Document wgmMnmYKQYdYg724Q3aRGO9dD

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 2 1601 (410) 822-8600 Page 1 of46 WiIdIife InternationaI , 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 1989and OECD Principles of Good 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. SPONSOR APPROVAL: DATE WiId1tfe Internationa1, Ltd. -3- Proiect 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: February 7,2001 February 14 and 15,2001 May 27,2003 DATE REPORTED TO: STUDY DIRECTOR MANAGEMENT: February 7,2001 February 15,2001 May 27,2003 February 9,200 1 February 15,2001 May 27, 2003 Susan L. Coleman, B.A.' Senior Quality Assurance Representative DATE WildlifeInternational, Ltd. -4- Project Number 454C-120 REPORT APPROVAL SPONSOR: 3M Corporation TITLE: Analytical Method Validation for the Determination of Perfluorooctmesulfonate, Potassium Salt (PFOS) in Artificial Soil WILDLIFE INTERNATIONAL, LTD. PROJECT NUMBER: 454C-120 3M ENVIRONMENTAL LAB PROJECT NUMBER: U2723 STUDY DIRECTOR: Scientist MANAGEMENT: Willard B. Nixon, P h d Director of Chemistry 5/3 ,Io:+ DATE DATE WiId1ife Internationa1. Ltd. -5- Project Number 454C-120 TABLE OF CONTENTS TitlelCover 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 Non-Target Terrestrial Plant Soil ............................................................................................ 10 Analytical Validation Procedures ................................................................................................. 11 Primary and Secondary Stock Solutions....................................................................................... 1 1 Calibration Standards and Curves ............................................................................................... 11 Matrix Blanks and Matrix Fortifications ..................................................................................... 12 Limit of Quantitation.................................................................................................................... 12 Results .................................................................................................................................................. 12 Reagent and Matrix Blank Samples ............................................................................................. 12 Method Validation Samples ........................................................................................................ 12 Conclusions ........................................................................................................................................... 13 References ............................................................................................................................................. 14 WildlifeInternational. Ltd. -6- Proiect 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 Data .............................................................................. 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 PFOS ......................................................................... 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 Soil Matrix Blank ..................... 40 Appendix 4.8 Ion Chromatogram of an Invertebrate Testing Soil Matrix Blank ................................ 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 WildlifeInternational, Ltd. -7- Project Number 454C-120 TABLE OF CONTENTS - Continued - APPENDICES Appendix 4.11 Ion Chromatogram of a Low-level Invertebrate Tes ing Soil Fortificz ion Sa.mple.....44 Appendix 4.12 Ion Chromatogram of a High-level Invertebrate Testing Soil Fortification Sample....45 Appendix 5 Personnel Involved in the Study....................................... ........................................... 46 WiId1ife Internationa1, Ltd. -8- Project Number 454C-120 SUMMARY SPONSOR: 3M Corporation TITLE: Analytical Method Validation for the Determinationof Peffluorooctanesulfonatae,Potassium Salt (PFOS) in Artificial Soil WILDLIFE INTERNATIONAL, LTD. PROJECT NUMBER: 4546-120 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 (mg a.i./Kg) 2.00 (2.37) 10.0 (11.8) 100 (118) 1000 (1180) Invertebrate Testing - Soil (mg a.i./K& 2.00 (2.317) 10.0 (lL8) 100 (118) 1000 (1 180) RESULTS: Non-target terrestrial plant soil samples and invertebrate testing soil samples were each fortified in triplicatewith PFOS to reflect nominal concentrations of 2.00,10.0,100and 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 chromatographytriple quadruple mass spectrometry (HPLCMSMS). 