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39) Analytical method validation for determination of PFBS-K in saltwater and algal media, 454C-117 . .. ANALYTICAL METHOD VALIDATION FOR THE DETERMINATION OF PERFLUOROBUTANE SULFONATE, POTASSIUM SALT (PFBS) IN SALTWATER AND ALGAL MEDIA SANITIZED WILDLIFE INTERNATIONAL, LTD. PROJECT NUMBER: 454C-117 DEC 0 9 2003 3M EnvironmentalLab Project No. E00-1429 AUTHORS : Raymond L. Van Hoven, PbD. Jon A. MacGregor, B.S. Willard B. Nixon, Ph.D. STUDY INITIATION DATE: November 7,2000 STUDY COMPLETION DATE: JUlyl3,2001 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 56 Wild1ife Intemational, Ltd. -2- Project Number 454C-117 GOOD LABORATORY PRACTICE COMPLIANCESTATEMENT SPONSOR: 3M Corporation TITLE: Analytical Method Validation for the Determination of PerfluorobutaneSulfonate,Potassium Salt (PFBS) in Saltwater and Algal Meda WILDLIFE INTERNATIONAL, LTD. PROJECT NUMBER: 454C-117 3M ENVIRONMENTAL LAB PROJECT NUMBER: E00-1429 STUDY.COMPLET~ON:J U ~ Y 13,2001 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 andor 792 and OECD Principles of Good Laboratory Practice (ENV/MC/CHEM (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: Wildlife International, Ltd. 7- 13-b I DATE SPONSOR APPROVAL: WildlifeInternational, Ltd. -3- Proiect Number 454C- 117 QUALITY ASSURANCE STATEMENT This study was examinedfor compliancewith Good Laboratory PracticeStandardsas publishedby the U.S. Environmental Protection Agency in 40 CFR Part 160 andor 792, 17 August 1989 and OECD Principles of Good Laboratory Practice (ENV/MC/CHEM (98) 17). The dates of all inspections and audts and the dates that any findings were reported to the Study Director and Laboratory Management were as follows: ACTMTY ~ a t r i xfirtifications Sample preparation Raw data, draft report Final report DATE CONDUCTED: November 9,2000 November 13,2000 November 28-30,2000 July 13,2001 DATE REPORTED TO: STUDY DIRECTOR MANAGEMENT: November 9,2000 November 13,2000 November 30,2000 July 13,2001 November 13,2000 November 16,2000 December 1,2000 July 13,2001 - I James H. Coleman, B.S. Quality Assurance Representative 7-13-01 DATE W iId1ife Intemationa1, Ltd. -4- Project Number 454C-117 REPORT APPROVAL SPONSOR: 3M Corporation TITLE: Analytical Method Validation for the Determinationof PerfluorobutaneSulfonate,Potassium Salt (PFBS) in Saltwater and Algal Media WILDLIFE INTERNATIONAL,LTD. PROJECT NUMBER 454C-117 3M ENVIRONMENTAL LAB PROJECT NUMBER: E00-1429 STUDY DIRECTOR: an Hoven, Ph.D. Scientist . MANAGEMENT: Willard B. Nixon, P h d Director of Analyhcal Chemistry 43-0 1 - WildlifeInternational. Ltd. -5- Project Number 454C- 117 TABLE OF CONTENTS TitldCover 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 Saltwater ...................................................................................... :................................................ 10 - Algal Medium ............................................................................................................................... 10 Analytical Validation Procedures ..................................................................................................... 11 Primary Stock Solution, Calibration Standards and Curves ............................................................ 11 Fortification Stock Solutions............................................................................................................ 11 Limit of Quantitation............................................................................................... ........................ 11 Results ...................................................................................................................................................... 12 . Matrix Blank Samples...................................................................................................................... 12 Method Validation Samples ............................................................................................................. 12 Conclusions................................................................................................................................................. 12 References ................................................................................................................................................... 13 W iId1ife International?Ltd. Proiect Number 454C-117 TABLE OF CONTENTS - Continued - TABLES Table 1 Method Validation Recoveries for PFBS in Algal Medium..............................1.4.............. Table 2 Method Validation Recoveries for PFBS in Saltwater .............................................................. 15 APPENDICES -Appendix 1 Protocol and Protocol Amendments .................................................................................. 16 Appendix 2 Certificates of Analysis..................................................... .(................................................ 32 Appendix 3 Matrix Water Parameters................................................................................................... 36 Appendix 3.1 Salinity and pH of Saltwater Measured During the 4-Week Period Immediately Preceding the Test .............................................................................................................. 37 Appendix 3.2 Analyses of Pesticides, Organics and Metals in Wildlife International, Ltd. Well Water ......................................................................................................................... 38 Appendix 3.3 Analyses of Pesticides, Organics and Metals in Wildlife International, Ltd. Saltwater ................................................................................. ;.......................................... 40 Appendix 3.4 Freshwater Algal Medium Constituen.............................................................................. 42 Appendix 4 Analpcal Method Validation Data.............................. ...................................................... 43 Appendix 4.1 Analytical Method Flow Chart for the Analysis of PFBS in Filtered Saltwater and Freshwater Algal Medlum........................................'...................................................44 .. Appendix 4.2 Typical LC/MS Operational Parameters........................................................................... 45 Appendix 4.3 A Typical Calibration Curve for PFBS ............................................................................. 46 Appendix 4.4 Example Calculations for a Representative Sample.......................................................... 47 Appendix 4.5 Ion Chromatogram of a Low-level PFBS Standard........................................................... 48 Appendix 4.6 Ion Chromatogram of a High-level PFBS Standard.......................................................... 49 WildlifeInternational. Ltd. -7- Proiect Number 454C-117 TABLE OF CONTENTS .Continued . APPENDICES Appendix 4.7 Ion Chromatogram of an Algal Medium Matrix Blank ................................................. 50 Appendix 4.8 Ion Chromatogram of a Saltwater Matrix Blank ........................................................... 51 Appendix 4.9 Ion'Chromatogramof a Low-level Algal Medium Fortification Sample...................... 52 Appendix 4.10 Ion Chromatogram of a High-level Algal Medium Fortification Sample ..................... 