Document dDOBJMoajwvRqDOYEmYVq8OmB

37) Analytical method validation for determination of PFBS-K in fish tissues, 454C-116 SANlTIZED DEC 0 9 2003 ANALYTICAL METHOD VALIDATION FOR THE DETERMINATION OF PERFLUOROBUTANE SULFONATE, POTASSIUM SALT (PFBS) IN FISH TISSUES WILDLIFE INTERNATIONAL,LTD. PROJECT NUMBER: 454C- 116 3M EnvironmentalLab Project No. E00-1429 AUTHORS: Raymond L. Van Hoven, Ph.D. Jon A. MacGregor, B.S. Willard B. Nixon, Ph.D. STUDY INITIATION DATE: June 12,2000 STUDY COMPLETION DATE: August 30,2000 Submitted to: 3M Corporation Environmental Laboratory 935 Bush Avenue St. Paul, Minnesota 55106 WildlifeInternational, Ltd. J 8598 Commerce Drive Easton, Maryland 21601 (410) 822-8600 Page 1 of 36 WILDLI FE INTERNATIONALLT, D. c1 -L- DEC 0 9 2003 PROJECT NO.: 454C-116 .SANITIZED GOOD LABORATORY PRACTICE COMPLIANCE STATEMENT SPONSOR: 3M Corporation TITLE: Analytical Method Validation for the Determination of Perfluorobutane Sulfonate,Potassium Salt (PFBS) in Fish Tissues WILDLIFE INTERNATIONAL, LTD. PROJECT NUMBER: 454C-116 3M W E O N M E N T A L LAB PROJECT NUMBER: E00-1429 STUDY COMPLETION: August 30,2000 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 OECD Principles of Good Laboratory Practice ( W / M C / C H E M (98) 17) with the following exceptions: 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: Scientist WildIife International, Ltd. 6J3b DATE SPONSOR APPROVAL: DATE WILD LIFE INTERNATIoNAL , LTD. -3- PROJECT NO.: 454C-116 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 (ENVMCKHEM (98) 17). The dates of all inspections and audits and the dates that any furdmgs were reported to the Study Director and Laboratory Management were as follows: ACTIVITY: Matrix Fortification Preparation Data and Draft Report Final Report DATE CONDUCTED: June 12,2000 June 29,2000 August 30,2000 DATE REPORTED TO: STUDY DIRECTOR MANAGEMENT: June 12,2000 June 14,2000 June 30,2000 June 30,2000 August 30,2000 August 30,2000 MMan Susan L. Coleman, B.A. . Senior Quality Assurance Representative 8-30-00 DATE WILDLI FE INTER NATIoNAL, LTD. -4- PROJECT NO.: 454C-116 - REPORT APPROVAL SPONSOR: 3M Corporation TITLE: Analytical Method Validation for the Determination of Perfluorobutane Sulfonate,Potassium Salt (PFBS) in Fish Tissues WILDLIFE INTERNATIONAL, LTD. PROJECT NUMBER: 454C-116 3M ENVIRONMENTALLAB PROJECT " M B E R : E00-1429 STUDY DIRECTOR v R a y m b R d L ~ a Hn oven, Ph.D. Scientist s/so/63 DATE Willard B. Nkon, P b D . / Manager, Analytical Chemistry W I L D L I F E INTERNATIONAL. LTD. PROJECT .NO. 454C-116 TABLE OF CONTENTS TitleXover Page................................................................................................................................................. 1 Good Laboratory Practice ComplianceStatement............................................................................................. 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....................................................................................................... 10 . - Stock Solutions, Calibration Standards and Curves .......................................................................... 10 Fortification Stock Solutions.............................................................................................................. 10 L.m.t of Quantitation.......................................................................................................................... 11 Example Calculations......................................................................................................................... 11 Results ............................................................................................................................................................ 12 Reagent and Matrix Blank Samples................................................................................................... 12 Method Validation Samples............................................................................................................... 12 Conclusions ...................................................................................................................................................... 12 References ........................................................................................................................................................ 13 ~~ ~~ ~ ~ WILDLIFE INTERNATIONAL. LTD. -6- PROJECT NO .. 