Document e73L895XkMj8Md1BXavQKoYRE

38) Analytical method validation for determination of PFBS-K in freshwater, 454C-115 SANIT1ZED OEC 0 9 2003 ANALYTICAL METHOD VALIDATION FOR THE DETERMINATION OF, PERFLUOROBUTANE SULFONATE, POTASSIUM SALT (PFBS) M FRESHWATER WILDLIFE INTERNATIONAL, LTD. PROJECT NUMBER 454C-115 3M Environmental Lab Project No. E00-1429 AUTHORS: Raymond L. Van Hoven, Ph.D. Willard B. Nixon, Ph.D. STUDY INITIATION DATE: April 17,2000 STUDY COMPLETION DATE:- June 12,2000 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 43 W.ILDLIFE INTERNATIONAL, LTD. -2- PROJECT NO.: 454C-115 GOOD LABOMTORY PRACTICE COMPLIANCE STATEMENT SPONSOR: 3M Corporation TITLE: Analytical Method Validation for the Determination of Perfluorobutane Sulfonate, Potassium Salt (PFBS) in Freshwater WILDLIFE INTERNATIONAL, LTD. PROJECT NUMBER: 454C-115 3M ENVIRONMENTAL LAB PROJECT NUMBER: 00-1429 STUDY COMPLETION: June 12,2000 This study was conducted in compliance with Good Laboratory Practice Standards as published by the U.S.Environmental Protection Agency in 40 CFR Part 792, 17 August 1989 and OECDPrinciples of Good Laboratory Practice (ENV/MC/CHEM (98) 17) with the followingexceptions: The test substance was not characterized in accordance with Good Laboratory Practice Standards. 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: .. Ra&gJ&L n L. Van Hoven, PbD. Scientist Wildlife International, Ltd. Q -13-00 DATE SPONSOR APPROVAL: WILDLIFE INTERNATIONAL, LTD. -3- PROJECT NO.: 454C-115 QUALITY ASSURANCE STATEMENT T h s study was examined for compliance with Good Laboratory Practice Standards as published by the U.S. Environmental Protection Agency in 40 CFR Part 792, 17 August 1989 and OECD Principles of Good Laboratory Practice (ENVIMCKHEM(98) 17). The dates of all inspections and audits and the dates that any findings were reported to the Study Director and Laboratory Management were as follows: ACTIVITY: Matrix Fortification Preparation Data and Draft Report Final Report DATE CONDUCTED: April 18,2000 May 2 and 3,200 June 12,2000 , DATE REPORTED TO: STUDY DIRECTOR: MANAGEMENT: April 18,2000 April 20,2000 May 3,2000 May 4,2000 June 12,2000 June 12,2000 Susan L. Coleman, B.A. Senior Quality Assurance Representative 6-la- m DATE WILDLIFE INTERNATIONAL,LTD. -4- PROJECT NO.: 454C-115 REPORT APPROVAL SPONSOR: 3M Corporation TITLE: Analytxal Method Validation for the Determination of PerfluorobutaneSulfonate,Potassium Salt (PFBS) in Freshwater WILDLIFE INTERNATIONAL, LTD. PROJECT NUMBER: 454C-115 3M ENVIRONMENTALLAB PROJECT NUMBER: E00-1429 STUDY DIRECTOR: _- Ra&n&. Van Hoven, PbD. Scientist MANAGEMENT: - ' I Willard B. Nixon, PbD. Manager, Analfical Chemistry 6-u-00 DATE .~ &Z$& DATE WILDLIFE INTERNATIONALLT. D. -5- PROJECT .NO. 454C-115 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............................................................................................................................................................. 7 Introduction ........................................................................................................................................................ 8 Purpose............................................................................................................................................................... 8 ExperimentalDesign.......................................................................................................................................... 8 Materials and Methods....................................................................................................................................... 8 Test Substance....................................................................................................................................... 9 Reagents and Solvents.......................................................................................................................... 9 Test System.......................................................................................................................................... 9 Analytical Validation Procedures......................................................................................................... 9 . Primary Stock Solution, Calibration Standards and Curves .............................................................. 10 Fortification Stock Solutions............................................................................................................... 10 Limit of Quantitation.......................................................................................................................... 10 Example Calculations......................................................................................................................... 10 Results ............................................................................................................................................................ 11 Matrix Blank Samples........................................................................................................................ 11 Method Validation Samples............................................................................................................... 12 Conclusions ...................................................................................................................................................... 12 References ........................................................................................................................................................ 