53 Appendix 4.11 Ion Chromatogram of a Low-level Saltwater Fortification Sample.............................. 54 Appendix 4.12 Ion Chromatogram of a High-level Saltwater Fortification Sample ............................. 55 Appendix 5 Personnel Involved inthe Study..................................................................................... 56 ... WildlifeInternational, Ltd. -8- Project Number 454C-117 SUMMARY SPONSOR: 3M Corporation TITLE: Analytical Method Validation for the Determinationof PeffluorobutaneSulfonate,Potassium Salt (PFBS) in Saltwater and Algal Me&a WILDLIFE INTERNATIONAL, LTD. PROJECT NUMBER: 454C-117 3M ENVIRONMENTALLAB PROJECT NUMBER: E00-1429 TEST DATES: Study Initiation: November 7,2000 ExperimentalStart (OECD): November 9,2000 ExperimentalStart (EPA): November 9,2000 ExperimentalTermination: November 14,2000 TEST SYSTEM: FreshwaterAlgal Medium and Filtered Saltwater FORTIFIED TEST CONCENTRATIONS : Algal Medium(mn a.i./L) 100 1000 5000 10000 Filtered Saltwater (mg a.i./L) 1.00 10.0 100 1000 RESULTS: Freshwater algal medium samples were fortifed with PFBS to reflect nominal concentrations of 100, 1000,5000 and 10000mg a.i./L. Filtered saltwater sampleswere fortified with PFBS to reflect nominal concentrations of 1.00,10.0,100and 1000mg a.i./L. PFBS stock solutions used for the cited matrix fortificationpreparations were adjusted for the purity of the potassium salt only. The samples were diluted into the instrumental calibration-range with dilution solvent (50% methanol : 50% NANOpure@water) and analyzed by liquid chromatography mass spectrometry (LCMS). Recoveries of PFBS in freshwater algal medium yielded an overall mean percent recovery of 107% (SD = 3.03; CV =2.83%; N = 12). Recoveries of PFBS in filtered saltwater yielded an overall mean percent recovery of 106% (SD = 3.14%; CV = 2.96%; N = 12). Matrix blank samples, consisting of unfortified fieshwater algal medium and filtered saltwater, were devoid of interfering components. WildlifeIntemational, Ltd. -9- Project Number 454C- 117 INTRODUCTION This study was conducted by Wildlife International, Ltd. for 3M Corporation at the Wildlife International,Ltd. analytxal chemistry facility in Easton, Maryland. Validation samples were fortifiedwith Peduorobutane Sulfonate, Potassium Salt (hereafter referred to as PFBS) and analyzed with liquid chromatographymass spectrometry (LC/MS) to evaluate the performanceof a method for the determination of PFBS in filtered saltwater and freshwater algal medium. Validation samples were prepared and analyzed between November 9,2000 and November 14,2000. Raw data generatedby WildlifeInternational,Ltd. and a copy of the final report are filed under Project Number 454C-117 in archives located on the Wildlife International, Ltd. site. OBJECTIVE The objectiveof thsstudy was to verify the perfonnance of a LCMS methodologyfor the analysesof PFBS in filtered saltwater and freshwater algal medium. The validated method will be used in support of aquatic toxicology and product chemistry tests to be conducted with the test substance. EXPERIMENTAL DESIGN Filtered saltwater and freshwater algal medium were each fortified at four different concentrations in triplicate and analyzed based on LC/MS methodology developed at Wildlife International, Ltd. The fortification levels bracketed the anticipated concentrations of samples from hture aquatic toxicity tests. Matrix blank samples in each matrix were analyzed concurrently to evaluate potential analytical method interferences. A calibration curve was generated from analyses of standard solutions of PFBS with each series of samples analyzed. MATERIALS A N D METHODS This study was conducted according to the procedures outlined in the protocol, "Analytical Method Validation for the Determinationof Perfluorobutane Sulfonate,Potassium Salt(PFBS) in SaltwaterandAlgal Media" (Appendix 1). Test Substance The test substance was received fiom 3M Environmental Technology and Safety Services on March 27,2000, assignedWildlife International, Ltd. Identificationnumber 5216, and storedunderambient conditions. The test substance, a white powder, was identified as: Potassium Perfluorobutane Sulfonate, WiId 1{fe Intemationa1, Ltd. - 10- Project Number 454C- 117 - Lot #2, CAS # 29420-49-3. The test substance was further identified with the 3M Environmental Laboratory test control and referencenumber Momation providedby the Sponsor when the protocol was issued indicated a purity of 97.90% and an expiration date of March 2010. A subsequent revision of the certificate of analysis indicated a purity of 97.3% and an Expiratiofleassessment Date of January 17, 2002 (Appenhx 2). Nominal and measured concentration values were not recalculated based on the relatively minor change in purity. The test substance was stored under ambient conditions. Reagents and Solvents All solventsused in this study were HPLC grade or equivalent. All reagents were ACS reagentgrade or equivalent. NAKOpure@water (equivalent to ASTM Type IIDesignation D1193-91) was used (1). Test Systems Saltwater Natural seawatercollected at Indian River Met, Delaware was filtered through a sandfilterprior to delivery into a 37,800-L storage tank. The salinity of the seawater was diluted to approximately 20%0 (parts per thousand) with Wildlife International, Ltd. well water in the holding tank to produce the saltwater matrix. The 20 % saltwater was aerated using spray nozzles and filtered ( 0 . 4 5 ~p)rior bits use in the method validation. Salinity and pH of the filtered saltwater during the four-week period - irnmediately preceding the test are presented in Appendix 3.1. The results of the most recent GLP-compliant analysesperformed to measure the concentrationsof selectedcontaminants in well water and saltwater used by Wildlife International, Ltd. are presented in Appendices 3.2 and 3.3, respectively. Aleal Culture Mehum Culturemedium used for the method validation was prepared accordingto WildlifeInternational, Ltd. Standard Operating Procedures. The concentrations of the components are presented in Appendix 3.4. Stock nutrient solutions were prepared by adding reagent-gradeor better chemicals to Wildlife International,Ltd. well water purified by reverse osmosis. Appropriate volumes of the stock nutrient solutions were then diluted with purified well water to prepare the medium. The medium was filter sterilized (0.2 p)prior to use. WildlifeInternational, Ltd. - 11 - Proiect Number 454C- 117 Analytical Validation Procedures Wildlife 'International, Ltd. conducted a validation trial using procedures developed at Wildlife International, Ltd. (Appendix 4.1). Validation samples were prepared in filtered saltwater and freshwater algal medium. Dilutions 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 PFBS were determined by reverse-phase high performance liquid chromatographyusing a Hewlett-PackardModel 1100 High Performance Liquid Chromatograph ("LC) with a Perkin-Elmer API 3000LC Mass Spectrometer equippedwith a Perkm-Elmer TurboIonSprayion source. Chromatographicseparations were achievedusing a Keystone Prism column (30 mrn x 1.5mm) fitted with a Keystone Prism guard column. A method flow chart is provided in Appendix 4.1 and the instrument parameters are summarized in Appendix 4.2. Primary 