Document RJyjMZ6y8vg04e7V9eVwD0xbX

ANALYTICAL METHOD VALIDATION FOR THE DETERMINATION OF PERFLUOROOCTANESULFONATE, POTASSIUM SALT (PFOS) IN ARTIFICIAL SOIL WILDLIFE INTERNATIONAL, LTD. PROJECT NUMBER: 454C-120 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: February 5, 2001 STUDY COMPLETION DATE: May 27, 2003 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 46 Wildlife International. Ltd -2 - Project Number 454C-120 GOOD LABORATORY PRACTICE COMPLIANCE STATEMENT SPONSOR: 3M Corporation TITLE: Analytical Method Validation for the Determination of Perfluorooctanesulfonate, Potassium Salt (PFOS) in Artificial Soil WILDLIFE INTERNATIONAL, LTD. PROJECT NUMBER: 454C-120 3M ENVIRONMENTAL LAB PROJECT NUMBER: U2723 STUDY COMPLETION: May 27, 2003 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: Scientist Wildlife International, Ltd. SPONSOR APPROVAL: DATE Sponsor DATE Wildlife International. Ltd. -3 - Project Number 454C-120 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 Data and Draft Report Final Report DATE CONDUCTED: DATE REPORTED TO: STUDY DIRECTOR: MANAGEMENT: February 7, 2001 February 14 and 15, 2001 May 27, 2003 February 7, 2001 February 15, 2001 May 27, 2003 February 9, 2001 February 15, 2001 May 27, 2003 vSc>u>cun (jy^JU^cnn__________ Susan L. Coleman, B.A. Senior Quality Assurance Representative 5-7- 03 DATE Wildlife International. Ltd._______ -4 - Project Number 454C-120 REPORT APPROVAL SPONSOR: 3M Corporation TITLE: Analytical Method Validation for the Determination ofPerfluorooctanesulfonate, Potassium Salt (PFOS) in Artificial Soil WILDLIFE INTERNATIONAL, LTD. PROJECT NUMBER: 454C-120 3M ENVIRONMENTAL LAB PROJECT NUMBER: U2723 STUDY DIRECTOR: MANAGEMENT: Willard B. Nixon, Ph. Director of Chemistry s/si/o z DATE 0 /0 DATE Wildlife International. Ltd.__________ ______ Project Number 454C-120 -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 Invertebrate Testing Soil............................................................................................................... 10 N on -T arget Terrestrial Plant S o i l .........................................................................................................................10 Analytical Validation Procedures.............................................................................................. Primary and Secondary Stock Solutions........................................................................................... 11 Calibration Standards and Curves............................................................................................. Matrix Blanks and Matrix Fortifications.......................................................................................... 12 Limit of Quantitation.................................................................................................................. 11 11 12 Results .......................................................................................................................................... 12 Reagent and Matrix Blank Samples................................................................................... 12 Method Validation Samples.............................................................................................................. 12 Conclusions...................................................................................................................................................13 References 14 Wildlife International. Ltd. 6- - Project Number 454C-120 TABLE OF CONTENTS - Continued - TABLES Table 1 Method Validation Recoveries for PFOS in Non-Target Terrestrial Plant Soil....................15 Table 2 Method Validation Recoveries for PFOS in Invertebrate Testing Soil..................................16 APPENDICES Appendix 1 Protocol and Amendments.................................................................................. 17 Appendix 2 Certificate of Analysis......................................................................................... 29 Appendix 3 Analyses of Pesticides, Organics and Metals in Wildlife International, Ltd. Greenhouse Soil................................................................................................... 32 Appendix 4 Analytical Method Validation D ata................................................................... 33 Appendix 4.1 Analytical Method Flowchart for the Analysis of PFOS in Artificial Soil..... ...............34 Appendix 4.2 Typical HPLC/MS/MS Operational Parameters.............................................................. 35 Appendix 4.3 A Typical Calibration Curve for PFO S............................................................. 36 Appendix 4.4 Example Calculations for a Representative Sample......................................... 37 Appendix 4.5 Ion Chromatogram of a Low-level PFOS Standard......................................... 38 Appendix 4.6 Ion Chromatogram of a High-level PFOS Standard....................................... 39 Appendix 4.7 Ion Chromatogram of a Non-Target Terrestrial Plant SoilMatrix B lank..... ................ 40 Appendix 4.8 Ion Chromatogram of an Invertebrate Testing Soil Matrix B lank................ 41 Appendix 4.9 Ion Chromatogram of a Low-level Non-Target Terrestrial Plant Soil Fortification Sample................................................................................................................................ 42 Appendix 4.10 Ion Chromatogram of a High-level Non-Target Terrestrial Plant Soil Fortification Sample..........................................................................................................43 Wildlife International. Ltd. -7- Project Number 454C-120 TABLE OF CONTENTS - Continued APPENDICES Appendix 4.11 Ion Chromatogram of a Low-level Invertebrate Testing Soil Fortification Sample.... 