13 WILDLIFE INTERNATIONAL, LTD. -6- PROJECT NO.: 454C-115 TABLE OF CONTENTS - Continued - TABLES Table 1- Typical LC/MS Operational Parameters......................................................................................... 14 Table 2 - Matrix Blanks Analyzed Concurrently During Sample Analysis ................................................... 15 Table 3 - Method Validation Recoveries for PFBS in Freshwater................................................................. 16 FIGURES Figure 1- Analytical method flow chart for the analysis of PFBS in freshwater............................................ 17 Figure 2 - A typical calibration curve for PFBS .............................................................................................. 18 Figure 3 - A representative ion chromatogramof a low-level (0.00100 mg a.i./L) standard ......................... 19 Figure 4 - A representative ion chromatogramof a high-level (0.0100mg a.i./L) standard ..........................20 . Figure 5 - A representative ion chromatogramof a freshwater matrix blank sample (454C-115-VMAB-4)..................................................................................................................... 21 . -- Figure 6 - A representative ion chromatogramof a low-level freshwater matrix fortification sample (454C-115-VMAS-2,norninal concentration of 0.500 mg a.i./L) .................................... 22 Figure 7 - A representative ion chromatogramof a high-level freshwater matrix fortification sample (454C-115-VMAS-12,nominal concentration of 1000 mg a.i./L) ................................... 23 APPENDICES Appendix I - Protocol, .Amendmentand Deviation ...........;..................................................................._.2.4 Appendix II - Certificateof Analysis......................................................................................................... 40 Appendix 111- Specific Conductance, Hardness, Alkalinity and pH of FreshwaterMeasured During the 4-Week Period Immediately Preceding the Test...............................................4 1 Appendix IV - Analyses of Pesticides, Organics, Metals and Other Inorganics in Wildlife International, Ltd. Freshwater............................................................................... 42 Appendix V - Personnel Involved in the Study.......................................................................................... 43 WILDLIFE INTERNATIONAL, LTD. -7- PROJECT NO.: 454C-115 SUMMARY STUDY TITLE: WILDLIFEINTERNATIONAL, LTD. PROJECT NUMBER: SPONSOR: TESTING FACILITY: LOCATION OF STUDY, RAW DATA AND A COPY OF THE FINAL REPORT: Analytical Method Validation for the Determination of Perfluorobutane Sulfonate, Potassium Salt (PFBS) in Freshwater 454C-115 3M Corporation Wildlife International, Ltd. Easton, Maryland 2 1601 Wildlife International, Ltd. Easton, Maryland 21601 TEST SUBSTANCE: PFBS FORTIFIED TEST CONCENTRATIONS FOR METHOD VALIDATION: 0.500, 10.0, 100 and 1000mg a.i./L TEST DATES: ExperimentalStart - April 18,2000 ExperimentalTermination- April 19,2000 - TEST SYSTEM: Freshwater .- I SUMMARY: Freshwater samples were fortified with PFBS to reflect nominal concentrations of 0.500, 10.0,lOOand 1000mg a.i./L. The sampleswere diluted into the instrumental calibration range with dilution solvent (50% methanol: 50% NANOpure@water) and analyzed by liquid chromatographymass spectrometry (LCMS).Recoveries of PFBS in freshwater yielded an overall mean percent recovery of 106% (SD = 5.72%; CV = 5.41%; N = 12). Matrix blank samples, consisting of unfortified freshwater, were devoid of interfering componentswhen a base-acid wash on preparatory apparatus was incorporated into the methodology. W I L D L I F E INTERNATIONAL, LTD. -8- PROJECT NO.: 454C-115 INTRODUCI'ION 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 Perfluorobutane Sulfonate, Potassium Salt (hereafter referred to as PFBS) and analyzed with liquid chromatography mass spectrometry (LCMS) to evaluate the performanceof a method infreshwater. Validation samples wereprepared and analyzed betweenApril 18 and 19,2000. Raw data generated by Wildlife International, Ltd. and a copy of the final report are filed under Project Number 454C-115 in archives located on the Wildlife International, Ltd. site. PURPOSE The purpose of this study was to venfy the performance of a LCMS methodology for the analysesof PFBS in freshwater. The validated method will be used in support of aquatictoxicology and product chemistryteststo be conducted with the test substance. EXPERIMENTAL DESIGN .. - Freshwater was fortified at four different concentrations in triplicate and analyzed based on LCMS methodology developed at Wildlife International, Ltd. The fortification levels bracketed the anticipated concentrations of samples fiom future aquatic toxicity tests. Matrix blank sampleswere analyzed concurrentlyto evaluate potential analyhcal method interferences. A calibration curve was generated from analyses of standard solutions of PFBS with each series of samples analyzed. MATERIALS ANTI METHODS This study was conducted according to the procedures outlined in the protocol, "Analyhcal Method Validation for the Determination of Perfluoro Butane Sulfonate, Potassium Salt (PFBS) in Freshwater" (Appendix I). WI LD LIFE INTE RNATI oNAL , LTD. -Y- PROJECT NO.: 454C-115 Test Substance The test substance was received from 3M Environmental Technology and Safety Services on March 27, 2000, assigned Wildlife International, Ltd. Identification