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, weire devoid of interfering components. The precision of triplicate fortifications in artificial soil matrix taken through the methodology was typically three percent relative, or better. WiId1ife Internationa1, Ltd. -9- Project Number 454C-120 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 perfclrmanceof 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 ProjectNumber 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 anticipatedconcentrationsof samples from fbture 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 calibratiori 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 WildltfeInternational, Ltd. - 10- Project Number 454C-120 was described as a white powder. It was identified as FC-95 from lot number 2 17,with an expirationdate of August 31,2001. 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 11Designation D1193-91) was used (1). Test Systems Invertebrate Testing Soil The artificial soil used for validating the methodology for invertebratetesting 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 :mixturewas adjusted to 5.5 with calcium carbonate powder prior to hydration. After hydration, the moisture content of the soil (30.5%) was determined by weighing soil samplesbefore and after dryingat 105OC. Non-Target Terrestrial Plant Soil The artificial soil used for validating the methodology for plant testingwas 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 arlchives at the Wildlife International, Ltd. site in Easton, Maryland. The results of the most recent GL:P-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. WildlifeInternational, 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 nontarget 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 sampleswere 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 chromatographyusing 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 CIScolumn (50 mm x 2.0 mm, 3-pm particle size) fitted with a Keystone Javelin CISguard 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 tes8tsubstance (86.9%). A 10.0 mg a.i./mL primary stock solution of PFOS in methanol was prepared by weighing 1.1508 g of the test substance and bringing to a final volume of 100 milliliters with methanol. Secondary stock solutions (1000,100,10.0,1.00, and 0.100 mg a.i./L) 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 appropriatedilutions of the 1.00 mg a.i./L stock solution of PFOS in methanol. Calibration standards of PFOS, 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 studysamples. 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 WildlifeInternational, 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 methanolusing a gas-tight syringe. The fortified soilswere 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.OO mg a.i./Kg on a wet-weight basis calculated as the product of the lowest calibrationstandalrd (0.00100 mg a.i./L) and the overall dilution factor of the matrix blank samples (1000 LKg). 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 detemune 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%, respectiveky (Table 1). Representative ion chromatograms of low and high-level fortifications in non-target terrestria1plant soil WildlifeInternational, 1:td. - 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 4.1 1 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 fortificationin 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 1000mg 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 it 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 lof solvents, and matrix blank samples, consisting of unfortified artificial soils, were devoid of interferingcomponents. WiId1[fe International, Ltd. - 14- Project Number 454C-120 REFERENCES 1. American Society for Testing and Materials. 1991. Standard Specificationfor Reagent Water. D1193-91, ASTM Section II Water and Environmental Technology, Vol. 11.01:45-47. WiIdItfe InternationaI-, 1:td. - 15 - Project Number 454C-120 Table 1 Method Validation Recoveries for PFOS in Non-Target Terrestrial Plant Soil -Number Sample (454C- 120-) Type PFOS Concentration (mg a.i./JSg) c Fortified Measured Percent Recovered' Mean Measured (mg a.i./Kg:i VREB-1 VMAB- 1 VMAB-2 VMAB-3 Reagent Blank Matrix Blank Matrix Blank Matrix Blank 0 < 1.18* -- 0 < 1.18 -- 0 < 1.18 -- 0 < 1.18 -- 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 f 0.0300 VMAS-3 Matrix Fortification 2.37 2.36 99.4 1.29% VMAS-4 Matrix Fortification 11.8 12.0 101 11.9 101 VMAS-5 Matrix Fortification 11.8 12.0 101 f 0.23 1 VMAS-6 Matrix Fortification 11.8 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 f 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 1180 1159 97.9 f 73.0 VMAS- 12 Matrix Fortification 1180 1062 89.7 6.40% Overall Mean = 98.1 Standard Deviation = cv = f 3.27 3.33% ' N= 12 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. 