44 Appendix 4.12 Ion Chromatogram of a High-level Invertebrate Testing Soil Fortification Sample....45 Appendix 5 Personnel Involved in the Study.......................................................................................46 Wildlife International. Ltd. 8- - Project Number 454C-120 SUMMARY SPONSOR: 3M Corporation TITLE: Analytical Method Validation for the Determination of Perfluorooctanesulfonate, Potassium Salt (PFOS) in Artificial Soil WILDLIFE INTERNATIONAL, LTD. PROJECT NUMBER: 454C-120 3M ENVIRONMENTAL LAB PROJECT NUMBER: U2723 TEST DATES: Study Initiation: February 5, 2001 Experimental Start (OECD): February 6, 2001 Experimental Start (EPA): February 7, 2001 Experimental Termination: February 8, 2001 TEST SYSTEM: Non-Target Terrestrial Plant Soil and Invertebrate Testing Soil FORTIFIED TEST CONCENTRATIONS : wet-weight (dry-weight) basis Non-Target Terrestrial Plant Soil (me a.i./Kel 2.00 (2.37) 10.0 ( 11.8) 100(118) 1000(1180) Invertebrate Testing Soil (mg a.i./Kg) 2.00 (2.37) 10. 0 ( 11.8) 100(118) 1000(1180) RESULTS: Non-target terrestrial plant soil samples and invertebrate testing soil samples were each fortified in triplicate with PFOS to reflect nominal concentrations of 2.00,10.0,100 and 1000 mg a.i./Kg on a wet-weight basis. The samples were extracted with methanol, vacuum filtered, centrifuged, diluted into the instrumental calibration range with dilution solvent (50% methanol : 50% NANOpure water) and analyzed by high performance liquid chromatography triple quadruple mass spectrometry (HPLC/MS/MS). Recoveries of PFOS in non-target terrestrial plant soils yielded an overall mean percent recovery of 98.1% (SD = 3.27; CV =3.33%; N = 12). Recoveries of PFOS in invertebrate testing soils yielded an overall mean percent recovery of 96.9% (SD = 3.61; CV = 3.73%; N = 12). Reagent blanks, consisting of solvents, and matrix blank samples, consisting of unfortified non-target terrestrial plant soil and invertebrate testing soil, were devoid of interfering components. The precision of triplicate fortifications in artificial soil matrix taken through the methodology was typically three percent relative, or better. Wildlife International. Ltd. -9- Project Number 454C-12Q 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 non-target terrestrial plant soils and invertebrate testing soils. Validation samples were prepared and analyzed between February 7, 2001 and February 8, 2001. Raw data generated by Wildlife International, Ltd. and a copy of the final report are filed under Project Number 454C-120 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 non-target terrestrial plant soils and invertebrate testing soils. The validated method will be used in support of plant and invertebrate toxicology tests to be conducted with the test substance. EXPERIMENTAL DESIGN Non-target terrestrial plant soils and invertebrate testing soils 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 future plant and invertebrate 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 Artificial Soil" (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-120 was described as a white powder. It was identified as FC-95 from lot number 217, with an expiration date of August 31, 2001. Information from the most recent Certificate of Analysis provided by the Sponsor indicated a purity o f 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 Invertebrate Testing Soil The artificial soil used for validating the methodology for invertebrate testing was composed in a ratio of approximately 10 parts sphagnum peat, 20 parts kaolin clay, and 70 parts industrial quartz sand. The dry constituents of the soil were mixed (Soil Mixer #2) and the pH of the dry mixture was adjusted to 5.5 with calcium carbonate powder prior to hydration. After hydration, the moisture content o f the soil (30.5%) was determined by weighing soil samples before and after drying at 105C. Non-Tareet Terrestrial Plant Soil The artificial soil used for validating the methodology for plant testing was composed in a ratio of approximately 4 parts kaolin clay, 50 parts industrial quartz sand and 5 parts peat. Crushed limestone was added to buffer the pH of the soil, and a slow-release fertilizer was added to provide nutrients essential for plant growth. A soil sample representative of that used in this study was sent to Agvise Laboratories, Inc., in Northwood, North Dakota, for analysis of the particle size distribution and organic content of the soil. Reports for the latest soil characterization are stored in the archives at the Wildlife International, Ltd. site in Easton, Maryland. The results of the most recent GLP-compliant analyses performed to measure the concentrations of selected contaminants in soil used for non-target terrestrial plant testing at Wildlife International, Ltd. is presented in Appendix 3. No analytes were measured at levels that were expected to have an impact on the study. Wildlife International. Ltd. - 11 - Project Number 454C-120 Analytical Validation Procedures Wildlife International, Ltd. conducted a validation trial using procedures developed at Wildlife International, Ltd. (Appendix 4). Matrix blanks and matrix fortification samples were prepared in non target terrestrial plant soils and invertebrate testing soils. A reagent blank (containing everything except PFOS and soil) was carried through the entire procedure for each soil matrix. The samples were extracted with methanol. The samples were then vacuum filtered, diluted with methanol, and 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 of PFOS 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 Ci8 guard column (20 mm x 2.0 mm). A method flowchart is provided in Appendix 4.1 and the instrument parameters are summarized in Appendix 4.2. 