number 5216,and storedunder ambientcondtions. The test substance, a white powder, was identified as: Potassium Perfluorobutane Sulfonate (L-7038, Lot 2), expiration date: March 20 10. The test substance was m e r identified with the 3M EnvironmentalLaboratory test control and reference number TCR# 99131-39. After the protocol was issued, the test substance purity was re-determined and had a reported purity of 97.9% (Appendix 11). ReaPents 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 DesignationD1193-91) was used (1). Test System The water used as matrix was freshwater obtained from a well approximately40 meters deep located on the Wildlife International, Ltd. site. The water was passed through a sand filter and pumped into a 37,800-L storage tank where the water was aerated with spray nozzles. Pnor to use, the water was filtered to 0.45 p min order to remove h e particles. The freshwater is characterized as moderately-hardwater. Specificconductance,hardness, _. alkalinity and pH of freshwater during the four-week period immediately preceding the test are presented in Appendix III. The results of analyses performed to measure the concentrations of selected contaminants in freshwater used by Wildlife International, Ltd. are presented in Appendix N. Analytical Validation Procedures Wildlife International, Ltd. conducted a vahdationtrial using procedures based onmethodologydeveloped at Wildlife International, Ltd. Validation sampleswere prepared in freshwater. Dilutions were performedwith a solution of 50% methanol (HPLC grade, 99.9+%) and 50% NANOpure@water. Sampleswere then analyzedby dxect injection. Concentrations of PFBS were determined by reverse-phase high performance liquid chromatographyusing a Hewlett-Packard Model 1100High PerformanceLiquid Chromatograph (HPLC)with a Perkin-Elmer API 3000LC Mass Spectrometer equipped with a Perk-Elmer TurboIonSpray ion source. Chromatographicseparations were achieved using a Keystone Prism column (30 mm x 1.5 mm, 3 - p n particle size). The instrument parameters are summarized in Table 1 and a method flow chart is provided in Figure 1. WILDLIFE INTERNATIONALL, TD. PROJECT NO.: 454C-115 Primarv Stock Solution. Calibration Standards and Curves A primary stock solution of PFBS was prepared in methanol. Calibration standards were prepared in 50% methanol: 50% NANOpure' water by appropriate dilutions of this stock solution. Calibration standards of PFBS, ranging in concentration from 0.00100 to 0.0100 mg a.i./L, were analyzed with each sample set. Five calibration standards (dfferent concentrations) were analyzed with the samples. 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 the peak area responses versus the respective concentrations of the calibration standards. A typical calibration curve is presented in Figure2. The concentration of PFBS in the samples was determined by substituting the peak area responses into the applicable linear regression equation. Representative ion chromatogramsof low andhighcalibrationstandardsarepresented in Figures 3 and 4, respectively. Fortification Stock Solutions Freshwater was fortified with stock solutions of PFBS prepared in methanol. Each stock solution was assigned a unique identification code that was recorded on a stock preparation log sheet. Limit of Ouantitation The method validation levels were well abovethe method capabilities for quantitationof thetestsubstance. -~ . I Therefore, the method limit of quantitation (LOQ) for the analyses was set at 0.100 mg a.i./L based upon the product of the lowest calibration standard(0.00 100mg a.i./L) and the ddution factor of thematrixblank samples (1 00). Examde Calculations Sample number 454C-115-WS-2, nominal concentration of 0.500 mg a.i./L in freshwater. Initial Volume: 0.100 mL Final Volume: 10.0 mL Dilution Factor: 1001 PFBS Peak Area: 1166071 WILDLIFE INTERNATIONAL, LTD. PROJECT NO.: 454C-115 Calibration curve equation. Slope: 2 14262096.0000 Intercept: 5379.9839 Curve is weighted (I/$. *eak area - (Y-intercept) PFBS (mg a.i./L) in sample = slope dilution factor - 1166071 - 5379.9839 - 2 14262096.0000 = 0.542 PFBS m a.i./L insam le Percent of Nominal Concentration= pFBS((mi x 100 --0S42 x 100 = 108% 0.500 Instrument Software: MacQuan, version 1.6. RESULTS Matrix Blank Samules Three matrix blank samples were analyzed to determine possible interferences. No interferenceswere observed at or above the LOQ (0.100 mg a.i./L) during the sample analyses (Table 2). However, a trace-level interfering component was evident in a preliminary trial that was eliminated through use of a base-acid wash procedure on preparatory apparatus. To prevent a potential source of bias in future definitivestudies,the baseacid wash procedure was incorporated into the methodology. A representativeion chromatogramof a fieshwater matrix blank sample is presented in Figure 5. W I L D L I F IENTERNATIONAL, LTD. - 12- PROJECT NO.: 454C-115 Method Validation Samples Freshwater was fortified in triplicate at 0.500,10.0,100 and 1000mg a.i./Lresultinginmeanrecoveriesof 112, 105, 103 and 103%, respectively (Table 3). Representative ion chromatograms of low and hgh-level freshwater matrix fortifications are presented in Figures 6 and 7, respectively. CONCLUSIONS Freshwater samples were fortifed with PFBS to reflect nominal concentrations of 0.500, 10.0, 100 and 1000 mg a.i./L. The samples were diluted into the instrumental calibration range with dilution solvent (50% methanoV50%NANOpureOwater) and analyzed by liquid chromatography mass spectrometry(LCMS). Recoveries of PFBS in freshwater yielded an overall mean percent recovery of 106%(SD=5.72%; CV =5.41%; N = 12). Matrix blank samples, consisting of unfortified freshwater, were devoidof intedehg componentswhen a base-acid wash on preparatory apparatus was incorporated into the methodology. WILDLIFE INTERNATIONALL, TD. - 13 - PROJECT NO.: 454C-115 REFERENCES 1. American Society for Testing and Materials. 