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 0.0100 to 0.0500 mg a.i./L, were analyzed with each sample set. Five calibration standards (different 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 Appendix 4.3. The concentrationof PFBS 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 calibrationstandards are presented in Appendices4.5 and 4.6, respectively. Fortification Stock Solutions Selected saltwater validation sampleswere fortified with stocksolutions of PFBS prepared in methanol (1.00,lO.O and 100 mg a.i./L levels). Each stock solution was assigned a unique identificationcodethat was recorded on a stock preparation log sheet. All algal medium validation samples and the high-level (1000 mg a.i./L) saltwater validation samples were fortified directly with the test substance. Limit of Quantitation The method validation levels in both the algal medium and saltwater matrices were well above the method capabilities for quantitationof the test substance. Therefore,the method limit of quantitation(LOQ) WiId1(fe International.,Ltd. - 12- Project Number 454C-117 in each matrix was calculated as the product of the lowest calibration standard and the dilution factor of the matrix blank samples. In algal medium, the LOQ was (0.0100 mg a.i./L) x (5000) = 50.0 mg a i & . In saltwater, the LOQ was (0.0100 mg a.i./L) x (50)= 0.500 mg a.i./L. RESULTS Matrix Blank Samples Three matrix blank samples in each matrix were analyzed to determine possible interferences. No interferences were observed at or above the LOQ (50.0 and 0.500 mg a.i./L in algal medium and saltwater, respectively) during the sample analyses (Tables 1and 2). Representative ion chromatogramsoffieshwater algal medium and filtered saltwater matrix blank samples are presented in Appendices 4.7 and 4.8, respectively. Method Validation Samples Freshwateralgal medium was fortified in triplicate at 100,1000,5000 and 10000mg a.i./Lresultingin mean recoveries of 108, 109, 107 and 105%,respectively (Table 1). Representativeion chromatograms of low and high-level freshwater algal medium matrix fortifications are presented in Appendices 4.9 and 4.10, respectively. Saltwater was fortified in triplicate at 1.00, 10.0, 100 and 1000 mg a.i./L resulting in mean recoveries of 106,109,107 and 103%, respectively (Table 2). Representativeion chromatogramsof low and high-level saltwater matrix fortifications are presented in Appendices 4.11 and 4.12, respectively. CONCLUSIONS Freshwateralgal medium sampleswere fortifiedwith PFBS to reflect nominal concentrations of 100, 1000, 5000 and 10000 mg a.i./L. Filtered saltwater samples were fortified with PFBS to reflect nominal concentrations of 1.00, 10.0, 100 and 1000 mg a.i./L. The samples were diluted into the instrumental calibration range with dilution solvent (50% rnethanoV50% NANOpure@water) and analyzed by liquid chromatographymass spectrometry (LCMS). Recoveries of PFBS in freshwater algal mehum yielded an overall mean percent recovery of 107% (SD = 3.03%; CV = 2.83%; N = 12). Recoveries of PFBS in saltwater yielded an overall mean percent recovery of 106% (SD = 3.14%; CV = 2.96%; N = 12). Matrix blank samples, consisting of unfortified freshwater algal medium and unfortified saltwater, were devoid of interfering components. WildlifeInternational, Ltd. - 13 - Proiect Number 454C- 117 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. Wildlcfe Xntemationa1, Ltd. - 14- Project Number 454C-117 Table 1 Method Validation Recoveries for PFBS in Algal Medium Sample Number (454C-117-) VMAB-I VMAB-2 VMAB-3 Type Matrix Blank Matrix Blank Matrix Blank PFBS Concentration (mg a.i./L) Fortified 0 0 0 Measured < LOQ' < LOQ <LoQ Percent Recovery - Mean Measured - (mg a.i./L) VMAS-I Matrix Fortification 100 VMAS-2 Matrix Fortification 100 VMAS-3 Matrix Fortification 100 103 103 108 111 111 109 109 Mean Percent Recovery - 108 VMAS-4 Matrix Fortification 1000 1110 111 1090 109 VMAS-5 Matrix Fortification 1000 1090 109 VMAS-6 Matrix Fortification 1000 1060 106 VMAS-7 Matrix Fortification 5000 5450 109 5340 107 VMAS-8 Matrix Fortification 5000 5290 106 VMAS-9 Matrix Fortification 5000 5270 105 VMAS-10 Matrix Fortification 10000 10400 104 10500 105 VMAS-11 Matrix Fortification 10000 10800 108 VMAS-12 Matrix Fortification 10000 10200 102 Overall Mean = 107 Standard Deviation = 3.03 C V = 2.83 N = 12 'The limit of quantitation (LOQ) was 50.0 mg a.i./L based upon the product of the lowest calibration standard (0.0100 mg a.i./L) and , the dilution factor of the matrix blank samples (5000). WildlifeInternational., Ltd. - 15 - Proiect Number 454C- 117 Table 2 Method Validation Recoveries for PFBS in Saltwater Sample Number (454C-117-) vMAB-4 VMAB-5' VMAB-6 Type Matrix Blank Matrix Blank Matrix Blank PFBS Concentration (mg a.i./L) Fortifed 0 0 0 Me- <LW' < LOQ <LoQ Percent Recovery - Mean Measured (mg a.i.lL) - VMAS-25 Matrix Fortification 1 .oo VMAS-26 Matrix Fortification 1 .oo VMAS-27 Matrix Fortification 1 .oo 1.08 108 1.06 1.07 107 1.03 103 Mean Percent - Recovery 106 VMAS-16 Matrix Fortification 10.0 VMAS-17 Matrix Fortification 10.0 VMAS-18 Matrix Fortification 10.0 10.5 105 10.9 109 10.8 108 11.3 113 VMAS-19 Matrix Fortification 100 VMAS-20 Matrix Fortification 100 VMAS-21 Matrix Fortification 100 106 106 107 107 109 109 107 107 VMAS-22 Matrix Fortification 1000 1060 106 1030 103 VMAS-23 Matrix Fortification 1000 1020 102 VMAS-24 Matrix Fortification 1000 I- 0_2_11_ 102 Overall Mean = 106 Standard Deviation = 3.14 C V = 2.96 N = 12 'The limit of quantitation (LOO) was 0.500 rng a.i./Lbased upon the product of the lowest calibration standard (0.0100 rng a.i./L) and the dilution factor of the matrix blank samples (SO). WildlifeInternational, Ltd. - 16- Appendix 1 Protocol and Amendments Project Number 454C-117 WildlifeInternational, Ltd. - 17- Project Number 454C-117 PROTOCOL ANALYTICAL METHOD VALIDATION FOR THE DETERMINATION OF PERFLUOROBUTANE SULFONATE, POTASSIUM SALT (PFBS) M SALTWATER AND ALGAL MEDIA 3M Lab Project No. E00-1429 Submitted to 3M Corporation Environmental Labratory 935 Bush Avenue St. Paul, Minnesota 55 106 Wildlife Xntemational, Ltd. 8598 Commerce Drive Easton, Maryland 2160 1 (410) 822-8600 July 17,2000 WiId1(fe Intemutiovtu'l,Ltd. - 18- wildlife International, Ltd. -2- SPONSOR: 3M Corporation Environmental Laboratory 935 Bush Avenue St. Paul, Minnesota 55 106 SPONSORS REPRESENTATIVE: -!, 3 TESTING FACLlTY: Wildlife International,Ltd. 8598 Commerce Drive Easton, Maryland 21601 STUDY DIRECTOR: Raymond L. VanHoven, Ph.D. Scientist LABORATORY MANAGEMENT: Willad B.Nixon, PbD. Manager of Analytical Chemistry FOR LABORATORY USE ONLY Project No.: 454C-117 Test SubstanceNo.: 5216 Experimental Termination Date: Test Concentrations: Reference Substance No.: NIA Project Number 454C-117 II- SPONSORS REPRESENTATIVE j DATE , PROTOCOL NO. 454/071700/MVAL'SUB454 3M LAB PROJECT NO. E00-1429 Wi1d1(fe InternationaI, Ltd. - 19- Project Number 454C-117 .. .. wildlife International, Ltd. -3 - INTRODUCTION Wildlife International, Ltd. will conduct analytical trials to validate the performance of a liquid chromatography mass spectrometry (LCMS) method for the determination of Perfluoro Butane Sulfonate, Potassium Salt (PFBS) in filtered saltwater and algal media. The study will be performed at the Wildlife International, Ltd. analytical chemistry facility in Easton, Maryland. The