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, and0.100mga.i./L) ofPFOS 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 mg a.i./L stock solution ofPFOS in methanol. Calibration standards ofPFOS, ranging in concentration from 1.00 to 10.0 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 Wildlife International. Ltd. - 12- Project Number 454C-120 in Appendix 4.3. The concentration of PFOS in the samples was determined by substituting the peak area responses into the applicable linear regression equation (Appendix 4.4). Representative ion chromatograms of low and high calibration standards are presented in Appendices 4.5 and 4.6, respectively. Matrix Blanks and Matrix Fortifications Selected 10-g aliquots of each artificial soil type were fortified with the appropriate primary or secondary stock solutions of PFOS prepared in methanol using a gas-tight syringe. The fortified soils were allowed to air dry then manually mixed prior to processing. The matrix blanks for each soil type were unfortified soils. Limit of Quantitation The method validation levels in both artificial soil types were well above the method capabilities for quantitation of the test substance. Therefore, the method limit of quantitation (LOQ) in each matrix was 1.00 mg a.i./Kg on a wet-weight basis calculated as the product of the lowest calibration standard (0.00100 mg a.i./L) and the overall dilution factor of the matrix blank samples (1000 L/Kg). For the non-target terrestrial plant artificial soil matrix with reported moisture content of 15.5%, the LOQ on a dry-weight basis was calculated as 1.18 mg a.i./Kg. For the invertebrate testing artificial soil matrix with reported moisture content of 30.5%, the LOQ on a dry-weight basis was calculated as 1.44 mg a.i./Kg. 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 respective LOQs (Tables 1 and 2). Representative ion chromatograms of non-target terrestrial plant soil and invertebrate testing soil matrix blank samples are presented in Appendices 4.7 and 4.8, respectively. Method Validation Samples Non-target terrestrial plant soils were fortified in triplicate at 2.37, 11.8, 118 and 1180 mg a.i./Kg (dry-weight basis) resulting in mean recoveries of 98.3, 101, 97.5, and 96.6%, respectively (Table 1). Representative ion chromatograms of low and high-level fortifications in non-target terrestrial plant soil Wildlife International. Ltd. - 13 - Project Number 454C-120 are presented in Appendices 4.9 and 4.10, respectively. Invertebrate testing soils were fortified in triplicate at 2.88, 14.4, 144 and 1440 mg a.i./Kg (dry-weight basis) resulting in mean recoveries of 99.7, 100, 93.1 and 95.1%, respectively (Table 2). Representative ion chromatograms of low and high-level fortifications in invertebrate testing soils are presented in Appendices 4.11 and 4.12, respectively. The precision of the methodology, as demonstrated by the analysis of triplicate samples within each fortification level, was three percent relative, or better, for all four levels of fortification in the invertebrate testing soil matrix. With the exception of one level (1180 mg a.i./Kg on a dry-weight basis = 6.40%), precision in the non-target terrestrial plant matrix was also three percent relative, or better. CONCLUSIONS Non-target terrestrial plant and invertebrate testing artificial soils were fortified with PFOS to reflect nominal concentrations of 2.00,10.0,100 and 1000 mg a.i./Kg on a wet-weight basis. The samples were extracted with methanol. The samples were then vacuum filtered, diluted with methanol, 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 then analyzed by high performance liquid chromatography mass spectrometry (HPLC/MS/MS). Recoveries of PFOS in non-target terrestrial plant soil yielded an overall mean percent recovery on a wet-weight basis of 98.1 % (SD = 3.27%; CV = 3.33%; N = 12). Recoveries of PFOS in invertebrate testing soil yielded an overall mean percent recovery on a wet-weight basis of 96.9% (SD = 3.61%; CV = 3.73%; N = 12). Reagent blanks, consisting of solvents, and matrix blank samples, consisting of unfortified artificial soils, were devoid of interfering components. Wildlife International. Ltd. - 14- Project Number 454C-120 REFERENCES 1. American Society for Testing and Materials. 1991. Standard Specification for Reagent Water. D1193-91, ASTM Section II Water and Environmental Technology, Vol. 11.01:45-47. Wildlife International. Ltd. - 15 - Project Number 454C-120 Table 1 Method Validation Recoveries for PFOS in Non-Target Terrestrial Plant Soil Sample Number (454C-120-) Type VREB-1 Reagent Blank VMAB-1 VMAB-2 VMAB-3 Matrix Blank Matrix Blank Matrix Blank PFOS Concentration (mg a.i./Kg) Fortified Measured 0 < 1.182 0 < 1.18 0 < 1.18 0 < 1.18 Percent Recovered1 Mean Measured (mg a.iTKg) Mean Percent Recovery -- __ -- -- _ VMAS-1 Matrix Fortification 2.37 2.33 98.7 2.33 98.3 VMAS-2 Matrix Fortification 2.37 2.30 97.1 0.0300 VMAS-3 Matrix Fortification 2.37 2.36 99.4 1.29% VMAS-4 Matrix Fortification 1 1 .8 12 .0 1 0 1 11.9 1 0 1 VMAS-5 Matrix Fortification 1 1 .8 12 .0 1 0 1 + 0.231 VMAS-6 Matrix Fortification 1 1 .8 11 .6 98.3 1.95% VMAS-7 Matrix Fortification 118 115 97.0 115 97.5 VMAS-8 Matrix Fortification 118 113 95.2 2.52 VMAS-9 Matrix Fortification 118 118 99.6 2.18% VMAS-10 Matrix Fortification 1180 1205 102 1140 96.6 VMAS-11 Matrix Fortification VMAS-12 Matrix Fortification 1180 1180 1159 1062 97.9 73.0 89.7 6.40% Overall Mean = Standard Deviation = CV = N= 98.1 3.27 3.33% 12 1 Percent Recovered values were calculated on a wet-weight basis using MacQuan, version 1.6 software. All other values were converted to dry-weight by dividing the wet-weight value by the percent solids for the matrix soil. 2 The limit of quantitation (LOQ) was 1.18 mg a.i./Kg on a dry-weight basis calculated as the product of the lowest calibration standard (0.001 mg a.i./L) and the overall dilution factor of the matrix blank samples (1000 L/Kg) divided by the percent solids for the soil (84.5%). Wildlife International. Ltd. - 16- Project Number 454C-120 Table 2 Method Validation Recoveries for PFOS in Invertebrate Testing Soil Sample Number (454C-120-) Type VREB-2 Reagent Blank VMAB-4 VMAB-5 VMAB-6 Matrix Blank Matrix Blank Matrix Blank PFOS Concentration (mg a.i./kg) Fortified Measured 0 < 1.442 0 < 1.44 0 < 1.44 0 < 1.44 Percent Recovered1 Mean Measured (mg a.i./Kg) -~ ---- -- -- Mean Percent Recovery - __ VMAS-13 Matrix Fortification 2 .8 8 2.82 98.1 2.87 99.7 VMAS-14 Matrix Fortification 2 .8 8 2.82 97.9 0.0808 VMAS-15 Matrix Fortification 2 .8 8 2.96 103 2.82% VMAS-16 Matrix Fortification 14.4 14.3 99.3 14.4 100 VMAS-17 Matrix Fortification 14.4 14.5 101 + 0 .1 0 0 VMAS-18 Matrix Fortification 14.4 14.4 1 0 0 0.694% VMAS-19 VMAS-20 VMAS-21 Matrix Fortification Matrix Fortification Matrix Fortification 144 144 144 136 94.6 134 93.1 133 92.5 2.08 132 91.4 1.56% VMAS-22 Matrix Fortification 1440 1370 95.2 1370 95.1 VMAS-23 Matrix Fortification 1440 1391 96.7 25.1 VMAS-24 Matrix Fortification 1440 1341 93.2 1.83% Overall Mean = Standard Deviation = CV = 96.9 3.61 3.73% N = 12 1 Percent Recovered values were calculated on a wet-weight basis using MacQuan, version 1.6 software. All other values were converted to dry-weight by dividing the wet-weight value by the percent solids for the matrix soil. 2 The limit of quantitation (LOQ) was 1.44 mg a.i./Kg on a dry-weight basis calculated as the product of the lowest calibration standard (0.001 mg a.i./L) and the overall dilution factor of the matrix blank samples (1000 L/Kg) divided by the percent solids for the soil (69.5%). Wildlife International. Ltd. - 17- Appendix 1 Protocol and Amendments Project Number 454C-120 Wildlife International. Ltd. - 18- Project Number 454C-120 PROTOCOL ANALYTICAL METHOD VALIDATION FOR THE DETERMINATION OF PERFLUOROOCTANESULFONATE, POTASSIUM SALT (PFOS) IN ARTIFICIAL SOIL Environmental Laboratory Project Number U2723 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 December 7, 2000 Wildlife International. Ltd. - 19- Proiect Number 454C-120 W ildlife International, Ltd. - 2- ANALYTICAL METHOD VALIDATION FOR THE DETERMINATION OF PERFLUOROOCTANESULFONATE, POTASSIUM SALT (PFOS) IN ARTIFICIAL SOIL SPONSOR: 3M Corporation Environmental Laboratory 935 Bush Avenue St. Paul, Minnesota 55106 SPONSOR'S REPRESENTATIVE: Rochelle R. Robideau TESTING FACILITY: Wildlife International, Ltd. 8598 Commerce Drive Easton, Maryland 21601 STUDY DIRECTOR. Raymond L. Van Hoven, Ph.D. Scientist Wildlife International, Ltd. LABORATORY MANAGEMENT: Willard B. Nixon, Ph.D. T hfuti or Manager-of Analytical Chemistry -oj Wildlife International, Ltd. FOR LABORATORY USE ONLY Proposed Dates: Experimental Experimental Start Date: Termination Date: Project No.: Test Concentrations: h. y A m 1 , 1 0 . t; i o n , i n n o ..Jm UA + Test Substance No.: 4675_______ Reference Substance No. (if applicable): 4526 na D w W nl DATE (tt/m DA ITIV -- /p /y /o DATE / PROTOCOL NO.: 454/I20700/MV-S/SUB454 Environmental Laboratory Project Numlrcr U2723 Wildlife International. Ltd. -20- Project Number 454C-120 W ildlife International, Ltd. -3- INTRODUCTION Wildlife International, Ltd. will conduct analytical trials to validate the performance o f a liquid chromatography mass spectrometry (LCMS) method for the determination o f Perfluorooctanesulfonate, Potassium Salt (hereafter referred to as PFOS) in artificial soil used for earthworm and non-target terrestrial plant tests. The study will be performed at the Wildlife International, Ltd. analytical chemistry facility in Easton, Maryland. The method developed by Wildlife International, Ltd. will be validated by fortifying artificial soil and quantifying the recoveries o f PFOS. Raw data for all work performed at Wildlife International, Ltd. and a copy o f the final report will be filed by project number in archives located on the Wildlife International, Ltd. site, or at an alternative location to be specified in the final report. OBJECTIVE The objective o f this study is to verify the performance o f LCMS methodology for analyses of PFOS in artificial soil used by Wildlife International, Ltd. to conduct non-target terrestrial plant and invertebrate testing. EXPERIMENTAL DESIGN Wildlife International Ltd. artificial soils similar to those used in earthworm and non-target terrestrial plant tests will be fortified at four different concentrations and analyzed using LCMS methodology based upon that developed at Wildlife International Ltd. Modifications to the methodology will be made as necessary to achieve quantitation o f PFOS in the test matrix. Matrix and/or reagent blanks will be analyzed concurrently to evaluate potential analytical interferences. The levels o f fortification will bracket the anticipated concentrations o f samples from earthworm and non-target terrestrial plant toxicity tests. Reagent and matrix blanks will be analyzed to evaluate potential analytical interferences. One calibration curve will be prepared and analyzed with each series of matrix fortification samples. The accuracy, precision, and limit o f quantitation o f the method will be