454C-116 TABLE OF CONTENTS .Continued . TABLES Table 1 .Typical LC/MS Operational Parameters......................................................................................... 14 Table 2 .Reagent Blank'and Matrix Blanks Analyzed ConcurrentlyDuring Sample Analysis................... 15 Table 3 .Method Validation Recoveries for PFBS in Fish Tissue ................................................................ 16 FIGURES Figure 1 .Analytical method flow chart for the analysis of PFBS in fish tissue ............................................ 17 Figure 2 .A typical calibrationcume for PFBS .............................................................................................. 18 Figure 3 .A representative ion chromatogram of a low-level (0.500 pg a.i./L) standard.............................. 19 Figure 4 .A representative ion chromatogramof a high-level (5.00 pg a.i./L) standard............................... 20 Figure 5 .A representative ion chromatogramof a reagent blank sample (454C-116-VREB-N-2) .............21 Figure 6 .A representative ion chromatogramof a fish tissue matrix blank sample (454C-1 16-VMAB-N-4) ................................................................................................................... . 22 Figure 7 .A representative ion chromatogramof a low-level fish tissue matrix fortification sample (454C.116.VMAS.N.13. nominal concentrationof0.100 mg a.i./Kg)............................ 23 Figure 8 .A representative ion chromatogramof a high-level fish tissue matrix fortification sample (454C.116.VMAS.N.22. nominal concentrationof 100 mg a.i./Kg)............................... 24 APPENDICES Appendix I . Protocol and Amendment.................................................................................................... 25 Appendix II . Certificate of Analysis......................................................................................................... 35 Appendix I11 . Personnel Involved in the Study....................................... :.................................................. 36 WILDLIFE INTERNATIONAL,LTD. -7- PROJECT NO.: 454C-116 I STUDY TITLE: WILDLIFEINTERNATIONAL, LTD. PROJECT W E R : SPONSOR: I TESTING FACILITY: LOCATION OF STUDY, . RAW DATA AND A COPY OF THE FINALREPORT: SUMMARY ~ Analytical Method Validation for the Determination of Perfluorobutane Sulfonate,Potassium Salt (PFBS) in Fish Tissues 454C- 116 3M Corporation Wildlife International, Ltd. Easton, Maryland 2 1601 Wildlife International, Ltd. Easton, Maryland 2 1601 TEST SUBSTANCE: FORTIFIED TEST CONCENTRATIONS FOR METHOD VALIDATION: TEST DATES: PFBS Negative Control, 0.100, 1.00, 10.0 and 100 mg a.i./Kg Experimental Start - June 12,2000 ExperimentalTermination - June 14,2000 TEST SYSTEM: .. Nonedible bluegll sunfish (Lepomis macrochirus) tissue SUMMARY: Nonedible bluegill sunfish tissues were homogenized and fortified (in triplicate) with PFBS to reflect nominal concentrations of 0.100,1.00,10.0 and 100 mg a.i./Kg. Three matrix blanks (negative controls) and a reagent blank were also prepared The 1-gsample homogenates and reagent blank were extracted with methanol, diluted into the instrumental calibration range with dilution solvent (50% methanol: 50% NANOpure" water), and analyzedby liquid chromatography mass spectrometry (LCMS). Recoveries of PFBS in nonedible fish tissue yielded an overallmean percent recovery of 80.8%(SD= 8.24%; CV = 10.2%; N = 12). Matrix blank samples, consisting of unfortifiednonedible fish tissue, and the reagent blank were devoid of interfering components. WILDLIFE INTERNATI oNAL, LTD. -8- PROJECT NO.: 454C-116 - 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 performance of a method in fish tissue. Validation samples were prepared and analyzed on June 14,2000. Raw data generated by Wildlife International,Ltd. and a copy of the finalreport are filed under Project Number 454C-I 16 in archives located on the Wildlife International, Ltd. site. PURPOSE The purpose of this study was to validate the performance of a LCMS methodology for the analyses of PFBS in fish tissue. The validated method will be used in support of aquatic toxicology tests to be conducted with the test substance. EXPERIMENTAL DESIGN Nonedible bluegill sunfish (Lepomis macrochirus) tissue was fortified at four different concentrationsin - triplicate and analyzedbased on