method will be verified by fortifying each water qtrk with the test substance and determining remvenes 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 the Wildlife International, Ltd. site, or at an alternative location to be specified in the final report. , OBJECTIVE ' The objective of this study is to verify the performance of LCMS methodology for \ analyses of PFBS in filtered salt water and algal media. The method to be validated will be used in support of aquatic toxicology and product chemistry tern to be conducted with the test substance. EWERlMENTAL DESIGN Wildlife International, Ltd. filtered saltwater and algal media will each be fortified at four different concentrations and analyzed using LCMS methodology based upon that developed at Wildlife International, Ltd. Mcdifications to the methodology will be made and documented as neqessary to achieve quantitation of PFBS in each of the test rnatrims. Matrix andor reagent blanks will be analyzed concurrently to evaluate potential analytical interferences. The levels of fortification will bracket the anticipated concentrations of samples from aquatic toxicity tests. One or more calibration curves willbe generated from analyses of standard solutionsof PFBS to be analyzed with each series of matrix fortification samples. PROTOCOLNO. 4541071700/MVAuSUl3454 3M LAB PROJECT NO.E00-1429 Wild1ife International, Ltd. - 20 - Project Number 454C- 117 .. wildlife International, Ltd. -4- MATERIALS AND METHODS Test and Reference Substances The test substance kill be PFBS. Information on the characterization of test and reference substances is required by Good Laboratory Practice Standards (GLP). The Sponsor is responsible for providing Wildlife International, Ltd.written verification that the test and reference substances have been c h a r a c t e d accordingto GLPs prior to initiation of the study. If written verification of GLP test and/or reference' substance characterization is not provided to Wildlife International, Ltd., it will be noted in the compliance statement of the final report. Reagents and Solvents All solvents used in the method or procedure will be HPLC grade or equivalent. All reagents will be ACS reagent grade or higher quality. Nanopure water (equivalent to ASTM Type I1 Designation D1193-91)will be used (1). The solvents and reagents are not expected to contain contaminants capable of interfering with the purpose of this study. Algal Culture Medium Culture medium to be used for the method validation will be prepared according to Wildlife International, Ltd. Standard Operating Procedures. The concentrations of the components in the medium are presented in Table 1 (2). Stock nutrient solutions will be prepared by adding reagent-grade or better chemicals to Wildlife International, Ltd. well water purified by revenearnosis. Appropriate volumes of the stock nutrient solutions will then be diluted with purified well water to preparc the medium. The medium will be filter sterilired (0.2 pm) or autoclaved prior to use. The pH of the medium should be 7.5 * 0.1. Saltwater Natural seawater collected at Indian River Met, Delaware will be filtered through a sand filter prior to delivery to a 37,800-L holding tank. The salinity of the seawater will be diluted to approximately 20 "/, @arts per thousand) with Wildlife International, Ltd. well water in the holding rank to produce the saltwater matrix. The 20 "/, saltwater will be aerated using spray nozzles and filtered (0.45 pm) prior to its use in the method validation. PROTOCOL NO. 454/071700/MVAL/SUB454 3M LAB PROJECT NO.E00-1429 Wildlife Intemational, Ltd. - 21 - Proiect Number 454C- 117 Wildlije International, Ltd. -5 - Salinity and pH will be measured weekly to monitor the consistency of the saltwater. Means and ranges of the measured parameters for the four-week period preceding the test will be provided in the fmal report. Analyses will be performed at least once annually to determine the concentrations of selected organic and inorganic constituents of the well water and seawater. Results of tbe most recent GLP- compliant analyses will be Summarized in the final report. Specifications for acceptable levels of contaminants in culture medium, well water, and seawater have not been established. However, there are no known levels of contaminants reasonably expected to be present in these well matrices that are considered to interferewith the purpose or conduct of the test. Analytical Validation Procedures Wildlife International, Ltd. will conduct validation trials using procedures based on LCMS methodology developed at Wildlife International, Ltd. The method may be m&ed to yield a method capable of quantitating PFBS at the desired concentrations in Siltered salt water and algal media. The method used, including any modifications, will be documented in the raw data and summarized in the final report. Reference Stock Solution(s), Calibration Standards and Curves A primary stock solution(s) will be prepared !?om PFBS reference substance, if available, or directly from the test substance in an appropriate solvent. Calibration standards will be prepared by appropriate dilution of this primary stock solution(s). The concentration range of the calibration standards will bracket the instrumental concentrations of the study samples. A minimum of five concentrations of calibration standards will be prepared and analyzed along with each analysis set of validation samples. The calibration standard series will be injected at the beginning and end of the analytical run kth, in addition, a minimum of one mid-level standard injected following every five samples. One or more calibration curves will be derived from regression analysis of the instrumental responses of the standards. Fortification Stock Solution(s) Saltwater and algal media will each be fortified with a stock solution(s) of PFBS. The fortification levels used will bracket the levels expected in anticipated aquatic toxicology studies PROTOCOL NO. 4541071700/MVAL'SUB454 3M LAB PROECT NO. E00-1429 WildlifeInternational, Ltd. - 22 - Project Number 454C-117 wildlife International, Ltd. with PFBS. Each stock solution will be assigned a unique identification code which will be recorded on a stock preparation log sheet Evaluation of Interferences Matrix and reagent blanks, if applicable, will be analyzed to assess the presence of potential interferences. Matrix blanks will consist of saltwater and algal media without addition \ of test substance. Reagent blanks, if applicable, will consist of reagents processed through the method. Validation - Analyses Method Performance Matrix fortifications (fortified saltwater and algal media) prepared at known concentrations of PFBS,will be analyzed to determine recoveries and to evaluate method performance. The anticipated validation series willconsist of the following analyses: SUMMARY OF PROPOSED VALIDATION ANALYSIS SCHEME Number of Samoles I Matrix Blank Matrix Fortifications Matrix fortificationswill span the range of concentrationsthat are anticipated to be used in subsequent environmental effects tests. Individual fortification samples (identified by project number and a unique sample identificationnumber) will be prepared and analyzed. If difficulties arise in the validation process (e.g., low recoveries or interferences), the Sponsor will be notified