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 (0.00 1 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%). WiId1{fe International, LEd. - 16- Proiect Number 454C-120 Table 2 Method Validation Recoveries for PFOS in Invertebrate Testirg Soil Sample Number (454C- 120-) -* Type VREB-2 VMAB-4 vMAr3-5 VMAB -6 Reagent Blank 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 Recovered' -- -- --- Mean Measured (mg a.i./K,g) Mean Percent Recovery VMAS- 13 Matrix Fortification 2.88 2.82 98.1 2.87 99.7 VMAS- 14 Matrix Fortification 2.88 2.82 97.9 f 0.0808 VMAS- 15 Matrix Fortification 2.88 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 +0.100 VMAS- 18 Matrix Fortification 14.4 14.4 100 0.694% VMAS- 19 Matrix Fortification 144 136 94.6 134 93.1 VMAS-20 Matrix Fortification 144 133 92.5 f 2.08 VMAS-21 Matrix Fortification 144 132 91.4 1.56% VMAS-22 VMAS-23 VMAS-24 Matrix Fortification Matrix Fortification Matrix Fortification 1440 1440 1440 1370 1391 1341 95.2 1370 95.1 96.7 +25.1 93.2 1.83% Overall Mean = Standard Deviation = cv = N= 96.9 3.61 3.73% 12 1 Percent Recovered values were calculated on a wet-weight basis using MacQuan, version 1.6 soltware. All other values were converted to dry-weight by dividing the wet-weight value by the percent solids for the matrix soil. The limit of quantitation(LOQ) was 1.44mg a.i./Kg on a dry-weightbasis calculated as the product of the lowest calibration standard (0.00 1 mg a.i./L) and the overall dilution factor of the matrix blank samples (1000 LKg) divided by the percent solids for the soil (69.5%). WiId1ijfe International, Ltd. -17- Appendix 1 Protocol and Amendments Project Number 454C-120 WildlifeInternational, Ltd. - 18- Proiect Number 454C-120 PROTOCOL ANALYTICAL METHOD VALIDATION FOR THE DETERMINATION OF PERFLUOROOCTANESULFONATE, POTASSIUM SALT (PFOS) IN ARTIFICLAL SOIL Environmental Laboratory Project Number U2723 Submitted to 3M Corporation Environmental Laboratory 935 Bush Avcnuc st. Paul, Minnesota 55 106 WildlijeInternational, Ltd. 8598 Cornmcrcc Dnvc Easton, Maryland 2 1601 (410) 822-8600 Dccember 7,2000 WildlifeInternational, Ltd. - 19- Project Number 454C-120 wildlife 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 55 106 SPONSOR'SREPRESENTATIVE: Rochelle R. Robideau TESTING FACILITY: Wildlife International, Ltd 8598 Commerce Drive Easton, Maryland 21601 STUDY DIRECTOR: RaymondL. Van Hoven, Pb.D. Scientist Wildlife International, Ltd. LABORATORY MANAGEMENT: Willard B. Nixon, Ph.D. 3 m A r -PArugsi.of Analytical Chemistry @ mi-+qi Wildlife International, Ltd. FOR LABORATORY USE ONLY PROTOCOL APPROVAL PROTOCOLNO.: 454/I20760/MV-S/SUB454 Environmental Laboratory Projcct N m I w U2723 WiIdIife InternationaI , Ltd. -20- Proied. Number 454C-120 wildrife International, Ltd. -3- INTRODUCTION Wildlife International, Ltd. will conduct analytical trials to validate the performance of a liquid chromatographymass spectrometry(LCMS)method for the detcrminationof Perfluorooctanesulfonate, Potassium Salt (hereafter referred to as PFOS) in artificd soil used for earthworm and non-target terrestrial plant tests. The study will be performed at thc Wildlifc International, Ltd. analytical chemistry facility in Easton, Maryland. The method developed by Wildlife International, Ltd. will bc validated by fortifying artificial soil and quantifying the recoveries of PFOS. Raw data for all work performed at Wildlife International, Ltd. and a copy ofthe finalrcporf willbe-filed by projea number in archives located on the Wildlife Intcrnational, Ltd. site, or at an alternative location to be specified in the final report. OBJECTIVE The objcctivc of this study is to verify thc pcrformancc of LCMS methodology for analyses of PFOS in artificial soil used by Wildlife Intcrnational, Ltd. to conduct non-target terrestrial plant and invertebrate testing. EXPERIMENTAL DESIGN Wildlifc International Ltd. artificial soils similar to those used in earthworm and non-target terrestrial plant tests will bc fortdied at four different concentrations and analyzed using LCMS methodology based upon that developed at Wildlife International Ltd. Modifications to the. methdology will be made. as neceSSafyto achicvc quantitation of PFOS in the test matrix. Matrix d o r reagent blanks will be analyzed w n d y to evaluate potential analytical intedemccs. The levels of fortification