LCMS methodologydeveloped at Wildlife International, Ltd. The fortification levels bracketed the anticipated concentrations of samples from future aquatic toxicity tests. A reagent blank and matrix blank samples were analyzed concurrently to evaluate potential analytical method interferences. A calibration curve was generated from analyses of standard solutions of PFBS 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 Perfluoro Butane Sulfonate, Potassium Salt (PFBS) in Fish Tissues" (Appendx 0. PROJECT NO.: 454C-116 -9- DEC 0 9 2003 Test Substance SANITIZED The test substance was received from 3M Environmental Technology and Safety Services on March 27, 2000, assigned Wildlife International, Ltd. Identification number 52 16,and storedunder ambient conditions. The test substance, a whte powder, was identified as: Potassium Peduorobutane Sulfonate Lot 21, expiration date: March 20 10. The test substance was further identified with the 3M EnvironmentalLaboratory test control and reference number TCR # The test substance had a reported purity of 97.90% (Appendm II). Reacents and Solvents All solvents used in this study were HPLC grade or equivalent. All reagents were ACS reagent grade or equivalent. NANOpure@water (equivalent to ASTM Type I1 Designation D1193-91) was used (1). Test System Nonediblebluegill s d i s h (Lepomis macrochirus) tissues were obtained5om a breeding stockmaintained at Wildhfe International, Ltd. Approximately 80 individual fish were removed from a breeding tank and euthanized by severing the spinal cord above the opercular region. Dissection was accomplished by making an incision fromjust posterior to the base of the pectoral fin dorsally throughthe spinal column. Heads, fins and viscera were removed from the body and were considered to be nonedible tissue. The balance of the tissue was considered to be edible tissue. Prior method development work (Wildlife International, Ltd. Project No. 454C-114) with PFBS in bluegill sunfish indicated that the nonediblematrix was more likelyto presentpotential spectral interference problems for the LCMS analyses. Therefore, the nonedible fraction was chosen for the method validation study. The nonedible tissues were transferred to a 1-quart glass bottle and blended with an Ultra-Turrax homogenizer (Janke & Kunkle, Model T25) set at approximately 9500 rpm. An appropriate number of 1-gram aliquots of the homogenate were weighed into separate, 20-mL glass scintillation vials. Each vial was uniquely identified and labeled with a facility log identification number. The aliquots were stored frozen until preparation and analysis of validation samples. ~ ~~ WILD LI FE I NTERNATI oNAL, LTD. PROJECT NO.: 454C-116 Analvtical Validation Procedures Wildlife International, Ltd. conducted a validation trial using procedures based on methodologydeveloped at Wildlife International, Ltd. Matrix blanks and matrix fortification samples were prepared in homogenized, nonehble bluegill sunfish tissue. A reagent blank (containing everythmg except PFBS and fish tissue) was carried through the entire procedure. The samples were extracted with methanol. Dilutions were performedwith a solution of 50% methanol (HPLC grade, 99.9+%) and 50% NANOpure@water. Sampleswere then analyzedby direct injection. Concentrations of PFBS were determined by reverse-phase high performance liquid chromatography using a Hewlett-Packard Model 1100High PerformanceLiquid 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 Prism coIumn (50 mm x 2.0 mm, 5 - p n particle size) fitted with a Betasil CI8guard column (20 mm x 2.0 mm). The instrument parameters are summarized in Table 1 and a method flow chart is provided in Figure 1. Stock Solutions. Calibration Standards and Curves Calibration standards were prepared in 50% methanol: 50%NANOpure@water by appropriatedilutionsof a 1.00 pg a.i./mL stock solution of PFBS in methanol. Calibration standards of PFBS, ranging in concentration from 0.500 to 5.00 pg a.i./L, were analyzed with each sample set. Five calibration standards (different concentrations) were analyzed with the samples. The calibration standard serieswas injected at thebeginningand 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 Figure 2. The concentration of PFBS in the samples was determined by substituting the peak area responses into the applicable linear regressionequation. Representative ion chromatograms of low and high calibration standards are presented in Figures 3 and 4, respectively. Fortification Stock Solutions Fish tissue homogenates were fortified with the appropriate stock solution of PFBS prepared inmethanol. Each stock solution was assigned a unique identification code that was recorded'on a stock preparationlogsheet. WILD LIFE INTERNATIONAL, LTD. - 11 - PROJECT NO.: 454C-116 - Limit of Ouantitation The method limit of quantitation (LOQ) for the analyses was set at 0.0500 mg a.i.Kg based upon the product of the lowest calibration standard (0.000500 mg a.i./L) and the overall dilution factorof the matrixblank samples (100 LKg) .