determined. PROTOCOL NO.: 454/120700/MV-S/SUB454 Environmental Laboratory Project Number U2723 Wildlife International. Ltd. -21 - Project Number 454C-120 W ildlife International, Ltd. -4 MATERIALS AND METHODS Test Substance The test substance will be Pcrfluorooctanesulfonate, 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 Wildlife 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 will be the test substance. If written verification o f GLP test substance characterization is not provided to Wildlife International, Ltd., it will be noted in the compliance statement o f the final report. The 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. Reagents and Solvents All solvents used in the method or procedure will be o f reagent grade or better. All reagents will be o f ACS grade or better. Test Soil - E arthw orm Soil The artificial soil used for earthworm tests will be composed o f approximately 10% sphagnum peat, 20% kaolin clay and 70% industrial quartz sand. The dry constituents o f the soil will be mixed in a PK Twinshell or equivalent mixer. Calcium carbonate powder will be added during the mixing of the dry soil as needed to adjust the pH to 6.0 0.5, prior to hydration. The moisture content o f the artificial soil will be determined by weighing samples o f the soil before and after drying at approximately 105C. The moisture content is calculated using the following formula: ' % moisture = [(net wet weight-net dry weight) + net wet weight] X 100 The soil will be adjusted to approximately 33% moisture content during the preparation o f the test soils. PROTOCOL NO.: 454/120700/MV-S/SUB454 Environmental Laboratory Project Number U2723 Wildlife International. Ltd. -22- Project Number 454C-120 W ildlife International, Ltd. -5- Test Soil - Non-Target T errestrial Plants Test plants will be grown in plastic pots approximately 16 cm in diameter and 12 cm in depth. An unsterilized artificial sandy-loam or sand soil substrate will be used for testing. The soil will be composed o f kaolinite clay, industrial quartz sand and peat mixed in a 4:50:2.5 ratio (w:w:w). Crushed limestone will be added to buffer the pH o f the soil, and a slow-release fertilizer will be added to provide nutrients essential for plant growth. Typically, the soil consists o f approximately 90 % sand, 2-3 % silt, and 7-10 % clay, with a neutral pH and an organic matter content less than 1.5 %. A sample o f soil representative of that used in this study will be sent to Agvise Laboratories, Inc., in Northwood, North Dakota, for analysis o f the particle size distribution and organic matter content of the soil. Soil characterization will include, but may not be limited to, the determination of particle size distribution, organic matter content, and pH. Those items relevant to the conduct o f the study will be discussed in the final report. The complete report from Agvise Laboratories, Inc. will be filed in the archives located at Wildlife International, Ltd. The results of the characterization will be stored in the archives located at the Wildlife International, Ltd. site, and those items relevant to the conduct of the study will be discussed in the final report. Analytical Validation Procedures The analytical method to be used will be LC/MS based upon procedures developed at Wildlife International, Ltd. The method used will be documented in the raw data and summarized in the final report. Prior to the analysis o f analytical samples, primary stock solution(s) will be prepared directly from the test substance. Calibration standards will be prepared by appropriate dilution o f the primary stock solution(s). Primary Stock Solution(s), Calibration Standards and Curves A minimum o f five calibration standards (of different concentrations) will be prepared and analyzed along with each analysis set. The calibration standard series will 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 will be injected with the analysis set. One calibration curve and regression equation will be prepared from the series o f calibration standards. PROTOCOL NO.: 454/120700/MV-S/SUB454 Environmental Laboratory Project Number U2723 Wildlife International. Ltd. -23- Project Number 454C-120 W ildlife International, Ltd. -6 - Fortification Stock Solutions The soil will be fortified with test substance in a manner consistent with the method anticipated for the toxicology studies. Evaluation of Interferences One reagent blank and three matrix blanks will be analyzed to detect possible interferences. The reagent blank will contain everything except the matrix and the test substance. The matrix blanks will contain everything except the test substance. Verification Analyses - Method Performance Matrix fortifications (spiked artificial soil from earthworm and non-target plant studies), prepared at known concentrations o f the test substance, will be analyzed to determine the recovery of the analyte and to evaluate method performance. The anticipated verification series will consist o f the following analyses: SUMMARY OF PROPOSED VERIFICATION ANALYSIS SCHEME Analysis Type Concentration Reagent Blank (Solvent) Matrix Blank (Soil) Matrix Fortifications 0 (Control) 0 (Control) Level 1 - Low Level 2 Level 3 Level 4 - High Total Number of Analyses Number o f Samples 1 Earthworm Soil Non-Target Plant Soil | 11 33 33 33 33 33 16 16 Matrix fortifications will represent the range o f concentrations that are anticipated to be used in subsequent environmental fate and/or effects tests and will be selected in consultation with the Sponsor. Individual samples (identified by project number and a unique sample identification number) will be prepared and analyzed for verification