1991. Standard Specification for Reagent Water. D1193-91, ASTM Section I1 Water and Environmental Technology, Vol. 11.01:45-47. WILDLIFE INTERNATIONALL, TD. -14- PROJECT NO.: 454C-115 Table 1 Typical LC/MS Operational Parameters INSTRUMENT: Hewlett-Packard Model 1100 High Performance Liquid Chromatograph with a Perkin-Elmer API 3000LC Mass Spectrometer equipped with a Perlun-Elmer TurboIonSpray ion source. Operated in selective ion monitoring mode (SIM). ANALYTICAL COLUMN: Keystone Prism Column (30 mm x 1.5 mm, 3 - p particle size) OVEN TEMPERATURE: 40C STOP TIME: 3.00 minutes FLOW RATE: 0.150 mL/minute MOBILE PHASE: 75.0% Methanol : 25.0% NANOpure' Water containing 0.1% Ammonium Formate INJECTION VOLUME: 2.5 pL PFBS RETENTION TIME: Approximately 1.5 minutes PFBS MONITORED h4ASS: 299.0 m u W I L D L I F EI N T E R N A T I O N A L , LTD. - 15- PROJECT NO.: 454C-115 Table 2 Matrix Blanks Analyzed Concurrently During Sample Analysis Number (454C-1 IS-) VMAB-4 Sample Type Matrix Blank Measured PFBS Concentration' (mg a.i./L) < LOQ VMAB-5 Matrix Blank -Z LOQ VMAB -6 Matrix Blank < LOQ 'The limit of quantitation (LOQ) was 0.100 mg a.i./L based upon the product of the lowest calibration standard (0.00100 mg a.i./L) and the dilution factor of the matrix blank samples (100). WILDLIFE INTERNATIONAL,LTD. PROJECT NO.: 4542115 Table 3 Method Validation Recoveriesfor P-FBS In Freshwater Sample Number (454C-115-) Type VMAS-1 Matrix Fortification VMAS-2 Matrix Fortification VMAS-3 Matrix Fortification PFBS Concentration(mg a.i./L) Fortified 0.500 Mesued 0.596 0.500 0.542 0.500 0.537 Percent Recovery 119 108 107 Mean Measured (mg a.i./L) 0.558 Mean Percent Recovery 112 VMAS-4 Matrix Fortification 10.0 VMAS-5 Matrix Fortification 10.0 VMAS-6 Matrix Fortification 10.0 9.89 98.9 10.5 105 10.5 105 11.2 112 VMAS-7 Matrix Fortification 100 VMAS-8 Matrix Fortification 100 VMAS-9 Matrix Fortification 100 106 106 103 103 102 102 102 102 VMAS-10 Matrix Fortification 1000 981 98.1 1030 103 .. VMAS-11 Matrix Fortification 1000 1039 104 VMAS-12 Matrix Fortifcation 1000 1071 107 O v d M e a n = 106 Standard Deviation= 5.72 C V = 5.41 N= 12 w1LDLl FE INTERNATIONAL, LTD. - 17- PROJECT NO.:454C-115 METHOD OUTLINE FOR THE ANALYSIS OF PFBS IN FRESHWATER Base-acid wash preparatory apparatus. Prepare matrix fortification samples by spiking the requisite volume of PFBS stock solutions directly into freshwater. Perform fortifications with gas-tight syringes and Class A volumetric flasks. J Dilute samples into the calibration range of the PFBS LCMS methodology: Partially fill Class A volumetric flasks with dilution solvent (50% methanol: 50% NANOpure' water). Add appropriate volume of sample and bring to volume with dilution solvent. Process matrix blank samples using the same dilution and aliquot volumes as for the lowest fortification level. Mix well by several repeat inversions. Ampulate samples and submit for LCMS anaIysis. Figure 1. Analyhcal m e t h d flow chart for the analysis of PFBS in freshwater. WILDLIFE INTERNATIONAL,TD. -18- Area 1 PROJECT NO.:454C-115 Conc. mg a.iA Figure2. A typical calibration curve for PFBS. Slope = 214262096.0000; Intercept = 5379.9839; r = 0.9985. W I L D L I F E INTERNATIONAL, LTD. - 19- PROJECT NO.: 454C-115 intensity: 300000 cps 1 0- 6 34 51 63 131 155 171 Figure 3. A representative ion chromatogram of a low-level (0.00100 mg a.i./L) standard. WILD LIFE I NTERNATIoNAL, LTD. - 20 - PROJECT NO.:454C-115 1009 080 7 0. 60 50. 4 0, 30 20 10 intensity: 300000 cps 89 0.52 1.02 1.52 2.03 2.53 Time Figure 4. A representative ion chromatogram of a high-level (0.0100 mg a.i./L) standard. 1 WILD LIFE INTERNATIONAL, LTD. - 21 - PROJECT NO.:454C-115 intensity: 300000 cps 100 90 80 70 60 50 40 30 18 34 49 8593 111 124 152 171 scan O ' l I 1 1 1 31 6 1 1 1 1 1 9I1 ' ' 1121 1 1 I I 151 0.52 1.02 1.52 2.03 2.53 Time _- Figure 5. A representative ion chromatogram of a freshwater ma& blank sample (454C-115-VMAB-4). The arrow indicates the retention time of PFBS. WI LDLlFE 1 NTERNATIONAL, LTD. -22- PROJECT NO.:454C-115 intensity: 300000 cps 100. 9 0. 80 7 0, 6 0, 09 50 40 30 20 10 scan 0 31 61 91 121 151 .- 0.52 1.02 1.52 2.03 2.53 Time Figure 6 . A representative ion chromatogram of a low-level freshwater matrix fortification sample (454C-115-VIbIAS-2, nominal concentration of 0.500 mg a.i./L). Wi LD LIFE INTERNATI ONAL, LTD. - 23 - 100- 9 0. 