will bracket thc anticipated concentrations of samples from carthworm and non-target terrestrial plant toxicity tests. Reagent and matrix blanks will be analyzed to evaluatc potential analfical interferences. One calibration curve will be prepared and analyzed with cach series of matrix fortification samples. The accuracy, precision, and limit of quantitation of the method will bc dcte&ed. PROTOCOLNO.: 454/120700/MV-S/SUB454 F ~ v i r m t aLlaboratory Project Number U2723 WildlifeInternational, Ltd. -21 - Project Number 454C-120 -4- MATERIALS AND METHODS Test Substance The test substance will bc Pcdluorocctanesulfonate, Potassium Salt. Information on the characterizationof test, control or referencesubstances is required by Good Laboratory Practice Standards (GLP). The Sponsor is responsible for providing Wildlife International, Ltd. written vcdication that the test substance has been characterizedaccordingto GLPs prior to initiationof the study. The reference material in this study will be the test substance. If written verification of GLP test substance characterization is not providcd to Wildlife International, Ltd., it will be noted in the compliancestatementof the final rcport. The Sponsor is responsible for all informationrclatcd to the test substance and agrees to acccpt any unused test substance and/ortest substancc containers remainingat the end of the study. Reagents and Solvents All solvents used in the mcthod or procedure will be of reagent grade or better. All reagents will be of ACS grade or bcttcr. Test Soil - Earthworm Soil The artificial soil used for carthworm tests will be composed of approximately 10% sphagnum peat, 20% kaolin clay and 70% industrial quartz sand. T h e dry constituents of the soil will be mixed in a PK Twinshell or equivalent mixcr. Calcium carbonate powder will be added during thc mixing of the dry soil as needed to adjust the pH to 6.0 i 0.5. prior to hydration. The moisture cxmtent of the artificial soil will be determined by weighmg samples of the soil before and after drying at approximately 105C. The moisture contat is calculated usingthe following formula: * YOmoisturc= [(nct wet weightaet dry weight) + net wet weight] X 100 The soil will be adjusted to approximately 33% moisture contcnf during thc prcpadon of the test soils. PROTOCOL NO.: 454/120700/Mv-S/SUB454 Environmental Laboratory Project Number U2723 WiIdltfe InternationaI , Ltd. - 22 - Project Number 454C-120 wildlife International, Ltd. -5 Test Soil - Non-Target Terrestrial Plants Test plants will be grown in plastic pots approximatcly 16 cm in diamctcr and 12 cm in depth. An unsterilized artificial sandy-loam or sand soil substrate will be used for testing. The soil will be composed of kaolinite clay, industrial quartz sand and pcat mixcd in a 4:50:2.5 ratio (w:w:w). Crushcd limestone will be added to buffer the pH of the soil, and a slow-release fertilizer will be added to provide nutrients essential for plant growth. Typically, the soil consists of approximately 90 % sand, 2-3 % silt, and 7-10 % clay, with a neutral pH and an organic matter content less than 1.5 %. A samplc of soil representative of that used in this study will be sent to Agvise Laboratories, Inc., in Northmod, North Dakota,for analysis of the particle size distribution and organic matter content of the soil. Soil charactcrization will include, but may not be limited to,the determination of particle size distribution, organic matter content, and pH. Those itcms rclcvant to thc conduct of thc study will be discusscd in the final report. The complete report from Agvisc Laboratories, Inc. will be filed in the archives located at Wildlife International,Ltd.The results of thc charactcrization will bc storcd in the archives located at the Widlite Intcrnational, Ltd. site, and those items relevant to the conduct of the study will bc &scussed in the final report. Analytical Validation Procedures The analyhcalmethcd to bc used w i i l be L C M S based upon procedures developed at Wildlife International, Ltd. The methodused will be documented in the raw data and summarid in thc ha1 report. Prior to thc analysis of analytical samples, primary stock solution(s) will be prepared directly from the test substance. Calibration standards will be prcparcd by appropriate dilution of the primary stock solution(s). Primary Stock Solution(s),Calibration Standards and Curves A minimum of five calibration standards (of different concentrations) will be prepared and analyzed along with each analysis set. Thc calibration standard series will be injected at the beginning and