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SOLUTIONS Weston Solutions, Inc. 1400 Weston Way P.O. Box 2653 West Chester, Pennsylvania 19380 610-701-3000 Fax 610-701-3186 www.westonsolutions.com September 1, 2005 Mr. Michael A. Santoro Director, Regulator/ Affairs 3M Environmental Health Safety & Regulations 3M Center Building 236-1B-10 St. Paul, MN 55144 W. O. No. 02181.129.081 Dear Mike: Enclosed are Exygen's Interim Report #3, "Analysis of Perfluorobutanesulfonate (PFBS), Perfluorohexanesulfonate (PFHS) and Perfluorooctanesulfonate (PFOS) in Water, Soil, Sediment, Fish, Clams, Vegetation, Small Mammal Liver and Small Mammal Serum Using LCMSMS", and raw data packages for the Decatur, Alabama, Monitoring Progfirrr'Please'S'tgfi^he marked pages and then forward the complete package to the 3M Laboratory for archival. Shoultryou have any auditions, please do not hesitate to contact me at 610-701-3761. Very truly yours, WES )N SOLUTIONS, INC. Jaifthha Kesari, P.E., DEE Principal Project Manager Enclosures P'ECON\ECON_530'FOLDERSA)-9.3M-DECATSamoro-13.doc INTERIM REPORT # 3 - Analysis of Ground Water Samples STUDY TITLE Analysis of Perfluorobutanesulfonate (PFBS), Perfluorohexanesulfonate (PFHS), and Perfluorooctanesulfonate (PFOS) in Water, Soil, Sediment, Fish, Clams, Vegetation, Small Mammal Liver and Small Mammal Serum Using LC/MS/MS for the 3M Decatur Monitoring Program DATA REQUIREMENTS EPA TSCA Good Laboratory Practice Standards 40 CFR 792 STUDY DIRECTOR Jaisimha Kesari P.E., DEE Weston Solutions, Inc. 1400 Weston Way West Chester, PA 19380 Phone: 610-701-3761 INTERIM REPORT COMPLETION DATE August 30, 2005 PERFORMING LABORATORY Exygen Research 3058 Research Drive State College, PA 16801 Phone: 814-272-1039 STUDY SPONSOR 3M Company 3M Building 0236-01-B-10 St. Paul, MN 55144 Phone: 651-733-6374 PROJECT Protocol Number: P0001131 Exygen Study Number: P0001131 Total Pages: 105 Interim Report #3-Analysis o f Ground Water Samples Exygen Study No.: P0001131 GOOD LABORATORY PRACTICE COMPLIANCE STATEMENT Exygen Study Number P0001131, entitled "Analysis of Perfluorobutanesulfonate (PFBS), Perfluorohexanesulfonate (PFHS), and Perfluorooctanesulfonate (PFOS) in Water, Soil, Sediment, Fish, Clams, Vegetation, Small Mammal Liver and Small Mammal Serum Using LC/MS/MS for the 3M Decatur Monitoring Program," conducted for 3M Company, is being performed in compliance with EPA TSCA Good Laboratory Practice Standards 40 CFR 792 by Exygen Research. Principal Investigator Exygen Research Jaisimh Study Director Weston Solutions, Inc. /M jJ Michael A. Sam Sponsor Repres 3M Company Exygen Research Date Page 2 o f 105 Interim Report #3-Analysis o f Ground Water Samples Exygen Study No.: P0001131 QUALITY ASSURANCE STATEMENT Exygen Research's Quality Assurance Unit reviewed Exygen Study Number P0001131, entitled, "Analysis of Perfluorobutanesulfonate (PFBS), Perfluorohexanesulfonate (PFHS), and Perfluorooctanesulfonate (PFOS) in Water, Soil, Sediment, Fish, Clams, Vegetation, Small Mammal Liver and Small Mammal Serum Using LC/MS/MS for the 3M Decatur Monitoring Program". All reviewed phases1 were inspected for conduct according to Exygen Research's Standard Operating Procedures, the Study Protocol, and all applicable Good Laboratory Practice Standards. All findings were reported to the Exygen Principal Investigator and Management and to the Study Director. Phase Date InsDected Date Reported to Date Reported to Principal Exygen Date Reported to Investigator Management Studv Director 9. Raw Data Review and Interim Analytical Report Review 14. Interim Analytical Report Review 20. Interim Analytical Report Review 04/07/05 07/06/05 08/29/05 06/07/05 07/07/05 08/30/05 6/30/05 07/07/05 08/30/05 07/08/05 07/08/05 08/30/05 'Note: All in-lab inspections will be documented in the QA statement for the final analytical report at the conclusion of the study. This QA statement involves only the review of the interim report and associated raw data. Exygen Research Page 3 of 105 Interim Report #3-Analysis of Ground Water Samples Exygen Study No.: P0001131 CERTIFICATION OF AUTHENTICITY This interim report, for Exygen Study Number P0001131, is a true and complete representation of the raw data. Submitted by: Exygen Research 3058 Research Drive State College, PA 16801 (814)272-1039 Principal Investigator, Exygen: Date Exygen Research Facility Management: Richard A. Grazzini President Exygen Research Study Director, Weston Solutions, Inc. Jaisimha|gjsari P.E., DEE Weston