8 0- 7 0- PROJECT NO.: 454C-115 intensity: 300000 cps 0.52 1.02 1.52 2.03 2.53 Time Figure 7. A representative ion chromatogram of a high-level freshwater matrix fortification sample (454C-115-VMAS-12, nominal concentration of 1000 mg a.i./L). WI LDLlFE INTERNATIONAL, LTD. -24 - APPENDIX I Protocol, Amendment and Deviation PROJECT NO.:454C-115 WILD LIFE INTER NATI ONAL, LTD. -25 - PROJECT NO.:454C-115 PROTOCOL ANALYTICAL METHOD VALIDATION FOR THE DETERMINATION OF PEWLUORO BUTANE SULFONATE,POTASSIUM SALT (PFBS) IN FRESHWATER, SALTWATERAND ALGAL MEDIA 3M EnvironmentalLab Project No.EW-1429 Submitted to 3M Corporation El- wry 935 Bush A ~ r m c Si Paul, Minncsata 55 106 Wild1v e Intemationul, Ltd. 8598 Commerce Drive EastOs Maryland 21601 (4 10) 822-8600 March 29,2000 WILDLIFE INTERNATIONALT, D. - 26 - PROJECT NO.:454C-115 ' WildliJe International, Ltd. -2- ANALYI'ICAL MFIHOD VALIDATION FOR THE DETERMINATION OF PERFLUORO BUTANE SULFONATE. POTASSIUM SALT (PFBS) IN FRESHWATER, SALTWATER AND ALGAL MEDIA SPONSOR: 3M Corporati00 Enviromcntal Laboratory 935 Bush Avcnue St Paul, Minnesota 55106 SPONSOR'S REPRESENTATIVE: TESTING F A C I L W Wddlifk Intcmatiooal, Ltd. 8598 Commerce Drive Easton, MKytand 21601 STUDY DIRECTOR Raymond L.VanHoven, PhD. SCicotist LABORATORY MANAGEMENT: W M B. N i x q PhD. Manager of ADalytical Chanimy FOR LABORATORY USE ONLY Proposed Dates: %* Start Datc: a a - Projcct No.: %4@ f 5 1Test Substance No.: 5916 TcrminationDatc: TatGx~eons: Rcfcrcllu Substance No.: p4h 65- E - 0 0 . .164.I(Xldlrn lMs L l J L PROTOCOL APPROVAL d U * L WBORATORY MANAGEMENT I-17-on DATE PROTOCOL NO. 454/032900/MvAUSUB454 3M PROJECTNO. E00-1429 WILDLIFE INTERNATIONAL,TD. - 27 - PROJECT NO.:454C-115 wildlife International, Ltd. -3- INTRODUCTION Wildlife Intcmational, Ltd. will conduct analytical trials to validate the performance of a liquid chromatography mass spectrometry (LCMS)method for the determination of Perfluoro Butane S u l f o ~ t eP, otassium Salt (PFl3S) in freshwater, filtered saltwater, and algal media. The study will be performed at the Wildlife International, M. analytical cbdnistry facility in Easton, Maryland. The mcthod will be verified by fo- cach watcr matrix with thc test substance and determining rccovcries of PFBS. Raw data for all work performed at Wildlifie International, Ltd. and a copy of the final report will be filed by project numbcr in archives located on the Wildlife International, Ltd.site, or at an altematin location to be specified in the finalreport. OBJECrrVE The objective of this study is to verify the pcrfonnancc of LCMS methodology for analyses of PFBS in Ecshwater, filtered salt water, and algal media. Thc method to bc validated will be used in support ofaquatic toxicology and product chemistry teststo be conducted withthe tcst substance. WERIMENTAL. DESIGN Wildlife I n t e r d o 4 Ltd. fi-cshwater,filtered saltwater, and algal media will cach be fortified at four clxcrmt-oc '011s and analyzed usingLCMS mtbodology based upon that dcvelopcd at Wddlife International, Ltd. Modifications to tk mctkdology will be made as ncccssary to achimguantitation of PFJ3S in cach ofthe testmatrios Matrix andfor magent blanks will be analyzd coacxlrrcntlyto cvaluatcpdmtd . aaalyticalkrhhuxa r n l m l s o f fortificationwill bracku tiic anticipated CoactntratiOaS of samples firan aquatic toxicity tests. One or more calibration awes will bc g c n d firom analysts of standard solutionsof PFBSto MATERIALS AND METHODS Test Substance I n f o d o n on the characterizatoa oftest, control and refercna substances is required by Gccd Laboratory P d c c Standards (GLP). 40 CFR Parts 160.31 and 792. Tbc Sponsor is responsible for providingWildlife International, Ltd.written validation thattbc tstsubstancehas ban characterized accordingto GLPs prior to its use in the study. If written validationof GLP PROTOCOL NO. 454/0329OO/MVAuSUB454 3M PROJECTNO.E00-1429 :: -. . . WILDLIFE INTERNATIONAL, LTD. - 28 - PROJECTNO.:454C-115 Ml'ildlije International, Ltd. -4- tcst substance charaderization is not provided to Wildlife International, Ltd., it will be noted in the compliance statement of the final report. The attachedform IDENTIFICATION OF TEST SUBSTANCEBY SPONSOR ( A p p d k r) is to be used to provide 'hfotmation nwssary for GLP wmpliance. The Sponsor is rrspoasible for all i n f o d o n relatcd to test and refcrcncc substances and agrces to acccpt any unused test or rcfercnce substance andor tcst or reference substance containersnmaining at the a d of the study. Reagents and Solvents All solvcnts used in the method or procedure will be HPLC gmde or equivalent. AU reagents will be ACS reagent grade or higher quality. Nanopun water (equivalent to ASTM Type 11 Designation D1193-91) will be used (1). The solvcnts and reagents arc not cxpectod to contain conoLminants capable of intafcring with the purpose of this study. Algal Culture Medium Culture medium to be used for the method validation will be prepad according to Wildlife Internationat, Ltd Standard Operating Proc4durcs. The concentdons of the componentsin thc d u m an p& in Table 1(2). Stocknutrid solutions will be pnpared _.. by adding rcagcnt-gradcor better chemicals to Wddlifc Intcmational,Ltd wll watcr purified by - .si- Appropriate volumez of thc stock rmtricnt saltions will thcn be diluted with purified W d watcrta pnpare thc mcdiua Thc medium will bc fztersttriliLed (0.2 I.m) or autoclaved pnor to use. Thc pH of thc medium W d be 7.5 f 0.1. Frcshwatcrtobe uscd forthcmdhodvaIidationwill be obtainedfi-om awcllapproxbatcly40- meters dcep l d on thc Wddlifc Intcmational,Ltd site. The water will be passed through a sand filter and pumped into a 37,800-L storage tank whcrc the water will be aerated with splay n o d e s . Prior to use, the water will be iiltercd to 0.45 pin order to m o v e h e particles. The resultingwater is charaderiz+d u moderatdy hard. A p p r o h t c values for hardness. -, pH and specific ' wndudtancc are: ...... ., - 'fl.:c- . - .