and the need for additional validation a d o r method development will be determined through discussions with the Sponsor. Upon completion of the method PROTOCOL NO. 454/071700RvNAuSUB454 3M LAB PROJECT NO.E00-1429 WiId1i,fe Internationa1, Ltd. Proiect Number 454C-117 W i l d 1fe International, Ltd. -7- validation, the Sponsor will review the results and authorize the use of the methodology for analysis of samples, or will authorize further method development trials. Recovery v;?ues in the range of 80 to 120% will be used as criteria for method acceptability. Data Analysis One calibration curve for each analytical run @I be derived from the instrumental responses of PFBS standards of hown concentration using regression analysis. The concentrations of PFBS in fortified samples will be determined by substituting the respective instrumental response into the regression equation If the fort5cation range and calibration curve standard concentrations are significantly higher than method capabilities for quantitation, the limit of quantitation (LOQ)will be defined 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 analyte, the LOQ will be determined from the responses of matrix blank samples. The mean (Sb) and standard deviation (a) of responses will be determined for the replicate control sample and converted to ppm equivalents using a standard curve and method dilution factors. The LOQ will be expressed as the concentration equivalent of Sb + 100. The limit of detection (LOD), defined as the lowest concentration of analyte which can be injected and produce a measurable response above background, will be expressedas the amount equivalentto Sb +30. RECORDS TO BE MAINTAINED Records, to be maintajned for data generated by Wildlife International, Ltd., will include: I. A copy of tie signed protocol. 2. Identhation and characterization of test and reference substances, if provided by the Sponsor. 3, Dates of initiation and termination of the test. 4. Storage conditions for test and reference substances, and samples. 5 . Test and reference substance use logs. 6. Concentration calculations and rccords of solution preparation. PROTOCOL NO. 454/07 1700/MVAuSUB454 3M LAB PROJECT NO.E00-1429 W iId1{fe International., LEd. -24 - Proiect Number 454C-117 w i l d l i f e International, Ltd. -8- 7. Instrument operating conditionsand chromatograms. 8. Statistical calculations. 9. A copy of the tinal report. FINAL REPORT The report will summarize the findings of the validation, the fortification recoveria obtained, and method($ and instrumentation employed. Upon receipt of these &dings, the Sponsor will review the methods and remits, and cvaluate the results for acceptability. The finalreport will include, but not be limitedto,the following: 1. Name and address of the facility performing the study. ' 2. Dates on which the study was initiated and completed. 3. A statement of compliance signed by the Study Director addressing any exceptions to Good Laboratory Practice Standards. 4. Objectives and procedures stated in the approved protocol, including any changes in the original protocol or deviations h m the protocol. 5 . Identification of test and referencz substances, including name, chemical abstract number or code number, strength, purity, composition, date of receipt, lot number, storage conditions, physical cbaracteristics, stability and method of preparation of test concentrations, if provided by the Sponsor. 6 . A brief summary of the analytical methodology to include, where applicable, a description of the experimental measurements, example calculations, sample preparation (sample weights andor dilutions), instrumentation employed, reagents used, purity of reagents and solvents, and any major modilications to the method. 7. A description of any circum.stances that may have affected the quality or integrity of the data. 8. The name of the -Study Director, the names of other scientists or professionals, and the names of all supervisory personnel involved in the study. 9. A description of the transformations, calculations, or operations performed on the data. 10. The signed and dated reports of each of the individual scientists or other professionals involved in the study, if applicable. 11. The location where raw data and final report are to be stored. PROTOCOL NO. 454/071700/MVAL'SuB454 3M LAB PROJECTNO. E00-1429 WildlifeInternational, Ltd. - 25 - Project Number 454C-117 Wildlife International, Ltd. -9- 12. A statement prepared by the Quality Assurance Unit listing the dates that study inspections and audits were made and the dates of any findings reported to the Study Director and Management. CHANGING OF PROTOCOL Planned changes to the protocol will be in the form of written amendments signed by the Study Director and the Sponsor's Representative. Amendments will be considered as part of the protocol and will be attached to the finalprotocol. Any other changes will be in the form of written deviations signed by the Study Director and filed with the raw data. All changes to the protocol will be indicated in the final report. GOOD LABORATORY PRACTICES This study will be conducted in accordance with Good Laboratory Practice Standards for EPA (40 CFR Part 160 andor 792) and OECD Principles of Good Laboratory Practices (ENVMUCHEM (98) 17). Each study conducted by Wddlife International, Ltd.is routinely examined by the Wildlife International, Ltd. Quality Assurance Unit for compliance with Good Laboratory Practices, Standard Operating Procedures and the specified protocol. A statement of compliance with Good Laboratory Practices will be prepared for all portions of the study conducted by Wildlife International, Ltd. The Sponsor will be responsible for compliance with Good Laboratory Practices for procedures performed by other laboratories. PROTOCOL NO. 4S4/0717OO/MVAYSUB4S4 3M LAB PROJECT NO. E00-1429 WildlifeInternational, Ltd. -26 - Project Number 454C-117 ' Wildlife International, Ltd. - 10- REFERENCES 1 American Society for Testing and Materials. 1991. Standard Specification for Reagent Water. D1193-91, ASTM SectionIIWater and Environmental Technology, Vol. 11.01 2 American Society for Testing and Materials. 1990. Standard Guide for Conducting Static 96-Hour Toxicity Tests with Microalgae. E 1218-90. ASTM Section 11 Water and Environmental Techology, Vol. 11.05. 3 American Society for Testing and Materials. 1991. Standard Guide for Conducting Static Toxicity Tests with Lema gibbo G-3. E 1415-91. ASTM Seaion II Water and Environmental Technology, Vol. 11.05. PROTOCOL NO. 454/071700/MVAUSUB454 3M LAB PROJECT NO. E00-1429 WiId1(fe Internationa1, Ltd. -27 - Project Number 454C-117 wildlife International, Ltd. - 11 - TABLE 1 FRESHWATER ALGAL MEDIUM CONSTITLTENTS Component Nominal Concentration 12.16 mglL .. 