o f the analytical methodology. PROTOCOL NO.: 454/120700/MV-S/SUB454 Environmental Laboratory Project Number U2723 Wildlife International. Ltd. -24- Project Number 454C-12Q W ildlife International, Ltd. -7- If difficulties arise in the validation process (e.g., low recoveries or interferences), the Sponsor will be notified and the need for additional validation and/or method development will be determined through discussions with the Sponsor. Upon completion o f the method validation, the Sponsor will review the results and authorize the use of the methodology for analysis o f samples, or will authorize further method development trials. Recovery values in the range o f 80 to 120% will be used as criteria for method acceptability. Data Analysis One calibration curve will be established for each analytical run. A regression equation o f the concentration versus peak area for the calibration standards will be generated. The concentration o f 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 o f fortification. RECORDS TO BE MAINTAINED Records to be maintained for data generated by Wildlife International, Ltd. will include: 1. A copy o f the 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. Test 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. FINAL REPORT The report will 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 will PROTOCOL NO.: 454/120700/MV-S/SUB454 Environmental Laboratory Project Number U2723 Wildlife International. Ltd. -25 - Project Number 454C-120 W ildlife International, Ltd. 8- - review the methods and results and evaluate the results for acceptability prior to initiating any fate and/or effects studies. The final report will include, but not be limited to, the following: 1. Name and address o f the facility performing the study. 2. Dates on which the study was initiated and completed. 3. Objectives and procedures stated in the approved protocol, including any changes in the 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, composition, date o f receipt, lot number, storage conditions, physical characteristics, stability and method o f preparation o f test concentrations, if provided by the Sponsor. 5. A brief summary o f the analytical methodology: A description o f the experimental measurements, example calculations, sample preparation (sample weights and dilutions), instrumentation employed, reagents and solvents used, class of water used, and any major modifications to the method. 6. A description o f all circumstances that may have affected the quality or integrity o f the data. 7. The name o f the Study Director, the names o f other scientists or professionals, and the names o f all supervisory personnel involved in the study. 8. A description o f the transformations, calculations, or operations performed on the data. 9. The signed and dated reports o f each o f the individual scientists or other professionals involved in the study, if applicable. 10. The location where raw data and final report are to be stored. 11. A statement prepared by the Quality Assurance Unit listing the dates that study inspections were made and the dates o f any findings reported to the Study Dircctor/Management. CHANGING OF PROTOCOL Planned changes to the protocol will be in the form o f written amendments signed by the Study Director and the Sponsor's Representative. Amendments will be considered as part o f the protocol and will be attached to the final protocol. Any other changes will be in the form o f written deviations signed by the Study Director and filed with the raw data. All changes to the protocol will be indicated in the final report. PROTOCOL NO.: 454/120700/MV-S/SUB454 Environmental Laboratory Project Number U2723 Wildlife International. Ltd. 2 6- - Project Number 454C-120 W ildlife International, Ltd. -9 - GOOD LABORATORY PRACTICES This study will be conducted in accordance with Good Laboratory Practice Standards for EPA (40 CFR Part 160); and OECD Principles o f Good Laboratory Practice (ENV/MC/CHEM (98) 17). Each study conducted by Wildlife International, Ltd. is routinely examined by the Wildlife International, Ltd. Quality Assurance Unit for compliance with Good Laboratory Practices, Standard Operating Procedures and the specified protocol. A statement o f compliance with Good Laboratory Practices will be prepared for all portions o f the study conducted by Wildlife International, Ltd. The Sponsor will be responsible for certification o f compliance with Good Laboratory Practices for procedures performed by other laboratories. Raw data for all work performed at Wildlife International, Ltd. and a copy o f the final report will be filed by project number in archives located on the Wildlife International, Ltd. site, or at an alternative location to be specified in the final report. A copy of the study raw data for all work performed at Wildlife International, Ltd. will be sent to the Sponsor with the final report. PROTOCOL NO.: 454/120700/MV-S/SUB454 Environmental Laboratory Project Number U2723 Wildlife International. Ltd. -27- Project Number 454C-120 W ildlife International, Ltd. Project Number 454C-120 Page I o f 1 AMENDMENT TO STUDY PROTOCOL STUDY TITL E: ANALYTICAL METHOD VALIDATION FOR THE DETERMINATION OF PERFLUOROOCTANE SULFONATE, POTASSIUM SALT (PFOS) IN ARTIFICIAL SOIL PR O T O C O L NO.: 454/120700/MV-S/SUB454 SPONSOR: 3M Corporation SPO N SO R STUDY NO.: U2723 AMENDMENT NO.: 1 PR O JEC T NUMBER: 454C-120 E FFE C T IV E DATE: February 6, 2001 AM ENDM ENT: Page 5, Test Soil - Non-Target Terrestrial Plants CHANGE: "The soil will be composed o f kaolinite clay, industrial quartz sand and peat mixed in a 4:50:2.5 ratio (w:w:w)." TO: "The soil will