- PROTOCOL NO. 454/0329oo/MvAvSLfJ3454 3M PROJECT NO. E00-1429 WI LDLlFE INTERNATIONAL, LTD. - 29 - PROJECT NO.:454C-115 wildlife International, Ltd. -5- Hardness, m a as CaCOl 145 ALkalinity, m g L as CaCO, 190 PH 8.1 Specific Conductance, pmhodcm 330 Hardness, alkalinity, pH and specific conductance will bc measured weekly to monitor the consistencyof the well water. Mcans and ranges of the mcasund paramdm for the four-week period pnceding the tcstwill be provided in thc final rcpoct. Saltwater Natural seawater colleatd at Indian River Inlet,Delaware will bc filtered through a sand fdter prior to delivery to a 37,800-L holding tank. The salinity of the seawater will bc diluted to approximately 20 "/- (parts per thousand)with Wildlife Intcmafional, Ltd. well water in the holding tank to produce the saltwater matrix The 20 "1, salhvater will be aerated using spray n o d e s and fdknd (0.45 p)prior to its use in the method validation. Salinity and pH will be measured mckly to monitor thc consismcy of the saltwater. Means and ranges of the mcasured ~CGUUC~fCorKthc four-& pcricd pnctding thc testwill be provided in - thCfinalrcport. AnalyticalValidation Procedures Wildlife Intmdonal, ud. will conduct validaton trials using p d u r c s basai on LUMS mahodology dmlopcd at Wildlife hkmtional, Ltd. The &may bc modified to yield a mdhod capable of quaruitating PFBS at the d c s i i concentrations in freshwater, filtered salt water, and algal mcdia. The method used, includq any modifications, will bc donrmcnted in thc raw data and summariztd in the final report. PROTOCOL NO. 454/032900/MvAuSuB454 3M PROJECTNO. E00-1429 W I L D L I F E INTERNATIONAL, LTD. - 30 - PROJECT NO.: 454C-115 wildlife International, Ltd. Reference Stock SoIution(s), Calibration Standards and Curves A primary stock soiution(s) will be prcpand from PFBS reference substance, if available, or direaly from the tcst substance in an appropriate solvent. Calibration standards will bc prepared by appropriate dilution of this primary stcck solution(s). A minimum of fivc concentrations ofcalibration standards will be p n p d and analyzed along with cach aoalysis sd of validation samples. n c calibration standard scria will be injcctcd at thc beginning and end of the analytical run with, in addti04 a minimum of (XK; mid-level staudard injodcd following every five samples. One or more calibration cwes will bc dcrived fiom rcgrcssion analysis of the instrumental responses of the standards. Fortification Stock Solution(s) Frcshwatcr, saltwater, and algal mcdia will each be fortified with a stock solution(s) of PFBS. Each stock solution will be assigned a uniquc identiticationwdc which will be ncorded on a stock preparation log sh&. Evaluation of Interferences Matrix and rcagcnt blanks, if applicable, wiU be analyzd to assess the presence of potential intc.rfercnccs. Matrix blanks will consist of fnshwata. Sattwatcr, and algal media without addition of test substance. Rcagtnt blanks, if applicable, will consist of rcagcnts promscdthroughthCmdhod .- ~. .. . ~- -... PROTOCOL NO. 454/032900/MvAuSUB454 3M PROJECT NO.Em-1429 wl LDLIFE I NTERNATIONAL, LTD. -31 - PROJECT NO.: 454C-115 wildlife International, Ltd. SUMMARY OF PROPOSED VALIDATION ANALYSIS SCHEME I Analysis Type ti^^ 11 Number ofSamples Algal Freshwater Saltwater Mediiim Reagent Blank (iapplicable) Matrix Blank Matrix Fortifications ` Matrix fortiiidons will be prepared at four concentrations, the lowcst and highcst of Matrix fotiScations will span the range of ulncentrations that arc anticipated to bc uscd in subsequent environmental &eds tests. Individual fortification samples (dcntifitd by project number and a unique sample identificationnumber) will be prepared and anal+. If dificulties arise in the validation proccss (c.g., low recoveries or intafmcnces), the Sponsor will be notified and thc need for additional validation and/or method dcvclopmcnt will be dctcmincd through discussions with the Sponsor. Upon completion of thc m e h d validation, the Sponsor will review thc rrsults and authorize the llse of the mctfrodology for aaaly5s of samplcs, or will authorize furthcr mcthod h l o p m c n t trials. Rccovuy values in thc range of 80 to 120%willb e d as c r h h for mahodacfcptabaQ'. Data Analyst Onc or more calibration ~ u y e sfor each a d y t i d run will bc dcrivcd fkom the instnunental rcsponscs of PFBS standards of known oollctntration using regresim analysis. The concentrafions of PFBS in fortified samples will be ddcrmincd by substituting the nspativc instrumad response into the regrtssion equation If the fomficaticm range and calibration curve standard Concentrations arc significantly higher &an mahod capabilitiesfor q u a n t i ~ o qthe h i t of quantitatkm WQ)will be d e w -* ..... PROTOCOLNO. 454/032900lMV~SUB454 3M PROJECTNO. E00-1429 h'lLDLI FE I NTER N AT10N A L , LTD. -32 - PROJECT NO.:454C-115 WiId1ife InternationaI, Ltd. -8- as the concentration equivalent to the lowest standard accounting for dilutions and other manipulations in the method for matrix blanks. If the fortification range is near the method capability for accurate quantitation of the analytc, the LOQ will be determined from the responses of rcplicate control samples. The mcan (Sb) and staadard deviation (0)of responses will be determined for the replicate control sample and converted to ppm equivalents using a standard cum and method dilution fadon. Thc LOQ will be cxprsscd as the concentration equivalent of Sb + 1 0 ~ .The limit of dctecb'on (LOD),defined as the lowest coaccatratioa of analytc which can be injected and produce a rncasurable response above background, will be expressed as the amount tquidcnt to Sb +30. RECORDS TO BE MAINTAWED Rccords, to be maintained for data gcncrated by Wildlife International, Ltd., will include: 1. A copy of the sig;led protocol. 