4.40 mglL 0.1856 mg/L 0.416 mglL 3.28 p a 0.1598 xu@ 1.428 J&L 7.26 p g 5 0.012 pglL 0.300 mg/L 25.50 mglL 14.70 mglL 1.044 mgL 15.00 me/L The pH of the medium will be adjusted,as necessary,to 7.5 f 0.1 using 0.1 N NaOH or 10%HCl. PROTOCOL NO. 454/0717OO/MVAUSuB454 3M LAB PROJECT NO. E00-1429 WildlifeIntemational, Ltd. - 28 - Wildlve International, Ltd. Proiect Number 454C-117 Project Number: 454C-117 Page 1 of 1 AMENDMENT T O STUDY PROTOCOL STUDY TITLE: Analytical Method Verification for the Determination of Perfluorobutane Sulfonate, Potassium Salt (PFBS) in Saltwater and Algal Media PROTOCOL NO.: 454/071700/MVAUSUE3454 SPONSOR: 3M Corporation 3M LAB PROJECT NO.: E00-1429 AMENDMENTNO.: 1 PROJECT NUMBER: 454C-117 EFFECTIVE DATE: November 9,2000 AMENDMENT: Page 2, Proposed Dates and Test Concentrations Add: Experimental Start Date: 11/09/00 ' Experimental Termination Date: 12/31/00 Test Concentrations: (Algal Medium) 0.0, 100,1000,5000, and 10000 mg a.i./L REASON: The above information was required to complete the algal medium portion of the protocol. . _- I.. / A LABORATORY MANA~EMENT I -,-,,, DATE ' ' Wildlife International?Ltd. - 29 - `WildlifeIntemational, Ltd. Project Number 454C-117 Project Number: 454C-117 Page 1 of 1 AMENDMENT TO STUDY PROTOCOL STUDY TITLE: Analytical Method Verification for the Determination of Perfluorobutane Sulfonate, Potassium Salt (PFBS) in Saltwater and Algal Media PROTOCOL NO.: 4541071700/MVAUSUB454 SPONSOR: 3M Corporation 3M LAB PROJECT NO.: E00-1429 AMENDMENT NO.: 2 PROJECT NUMBER:454C-117 EFFECTIVE DATE: November 9,2000 AMENDMENT: Page 5 , Reference Stock Solution(s), Calibration Standards and Curves Change: `The calibration standard series will be injected at the beginning and end of the analytical run with, in addition, a minimum of one mid-level standard injected following every five samples." To: "The calibration standard series will be injected at the beginning and end of the analytical run with, in addition, a minimum of one standard injected following every five samples." REASON: The revised wording is compatible with current Wildlife International, Ltd. SOPS WildlifeInternational, Ltd. - 30 - wildlife International, Ltd. Project Number 454C-117 Project Number: 454C-117 Page 1 of 1 AMENDMENT TO STUDY PROTOCOL STUDY TITLE: Analytical Method Verification for the Determination of Perfluorobutane Sulfonate,Potassium Salt (PFBS) in Saltwater and Algal Meda . PROTOCOL NO.: 454/07170O/MVAUSUB454 AMENDMENT NO.: 3 SPONSOR: 3M'Corpontion PROJECT NUMBER: 454C-117 3M LAB PROJECT NO.: E00-1429 ~ ~~ EFFECTIVE DATE: November 13,2000 AMENDMENT: Page 2, Proposed Test Concentrations Add: Test Concentrations: (Saltwater) 0.0, 1.00, 10.0, 100, and 1000 mg ai.& REASON: The above information wds required to complete the saltwater portion of the protocol. LABORATORY M ~ A G E M E N T If -13- ow DATE DAT~ ' 11 j 3 0 l b Z . DATE WiId1ife InternaEional?LEd -31 - Wildlife International, Ltd. Proiect Number 454C.117 Project Number: 454C-117 Page 1 of 1 AMENDMENT TO STUDY PROTOCOL STUDY TITLE: Analytical Method Validation for the Determination of Perfluorobutane Sulfonate, Potassium Salt (PFBS) in Saltwater and Algal Media PROTOCOL NO.: 454/071700/MVAuSUB454 SPONSOR: 3M Corporation 3M LAB PROJXCT NO.: E00-1429 AMENDMENT NO.: 4 PROJECT NUMBER: 454C-117 EFFECTIYE DATE: November 13,2000 AMENDMENT: Amendments 1.2, and 3 - STUDY TITLE Change: Analytical Method Verification for the Determination of Perfluorobutane Sulfonate, Potassium Salt (PFBS) in Saltwater and Algal Media. To: Analytical Method Validation for the Determination of Perfluorobutane Sulfonate, Potassium Salt (PFBS) in Saltwater and Algal Media. REASON: Typographical error. LABORATORY MANACfkMENT DATE ' Wildlife IntemationnI, Ltd. - 32 - Proiect Number 454C-117 Appendix 2 Certificates of Analysis ip"":(814) 2314032 v. . ..I-_ Fpy:(al;) 231-1253 01 (814) P1-15W INTERIM CERZTTCATE OF ANALYSIS Revhion I CentrcAnalytical Laboratories COARticrence #: 023-032 3M Product: P'FBS, Lot 2 Test Contrd Reftrcnca #: TCR-00017-71 Purity: 973% T a t Nanw I SpcuGmUom I Rcsult 973 Y. 5. N i C M 6. Iron 1. 0.O03wthtOX 2. O.O01wth.t% 3. 0.768 wthrt% 4. 10.718wthL% 5. 4].001wt/nt% 6. O.W2v&wL%' I n w e Anions (IC) 1. Chlm-ds 2. Pluoridc 3. Bromide . . 4. 5. Nitc Nitrite 6. Phosphuts ..- ac, 7. Sulfate Aadr' - Elcnrnbl Analysis': 1.crrbon 2. Hydrogm 3. Mtrogm 4. sulfur 5. FhLorinc - Tharelical Vduo 14.2% 'IheortricolVduc = 0.0% Thcorctiul Vduc = 0.0% - Tbcorctid Vdue 9.5% 'Iheomtical VsIuc-M.6% I 1. O.Dl6wthtX 2. co.ooswhvL% 3. - = . O o . D 4 O ~ % 4. a . o O 9 ~ y . 5. 4).006~!-)wt.% 6. <0.030wrJwtY~ 7. 4).044Wtht% 2I. 44 .11 VwdrW )aLtK% . 3. <O.l wtlwt% 1 . 11.0 at/*% 2. oJa2plt/ntyI * 3. 0.465 WVwt.% 4. llSWt/wt% s. 48.2 arthvls/. WildlifeInternationa1, Ltd. - 33 - Project Number 454C- 117 w: (814) 231-8032 FEC(EII~a) i - 1 2 ~w1pi~)mi-ism DVTFkIM CERlTFICATE OF ANALYSIS Revirion I Centre Analytical Labontorlsc COA Reference #: 023-032 3M Product: P D S ,Lot 2 Tut Control Reference #: TCR-00017-71 Dare ofLast Analysis: 01/17/01 ExpkdionDalc: 01/17/02 Storago Conditions: <-10'C Reassessment Datu: 01/17/02 - - 'purity a 1O W (Total NMR LnpUriti~2~.0,7% + T ~ t Ldc/Msknpuritie~,(5.56% 4.99%*) +Total Metal impurities, 0.006% +Total inorganic anions, 0.016%) - TotaI impurity 6com all tub 2.662% - Purity 9 100% 2.662% 9 973% 1.93 X of the NMR total impurity value is duo to thc pnsmcc of i This impurityia also obsemed in the Lc/MS analyms at 4.99%. The kpurity value is taken f h m the NhiR analysisas it is deerridthe more quantitative techniqueinthe abscncc of standad~(See impurity profile on pago 3). 7.Potassium and sodium are present as counterions and thus are cxcludcd from the purity calculation. . .. . %vorcticd value calculationsbased onthe empirical formula, C&SQX+ (Mw=3383) 0 CO.4023032-7 1.des h g C 2 Of 3 Wi1d1ife Inte mational., Ltd. -34- . Project Number 454C- 117 I'TE& CERTIFICATE OFANALYSIS Revission I Cenwe Analytical Laboratories COAReference #; 023-032 3M Product: PFBS,'Lot 2 Test Control Reference #: TCR-00017-71 Peak# RcttntionTimc (min) I M u & 1 2 3 Total 1 9.508 I 298.9 I I Identity I M e n I %Impurity' i I PFBS 1308351-00 I 132651678 0-57 -4.99 .. 5.56 N M R PurityProfile: Component I c~I-cF2-arcF;-so3', I Molecnlar Weight 298.9 I Relative We&ghPt ercent I 97.9 - I Total Impwity I 1.93 0.13 0.01 2.07 This work was conductd&dm EPA Good Laboratory Practice Standards (40 CFR 160). COA013032-7I .&c Page 3 of 3 WiId1ife InternationaI , Ltd. -35- Project Number 454C-117 Original Certificate of Analysis c~F$o~-K+( Lot 1; PFBS) , Lot 2 (1999) April 17,2000 (Supencda OrtLfiulc of Analysis&idApril 11,2oOO) This sample was a n w using ,and elemental analyses techniques. The results of these tests show the sample to contain the following weight percent composition: 0.13 % 1.96 % i Additionally, the isomer distriiution of the sample was determined using contain the following weight percent composition: .techniques and found to CE;(CF&-S@' K' (Normal chain) 97.86 % 0.04 % Analpal Study Number: 60488 WildlcfeInternutional, Ltd. -36- Appendix 3 Matrix Water Parameters Proiect Number 454C-117 W iId1ife InternationaI, Ltd. -37- Proiect Number 454C-117 Salinity (ppt) PH Appendix 3.1 Salinity and pH of Saltwater Measured During the 4-Week Period Jinmediately Preceding the Test Mean 20(N=4) 8.3 (N= 4) Range 20 - 20 8.2 - 8.5 WildlifeInternational, Ltd. -38- Project Number 454C-117 Appendix 3.2 Analyses of Pesticides, Organics and Metals in Wildlife International,~ t dW. eU Water' Pesticides and Organics Component Measured Concentration Component Measured Concentration Aclonifen 4.03Ta Dichlorvos xo.01 p g n Alachlor <0.01 pg/L Dicofol c0.25 pg/L Ametryn 4.01 pgn Diethyltoluamide -=0.02 pg/L Atrazine 4 . 