Solutions, Inc. Sponsor Representative, 3M Company: t//A/C/kr<f <h Michael A. Santolo Director of Regulatory Affairs Exygen Research Date 1h i t s Ditte/ Page 4 of 105 Interim Report #3-Analysis o f Ground Water Samples Exygen Study No.: P0001131 STUDY IDENTIFICATION Analysis of Perfluorobutanesulfonate (PFBS), Perfluorohexanesulfonate (PFHS), and Perfluorooctanesulfonate (PFOS) in Water, Soil, Sediment, Fish, Clams, Vegetation, Small Mammal Liver and Small Mammal Serum Using LC/MS/MS for the 3M Decatur Monitoring Program PROTOCOL NUMBER: P0001131 EXYGEN STUDY NUMBER: P0001131 TYPE OF STUDY: Residue SAMPLE MATRIX: Ground Water TEST SUBSTANCE: Perfluorobutanesulfonate (PFBS), Perfluorohexanesulfonate (PFHS), and Perfluorooctanesulfonate (PFOS) SPONSOR: 3M Company 3M Building 0236-01-B-10 St. Paul, MN 55144 STUDY DIRECTOR: Jaisimha Kesari P.E., DEE Weston Solutions, Inc. 1400 Weston Way West Chester, PA 19380 STUDY MONITOR: Michael A. Santoro 3M Company 3M Building 0236-01-B-10 St. Paul, MN 55144 PERFORMING LABORATORY: Exygen Research 3058 Research Drive State College, PA 16801 ANALYTICAL PHASE TIMETABLE: Study Initiation Date: 11/05/04 Interim Analytical Start Date: 02/16/05 Interim Analytical Termination Date: 03/04/05 Interim Report Completion Date: 08/30/05 Exygen Research Page 5 o f 105 Interim Report #3-Analysis o f Ground Water Samples Exygen Study No.: P0001131 PROJECT PERSONNEL The Study Director for this project is Jaisimha Kesari at Weston Solutions, Inc. The following personnel from Exygen Research were associated with various phases of this interim portion of the study: Name John Flaherty Karen Risha Paul Connolly Chrissy Edwards Mark Ammerman Amy Sheehan Eric Edwards Brittany Kravets Title Vice President Scientist Technical Lead-LC/MS Technician Sample Custodian Associate Scientist Sample Custodian Technician Exygen Research Page 6 of 105 Interim Report #3-Analysis o f Ground Water Samples Exygen Study No.: P0001131 TABLE OF CONTENTS Page TITLE PAGE.......................................................................................................................1 GOOD LABORATORY PRACTICE COMPLIANCE STATEMENT............................. 2 QUALITY ASSURANCE STATEMENT......................................................................... 3 CERTIFICATION OF AUTHENTICITY.......................................................................... 4 STUDY IDENTIFICATION............................................................................................... 5 PROJECT PERSONNEL.................................................................................................... 6 TABLE OF CONTENTS.................................................................................................... 7 LIST OF TABI.ES.............................................................................................................. 8 LIST OF FIGURES............................................................................................................. 9 LIST OF APPENDICES....................................................................................................10 1.0 SUMMARY................................................................................................................11 2.0 OBJECTIVE...............................................................................................................11 3.0 INTRODUCTION.......................................................................................................11 4.0 ANALYTICAL TEST SAMPLES..............................................................................12 5.0 REFERENCE MATERIAL........................................................................................12 6.0 DESCRIPTION OF ANALYTICAL METHOD........................................................13 6.1. Extraction Procedure...............................................................................................13 6.2 Preparation of Standards and Fortification Solutions.............................................. 