be composed o f kaolinite clay, industrial quartz sand and peat mixed in a 4:50:5 ratio (w:w:w)." REASON: To use a ratio of components in the validation study in line with current standard practice for plant studies. W iC u )e d bij QA (suj) -(n-OI Wildlife International. Ltd. -28- Project Number 454C-120 W ildlife International, Ltd. Project Number 454C-120 Page 1 o f 1 AMENDMENT TO STUDY PROTOCOL STUDY T IT L E : Analytical Method Validation For The Determination O f Perfluorooctane Sulfonate, Potassium Salt (PFOS) In Artificial Soil PR O TO C O L NO.: 454/I2070Q/MV-S/SUB454 SPONSOR: 3M Corporation SPONSOR STUDY NO.: U2723 AM ENDMENT NO.: 2 PRO JECT NUMBER: 454C-120 EFFECTIV E DATE: May 16, 2003 AM ENDM ENT: Page 2, Sponsor's Representative Change: Rochelle R. Robideau To: Susan A. Beach REASON: Reassignment o f Sponsor's Representative responsibilities at the request o f the Sponsor. AM ENDM ENT: Page 8, Changing o f Protocol Change: "Planned changes to the protocol will be in the form o f written amendments signed by the Study Director and the Sponsor's Representative." To: "Planned changes to the protocol will be in the form o f written amendments signed by the Study Director and approved by the Sponsor's Representative." REASON: To expedite finalization o f the study. S ' tf '0 3 Wildlife International. Ltd. -29- Project Number 454C-120 Appendix 2 Certificate of Analysis INTERIM CERTIFICATE OF ANALYSIS R evision 1(9/7/00) Centre Analytical Laboratories COA Reference #: 023-018A 3M Product: PFO S,Lot217 Reference#: SD-018 Test Name Purity1 Appearance Identification NMR Metals (ICP/MS) 1. Calcium 2. Magnesium 3. Sodium 4. Potassium2 5. Nickel 6. Iron 7. Manganese Total % Impurity (NMR) Total % Impurity (L C /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. Sulfate* Organic A cids5 (IC) 1. TFA 2. PFPA 3. HFBA 4. NFPA Elemental Analysis6: 1. Carbon 2. Hydrogen 3. Nitrogen 4. Sulfur 5. Fluorine Specifications ' W hite Crystalline Powder ' ::' : ?%'x'?? f "rii ^' :i: i :i: T |' | 2: ^ , , :: ' ''' ', '' t ', ' *. * ' ' ' ' '{'"< . . "" "* ":''U\ r T . '" i T ; ; nm m m H 1. Theoretical Value = 17.8% 2. Theoretical Value = 0% 3. Theoretical Value = 0% 4. Theoretical Value 5.95% 5. Theoretical Value - 60% Result 86.9% Conforms Positive 1. 0.005 wt./wt.% 2. 0.001 wt./wt.% 3. 1.439 wt./wt.% 4. 6.849 wt./wt.% 5. <0.001 wt./vrt.% 6. 0.005 wt./wt.% 7. <0.001 wt./wt.% 1.93 wt./wt.% 8.41 vrt./wt.% None Detected 0.33 wt7wt.% None Detected N ot Applicable3 1. <0.015 wt./w t.% 2. 0.59 wt7wt.% 3. <0.040 wtTwt.% 4. <0.009 wtAvt.% 5. <0.006 wt.Avt.% 6. <0.007 wt./wt.% 7. 8.76 wt./wt.% 1. <0.1 wt7wt.% 2. <0.1 wt7wt.% 3. 0.10 wtAvt.% 4. 0.28 wt7wt.% 1. 12.48 w t7w t.% 2. 0.244 wt./wt.% 3. 1.74 wt./wt.% 4. 8.84 wt7wt.% 5. 54.1w t7w t.% COA023-018A Page l of 3 Wildlife International. Ltd. -30- Project Number 454C-120 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 o f metal impurities, 1.45% +LC/MS impurities, 8.41%+Inorganic Fluoride, 0.59%+NMR impurities, 1.93%+organic acid impurities, 0.38%+PQAA, 0.33%) Total impurity from all tests = 13.09% Purity = 100% -13.09% = 86.9% Potassium is expected in this salt form and is therefore not considered an impurity. Purity by DSC is generally not applicable to materials o f low purity. N o endotherm was observed for this sample. 4Sulfiir 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. *TFA HFBA NFPA PFPA Trifluoroacetic acid Heptafluorobutyric add Nonofluoropentanoic acid Pentafluoropropanoic add `Theoretical value calculations based on the empirical formula, CsFnSOj'K* (MW=538) 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-120 Centre A nalytical Laboratories, Inc. 3046 Research Drive State College, PA 16801 www.centrelab.com ^ ^ F # ^ 0 6 S 5 B L . Phone: (814) 231-6032 Fax: (814) 231-1253 or (814) 231-1580 INTERIM CERTIFICATE OF ANALYSIS Revision 3 Centre Analytical Laboratories COA Reference #: 023-018A LC/MS Purity Profile: Im purity 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 result from the C4 and C6 standard curves. Likewise, the C7 value was calculated using the average result from the C6 and C8 standard curves. Prepared By; Charles Sirndtis Scientist, Centre Analytical Laboratories /o fi/o t Date L mtided:qReviewed By: John Flaherty 7 _ Date 'Laboratory Manager, Centre Analytical Laboratories COA023-018A Page 3 of 3 Wildlife International. Ltd. -32- Project Number 454C-120 Appendix 3 Analyses of Pesticides, Organics and Metals in Wildlife International, Ltd. Greenhouse Soil1 Component a-chlordane y-chlordane P,P'-D D E 0,P'-D D T A ldrin P.P'-D D T D ieldrin -endosulfan E n d o su lfan su lp h ate E n d rin E n d rin -k e to n a-H C H -H C H Lindane (y-HCH) 8-H C H H eptachlor -H ep tach lo rep o x id e H exachlorbenzene M ethoxychlor P,P'-tD E M agnesium Sodium C a lc iu m Iron Potassium Alum inum M anganese B e ry lliu m Chromium C o b alt Pesticides And Organics M easured Concentration C om ponent <0.01 m g/kg <0.01 m g/kg <0.01 mg/kg <0.01 mg/kg <0.01 mg/kg <0.01 mg/kg <0.01 mg/kg <0.01 mg/kg <0.01 m g/kg <0.01 mg/kg <0.01 m g/kg <0.01 mg/kg <0.01 mg/kg <0.01 m g/kg <0.01 m g/kg <0.01 m g/kg <0.01 m g/kg <0.05 mg/kg <0.05 mg/kg <0.01 m g/kg Azinphos-methyl C h lo rfen v in p h o s C h lo rp y rip h o s Chlorpyriphos-methyl D iazinon D ichlorvos Fenitrothion M alathion M evinphos (Cis+Trans) Parathion Parathion-methyl Pirimiphos-methyl Sulfotep PCB 28 PCB 52 P C B 101 PCB 118 PCB 138 PC B 153 PCB 180 3,000 mg/kg 60 m g/kg 4,700 mg/kg 6,100 mg/kg 220 mg/kg 5,200 mg/kg 51 m g/kg <0.04 mg/kg 6.0 mg/kg 0.50 mg/kg M etals N ickel Copper Z in c M olybdenum Silver C adm ium Arsenic M ercury Selenium M easured Concentration N ot