125- VMAB-8 Tue, Ocl 2, 2001 10:43 4.98 in 1 period Fla No Internal Standard use Area 1: 4.97 MRM, 298 scans 499.0->99.1 Noise Thres. 2.0 Quant Thres. 1 .O Min. Width 12 Mult. Width 10 Base. Width 50 RTWin.(secs) 10 Smooth 2 Expected RT 4.37 Area 0 Height 0 Start Time 0.00 End Time 0.00 Integration Width 0.00 Retention Time 0.00 Integration Type 304 24 52 intensity: 6000 cps I 5 133 168 lil 161 2.03 2.70 201 228 27fi 201 241 281 Scan 3.37 4.04 4.71 Time Sample number 454C-125-VMAB-8. The arrow indicates the approximate retention time of IPFOS. WildlifeInternational, Ltd. - 43 - Proiecl. Number 454C- 125 Appendix 3.1 1 . Ion Chromatogram of a Low-level Alfalfa Fortification Sample PFOS-9 454C-125- VMAS-49 Thu, Oct 4. 2001 12:39 m 4.98 in 1 period No lntemal Standard U s e Area 1: 4.97 MRM, 298 scans 499.0->99.1 Noise Thres. 2.0 Quant Thres. 0.5 Min. Width 12 Mull. Width 10 Base. Width 40 RT Win. (secs) 1 0 Smooth 2 ExpectedRT 3.72 Area 5449 Height 5 3 7 Start Time 3.55 End Time 4.17 Integration Width 0.62 Retention Time Integration Type -3.80 A VB intensity: 5000 cps 10 ~54 3 2 x8- 5 0 227 10- 0'8 2 2 95 168 I B I I I I I I I I 41 8 1 121 161 201 241 281 Scan 0.69 1.36 2.03 2.70 3.37 4.04 4.71 Time Sample number 454C- 125-VMAS-49,0.0500mg a.i./Kgnominal concentration,overall dilution factor = 100 LKg. WildlifeInternational, Ltd. - 44 - Proiecf. Number 454C-125 Appendix 3.12 . Ion Chromatogram of a High-level Alfalfa Fortification Sample PFOS-23 454C-125- VMAS-34 Tue, Oct 2. 2001 1201 4.98 in 1 period PFOS No lntemal Standard Use Area intensity: 6000 cps 1: 4.97 MRM, 298 499.0->99.1 Noise Thres. Quant Thres. Min. Width Mult. Width Ease. Width RTWin.(secs) Smooth ExpectedRT Area 18647 Height 1901 Start Time End Time Integration Width Retention Time Integration Type scans 2.0 1 .o 12 10 50 10 2 4.37 3.60 4.25 0.65 -3.89 A VB I 232 3 - 2 1 278 41 0.69 81 1.36 121 2.03 161 2.70 201 241 3.37 4.04 281 Scan 4.71 Time Samplenumber 454C-l25-VMAS-34,50.0 mg a.i./Kgnominal concentration,overall dilution factor = 25000 LKg. I WildlifeInternational, Ltd. - 45 - -_ Project Number 454C-125 Appendix 3.13 Ion Chromatogram of a Tomato Matrix Blank PFOS-10 454C-125- VMAB-2 Sat, Sep 1, 2001 12:04 m 4.98 In 1 period No internal Standard Use Area 1: 4.97 MRM, 298 scans 499.0->99.1 Noise Thres. 2.0 Quant Thres. 1 .o Min. Width 12 Mult. Width 10 Base. Width 40 RTWin.(secs) 1 0 Smooth 2 ExpectedRT 3.82 Area 816 Height 7 9 Starl Time 3.69 End Time 4.04 Integration Width 0.35 Retention Time Integration Type -3.80 A BB 1009080. 7060. 50. 40 30 201 12 41 41 0.69 intensity: 8000 cps r 92 81 1.36 135 159 184 $z7-, 285 , I 121 161 201, 241 281 Scan 2.03 2.70 3.37 4.04 4.71 Time Sample number 454C-125-VMAB-2. The arrow indicates the approximate retention time of E'FOS. b WildlifeInternational? Ltd. - 46 - ._ Proieci. 