14 6.3 Chromatography......................................................................................................14 6.4 Instrument Sensitivity..............................................................................................15 6.5 Description of LC/MS/MS Instrument and Operating Conditions..........................15 6.6 Quantitation and Example Calculation....................................................................16 7.0 EXPERIMENTAL DESIGN......................................................................................17 8.0 RESULTS...................................................................................................................17 9.0 CONCLUSIONS.........................................................................................................17 10.0 RETENTION OF DATA AND SAMPLES.............................................................17 Exygen Research Page 7 of 105 Interim Report #3-Analysis o f Ground Water Samples Exygen Study No.: P0001131 Table I. LIST OF TABLES Page Summary of PFBS, PFHS and PFOS in Ground Water Samples...................19 Table II. Matrix Spike Recovery of PFBS, PFHS and PFOS in Ground Water Samples............................................................................................... 21 Exygen Research Page 8 o f 105 Interim Report #3-Analysis o f Ground Water Samples Exygen Study No.: P0001131 Figure 1. LIST OF FIGURES Page Typical Calibration Curve for PFBS in Reagent Water................................. 26 Figure 2. Extracted Standards of PFBS in Reagent Water, 0 ng/L and 25 ng/L, Respectively................................................................................................... 27 Figure 3. PFBS in Reagent Water, 500 ng/L Fortified Reagent Water, and 5000 ng/L (DF=10) Fortified Reagent Water, Respectively.......................... 28 Figure 4. Chromatogram Representing a Ground Water Sample Analyzed for PFBS (Exygen ID: C0059083, Data Set: 021805C)................................ 29 Figure 5. Typical Calibration Curve for PFHS in Reagent W ater................................ 30 Figure 6. Extracted Standards of PFHS in Reagent Water, 0 ng/L and 25 ng/L, Respectively................................................................................................... 31 Figure 7. PFHS in Reagent Water, 500 ng/L Fortified Reagent Water, and 5000 ng/L (DF=10) Fortified Reagent Water, Respectively.......................... 32 Figure 8. Chromatogram Representing a Ground Water Sample Analyzed for PFHS (Exygen ID: C0059083, Data Set: 021805C)................................ 33 Figure 9. Typical Calibration Curve for PFOS in Reagent W ater................................ 34 Figure 10. Extracted Standards of PFOS in Reagent Water, 0 ng/L and 25 ng/L, Respectively....................................................................................................35 Figure 11. PFOS in Reagent Water, 500 ng/L Fortified Reagent Water, and 5000 ng/L (DF=10) Fortified Reagent Water, Respectively.......................... 36 Figure 12. Chromatogram Representing a Ground Water Sample Analyzed for PFOS, DF=50 (Exygen ID: C00059083, Data Set: 021805CR).............. 37 Exygen Research Page 9 of 105 Interim Report #3-Analysis of Ground W ater Samples Exygen Study No. : P0001131 LIST OF APPENDICES Appendix A Study Protocol P0001131 (Exygen Study No. P0001131) with Analytical Method: V0001780:"Method of Analysis for the Determination of Perfluorooctanoic Acid (PFOA) in Water by LC/MS/MS" .............................................................................. Page 38 Exygen Research Page 10 of 105 Interim Report #3-Analysis o f Ground Water Samples Exygen Study No.: P0001131 1.0 SUMMARY Exygen Research extracted and analyzed ground water samples for the determination of perfluorobutanesulfonate (PFBS), perfluorohexanesulfonate (PFHS), and perfluorooctanesulfonate (PFOS) according to Exygen Method V0001780 (Appendix A). The limit of quantitation for PFBS, PFHS and PFOS in water was 50 ng/L and the limit of detection was 25 ng/L. Analytical results for the analysis of PFBS, PFHS and PFOS found in ground water samples are summarized in Table 1. The overall average percent recoveries standard deviations for field and laboratory spikes for PFBS, PFHS, PFOS in ground water samples were 165% 21%, 104% + 15%, and 112% 28%, respectively. 2.0 OBJECTIVE The objective of the analytical part of this study was to determine levels of perfluorobutanesulfonate (PFBS), perfluorohexanesulfonate (PFHS), and perfluorooctanesulfonate (PFOS) in ground water according to Protocol P0001131 (Appendix A). 