one standard injected, at a minimum, after every fivc samplcs. A minimum of two injections of each standard in thc ahbration standard series will be injected with the analysis sct. One calibration cufvc and regression equation will be prepared from the series of calibration standards. PROTOCOL NO.:454/120700/MV-S/SUB454 EnvironmentalLaboratory Project Number U2723 WildlifeInternational, Ltd. -23- Project Number 454C-120 'wildlife International, Ltd. -6 - Fortification Stock Solutions The soil willbe fortified with test substance in a manner consistcnt with thc methtdanticipated for the toxicology studies. Evaluation of Interferences One reagent blank and three matrix blanks 4lbe analyzed to detect possible interferences. The reagent blank will contain everything except the matrix and the test substance. The matnx blanks \rill contain everythmg except the test substance. Verification Analyses - Method Performance Matrix fortifications (spiked artificial soil from earthworm and non-target plant stuhcs), prepared at known concentrations of the test substance, will be analyzed to determine the recovery of the analyte and to evaluate methcd p c r f o m c e . The anticipated verification series will consist of the following analyses: SUMMARYOF PROPOSEDVERIFICATION ANALYSIS SCHEME Matrix fortifications will represent the range of concentratioosthat are anticipated to be used in subsequentenvironmentalfitc and/or efFects fests and will be selected in consultation with the Sponsor. Individual samples ( i d c n ~ e dby project number and a unique sample identification number) will be prepared and analyzed for verification of the analytical methodology. PROTOCOLNO.:454/120700/MV-S/SUB454 EnvironmentalLaboratory Projcct Nurnbcr U2723 WildlifeInternational. Ltd. - 24 - Project Number 4546-120 'wildlife International, Ltd. __ -7- If difficulties arise in the validation process (e.g., low,rccovcrics or intcrfcrcnces), thc Sponsor will be notified and thc d for additional validation and/or method development will be determined through discussions with the Sponsor. Upon completion of the method validation, the Sponsor will review the results and authoriu: thc use of the methodology for analysis of samples, or will authorize furthcr method development trials. Recovely values in the range of 80 to 120% will bc uscd as critcria for method acceptability. Data Analysis One calibration curvc \viU be established for each analytical run. A regression equation of the conmtmtion versus peak arca for the calibration standards will be generated. The concentration of the samples will be determined by substituting the respcctivc pcak arca into the regrcssion cquation. Statistics to be determined and rcp0rte.d will include the mean and standard deviation for each level of fortification. RECORDS TO BE MAINTAINED Records to be maintainedfor data generated by Wildlife International, Ltd.wdl include: 1. A copy of thc signed protocol. 2. Identificationand charactcrizationof the test substanceandor analyticalstandard,if provided by the Sponsor. 3. Dates of initiation and termination ofthetest. 4. Storagc conditionsfor test substance,analytical standards,and/or samples. 5. Test substancc and/or analyticalstandarduse log. 6. Concentration calculationsand records of solutionpreparation. 7. Instrumentoperatingconditionsand chromatograms. 8. Statisticalcalculations. 9. A copy of the finalreport. 10. Documentation that the steps in thc method were followed. FINAL REPORT The report will summarize the f d q s of the verification, the procedural recoveries obtained, and thc methods and instrumentation employcd. Upon receipt of these fmdmgs, the Sponsor will PROTOCOLNO.: 454/12070O/MV-S/SUB454 Environmental Laboratory Project Number U2723 W i l d l i f eInternational? L t d . Project Number 454C-120 wildlife International, Ltd. -8- review the methods and rcsults and evaluate the results for q t a b i l i t y prior to initiatuy any fate andor effects studies. The final report will include, but not bc limited to, the following: 1. Name and address of the fkcilitypcrfonningthe study. 2. Dates on which the study was initiated and completed. 3. Objectivcsand procedures stated in the approvcdprotocol, includingany changes in thc original protocol or deviationsfrom the protocol. 4. The test substance andor analytical standard identification, includmg name, chemical abstract number or codc number, strength, purity, composition, date of receipt, lot number, storage conditions,physical charactcristics, stability and method of prcparationof test concentrations, if provided by the Sponsor. 