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 rn No Internal Standard Use Area 1: 4.97 MRM, 298 499.0->99.1 Noise Thres. Quant Thres. Min. Width Mull. Width Base. Width RT Win. (secs) Smooth Expected RT Area 8465 Height 972 Start Time End Time Integration Width Retention Time Integration Type scans 2.0 1 .O 12 10 25 10 2 3.82 3.57 3.99 0.42 -3.77 A* BB 10 1 40 ' 4'1 0.69 intensi1.y: 8000 cps 225 84 128 152 %1&,255 8'1 ' 141 ' 181 ' 201 241 1.36 2.03 2.70 3.37 4.04 , 281 Scan 4.71 Time Sample number 454C-125-VMAS-1,0.05m00g a.i./Kg nominal concentration, overall dilution factor = 100 L/Kg. WildlifeInternational?Ltd. - 47 - -- Proiect 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 m No Internal Standard Use Area 1: 4.97 MRM,298 499.0->99.1 Noise Thres. Quant Thres. Min. Width Mutt. Width Base. Width RTWin.(secs) Smooth Expected RT ' Area 24270 Height 2598 Start Time End Time Integration Width Retention Time Integration Type scans 2.0 1 .O 12 10 40 10 2 3.82 A 3.55 4.09 0.54 -3.79 VB Sat. Sep 1, 2001 13:28 10 intensity: 8000 cps 226 i4, 1 , , , , , , ,J. , 0 73 93 120 154 261285 41 8 1 121 161 201 241 281 Scan 0.69 1.36 2.03 2.70 3.37 4.04 4.71 T h e Sample number 454C-125-VMAS-11,50m.0g a.i./Kgnominal concentration,overall dilution factor = 25000 LKg. WildlifeInternational., Ltd. -48 - Proiecl.Number 454C- 125 Appendix 3.16 . Ion Chromatogram of a Soybean Matrix Blank PFOS-9 454C-125- VMAB-4 Fri, Sep 7 , 2001 1238 4.98 in 1 period FFc6 No Internal Standard Use Area 1: 4.97 MRM, 298 scans 499.0->99.1 Noise Thres. 2.0 Quant Thres. 1.o Min. Width 12 Mult. Width 10 Base. Width 40 RT Win. (secs) 1 0 Smooth 2 ExpectedRT 3.82 Area 0 Height 0 Start Time 0.00 End Time 0.00 Integration Width 0.00 Retention Time 0.00 Integration Type :?a9j 7 4 3 2010- 22 66 a9 io9 intensity: 4000 cps 167 199 232 275 Sample number 454C-125-VMAB-4. The arrow indicates the approximate retention time of PFOS. c WildlifeInternational, 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 1256 4.98 in 1 period pR)s No Internal Standard Use Area 1: 4.97 MRM, 298 499.0->99.1 Noise Thres. Quant Thres. Min. Width M u l . Width Base. Width RTWin.(secs) Smooth ExpectedRT Area 4065 Height 416 Start Time End Time Integration Width Retention Time Integration Type scans 2.0 0.5 12 10 60 10 2 3.82 3.50 4.19 0.69 -3.87 A* BE _i intensity: 4000 cps 10 5 4 3 2 1 231 0.69 1.36 2.03 2.70 3.37 4.04 4.71 Time Sample number 454C-125-VMAS-13,0.0500 mg a.i./Kgnominal concentration, ovmall dilution factor = 100 L K g . 0 WildltfeInternational, Ltd. - 50 - -- Proiecf Number 454C-125 Appendix 3.18 . Ion Chromatogram of a High-level Soybean Fortification Sample PFOS-26 454C-125- VMAS-24 Fri, Sep 7, 2001 14:20 4.98 in 1 period Ffcs No Internal Standard Use Area 1: 4.97 MRM,298 Scans 499.0->99.1 Noise Thres. 2.0 Quant Thres. 1.o Min. Width 12 Mutt. Width 10 Base. Width 40 RTWin. (secs) 1 0 Smooth 2 Expected FIT 3.82 Area 15377 Height 1645 Start Time 3.60 End T i m 4.27 Integration Width 0.67 Retention Time Integration Type -3.84 A BB intensity: 4000 cps 10 I229 1 0.69 1.36 2.03 2.70 3.37 4.04 4.71 Time Sample number 454C- 125-VMAS-24,50.0 mg a.i./Kgnominal concentration, ove:rall dilution factor = 25000 L/Kg. 4 4 f WildlifeInternational?Ltd. -51 - -- Proiecl:Number 454C-125 Appendix 4 Personnel Involved in the Study The following key Wildlife International, Ltd. personnel were involved in the conduct or inanagement 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