3.0 INTRODUCTION This report details the results of the analysis for the determination of PFBS, PFHS and PFOS in ground water using the analytical method entitled, "V0001780: Method of Analysis for the Determination of Perfluorooctanoic Acid (PFOA) in Water by LC/MS/MS." The study was initiated on November 05, 2004, when the study director signed protocol number P0001131. The analytical start date for this interim report was February 16, 2005, and the analytical termination date for this interim report was March 04,2005. Exygen Research Page 11 o f 105 Interim Report #3-Analysis of Ground Water Samples Exygen Study No. : P0001131 4.0 ANALYTICAL TEST SAMPLES One hundred and twenty-one ground water samples (Exygen ID C0058994-C0059024, C0059033, C0059037, C0059039, C0059042, C0059044-C0059047, C0059049C0059055, C0059057-C0059061, C0059063-C0059067, C0059070-C0059074, C0059078-C0059137) from twenty-three ground water sampling sites along with two field blanks, were received on wet ice on January 27, 2005 and January 28, 2005 from Tim Frinak at Weston Solutions, Inc. The samples were logged in by Exygen personnel and placed in refrigerated storage. Sample log-in and chain of custody information is located in the raw data package associated with this interim report. Storage records will be kept at Exygen Research. 5.0 REFERENCE MATERIAL The analytical standards, PFBS and PFHS, were supplied by 3M. PFBS was received from 3M at Exygen on July, 06, 2000. PFHS was received from 3M at Exygen on January, 20, 2003. PFOS was purchased from Fluka Corporation and was received at Exygen on April, 23,2003. The available information for the reference materials is listed below. PFBS and PFHS were stored frozen and PFOS was stored refrigerated. Compound PFBS PFHS PFOS Exygen Inventory No.. SP0000252 SP0002401 SP0002694 Lot # 101 SE036 430180-1 Purity (%) Expiration Date 96.7 12/04/06 98.6 10/18/06 101.2 10/31/07 The molecular structures of PFBS, PFHS and PFOS are given below: PFBS Chemical Name: Perfluorobutanesulfonate Molecular Weight: 338 supplied as the potassium salt ^FgSOsTC*) Transitions Monitored: 299 -- 99 Structure: FF FF F SO3 F FF Exygen Research Page 12 of 105 Interim Report #3-Analysis o f Ground W ater Samples Exygen Study No.: P0001131 PFHS Chemical Name: Perfluorohexanesulfonate Molecular Weight: 438 supplied as the potassium salt (CeFnSOsTC1-) Transitions Monitored: 399 - 80 Structure: FFF FFF F SO3 FFF FFF PFOS Chemical Name: Perfluorooctanesulfonate Molecular Weight: 538 supplied as the potassium salt (CsFnSOa'K'1') Transitions Monitored: 499 - 80 Structure: FFFF FFFF F SO3 FFFF FFFF 6.0 DESCRIPTION OF ANALYTICAL METHOD The analytical method "V0001780: Method of Analysis for the Determination of Perfluorooctanoic Acid (PFOA) in Water by LC/MS/MS" was used for this study. 6.1. Extraction Procedure A 40 mL aliqxiot of the water sample was used for the extraction procedure. After fortification of appropriate samples, the samples were loaded onto a Ci8 SPE cartridge conditioned with 10 mL of methanol and 5 mL of water. The eluate was discarded. Approximately five milliliters of methanol was added to the cartridge. Five milliliters of eluate was collected into a graduated 15 mL polypropylene centrifuge tube. Each sample was analyzed by LC/MS/MS electrospray. Exygen Research Page 13 o f 105 Interim Report #3-Analysis o f Ground Water Samples Exygen Study No. : P0001131 6.2 Preparation of Standards and Fortification Solutions Individual stock standard solutions of PFBS, PFHS and PFOS were prepared as specified in Exygen method V0001780. The stock standard solutions were prepared at a concentration of 100 pg/mL by dissolving 10 mg of each of the standards (corrected for purity and salt content) in methanol. From these solutions, mixed 1.0 pg/mL fortification standard solutions were prepared by taking 1 mL of each of the appropriate stock solutions and bringing the volume up to 100 mL with methanol. By taking 10 mL of the mixed 1.0 pg/mL fortification standard and bringing the volume up to 100 mL with methanol, a mixed 0.1 pg/mL fortification standard was prepared. By taking 10 mL of the mixed 0.1 pg/mL fortification standard and bringing the volume up to 100 mL with methanol, a mixed 0.01 pg/mL fortification standard was prepared. A set of standards containing PFBS, PFHS and PFOS was prepared in water and processed through the extraction procedure, identical to samples. The following concentrations were prepared: Cone, of Fort Fort Solution Volume (ng/mL)1 (PL) 00 10 100 10 200 10 400 100 100 100 200 100 400 1of PFBS, PFHS and PFOS Volume of Fortified Sample (mL) 40 40 40 40 40 40 40 Final Cone, of Calibration Std. (ng/L) 0 25 50 100 250 500 1000 The stock standard solution and all fortification and calibration standard solutions were stored in a refrigerator (4 2C) when not in use. Documentation of standard preparation is located in the raw data package associated with this interim report. 