5 . A brief summary of the analytical methodology: A description of the expcrimcntal measurements, oramplc calculations, sample preparation (sample weights and dilutions), instrumentation anployed, reagents and solvents uscd, class of water used, and any major modificationsto thc method. 6 . A dcscriptionof all circumstancesthat may have affected the quahty or integrity of the data. 7. The name of the Study Director, the names of other scientistsor profcssionals,and the names of all supervisory personnel involved in the study. 8. A description of the transformations,calculations,or operationsperformed on the data. 9. The signed and dated rcports of each of the individualscientists or other profcssiuaals involved in the study, if applicablc. 10. The location where raw data and final report are to be stored. 11. A statementpreparcd by the Quality Assurance Unit listingthe dates that study inspectionswere madc and the dates of any findings reportedto the Study Dircctor/Managemcnt. CHANGING OF PROTOCOL Planned changes to the protocol -ill be in the form of written amendments s i w by the Study Director and the Sponsor's Representative. Amendments will be consideredas part of the protocol and will be attached to the final protocol. Any other changeswill be in the form of writtcn daiations 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.:454/12070O/MV-S/SUB454 En~konmentalLaboratory Projcct Number U2723 WiId1[fe Internationa1, Ltd. -26- Project Number 454C-120 wildlifeInternational, Ltd. -9- GOOD LABORATORY PRACTICES This study will be c o n d u d in accordance with Good Laboratory Practice Standardsfor EPA (40CFR Part 160);and OECD PMciples of Good Laboratory Practice (ENVMUCHEM (98) 17). Each study conducted by Wildlife International, Ltd. is routinely examined by thc Wildlifc International,Ltd. Quality Assurance Unit for compliancc with Good Laboratory Practices, Standard Operating Procedures and the specified protocol. A statanent of compliance with Good Laboratory Practiccs will be prepared for all portions of the study conductcd by Wildlifc Intcmationii, Ltd. Thc Sponsor will be responsible for certification of compliance with Good Laboratory Practices for p r d u r e s performed by other laboratories. Raw data for all work performcd at Wildlifc Imcmational, Ltd. and a copy of the final report will be tiled by project number in archives locatedon the Wildlife International, Ltd. site, or at an alternative location to bc spccificd in the fmal report. A copy of the study raw data for all work performcd at Wildlife International,Ltd. will be sentto the Sponsorwiththe final report. PROTOCOLNO.:454/1207OO/MV-S/SUE454 Envircumcntal Laboratory Project Number U2723 WiId1ife InternationaI? Ltd. - 27 - Project Number 454C-120 wildlife International, Ltd. Project Numbcr 454C-120' Page I of 1 AMENDMENT TO STUDY PROTOCOL STUDY TITLE: ANALYTICAL METHOD VALIDATION FOR THE DElTZMATION OF PERFLUOROOCTANE SULFONATE, POTASSIUM SALT (PFOS) 1N ARTIFICIAL SOIL PROTOCOL NO.: 454/120700/MV-S/SUB454 SPONSOR: 3M Corporation SPONSOR STUDY NO.: U2723 AMENDMENT NO.: 1 PROJECT NUMBER: 454C-120 EFFECTIVE DATE: February 6,2001 AMENDMENT: Page 5, Test Soil - Non-Target Terrestrial Plants CHANGE: ` m e soilwill be composcd of kaolinite clay, industrial quartz sand and peat mixed in a 4:50:2.5 ratio (w:w:w)." TO: "The soil will be composed of kaolinite clay, industrialquartz sand and peat mixedin 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. LABORATORY GEMENT 03-06 -01 DATE Wild1ife International, 1:td. -28- Project Number 454C-120 wildlife International, Ltd. Project Number 454C-120 Page 1 of 1 AMENDMENT TO STUDY PROTOCOL STUDY TITLE Analytical Methd Validation For The Determination Of Perfluorooche Sulfimate, Potassium Salt (PFOS)In Artificial Soil PROTOCOL NO.: 454/120700/MV-S/SUB454 SPONSOR: 3M Corporation SPONSOR STUDY NO.: U2723 AMENDMENI` NO.: 2 PROJECT NUMBER: 454C-120 EFFECTIVE DATE: May 16,2003 AMENDMENT: Page 2, Sponsor's Representative Change: Rochcllc R Robideau To: Susan A. Beach REASON: Reassignment of Sponsor's Representative responsibilities at thc request of the Sponsor. AMENDMENT: Page 8, changing ofProtocol Change: "Planned changes to thc protocol will be in the form of written amendments signed by the Study Director and the Sponsor's Representative." To: ''Planned changes to the protocol will be in the form of written amendments signed by the Study Director and approved by the Sponsor's Represeutative." REASON: To expdtc finalization of the study. LABORATORY &AGEMEN DATE WildlifeInternutional, Ltd. Project Number 