0 1 pg/L Difenoconazole 4.03 pg/L Azinphos-ethyl co.04 pg/L Dimethoate 4.02 pgn Azinphos-methyl 4.08p g n Dimethomorph <0.05 pg/L Azoxystrobin . c0.25 p g n Disulfoton <0.02 pg& Bifenthrin 4.05 p g n DMST -=0.05 pg/L Bioallethrin 4.05pg/L Dodemorph -=0.01 p g n Bitertanol <0.05 pglL Endosulfanu <0.01 pg/L Bromacil <0.05 pg/L Endosulfan+ <0.01 p g n Bromophos <0.02 p g n Endosulfan-sulfate c0.02 pg/L . Bromophos-ethyl 4.02 pglL Epoxiconazole <0.05 pg/L Bromopropylate Bupirirnate <0.02 p g n 4.05p g n EPEsfenvalerate -=0.02 p g n <0.02 pg/L Carbaryl 4.05 p g n Ethion <0.05 p g n Carbofuran <0.03 p g k Ethofurnesate -=0.02 p g n Carboxin 4.02 pg/L Ethoprophos <0.01 pg/L Chlorfenvinphos <0.02 p g n Etridiazole <0.02 pg/L Chloridazon 4.05 pg/L Etrimfos <0.05 pg/L Chlorpropham 4 . 0 2 pg/L FenarimoI <0.05 pglL .- Chlorpyrifos co.01 p g n Fenchlorphos Chlorpyrifos-methyl 4 . 0 1 pglL Fenitrothion <0.01 pg/L <0.03 pglL Chlorothalonil 4 . 0 4 pg/L Fenoxycarb 4.03 pg/L Coumaphos 4 . 0 2 pg/L Fen piclonil <0.05 pg/L Cyanazine <0.05 p g n Fenpropathrin 4 . 2 5 pg/L Cyfluthrin Cypermethrin 4.05 pglL <0.25 p g n Fenpropirnorph Fenthion (0.01 pg& <0.01 p g n Cyproconazole . 4.05 p g n Fenvalerate 4.02 pgn Deltamethrin <0.02 p g n Fluazifop-butyl 4.02 pgn Demeton C0.02 p g n Fluoroglycofen-ethyl <0.02 p g 5 Derneton-0 Desethylatrazine 4.02 pgn co.01 pg/L Fluroxypyr-meptyl Flutolanil <0.05 pg/L 4.02 pgn Desisopropylatrazine 4 . 0 2 pg/L Fonophos 4.01 pgn Desmetryn 4 . 0 1 pa Furalaxyl -=0.02 pg/L Diazinon 4 . 0 1 pg/L Heptenophos 4 . 0 2 pg/L Dichlobenil 4.01 pgn ImaZalil co.01 pg/L Dichloran Dichlorbemmide 4.03 pg/L 4 . 0 2 pg/L Iprodion Kresoxirn-rnethyl pa d.05 pg/L 4.02 Dichlorfenthion <0.01p g L Lenacil (0.05 pglL Dichlorfluanid 4 . 0 3 ppjL Lindane 4 . 0 2 pa 'Analyses performed by TNO Nutrition and Food Institute on samples collected on November 15,2000. WildlifeInternational, Ltd. -39- Project Number 454C- 117 Appendix 3.2 (Continued) Analyses of Pesticides, Organics and Metals in Wildlife International,Ltd.WeU Water' Pesticides and Organics ~ ~ Component Measured Concentration Component Measured Concentration Malathion Metalaxyl Metamitron Metazachlor Methidathion Paclobutazole ' Parathion Parathion-methyl Penconazole Pendimethalin Permethrin-cis Permethrin-trans Phosalone Phosmet Phosphamidon-cis Pirimicarb Pirimiphosethyl Pirimiphos-methyl Prochloraz Procymidon Prometryn Propachlor Propazine Propham Propiconazole Propoxur Propyzamide Prosulfocarb Pyrazophos 4 . 0 2 pg/L 4 . 0 5 pg/L 4 . 0 5 pg/L 4 . 0 2 pg/L 4 . 0 2 pg/L <0.05 pg/L 4 . 0 1 pglL 4 . 0 1 pg/L 4.05 pgn <0.03 p g L 4.01 pgn 4 . 0 1 pg/L 4 . 0 5 pg/L 4 . 0 2 pg/L <O.OS pg/L 4 . 0 1 pg/L 4.01 pg/L 4 . 0 1 pglL 4.02 pg/L 4 . 0 1 pg/L 4 . 0 1 pg/L 4.01 pa 4 . 0 1 pg/L co.02 p p n 4.05 ppn <0.03 pg/L 4 . 0 2 pg/L 4 . 0 2 pg/L 4.03 pg/L Methoxychlor Metolachlor Metribuzin Mevinphos Nitrothal-Isopropyl F'yrifenox-1 Pyrifenox-2 Pyrimethanil Quizalofopethyl Simazine Sulfotep Tebuconazole Tebufenpyrad Terbutryn Terbuthylazine Tetrachlorvinphos Tetrahydrofilimide Tetramethrin Thiabendazole Thiometon Tolclofos-methyl Tolylfluanid Triadimefon Triadimenol Triallate Triazophos Trifluralin Vamidothion Vinclozolin <0.01 pg/L 4 . 0 1 pg/L <0.02 pg/L 4 . 0 1 pg/L 4 . 0 5 pg/L 4 . 0 1 pg/L c0.01 pglL co.01 pg/L 4 , 0 2 pg/L <0.01 pg/L c0.02 pg/L co.05 pglL 4 . 0 5 pg/L 4 . 0 1 pg/L 4 . 0 1 pg/L <0.01 pg/L <0.05 pg/L <0.01 pg/L -=0.05 pa 4 . 0 4 pg/L co.01 pg/L 4.04 pg5 ~ 0 . 0 5pg/L ~ 0 . 0 5pg/L c0.02 pg/L c0.02 pg/L 4 . 0 2 pg/L <0.01 pg/L 4 . 0 1 pg/L Metals Magnesium Sodium Calcium Iron Potassium Aluminum Manganese Beryllium Chromium Cobalt 13.2 m g l l 21 mg/L 35 mg/L 4 . 0 2 mg/L 6.2 mg/L 4 . 0 9 mg/L 0.72 pg/L 4 . 3 Pg/L 4.6 ~ 0 . 4pg/L Nickel Copper Zinc Molybdenum Silver Cadmium Arsenic Mercury Selenium 'Analyses performed by TNO Nutrition and Food Institute on samples collected on November 15,2000. W iId1 fe Intemational?Ltd. Project Number 454C-117 Appendix 3.3 Analyses of Pesticides, Organics and Metals in Wildlife International,~ t Sdaltwater' ~ ~~~~~~~~ ~~ Pesticides and Organics Component Measured Concentration Component Measured Concentration Aclonifen AIachlor Ametryn Atrazine Azinp hos-ethyl Azinphos-methyl ' Azoxystrobin Bifenthrin Bioallethrin Bitertanol Bromacil Brornophos Brornophos-ethy1 Brornopropylate Bupirirnate Carbaryl Carbofuran Carboxin C hlorfenvinphos Chloridazon C hlorpropham Chlorpyrifos Chlorpyrifos-rnethyl Chlorothalonil Coumaphos C yanazine Cyfluthrin C ypermethrin Cyproconazole . Deltamethrin Demeton Derneton-0 Desethylatrazine Desisopropylatrazine Desrnetryn Diazinon Dichlobenil Dichloran Dichlorbenzamide Dichlorfenthion Dichlorfluanid 4.03 pg/L 4 . 0 1 pg/L -=0.01 pg/L <0.01 pg/L 4 . 0 4 pg/L 4 . 0 8 pg/L c0.25 pa 4 . 0 5 pg/L 4 . 0 5 pglL co.05 pg/L co.05 pg/L 4 . 0 2 pg/L c0.02 pg/L a 0 2p a ' co.05 pg/L <0.05 pg/L c0.03 pg/L c0.02 p g L <0.02 pg/L 4 . 0 5 pg/L c0.02 pg/L <0.01 pg/L <0.01 pg/L 4 . 0 4 pg/L c0.02 pg/L 4.05 pg/L -=0.05 pg/L 4.25 pg/L ~ 0 . 0 5pg/L c0.02 pg/L c0.02 pg/L 4 . 0 2 pg/L co.01 pg/L 4 . 0 2 pa <0.01 pg/L 4 . 0 1 pg/L co.01 pg/L ~ 0 . 0 3pg/L c0.02pg/L 4 . 0 1 pg/L 4 . 0 3 pg/L Dichlorvos Dicofol Diethyltoluarnide Difenoconazole Dimethoate Dimethornorph Disulfoton DMST Dodemorph Endosulfan-a Endosulfan-P Endosulfan-sulfate Epoxiconazole EPm Esfenvalerate Ethion Ethofurnesate Ethoprophos Etridiazole Etrirnfos Fenarirnol Fenchlorphos Fenitrothion Fenoxycarb Fenpiclonil Fenpropathrin Fenpropirnorph Fenthion Fenvalerate Fluazifopbutyl Fluoroglycofen-ethyl Fluroxypyr-rneptyl Flutolanil Fonophos Furalaxyl Heptenophos Imazalil Iprodion Kresoxirn-methyl Lenacil Lindane <0.01 pg/L 4 . 2 5 pg/L 4 . 0 2 pg/L 4.03 pg/L <0.02 pg/L 4 . 0 5 pg/L c0.02 pg/L c0.05 pg/L (0.01 pg/L co.01 pg/L 4.01 pgL 4 . 0 2 pg/L <0.05 pg/L <0.02 pg/L <0.02 pg/L co.05 pg/L c0.02 pg/L <0.01 pg/L c0.02 p g L co.05 pg/L co.05 pg/L co.01 pg/L 4.03 pg/L 4.03 pg/L 4 . 0 5 pg/L 4 . 2 5pg/L co.01 p g L 4 . 0 1 pg/L 4.02 pg/L c0.02 pg/L c0.02 pg/L co.05 pg/L 4 . 0 2 pg/L CO.01 pg/L C0.02 pg/L 4.02 pg/L 4 . 0 1 pg/L 4 . 0 5 pB/L 4 . 0 2pfl .co.os pg/L 4.02 pgL 'Analyses performed by TNO Nutrition and Food Institute on samples collected on November 15,2000. WiId1(fe IntemationaI, Ltd. -41 - Project Number 454C-117 Appendix 3.3 (Continued) Analyses of Pesticides,Organics and Metals in WildlifeInternational,~ t dS.altwater' ~ ~ ~~ ~~ ~~~ Pesticides and Organics Component Malathion Metalaxyl Metamitron Metazachlor Methidathion Paclobutazole ' Parathion Parathion-methyl Penconazole Pendimethah Permethrin-cis Permethrin-trans Phosalone Phosm et Phosphamidon4 Pirimicarb Pirimiphoscthyl Pirimiphos-methyl Prochloraz Procymidon Prometryn Propachlor Propazine Prophk Propiconazole Propoxur Proppmide Prosulfocarb Fyrazophos Measured Concentration Component 4 . 0 2 pgiL 4 . 0 5 pg/L 4 . 0 5 pg/L 4.02 ppn 4.02 ppn 4 . 0 5 pg/L 4 . 0 1 pg/L 4 . 0 1 pg/L 4 . 0 5 pg/L d.03 p g 5 4 . 0 1 pg/L 4 . 0 1 pg/L 4 . 0 5 pg/L 4 . 0 2 pg/L 4 . 0 5 pg/L 4 . 0 1 pg/L 4.01 pgL 4.01 pg/L 4.02pgL 4 . 0 1 pg/L 4 . 0 1 pg/L 4 . 0 1 pg/L 4 . 0 1 pg/L 4.02 pg/L 4 . 0 5 p& 4.03 pg/L 4 . 0 2 pg/L 4 . 0 2 pg/L 4.03 pg/L Methoxychlor Metolachlor Metribuzin Mevinphos Nitrothal-Isopropyl Pynfenox-1 Pyrifenox-2 Pyrimethanil Quizalofopethyl Simazine Sulfotep Tebuconazole Tebufenpyrad Terbutryn Terbuthylazine Tetrachlorvinphos Tetrahydroftalimide Tetramethrin Thiabendazole Thiometon Tolclofos-methyl Tolylfluanid Triadimefon Triadimenol Triallate Triazophos Trifluralin Vamidothion Vinclozolin Metals Measured Concentration CO.01 pglL CO.01 p. g_/L 4.02 pgn 4 . 