- Example Calculations Sample number 454C-116-VMAS-N-21, nominal concentration of 10.0 mg a.i./Kg in fish tissue. Sample Weight: 1:OO g Extraction Volume: 10.0 mL Initial Volume: 0.100 mL Final Volume: 50.0 mL Overall Dilution Factor: 5000 mL/g = 5000 L K g PFBS Peak Area: 1309436.13 Calibration curve equation. Slope: 758795.25 Intercept: -3605.85 _. Curve is weighted (Ux). - PFBS (mg a.i./Kg) in sample = Pe* areaslo'-pinetercept' x dilution factor x unit conversion factor - 1309436.13 - (-3605.851 -.k?L 758795.25 5000 1000 pg = 8.65 PFBS m a.i.K insam le- Percent of Nominal Concentration= PFB~(&g a.i.K$ x 100 ---81.06.50 x 100=86.5% Instrument Software: MacQuan, version 1.6. WILDLIFE INTERNATIONALL, TD. - 12- PROJECT NO.: 454C-116 RESULTS Reagent and Matrix Blank Samules A reagent blank and three matrix blank samples were analyzed to determine possible interferences. No interferences were observed at or above the LOQ (0.0500 mg a.i./Kg) during the sample analyses (Table 2). Representative ion chromatograms of a reagent blank sample and a nonelble fish tissue matrixblank sampleare presented in Figures 5 and 6, respectively. Method Validation Samules Nonedible bluegill sunfish tissue was fortified in triplicate at 0.100, 1.00,10.0and 100mg a.i./Kgresulting in mean recoveries of 75.7,72.1,84.1and 91.5%, respectively (Table 3). Representative ion chromatogramsof low and high-level fish tissue matrix fortifications are presented in Figures 7 and 8, respectively. CONCLUSIONS Nonedible bluegill sunfish tissues were fortified with PFBS to reflect nominal concentrations of 0.100, 1.00, 10.0 and 100 mg a.i./Kg. The samples were extracted with methanol and diluted into the instrumental calibration range with dilution solvent (50% methanoV50% NANOpure@water) and analyzed by liquid chromatography mass spectrometry (LCMS). Recoveries of PFBS in fish tissue yielded an overallmeanpercent recovery of 80.8% (SD = 8.24%; CV = 10.2%; N = 12). Matrix blank samples, consisting of unfortified nonedible bluegill sunfish tissue, were devoid of interfering components. WILDLIFE INTERNATIONALLT, D. - 13 - PROJECT NO.: 454C-116 REFERENCES 1. American Society for Testing and Materials. 1991. Standard Specification for Reagent Water. D1193-91, ASTM Section II Water and Environmental Technology, Vol. 11.01:45-47. WILDLIFE INTERNATIONAL,LTD. - 14- PROJECT NO.: 454C-116 Table 1 Typical L C N S Operational Parameters INSTRUMENT: Hewlett-Packard Model 1100 High Performance Liquid Chromatograph with a Perkin-Elmer API 3000LC Mass Spectrometer equipped with a Perkin-Elmer TurboIonSpray ion source. Operated in selective ion monitoring mode (SIM). ANALYTICAL COLUMN: GUARD COLUMN Keystone Prism Column (50 mm x 2 mm, 5 - p particle size) Keystone Javelin Betasil Guard Column (20 mm x 2 mm) OVEN TEMPERATURE: 40 "C STOP TIME: 5.00 minutes FLOW RATE: 0.200 mL/minute MOBILE PHASE: 90% Methanol : 10%NANOpure@Water containing 0.1% Ammonium Formate INJECTION VOLUME: 5.0 pL PFBS RETENTION TLME: Approximately 3.5 minutes PFBS MONITORED MASS 299.0 amu WI LDLI FE INTERNATI oNAL , LTD. - 15- PROJECT NO.: 454C-116 Table 2 Reagent Blank and Matrix Blanks Analyzed Concurrently During Sample Analysis Sample Number (454C-116-) Type - Measured PFBS Concentration' (mg a i / Kg) VREB-N-2 Reagent Blank c LOQ VMAB-N-4 VMAB-N-5 VMAB-N-6 Matrix Blank Matrix Blank Matrix Blank c LOQ .=LOQ -= LOQ 1The limit of quantitation (LOQ) was 0.0500 mg a.i./Kg based upon the product of the lowest calibration standard (0.000500 mg a.i./L) and the overall dilution factor of the matrix blank samples (100 LKg). ~ ~ ~~ WILDLI FE I NTERNATI ONAL, LTD. - 16- PROJECT NO.:454C-116 Table 3 Method Validation Recoveries for PFBS In Fish Tissue Number (454C-116-) Sample Type VMAS-N-13 Matrix Forhfication VMAS-N-14 Matrix Fortification VMAS-N-15 Matrix Fortification PFBS Concentration (mg a.i./Kg) Fortified Measured 0.100 0.0742 0.100 0.0806 0.100 0.0722 Percent Recovery 74.2 80.6 72.2 Mean Measured (mg a.i./Kg) 0.0757 Mean Percent Recovery 75.7 VMAS-N-16 Matrix Fortification 1.oo VMAS-N-17 Matrix Fortification. 