454C-120 Appendix 2 Certificate of Analysis INTERM CERTIFICATE OF ANALYSIS Revision I (9fl!OO) Centre Analytical Laboratories COA Reference #: O23-018A 3M Product: PFOS,Lot 217 Reference #: SD-018 COA023418A Page 1of 3 WiIdItfe InternationaI , Ltd. - 30 - Project Number 454C-120 INTERIM CERTIFICATE OF ANALYSIS Centre Analytical Laboratories COA Reference #: 023-018A Date of Last Analysis: 08/31/00 Expiration Date: 08/31/01 StorageConditions: Frozen ClO'C Re-assessment Date: OW31/01 - 'Purity = 100% (sum of metal impurities, 1.45% +LC/MS impurities,8.41%+Inorganic Fluoride, 0.59%+NMR impurities, 1.93%+0rganicacid impurities,0.38%+POAq 0.33%) - Totalimpurityfrom all tests = 13.09% Purity = 100% 13.09% = 86.9% 'Potassium is expected in this salt form and is therefore not considered an impurity. 9urity by DSC is generally not applicableto materials of low purity. No endothermwas observed for this sample. 4Sulfurin the sampleappearsto be converted to SO4 and hence detectedusing 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. %A HFBA NFPA PFPA Trifluorodc acid Heptailuorobutyric acid Nonofluoropentanoic acid PenMuoropropanoic acid ?heoretical value calculations based on the empirical formula, C~F,,S03K'v=538) This work was conducted under EPA Good LaboratoryPractice Standards(M CFR 160) / COA023-018A Page 2 of 3 Wildlcfe International, Ltd. -31 - Project Number 454C-120 Centre Inc Analytikal labaratorks. 3048 Research Drive State College. PA 16801 www.centrelab.com F a (814) 231-1253or (814) 231-1580 INTERIM CERTIFICATE OF ANALYSIS Revision 3 Centre Analytical Laboratorks COA Reference #: 023-018A LClMS Purity Profile: Impurity c4 c5 W t J W t Ye 1.22 1.33 I C6 I 4.72 I c7 1.14 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 imm the C4 and C6 standard curves. Likewise,the C7 value was calculated Usingthe average result fromthe C6 and C8 standardcurves. Prepared By: scialtist, centreAnalytical Laboratories &IFlahatv / COAO23-018A Pkh -- Date Page 3 Of 3 WildlifeInternational, Ltd. - 32 - Project Number 454C-120 Appendix 3 Analyses of Pesticides, Organics and Metals in Wildlife International, Ltd. Greenhouse Soil' Component achlordane y-chlordane P,P'-DDE 0,P'-DDT Aldrin P,P'-DDT Dieldrin Pendosulfan Endosulfansulphate Endrin Endrin-keton a-HCH P-HCH Lindane (y-HCH) GHCH Heptachlor P-Heptachlorepoxide Hexachlorbenzene Methoxychlor P,P'-tDE Magnesium Sodium Calcium Iron Potassium Aluminum Manganese Beryllium Chromium Cobalt Pesticides And Organics Measured Concentration Component <0.01 mgikg <0.01 mgkg <0.01 mgkg <0.01 mgkg <0.01 mgikg <0.01 mgikg 10.01 mg/kg ~ 0 . 0 1mgkg <0.01 mgikg <O .O 1 mgikg <0.01 mg/kg <0.01 mg/kg ~ 0 . 0 1mgkg <0.01 mgikg <0.01 m a g 10.01 mgkg <0.01 mgikg <0.05 mgkg <0.05 mgkg <0.01 mgikg Azinphos-methyl Chlorfenvinphos Chlorpyriphos Chlorpyriphos-methyl Diazinon Dichlorvos Fenitrothion Malathion Mevinphos (Cis+Trans) Parathion Parathion-methyl Pirimiphos-methyl Sulfotep PCB 28 PCB 52 PCB 101 PCB 118 PCB 138 PCB 153 PCB 180 Metals 3,000 mgkg 60 w k g 4,700mgkg 6,100 mgkg 220 mgkg 5,200 mgkg 51 mgkg <0.04 mgkg 6.0 mgkg 0.50 mgkg Nickel coppa Zinc Molybdenum Silver Cadmium Arsenic Mercury Selenium Measured Concentration Not Determined <0.01 mg/kg <0.01 mgkg <0.01 mg/kg <0.01 mg/kg <0.01 mg/kg 10.01 mg/kg <0.01 mg/kg Not Determined <0.01 mg/kg ~ 0 . 0 1mg/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 . 0 1mg/kg c2.0 mgkg ~ 2 . m0 &g 6.1 mgkg < 1.2 mgkg <0.40 mgkg 12.0 tng/kg 3.8 mgkg 12.0 pgkg 0.11 mgcg 'Analyses performed by TNO Nutrition and Food Institute on samples collected on October 14 and 15, 1999 WildltfeInternational, Ltd. - 33 - Appendix 4 Analytical Method Validation Data Project Number 454C-120 WildltfeInternational, 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 8-0z French-square bottles. For matrix fortification samples, forti@ each sample with the appropriate volume of PFOS stock solution in methanol with a gas-tight syringe. Allow to air dry. 'I'he matrix blank is unfortified soil matrix. 