0 1 pg/L 4 . 0 5 pg/L CO.01 pg/L <0.01 p g n -=0.01 p g L 4 . 0 2 pg/L CO.01 pglL <0.02 pg/L ~ 0 . 0 5pg/L 4 . 0 5 pg/L 4 . 0 1 pg/L CO.01 pg/L 4.01 pgn 4 . 0 5 pglL CO.01 pg/L ~ 0 . 0 5pgiL <0.04 p g n 4.01 pgn <0.04 pg/L <0.05 pglL ~ 0 . 0 5pa 4.02 p a 4 . 0 2 pg/L C0.02 pg/L 4.01 pglL 4 . 0 1 pg/L Magnesium Sodium Calcium Iron Potassium Aluminum Manganese Beryllium Chromium Cobalt 730 m a 5,800 mg/L 270 mg/L 4.03m g L 200 mglL 4 . 1 8 mg/L 4 . 0 pg/L 4.0 PgL -6.0 Pg/L 4 0 pg/L Nickel Copper Zinc Molybdenum Silver Cadmium Arsenic Mercury Selenium 4 4 Pg/L -40.0 p g L (20.0 pg/L q . 0 PglL 4 . 0 clgn 4 . 0 Pg/L 1.2 Pg/L c0.025 p g L <1 Pg/L 'Analyses performed by TNO Nutrition and Food Institute on samples collected on November 15,2000. WiId1(fe InternationaI, 11,td. Project Number 454C-117 Appendix 3.4 Freshwater Algal Medum Constituents' Compound MgC12.6HzO CaC12.2H20 H3BO3 hhC12.4H20 ZnCl2 FeCl3.6H20 CoC12.6H20 Na2M004 2H20 CuC12.2H20 Na2EDTA.2H20 NaN03 MgS04.7H20 Kzm04 NaHC03 'The pH was adjusted to 7.5 f 0.1 after ureDaration. Nominal Concentration 12.16 4.40 0.1856 0.416 3.28 0.1598 1.428 mdL mb mg/L mg/L pg/L mg/L pg/L 7.26 0.012 0.300 25.50 14.70 1.044 15.0 pg/L pg/L mg/L mg/L mg/L mg/L mgiL WildltfeInternational? Ltd. - 43 - Appendix 4 . Analytical Method Validation Data Project Number 454C-117 WildlifeInternational, Ltd. - 44 - Project Number 454C-117 Appendix 4.1 Analytical Method Flow Chart for the Analysis of PFBS in Filtered Saltsiater and Freshwater Algal Medium. MXTHOD OUTLINE FOR THE ANALYSIS OF PFBS IN FILTERED SALTWATER AND FRESHWATER ALGAL MEDIUM Prepare matrix fortification samples in algal medium by weighmg the requisite amount of PFBS test substance on an analytical balance and transferring directly into a Class A volumetric flask partially filled with algal medium. Rinse weighmg paper and sides of the flask with repeat algal medium rinses. Swirl the flask to dssolve the test substance and then bring to final volume with algal medium. Sonicate as appropriate and mix with several repeat inversions. The matrix blank is unfortified algal medium. J Prepare matrix fortification samples in saltwater as follows: For target PFBS concentrations I 100 mg a.i./L, fortify the saltwater matrix with the appropriate stock solution of PFBS. For target PFBS concentrations >IO0 mg a.i./L, prepare by weighmg the requisite amount of PFBS on an analytical balance and transferringdirectly into a Class A volumetric flask partially filled with saltwater matrix. Rinse weighmg paper and the sides of the flask with repeat saltwater rinses. Swirl the flask to dissolve the test substance and then bring to final volume with saltwater. Sonicate as appropriate and mix with several repeat inversions. The matrix blank is unfortified saltwater. Dilute samples into the calibrationrange 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 LC/MS analysis. WiIdIife InternationaI? Ltd. -45- Proiect Number 454C- 117 INSTRUMENT: Appendix 4.2 Typical LC/MS Operational Parameters Hewlett-PackardModel 1100High PerformanceLiquidChromatographwith a Perkin-Elmer API 3000LC Mass Spectrometerequipped witha PerlunElmer TurboIonSpray ion source. Operated in selective ion monitoring mode (SIM). ANALYTICAL COLUMN: GUARD COLUMN: Keystone Prism Column (30 mm x 1.5 mm) Keystone Prism (20 mm x 2 mm) OVEN TEMPERATURE: 40C STOP TIME: 3.00 minutes FLOW RATE: 0.200 mL/minute MOBILE PHASE: 75.0% Methanol : 25.0% NANOpure@Water containing 0.1% Ammonium Formate INJECTION VOLUME: 5.0 pL PFBS RETENTION TIME: Approximately 2.2 minutes PFBS MONITORED MASS: 299.0 a m u WildlifeInternational, Ltd. -46- Appendix 4.3 A Typical CalibrationCurve for PFBS Project Number 454C-117 o~I,,ll,,lI,,,,,,II,Il,rll(. 10.0 20.0 30.0 40.0 50.0 Cona. pg a.i./L Slope = 508806048.00; Intercept = 1216876.63; r = 0.99863. WildlifeInternational, Ltd. -47- Proiect Number 454C-117 Appendix 4.4 Example Calculations for a Representative Sample Sample number 454C-117-VMAS-1, nominal concentration of 100 mg a.i./L in freshwater algal medium. First Initial Volume: 0.500 mL First Final Volume: 25.0 mL Second Initial Volume: 0.500 mL Second Final Volume: 50.0 mL Dilution Factor: 5000 PFBS Peak Area: 11667144 Calibration curve equation. Slope: 508806048.00 Intercept: 1216876.63 Curve is weighted (Ux). - -- 11667144 1216876.63 5ooo 508806048.00 = 102.7 PFBS m a.i./L in sam le Percent of Nominal Concentration = PFBS((mt a . i . i l x 100 ---110020.7 x 100 = 103% Instrument Software: MacQuan, version 1.6. WiId1[fe InternationaI? Ltd. -48- Project Number 454C- 117 Appendix 4.5 Ion Chromatogram of a Low-level PFBS Standard intensity: 4500000 cps 6 133 0 21 34 46 67 90 I 1 , , , , , , , , , 31 61 91 121 151 0.52 1.02 1.52 2.03 2.53 Scan Time Nominal concentration: 0.0100 mg a.i./L Wi1d1(fe InternationaI, Ltd. - 49 - Project Number 454C- 117 Appendix 4.6 Ion Chromatogram of a High-level PFBS Standard '"8 3 intensity: 4500000 cps Nominal concentration: 0.0500 mg a.i./L W iId1(fe Internationa1, Ltd. - 50 - Project Number 454C-117 Appendix 4.7 Ion Chromatogram of an Algal Medium Matrix Blank 1001 Intensity: 4500000 cps o! 1*0 , , 4, 4 . 6, 4 , 31 61 0.52 1.02 , 91, , 91 1.52 , . 1,33 , 15, 2 , 1,72 121 151 scan 2.03 2.53 Time Sample number 454C-117-VMAB-1T.he m o w indicates the approximate retention time of PFBS. WildlifeInternational, Ltd. -51 - Proiect Number 454C-117 Appendix 4.8 Ion Chromatogramof a Saltwater Matrix Blank intensity: 4500000 cps 100 '"180 60 1 20 48 69 85 113 132 o'iiiiazi i i i u t , , , , , , 31 61 91 121 151 0.52 1.02 1.52 2.03 2.53 Scan Time Sample number 454C-117-VMAB-4. The arrow indicates the approximate retention time of PFBS. Wildltfe InternationaI , Ltd. -52 - Proiect Number 454C-117 Appendix 4.9 Ion Chromatogram of a Low-level Algal Medium Fortification Sample intensity: 4500000 cps '6 3 45! 3 02 010- 714 41 64 133 101 Sample number 454C-117-VMAS-1, 100 mg a.i./L nominal concentration, dilution factor = 5000. WildlifeInternational, Ltd. - 53 - Project Number 454C-117 Appendix 4.10 Ion Chromatogram of a High-level Algal Medium Fortification Sample intensity: 4500000 cps 301401 302 0 - 133 10- 0 1 15 32 46 61 1 Il 11 .I 1I 1I 1I 1I , I Sample number 454C-117-VMAS-10, 10000mg a.i./L nominal concentration, ddution factor = 500000 -- WildlifeIntemational, Ltd. - 54 - Project Number 454C- 117 Appendix 4.11 Ion Chromatogram of a Low-level Saltwater Fortification Sample 100- 90- 80- 70- 60- 5 0- 40- 30130 20- 10- 0-1 - I I I I I I I I I I I r a I r 1 1 Sample number 454C-117-VMAS-25, 1.00 mg a.i./L nominal concentration, dilution factor = 50 WiId1(fe IntemationaI, Ltd. -55- Project Number 454C- 117 Appendix 4.12 Ion Chromatogramof a High-level Saltwater Fortification Sample intensity: 4500000 cps 2 0- pj,:.L 10- --I l l W b I G . r Sample number 454C-117-VMAS-22,1000mg a.i./L nominal concentration, dilution factor = 50000 c WiId1[fe Internationa1, Ltd. - 56 - Project Number 454C- 117 Appendix 5 Personnel Involved in the Study The followingkey Wildhfe International, Ltd.personnel were involved in the conduct or management of this study: 1. Willard B. Nixon, PbD., Director, Analybcal Chemistry 2. Raymond L. Van Woven, PbD., Scientist 3. Jon A. MacGregor, B.S.,Scientist 4. Frank J. Lezotte, B.S.,Chemist