1.oo VMAS-N-18 Matrix Fortification 1 .oo 0.712 0.724 0.726 71.2 0.721 72.1 72.4 72.6 VMAS-N- 19 Matrix Fortification 10.0 VMAS-N-20 Matrix Fortification 10.0 VMAS-N-2 1 Matrix Fortification 10.0 8.03 80.3 8.41 84.1 8.56 85.6 8.65 86.5 VMAS-N-22 Matrix Fortification 100 90.9 90.9 91.5 91.5 _. VMAS-N-23 Matrix Fortification 100 VMAS-N-24 Matrix Fortification 100 90.7 90.7 92.8 92.8 Overall Mean = Standard Deviation = cv= N= 80.8% 8.24% 10.2% 12 WILDLI FE INTERNATIONAL, LTD. - 17- PROJECT NO.: 454C-116 METHOD OUTLINE FOR THE ANALYSIS OF PFBS IN FISH TISSUE Prepare matrix fortification samples by spiking the requisite volume of appropriate PFBS stock solution drectly into homogenized control fish tissue. Perform fortifications with gas-tight syringes. The matrix blank samples consist of unfortified control tissue homogenates. The reagent blank is prepared starting with a clean, 20-mL glass.scintillation vial. J Add 10.0 mL of methanol to each test sample with a Class A volumetric pipette. Further homogenize each test sample for approximately one minute with a hand-held tissue shredder. Rinse the shredder with the appropriate solvent(s) in between samples. Cap the vials and shake well. Centrifuge the vials at 2000 rpm for approximately 5 minutes to complete the extraction 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 blanks and the reagent blank 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 analysis. Figure 1. Analytical method flow chart for the analysis of PFBS in fish tissue. WILDLIFE INTERNATIONAL, LTD. - 18- PROJECT NO.: 454C-116 Area i3600000 300000~ 240000411 1800000 I 1200000 1 I 3600000 0.0 ~ ~ ~ 1 ' 1 1 1 1 1 1 1 1 1 , 1 , , , , , i , , 0.60 1.20 1.80 2.40 3.00 3.60 4.20 4.80 Conc. ug a.i./L Figure 2. A typical calibration curve for PFBS. Slope = 758795.25; Intercept = -3605.85; r = 0.99780. WI LDLlFE I NTERNATlONAL, LTD. - 19- PROJECT NO.: 454C-116 intensity: 500000 cps 100 60 50 3 0- 2 0- 1 0- 206 01 I I 1 I 1 1 I I I I I I I I Figure 3. A representative ion chromatogram of a low-level (0.500 pg a.i./L) standard. WILDLIFE INTERNATIONALL, TD. - 20 - PROJECT NO.: 454C-116 intensity: 500000 cps 80 70 60 50 40 30 20 10 0 0.69 1.36 2.03 2.70 3.37 4.04 4.71 Time . _. Figure 4. A representative ion chromatogram of a high-level (5.00 pg a.i./L) standard. WILDLIFE INTERNATIONAL, LTD. PROJECT NO.: 454C-116 1009 080- 70- 6050- 40- 30- 201 0- 0 i intensity: 500000 cps 43 61 116 158 +209 252 286 I I 1 I I I I 1 I I I I I I 41 81 121 161 201 241 281 Scan 0.69 1.36 2.03 2.70 3.37 4.04 4.71 Time _. Figure 5. A representative ion chromatogramof a reagent blank sample (454C-116-VREB-N-2)T.he arrow - indicates the retention time of PFBS. WI LDLIFE INTERNATIONAL, LDI . - 22 - P 1 PROJECT NO.:454C-116 intensity: 500000 cps O ! I /I I r I I 1 I I r 1 I I I 41 81 1 2 1 1 6 1 201 2 4 1 2 8 1 Scan 0.69 1.36 2.03 2.70 3.37 4.04 4.71 Time Figure 6. A representative ion chromatogramof a fish tissue matrix blank sample (454C-116-VMAB-N-4). The m w indicatesthe retention time of PFBS. WILDLI FE INTERNATI ONAL, LTD. - 23 - PROJECT NO.:454C-116 10090807 06 0504 03 0- 2 0- 10- 1 intensity: 500000 cps i I Figure 7. A representative ion chromatogram of a low-level fish tissue matrix fortification sample (454C-116-WS-N-13n,ominal concentration of0.100mg a.i./Kg). WILDLIFE INTERNATIONALT, D. -24- ~~ ~~ PROJECT NO.:454C-116 intensity: 500000 cps 3 d 207 A 2 1 0.69 1-36 2.05 Figures. A representative ion chromatogram of a high-level fish tissue matrix fortification sample (454C-116-VMAS-N-22,nominal concentrationof 100 mg a.i./Kg). Wi LDLI FE iNTERNATIONAL, LTD. -25- APPENDIX I Protocol and Amendment PROJECT NO.: 454C-116 WI LDLI FE INTERNAT10NA L , LTD. - 26 - PROJECT NO.: 454C-116 PROTOCOL ANALYnCAL METHOD VALIDATION FOR THE DETERMINATION OF PERFWJORO BUTANE SULFONATE, POTASSIUM SALT (PFBS)IN FISH TISSUES 3M Environmental Lab Project No.E00-1429 Submitted to 3M Corporation EnviromcntalLaboratory 935 Bush Avenue St Pad, Minnesota 55106 Wildlife International Ltd. 8598 Commcnx Drive Easton,Maryland 21601 (410) 