Determined <0.01 mg/kg <0.01 mg/kg <0.01 mg/kg <0.01 mg/kg <0.01 mg/kg <0.01 mg/kg <0.01 mg/kg N ot Determined <0.01 mg/kg <0.01 mg/kg <0.01 mg/kg <0.01 mg/kg <0.01 mg/kg <0.01 mg/kg <0.01 mg/kg <0.01 mg/kg <0.01 mg/kg <0.01 m g/kg <0.01 mg/kg <2.0 m g/kg <2.0 mg/kg 6.1 m g/kg <1.2 m g/kg <0.40 mg/kg <2.0 mg/kg 3.8 mg/kg 12.0 pg/kg 0.11 mg/kg 'A nalyses perform ed b y T N O N utrition and Food Institute on sam ples collected o n O ctober 14 and 15, 1999. Wildlife International. Ltd. -33 - Appendix 4 Analytical Method Validation Data Project Number 454C-120 Wildlife International. Ltd. - 34- Project Number 454C-120 Appendix 4.1 Analytical Method Flowchart for the Analysis of PFOS in Artificial Soil Weigh approximately 10.0 grams of soil per sample into a tared weigh boat. Record weights. Transfer to 8-oz French-square bottles. For matrix fortification samples, fortify each sample with the appropriate volume of PFOS stock solution in methanol with a gas-tight syringe. Allow to air dry. The matrix blank is unfortified soil matrix. 4 For each soil sample, measure 100 milliters of methanol with a graduated cylinder and transfer into the French-square bottle. Add 100 milliliters of methanol into an empty French-square bottle and process as for the soil samples. The additional sample is the reagent blank. 4 Cap bottles and place on a shaker table. Allow the samples to shake for a minimum of 30 minutes at approximately 250 rpm. 4 Vacuum filter with qualitative filter paper and rinse retained soil three times with methanol into filtrate. 4 Transfer the filtrate into a 200-mL volumetric flask and bring to volume with methanol. Mix well with several repeat inversions. 4 Transfer approximately twenty milliliters of each sample into a separate glass centrifuge tube or scintillation vial and cap. Centrifuge samples for approximately five minutes at 2000 rpm. 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 volu m e o f sam ple w ith a gas-tight syringe and bring to volum e w ith 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. -35 - Project Number 454C-120 INSTRUMENT: Appendix 4.2 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 Perkin-Elmer 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 Cis column (50 mm x 2 mm I.D., 3-|im particle size) Keystone Javelin C|g 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. - 36- Appendix 4.3 A Typical Calibration Curve for PFOS PTOS 4 9 9 .0 -> 9 9 .1 No Internal Standard Weighted (1/x) Intercept = 741.01 Slope = 11944389.00 Correlation Coeff. = 0.99977 Project Number 454C-120 Cono, Jig a . i . / L Wildlife International, Ltd - 37- Project Number 454C-I20 Appendix 4.4 Example Calculations for a Representative Sample Sample number 454C-120-VMAS-1, nominal concentration of 2.00 mg a.i./Kg on a. wet-weight basis in plant soil Peak Area = 24317 Y-intercept = 741.01 Slope=11944389 Primary Dilution: Initial Weight (W,) = 10.0 g Original Final Volume (V0= 200 mL Secondary Dilution: Initial Volume (Vi) = 0.200 mL Final Volume (Vf) = 10.0 mL Overall Dilution Factor (Vi/Wi x Vf/Vi) = 1000 mL/g = 1000 L/Kg (peak area - y-intercept) PFOS at instrument (mg a.i./L) slope (24317-741.01) 11944389 = 0.0019738 mg a.i./L PFO S in sam ple (m g a.i./K g) = PFO S at instrument (m g a.i./L ) x overall dilution factor (L/K g) 0,0019738 mg a.i, 1000L PFOS in sample (mg a.i./Kg) = L X Kg = 1.97 mg a.i./Kg PFBS (mg a.i./L) in sample Percent of Nominal Concentration = PFBS (mg a.i./L) nominal X 1.97 j ^ x 100 = 98.7% Instrument Software: MacQuan, version 1.6. Wildlife International. Ltd. -38- Project Number 454C-120 Appendix 4.5 Ion Chromatogram of a Low-level PFOS Standard intensity: 10000 cps Nominal concentration: 0.00100 mg a.i./L Wildlife International. Ltd. -39- Project Number 454C-120 Appendix 4.6 Ion Chromatogram of a High-level PFOS Standard intensity: 10000 cps Nominal concentration: 0.0100 mg a.i./L Wildlife International. Ltd. -40- Project Number 454C-120 Appendix 4.7 Ion Chromatogram of Non-Target Terrestrial Plant Soil Matrix Blank intensity: 10000 cps Sample number 454C-120-VMAB-1. The arrow indicates the approximate retention time of PFOS. Wildlife International. Ltd. -41 - Project Number 454C-120 Appendix 4.8 Ion Chromatogram of an Invertebrate Testing Soil Matrix Blank intensity: 10000 cps Sample number 454C-120-VMAB-4. The arrow indicates the approximate retention time of PFOS. Wildlife International. Ltd. -42- Project Number 454C-120 Appendix 4.9 Ion Chromatogram of a Low-level Non-Target Terrestrial Plant Soil Fortification Sample intensity: 10000 cps Sample number 454C-120-VMAS-1, 2.00 mg a.i./Kg wet-weight nominal concentration, overall dilution factor = 1000 L/Kg. Wildlife International. Ltd. -43 - Project Number 454C-120 Appendix 4.10 Ion Chromatogram of a High-level Non-Target Terrestrial Plant Soil Fortification Sample intensity: 10000 cps Sample number 454C-120-VMAS-10,1000 mg a.i./Kg wet-weight nominal concentration, overall dilution factor = 200000 L/Kg. Wildlife International. Ltd. -44- Project Number 4S4C-120 Appendix 4.11 Ion Chromatogram of a Low-level Invertebrate Testing Soil Fortification Sample intensity: 10000 cps Sample number 454C-120-VMAS-13,2.00 mg a.i./Kg wet-weight nominal concentration, overall dilution factor = 1000 L/Kg. Wildlife International. Ltd. -45 - Project Number 454C-120 Appendix 4.12 Ion Chromatogram of a High-level Invertebrate Testing Soil Fortification Sample intensity: 10000 cps Sample number 454C-120-VMAS-22,1000 mg a.i./Kg wet-weight nominal concentration, overall dilution factor = 200000 L/Kg. Wildlife International, Ltd. - 46- Project Number 454C-12Q Appendix 5 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