1 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. L Cap bottles and place on a shaker table. Allow the samples to shake for a minimum of 30 minutes at approximately 250 rpm. L Vacuum filter with qualitative filter paper and rinse retained soil three times with methanol into filtrate. L Transfer the filtrate into a 200-mL volumetric flask and bring to volume with methanol. Mix well with several repeat inversions. L Transfer approximatelytwenty milliliters of each sample into a separate glass centrifuge tube or scintillation vial and cap. Centrifuge samples for approximately five minutes at 2000 rpm. L Dilute samples into the calibration range of the PFOS LC/MS methodology: Partially fill Class A volumetric flasks with dilution solvent (50% methanol: 50% NANOpure@water). Add ;appropriate volume of sample with a gas-tight syringe and bring to volume with dilution solvent. Process matrix L blank samples using the same dilution and aliquot volumes as for the lowest fortification level. Ampulate samples and submit for HPLC/MSiMS analysis. WiId1ife Internationa1, Ltd. - 35 - Project Number 454C-120 INSTRUMENT: Appendix 4.2 Typical HPLC/MS/MS Operational Parameters Hewlett-Packard Model 1100High 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: Keystone Betasil Clg column (50 mm x 2 mm I.D., 3-pm particle size) GUARD COLUMN: Keystone Javelin Clg column (20 mm x 2 mm I.D.) OVEN TEMPERATURE: 40C STOP TIME: 5.00 minutes FLOW RATE: 250 p,L/minute MOBILE PHASE: 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 WildltfeInternational, Ltd. - 36 - Appendix 4.3 A Typical Calibration Curve for PFOS K 6 4 9 9 . 0 4 9 . 1 No Internal Slandard Neighred (l/x) Intercept = 741.01 Slope = 11944389.00 Correlation Coeff. = 0.99977 Area Project Number 454C-120 a n a . pg a.i./L WildlifeInternational, Ltd. - 37 - Project Number 454C-120 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 = 243 17 Y-intercept = 741.01 Slope = 11944389 Primary Dilution: Initial Weight (Wi) = 10.0 g Original Final Volume (VI)=200 mL Secondary Dilution: Initial Volume (Vi) = 0.200 mL Final Volume (Vf)= 10.0 mL Overall Dilution Factor (Vl/Wi x V n i ) = 1000 mL/g = 1000 L/Kg eak area - y-intercept) PFOS at instrument (mg a.i./L) = (p slope - (24317 - 741.01) - 11944389 = 0.0019738 mg a.i./L PFOS in sample (mg a.i./Kg)= PFOS at instrument (mg a.i./L) x overall dihtion factor (LJKg) 0.0019738 mg a.i. lOOOL PFOS in sample (mg a.i./Kg) = L X- Kg = 1.97 mg a.i./Kg PFBS m a.i./L in Sam le Percent of Nominal Concentration = pFBs((2g nomin:l x 1100 - --21.*0907x 100 = 98.7% Instrument Software: MacQuan, version 1.6. Wildltfe International, Ltd. - 38 - Proiect Number 454C-120 Appendix 4.5 Ion Chromatogram of a Low-level PFOS Standard intensity: 10000 cps 1 24 1 27 4 8 I I 1 41 0.69 I I 81 1.36 126 I I 121 2.03 I 161 2.70 201 3.37 241 4.04 281 Scan 4.71 Time Nominal concentration: 0.00100 mg a.i./L Wild1[fe International, 1:td. - 39 - Project Number 454C-120 Appendix 4.6 Ion Chromatogram of a High-level PFOS Standard intensity: 10000 cps 10 9 8 7 6 5 4 3 2 1 41 0.69 81 1.36 121 2.03 161 2.70 201 3.37 241 4.04 281 Scan 4.71 Time Nominal concentration: 0.0100 mg a.i./L WildltfeInternational, Ltd. - 40 - Project Number 454C-120 Appendix 4.7 Ion Chromatogram of Non-Target Terrestrial Plant Soil Matrix 13lank intensity: 10000 cps li5 1 2 1 a! 16 I I I 41 0.69 82 119 150 187 211 r I I 1 I 8 1 121 161 201 1.36 2.03 2.70 3.37 241 I 241 4.04 280 I 281 Scan 4.71 Time Sample number 454C-120-VMAB-1. The arrow indicates the approximate retention time of PFOS. WildltfeInternational, Ltd. -41 - Project Number 454C-120 Appendix 4.8 Ion Chromatogram of an Invertebrate Testing Soil Matrix Blank intensity: 10000 Cp!; 100 90 80 70 60 50 40 30 20 Sample number 454C-120-VMAB-4. The arrow indicates the approximate retention time of PFOS. WiId1[fe Internationa1, 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 70 .3 4 20-1 1 ~ 240 b I 1 41 0.69 109 143 167 I 1 I I I I 81 121 161 1-36 203 2.70 I I 201 3.37 I I 241 4.04 283 1 281 4.71 Timie Sample number 454C-120-VMAS-1,2.00 mg a.i./Kg wet-weight nominal concentration, overall dilution factor = 1000 WKg. WildltfeInternational, 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 242 5 4 3 2 1 0.69 1.36 2.03 2.70 3.37 4.04 In 4.71 Time Sample number 454C-120-VMAS-10,lOOO mg a.i./Kg wet-weight nominalconcentration, ovleralldilution factor = 200000 WKg. WildlifeInternational, Ltd. - 44 - Project Number 454C-120 Appendix 4.11 Ion Chromatogram of a Low-level Invertebrate Testing Soil Fortification Sample intensity: 10000 cps 40 20 10 20 66 139 171 41 0.69 81 1.36 121 2.03 161 2.70 201 3.37 241 4.04 281 Scan 4.71 Time Sample number 454C-120-VMAS-13,2.00 mg a.i./Kg wet-weight nominal concentration, overall dilution factor = 1000 L/Kg. WiId1tfe Internationa1, Ltd. - 45 - Project Number 454C-120 Appendix 4.12 Ion Chromatogram of a High-level Invertebrate Testing Soil Fortification Sample Sample number 454C-12O-VMAS-22,lOOOmg a.i./Kg wet-weight nominal concentration,overalldilution factor = 200000 L/Kg. WildltfeInternational, Ltd. - 46 - Proiect Number 454C-120 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