822-8600 May 25,2000 WI LD LI FE I NTERNATI oNAL , LTD . - 27 - PROJECT NO.:454C-116 SANITIZED W i l d l g e International, Ltd. DEC 0 9 2003 -2- ANALYnCAL METHOD VALIDATION FOR THE DFTERMMATION OF PERFLUORO BUTANE SULFONATE, POTASSIUM SALT (PFBS) M FISH TISSUES SPONSOR: 3M Corporation Environmental Laboratory P.O.Box 33331 St Paul, Minnesota 55133 SPONSOR'S REPRESENTATIVE: TES?MG FACILITY: wildlife International,Ltd. 8598 CommerceDrive Easton, Maryland 21601 STUDY DIRECTOR Raymond L. VanHova~,Ph.D. Scientist . Wildlife International,Ltd. LABORATORY MANAGE= Willard B. Nixon, Ph.D. Manager of Analydcal Chemistry Wildlife International,Ltd. FOR LABORATORY USE ONLY Tcst Substana NO.: 521C _PROTOCOLAPPROVAL I &&/&- - 1.A RORATORY M A N A G E ~ PROTOCOL NO.: 454/0S25001Mv-FT/SUB4S4 3 M LAB REQUEST NO.EW-1429 WILDLI FE I NTERNATIONAL, LTD. - 28 - PROJECT NO.:454C-116 wi1d1ife InternationaI, Ltd. -3- INTRODUCTION Wildlife International, Ltd. will conduct analytical trials to validate the performance of a liquid chromatography mass spectrometry ( L W S ) method for the determination of Perfluoro Butane Sulfonate, Potassium Salt (PFBS) in fish tissues. The study will be performed at the Wildlife International, Ltd. analytical chemistry facility in Easton, Maryland. The method proLided will be validated by fortifying fish tissues and quantifying thc recoveries of PFBS. Raw data for all work performed at Wildlife International, Ltd and a copy of the final report will be filed by project number in archives located on thc Wildlifc International, Ltd. site, or at an alternative location to be specified in the final report. OB.TE(XIVE The objective of this study is to validate the performance of LclMS methodology for determination of PFBS residues in fish tissues used by Wildlife International, Ltd. to perform environmental effects studies. EXPERIMENTAL DESIGN Fish tissues will be faded at least three diffcrcnt concentrations and analyzed using L M S methodology developed at Wddlife Intcmational,Ltd. Rcagcnt and matrix blanks will be analyzed to evaluate potential analytical interfernas. One or morc calibration curves will be prepared and analyzed with cach series ofmatrix foomficationsamples. MATERIALSAND METHODS T a t Substance Thc test substance will be Pcduoro Butanc Sulfonate, Potassium Salt, hexafter ref& to as PFBS. Informationon the charadcriration of test, co&ol or nfcrence substanas is raquindby Good Laboratory Practice Standards (GLP).?bc Sponsor is nsponsible for providingWddlife Inttmational, Ltd. written verification that the tat substance has bea~c h a r a c k M according to GLPs prior to initiation of the study. If written verification of GLP test substance characterization is not provided to Wildlife International, Ltd, it willbe nottd in the compliice statement of the final report. The Sponsor is responsible for all information related to the test substance and agrees to acccpt any unused testsubstance andor tat substance containersranaining at thc a d of the study. PROTOCOL NO.:4S4/052SOO/Mv-~/SuB4S4 3M Lab Project No. EOO-1429 WILDLIFE INTERNATIONAL, LTD. - 29 - PROJECT NO.:454C-116 wildlife IntemationaI, Ltd. -4. Reagents and Solvents All solvents used in the method or procedure will be of reagent grade or better. All reagents will be of ACS grade or better. Fish Tssues Fish tiska will be provided using animals that have no known exposure to the Msubstance. There an no levels of contaminants reasonably expected to be present in the fish tissues that are considered to interfere with the purpose or conduct of the study. Analytical Verification Procedures The analytical method to be used will be LClMS b a d upon procedures dewclopad at Wildlife International, Ltd. ThC method used will be documented in the raw data and summaritd in the final repon Prior to the analysis of analytical samples, primary Stock solution(s) will be prcpared directly from the test substance. Calibration standards will be prepared by appropriate dilution of the primary stock solution(s). Primary Stock Solution(s), Calibration Standards and Curves A minimum of five calibration standards (of diffemt concatrations) will bc prepared and anal& dong with each analysis sa The caliiration standard series will be injected at the beginning and one standaml injcckd, a! a after every five samples. A minimum of two injections of each standard in the calibration standard