2. Identification and characterization of tat and reference substances, if provided by the Sponsor. 3. Dates of hitiationand tmnkation ofthe test. 4. Storage wnditiom for test and rcfauxx substances, and samples. 5. Test and nfcrcncc substance use logs. 6. Conccntratioacalahfions and rtcords of solution preparation 7. ~ a l t o p e r a t i n g m o d i t i o n s a o d ~ . 8. statisticalcalallafiions. 9. Awpyofthefinalrcport- FINAL REPORT . ?hc report will summarize ik findings of ik validation, the fortification rCCOVCriCS obtain4 and mcthod(s) and . . 5m cmployba Upon raccipt of thcse findingt, the Sponsor will rcvicwthe&and results, and cvaluakthe resultsfor acceptability. ?he 6nal rcport will include, but mt be limited to.the following: 1. Name and address of tfic ficility paforming the study. 2. D a t u on which the study was initiataiand cornplcrcd. PROTOCOL NO. 454/0329#/MVAysUB454 3M PROJECT NO.E00-1429 ~- . .- W I L D L I F E INTERNATIONAL, LTD. - 33 - PROJECT NO.:454C-115 wildlife International, Ltd. -9- 3 A statement of compliance signed by the Study Director addressing any exceptions to Good Laboratory Pmctice Standards 4. Objectives and procedures stated in the approved protocol, includq any changes in the original protocol or deviations from the protocol. 5 . Identification of test and reference substances, including name, chemical abstract number or d e number, sku@, purity, composition, date of receipt, lot number, storage conditions, physical charactcristicS, stability and mahod of p r c p d o n of tat concentrations,if provided by the Sponsor. 6. A brief summary of the analytical mcthcdology to include, where applicable, a description of the apaimmtal mcasuruncnts. example calcutations, sample preparation (sample weights and/or dilutions), instrummtationcmploycd, rcagmts used,purity of nag~ntsand solvents, and any major modifications to the method. 7. A description of any circumstances that may have affected the quality or integrity of the data. 8. The name of the Study Dircaor, the names of other scientists or professionals, and the names of all supervisory personnel involved in the study. 9. A descriptionof the transformations,calculations,or operations performed on the data. 10. The signed and daied reports of each of the individual scientists or 0 t h professionals involved in the study, if applicable. 11. Thc l o c a t i o n ~ amc w data and finalrcpoaarc to bc stored. 12. A datrmrrrt pnparod by the Qualay Assurance Unit listing the dates that study inspdons and audits wcrc made and the dates of any faings nportcd to the study Dim%xadhhxigemazt. CHANGING OF PROTOCOL .II . P W changa to the protocol will be in the form of writta~am!mdmatssigned by the Study Director and thc Sponsor's Rcprcscntah. hahunts will be considered as part of the protocol a d will be attached to thefinal protocol. Any other change, will bc in the form of writtcn deviations signed by the Study Dircctor and filed with the raw data. All changes tothe protocol will bc indicated in the final report. PROTOCOL NO. 454/032900/MvAuSUB454 3M PROJECT NO. E00-1429 WILDLIFE INTERNATIONAL,LTD. - 34 - PROJECT NO.:454C-115 WildlifeInternational, Ltd. - 10- GOOD LABORATORY PRACTICES This study will be conducted in accordance with Good Laboratory Practice Standards for EPA (40 CFR Part 160 andlor 792) and OECD Principles of Good Laboratory Practices (ENVMUCHEM (98) 17). Each study conducted by Wddlifc Inte.mational, Ltd. is routinely examined by thc Wddlift htcmational, Ltb QualivAssurance Unit for compliancewith G o d Laboratory Practices, Standard Operating Procedures and thc specified protocol. A statanent of compliance with Good Laboratory Practices will be prepared for dl portions of the study conducted by Wildlife Intcrnatiooal, Ltd The Sponsor will be rsponsiblc for compliance with Good LabolatoryPraCticcs for proc#lurs performed by othcr laboratories. PROTOCOLNO 454/03290OlMVAIJSUB454 3M PROECT NO. E00-1429 WILDLIFE INTERNATIONAL, LTD. - 35 - PROJECT NO.: 454C-115 wildlife International, Ltd. -11REFERENCES 1 American Society for Testing and Materials. 1991. Standard Specification for Reagent W a r . D1193-91,ASTMSecrionIIWatcrandEnvironmcntalTcchnology,Vol. 11.01 2 American Society for Testing and Materials. 1990,Standard Guide for conducting Static 96-Hour Toxicity Tcsts with Mcraalgac. E 1218-90. Asru Section II Water and Environma~taTl cchnology, Vol. 11.OS. 3 American Society for Tating and Materials. 1991. Standard Guide for Conducting Static Toxicity Tcsts with L . e m gibba G-3. E 1415-91. MTh4 Section II Water and Environmental Tcchnology, Vol. 1 1.05. PROTOCOL NO. 454/032900fMVAL6uB454 3 M PROJECTNO.EM-1429 WILDLIFE INTERNATIONALL, TD. - 36 - PROJECT NO.:454C-115 wi1dlife International, Ltd. - 12- TABLE 1 FRESHWATER ALGAL M E D m CONSTITUENTS Component Nominal Concentration 12.16 xr@ 4.40 XI@ 0.1856 mg/L 0.416 mg/L 3.28 pg/L 0.1598 m g 5 1.428 pglL 7.26 pglL 0.012 pg/L 0.300 mglL 25.50 XI@ 14.70 XI@ 1.044 mgn 15.00 mg/L ~ _ _ ~ The pH ofthe medium will bc adjusted, as necessary. to 7.5 f 0.1 using 0.1 N NaOH or 10% HCI. PROTOCOL NO. 454/032900/MVAEXJB454 3M PROJECT NO. E00-1429 * WILDLIFE INTERNATIONAL, LTD. -37- PROJECT NO.:454C-115 wi1dlife InternutionuI, Ltd. - 13 - APPENDIX I IDWrmCATlON OF TEST SUBSTANCEBY SPONSOR To bc Completed by Spcnsor I. Test Substance Identity (name to be used in the report) Perfluoro Butane Sulfonate. Potassium Salt PFBS) Tcst Sub- Sample Code or Batch Number: Tcst SubstancePunty rA Active Ingredient): ILTatSukatxz@. ' . '. 