series will be injedcd with the analysis set. One or more caliiratioo curyts and rcgnssion equations will be prcparcd k r n the series of calibration standardr. .. !I Fortification Stock Solutions Fish tissues will be fortified with a stock solution(s) of PFBS at levels anticipated to bracket levels in the bioconccntration study. PROTOCOL NO.: 454/0525001Mv-~/SUB454 3M Lab Project No.E00-1429 WI LDLIFE INTERNATI oNAL , LTD. - 30 - PROJECT NO.: 454C-116 Wildrife International, Ltd. Evaluation of Interferences Reagent blanks and matrix blanks will be analyzed to detect possible interferences. The reagent blank will contain everytl-ung except the matrix and the test substance. The matrix blank will contain everything except the test substance. Verification Analyses - Method Performance Matrix fortifications (spiked fish tissues), prepared at hown conmtrations of the test substance, will be anal* to determine the recovery of the anal@ and to naluatc method performance. The anticipated verification series will comist of the following analyses: SUMMARY OF A W P I W VERIFICATION ANALYSIS SCHEME Level 1 - LOW' Level 2 LNcl3 Matrix fortifications will npnscnt the range of biownmtration that is anticipated to be found in fish tissue in subsequent c&cts tcsts and will be selected in c w s u l ~ o nwith the Sponsor. Individual samples fidcxdiiied by pmjtd number and a unique sample identification number) will be . I .1 prepared and anal* for verification of the analytical ut&.odology. If difficultia arise in the verification proccss (e.g. low recoveries or interferences), the Sponsor will be notified and the need for additional verification or method developmentwill be determind through discussion with the Sponsor. Upon completion of the method verificatioa, the Sponsor will rcvicw the results and authorize use of thc methodology for analysis of samples, or will PROTOCOL NO.: 454/052500/MV-~lSUB454 3M Lab Projcct No. EOO-1429 WILDLIFE INTERNATIONAL, LTD. -31 - PROJECT NO.:454C-116 wildlife International, Ltd. -6- authorize further method development trials. The target range of recoveries is 70 to 120% of the nominal concentrations. Data Analysis One or more calibration curves will be established for each analytical run. A regression equation of the concentration versus peak area for the calibration standards will be generated. The concentxation of the samples will be determined by substituting the respativc peak area into the regrcssion equation RECORDS TO BE MAINTAINED Records to be maintained for datagenerated by WJdlife International, JA.will include: 1. A copy of the signed protocol. 2. Identification and characterization of the test substance and/or analytical standard, if provided by the Sponsor. 3. Data of initiation and termination of the test. 4. Storage conditions for lestsubstance, analytical standards,and/or samples. 5. Test substance and/or analytical standard use log. 6. Conccntntion calculations and rccords ofsolutionprcpatatioe 7. Instrument operating conditions and chromatograms. 8. statisticalcalculations. 9. A copy ofthc final report 10. Documentationthat thesteps in the method wtn followed. FINAL REPORT The report will summakc the findings of the veriiidoa, the procedural m v e r i c s obtained, and the methods and instrumentation cmployed. Upon d p t of these findings, the Sponsor will rmicw thc methods and results and evaluate the rcsults for acceptability prior to initiating any fate andlor cffeds studia. The final report will include, but not be limitedto. the following: 1. Name and address of the acility performingthe study. 2. Dates on which the study was initiated and completed. PROTOCOL NO.: 454/052500/MV-FT/SUB454 3M Lab Projed No. E00-1429 WILDLIFE INTERNATIONAL, LTD. - 32 - PROJECT NO.:454C-116 Wildzife Internationa1, Ltd. .~ . .. ,. 3 . objectives and procedures stated in the approved protocol, including any changes in the original protocol or deviations from the protocol. 4. The test substance andor analytical standard identification, including name, chemical abstract number or code number, strength, purity, composition, date of receipt, lot number, storage conditions, physical characteristics, stability and mahod of prepaxation of test concentrations, if provided by the Sponsor. 5 . A brief summary of the analytical methodology. A description of the experimental measurements, example calculations, sample preparation (sample weights and dilutions), instrumm~onemployed, rcagmts and solvcnts used, class of water used, and any major m & d m to the method. 6 . A description of cirwnst;tnccs that may have aE:Ccted the quality or integriq of the dataI. The name of the Study Director, the names of other scientists or professionals, and the names of all supervisory pmonnel involvtd in the study. a. A description of the transformations, calculations, or operations performed on the data. 9. The signal and dated reports of each of the individual scientists or other professionals involved in the study, if applicable. 10. The location where raw data and finalreport arc stored at the conclusion of the study. 11. A statanent prepared by the Quality Assurance Unit listing the dates that study inspections werc made and the datcs of any hdqs rcportcdto the Study DirectorlManagement. CHANGING OF PROTOCOL Planned changes to the protocol will be in the form of wriatn ammdmmts signed by the Study Director and the Sponsor's RcpnscntativC. Ammdmmts will be considered as part of the protocol and will be attachedto the h a l protocol Any 0th- changes will be in thc form of writtra deviations signed by the Study Dircdor and filed with the raw data. All changes to the protocol will be indicated in tbe fins rcport PROTOCOL NO.: 454/052500flvN-FTISUB454 3M Lab Project No. E00-1429 WILDLIFE INTERNATIONAL,LTD. - 33 - wildlife International, Ltd. PROJECT NO.:454C-116 GOOD LABORATORY PRACIICES This study will be conducted in accordance with Good Laboratory Practice Standards for EPA (40 CFR Part 160 andor Part 792); and OECD Principles of Good Laboratory Practice (ENVIMUCHEM (98) 17). Each study conducted by Wildlife International, Ltd. is routinely examined by the Wildlife International, Ltd. Quality Assurance Unit for compliance with Good Laboratory Practices, Standard Operating Procedures and the specified protocol. A statanent of compliance with Good Laboratory Practices will be prepared for all portions of the study c o n d u d by Wddlife International, Ltd. The Sponsor will be responsible for d a t i o n of compliance with Good Laboratory Practices for procbdurcs performed by other laboratories. Raw data for a l l work performed at Wildlife International,Ltd.and a copy of the final report will be filed by pro@ number in archives lccatrd on the Wildlife International, Ltd. site, or at an alternative location to be spcc5d in the final report. :i PROTOCOL NO.: 454/052500/MV-FT/SUB454 3M Lab ProjectNo.E00-1429 WILDLIFE INTERNATIONAL,LTD. - 34 - DEC 0 9 2003 PROJECT NO.:454C-116 SANITIZED WILDLIFE INTERNATONAL LTD. PROJECT NO.:454C-116' Pagc 1 of 1 AMENDMENT TO STUDY PROTOCOL STUDY TITLE: Analytical Method Validation for the Determination of Perfluoro Butane Sulfonate, Potassium Salt (PFBS) in Fish Tissues PROTOCOL NO: 454/05250O/Mv-~/SUB454 AMENDMENTNO.: I SPONSOR 3M Corporation PROJECT NO.: 454C-116 EFFEC'UVE DATE: June 12,2000 3M ENVIRONMENTAL LAB PROJECTNO.: E00-1429 AMXNDMENT: Pagc2 Add: T a t Gnccntrations:. O.OO,O.lOO, 1.00, 10.0, and 100 rng a.i./Kg REASON: To specify the targct nominal conccntration levcls in the protocol. AMENDMENT: Pagc 2, Footcr Change: 3M LAB REQUEST NO.E00-1429 To: 3M LAB PROJECTNO.E00-1429 REASON To spccify current 3M project number nomenclature. AMENDMENT: Page 3, Introduction Changc: "Thc methodprovided willbc validated by fot-bfymg fish tissucs arid quantifying the rccovcrics of PFBS." To: -The mcthod dcvclopcd at Wddlife htunatioaal, Lid. willbc validated by fortifyingfish tissucSand quantifyingdle rccovcfies of PFBS." .. . REASON Thc methodology war dcvclopcd at WddE htanatiod, Ltd c- !I-0 0 DATE LABJ3RATORY MA&AGEMENT DATI~ ' WILDLIFE INTERNATIONAL, LTD. - 35 - DEC 09 22M0033 PROJECT NO.: 454C-116 SANITIZED APPENDIX I1 Certificate of Analysis C ~ F ~ S O ~ - K + 1 Lot 1; PFBS) ,Lot 2 (1999) April 17,2000 (Supcrttdcs ortificatc of Anaiysis &idApril 11,2OOO) W9S03X' 0.13 % 97.90% 1.96 % Additionally, the isomer distriiutionofthe sample was determined using contain the following weight percent composition: techniques and found to CFI(CF&-SW K' (Normal chain) 97.86 % 0.04 % WILDLIFE INTERNATIONAL,LTD. - 36 - PROJECT NO.: 454C-116 APPENDIX 111 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, PbD., Manager, Andyhcal Chemistry 2. Raymond L. Van Hoven, Ph.D., Scientist 3. Jon MacGregor, B.S., Scientist 4. Frank J. Lezotte, B.S., Chemist