99.82 Expiration Date: hbrch 2010 .- .... .. -- PROTOCOL NO. 454/032900/MvAuSUB454 3M PROJECT NO.E00-1429 W I L D L I F EINTERNATIONALL,TD. - 38 - PROJECT NO.: 454C-115 WILDLIFE hTERNATIONAL LTD. PROJECT NO.: 454'2-1 15 Page 1 of 1 AMENDMENTTO STUDYPROTOCOL STUDY TITLE: Analytical Method Validation for the Determination of Perfluoro Butane Sulfonate, Potassium Salt (PFBS) in Freshwater, Saltwatez and Algal Media PROTOCOL NO: 454/032900/MVAUSUB454 AMENDMENTNO.: 1 SPONSOR: 3M Corporation PROJECr NO.: 454C-115 EFFECTIVEDATE: April 17.2000 3M LAB FWJUEST NO.: E00-1429 AMENDMENT: Page I, Studv Title change: Analytical Method Validation forthe Dctamu' &'on o fPafluoro Butane S U l f o ~ tP~o,tassiumSalt (PFBS)in Freshwater, Saltwata and Algal Mcdia To: AnalyticalMcthcd Validation for the Detaminationof Pafluoro Butane Sulfonate,Potassium Salt (PFBS) in Freshwater REASON: At the Sponsor's request,the protocol was changedto encompass validation in fishwater only. AMENDMENT: P a m 1-13 Del&: All rcferencs to Saltwater and Algal Media REASON: At the Sponsor's quest, the protcal was changedto cncumpassvalidation mfnshwata only. - AMENDMENT Page 13, Aarw& I Changc: Test Substanceplrrity (?AActive Ingrediaxt): 99.82 To: Tcst Substance plaity (?AA& Ingrtdicnt): REASON Tbc CutScatc of- fortheTest Substsnce was rcvisd -c DATE .- SPONSOR'S REPRESENTATIVE W I L D L I F E INTERNATIONAL, LTD. -39- PROJECT NO.:454C-115 WILDLIFE INTERNATIONALTLD, WLI Project NO.:454C-1 I5 Page 1 of 1 DEVIATION TO STUDY PROTOCOL STUDY TITLE: ANALYTICAL MEIHOD VALIDATION FOR THE DEIERMINATION OF PEFSLUORO BUTANE SULFONATE, POTASSIUM SALT (PFBS)IN FRESHWATER PROTOCOL NO.: 454/032900/MvAuSUB454 DEVIATION NO.: 1 SPONSOR 3M Corporation PROJECT NO.: 454C-115 DATE OF DEFACTO DEVIATION: April 18,2000 DEVIATION: Thc protocol stats thatthe calibration standard series will be injcatd at the beginning and end ofthc analyticalrun with, in addition, a minimumof one mid-lcnl standard injected following C V C , ~five samples. Aftcr thc first five samples, a low-level standard was injected. After the next five samples a high-lcnl standard was injected. REASON: Typically the standard level injccted after five samples depends on the total number of samples rcquircdin betweensampla. For example, ifonly onc standardis requind,the mid-level standard is i n j d , iftwo standards arc required, thc low and high-lcvcl standardsarc injcacd, ifthree standardsarc rcquircd, the low, mid and high-level standards arc injectcq ctc. IMPACT:This deviation bad no impact OLI the study since all sample results were bracketedby time and~nccnpationThc standards injoctedinbctwtensampampls~ ~ e t h c p u posfe demonstrating instnunCnt stabilityin thc p r r x n c ~ofthc matrix .L s; W Q & E C T O R Ibymond L. Van Hovm, PCD. Willard B.N~on.P,h.D. 5-d-00 DATE WILDLI FE INTERNATIONAL, LTD. - 40 - PROJECT NO.:454C-115 APPENDIX LI Certificate of Analysis C ~ Fo~~S-K+ . . . ;PFBS) L-7038, Lot 2 (1999) April 17,2000 (Supercedes o r t i f i c a t c of Analysis dated April 11,2000) Cd9SQiX+ 0.13 Yo 97.90% 1.96 % Additionally, the isomer distriiution of the samplewas determined usiig "F-NMR techniques and found to contain the following weight percent composition: CF,( CF2h-SQ-K' 97.86 % . .. Analyrical Study Numbcr: M488 WILDLIFE INTERNATIONAL, LTD. -41 - PROJECT NO.: 454C-115 APPENDIX TI1 Specific Conductance, Hardness, Alkalinity and pH of Freshwater Measured During the 4-Week Period Immediately Preceding the Test Specific Conductance (pmhos/cm) Hardness ( m aas CaC03) Alkalinity ( m a as CaC03) pH 315 ( N = 4 ) 131 ( N = 4 ) 172 (N = 4) 8.2 (N= 4) 128 - 132 170 - 174 8.1 - 8.3 W I L D L I F EI N T E R N A T I O N A L , LTD. - 42 - PROJECT NO.:454C-115 - APPENDIX N Analyses of Pesticides, Organics, Metals and Other Inorganics in Wildlife International,Ltd. Freshwater' ANALYSIS MEASURED CONCENTRATION Miscellaneous Measurements Total Dissolved Solids Ammonia Nitrogen ~ o t aOl rganic Carbon2 Total Cyanide 286 C 0.050 < 1.0 < 10.0 0 1 inochlorines and PCBs Aldrin Alpha BHC Beta BHC Delta BHC Gamma BHC (Lindane) Chlordane DDD, PP,' DDE, PP DDT, pp' Die1dnn Endosulfan, A Endosulfan, B Endosulfan Sulfate Endrin Endrin Aldehyde Heptachlor Methoxychh Heptachlor Epoxide Toxaphene PCB-10 16 PCB-122 1 PCB-1232 PCB-1242 PCB-1248 PCB-1254 PCB-1260 < 0.005 < 0.005 < 0.005 < 0.005 < 0.006 < 0.025 < 0.006 < 0.005 < 0.008 < 0.005 < 0.005 < 0.005 < 0.018 < 0.010 < 0.005 < 0.005 < 0.007 < 0.005 < 0.500 < 0.260 < 0.260 < 0.260 < 0.720 < 0.720 < 0.720 < 0.720 Metals and Other qorganics Alurninym Arsenic Be Ilium3 caYmiuy3 Calcium gm~um~ COP$ Iron Lead3 Magnesiut~f Manganese EGnum3 Nickel. ~0tasslur4~ Selen um SilveS s*iym3 Zinc ~ < 100 < 25.0 < 0.50 < 1.o 35.0 < 2.0 < 1 .o < 20.0 < 100 < 10.0 13.5 < 1 .o < 0.20 < 2.0 < 2.0 6.62 < 25.0 < 1 .o 21.3 < . 20.0 .- ' Analyses performed by QST Environmental, Gainesville, Florida for samples collccted on November 3 through November 7, 1997. Analyses performed by Wildlife International, Ltd. for the sample collected on November 5 , 1997. Analyses performed by Wildlife International, Ltd. for samples collected on November 5 through 7, 1997. WILDLIFE INTERNATIONAL, LTD. - 43 - - PROJECT NO.: 454C-115 APPENDIX V Personnel Involved in the Study Th Followin k I Wildlife International, Ltd. personnel were involved in the conduct [r gement f this study: 1. Willard B. Nixon, Ph.D., Manager, Analytical Chemistry 2. Raymond Van Hoven, PbD., Scientist 3. Jon MacGregor, B.S., Scientist 4. Keith Keller, B.S.,Chemist