6.3 Chromatography Quantification of PFBS, PFHS and PFOS was accomplished by LC/MS/MS electrospray. The retention times of PFBS, PFHS and PFOS were ~ 4.7 mins, ~ 10.8 mins, and ~ 12.9 mins, respectively. Peaks above the LOD were not detected in any of the reagent blank samples corresponding to the analyte retention time. Exygen Research Page 14 of 105 Interim Report #3-Analysis o f Ground Water Samples Exygen Study No. : P0001131 6.4 Instrument Sensitivity The smallest standard amount injected during the chromatographic run had a concentration of 25 ng/L of PFBS, PFHS and PFOS. 6.5 Description of LC/MS/MS Instrument and Operating Conditions Instrument: API 4000 Biomolecular Mass Analyzer Interface: Turbo Ion Spray Liquid Introduction Interface Computer: DELL OptiPlex GX400 Software: Windows NT, Analyst 1.4.1 HPLC: Hewlett Packard (HP) Series 1100 HP Quat Pump HP Vacuum Degasser HP Autosampler HP Column Oven HPLC Column: Thermo Fluophase RP, 50 mm x 2.1 mm Column Temp.: 30 C Injection Voi.: 15 pL Mobile Phase (A): 2 mM Ammonium Acetate in water Mobile Phase (B): Methanol Time ('mini 0.0 1.0 8.0 10.0 11.0 18.0 Total run time: ~18min %A 65 65 25 25 65 65 %B 35 35 75 75 35 35 Flow Rate: 0.3 mL/min Ions monitored: Analvte PFBS PFHS PFOS Mode negative negative negative Transition Monitored 299 -> 99 399 -80 499 80 Approximate Retention Time ("mini -4.7 min. -10.8 min. -12.9 min. Exygen Research Page 15 of 105 Interim Report #3-Analysis o f Ground Water Samples Exygen Study No.: P0001131 6.6 Quantitation and Example Calculation Fifteen microliters of sample or calibration standard were injected into the LC/MS/MS. The peak area was measured and the standard curve was generated (using 1/x fit weighted linear regression) by Analyst software using six concentrations of standards, except in a few cases of PFBS, where only five were used when the 500 ng/L standard results were rejected as outliers. The concentration was determined from the equations below. Equation 1 calculated the amount of analyte found (in ng/L, based on peak area) using the standard curve (linear regression parameters) generated by the Analyst software program. Equation 1: Analyte found (ng/L) = (Peak area - intercept) x DF slope Where: DF = Dilution Factor, factor by which the final volume was diluted, if necessary. For samples fortified with known amounts of PFBS, PFHS and PFOS prior to extraction, Equation 2 was used to calculate the percent recovery. Equation 2: Recovery (%) = (analyte found (ng/L) - analyte in corresponding sample (ng/L)) xl00% amount added (ng/L) An example of a calculation using an actual sample follows (calculation is for PFOS only): Water sample Exygen ID: C0058999 Spk F (Set: 021605C), fortified at 50000 ng/L with where: peak area = 331767 intercept = 8700 slope = 466 dilution factor = 100 ng/L PFOA added (fort level) = 50000 amt in corresponding sample = 28600 From equation 1: Analyte found (ng/L) = [331767 - 87001 x 100 466 From equation 2: % Recovery 69328 ng/L (69328 ne/L - 28600 ng/LI x 100% 50000 ng/L = 81% Exygen Research Page 16 o f 105 Interim Report #3-Analysis o f Ground Water Samples Exygen Study No. : P0001131 7.0 EXPERIMENTAL DESIGN For samples designated as field matrix spikes, PFBS, PFHS and PFOS were added at a known concentration to the bottles in the laboratory before being shipped to the field. The samples were filled to a 200 mL volumetric fill line in the field. The samples were extracted in four sets. Each set included one reagent blank and two reagent blanks fortified at known concentrations. Three sets contained six sample sites each and the fourth set contained five sample sites, along with all the field blanks and field blank spikes collected for the ground water samples. For each site, a sample, a field duplicate and three-matrix field spikes were collected. Only the two lowest field matrix spikes were analyzed for each sample site. For each site, a laboratory duplicate was extracted and two laboratory matrix spikes were also extracted. 8.0 RESULTS Analytical results for PFBS, PFHS and PFOS in ground water samples are summarized in Table I. Fortification recoveries for PFBS, PFHS and PFOS in the water samples and the assessed accuracy for each sample are detailed in Table II. The average percent recoveries standard deviations for PFBS, PFHS, PFOS in water samples were 165% 21%, 104% 15%, and 112% 28%, respectively. 9.0 CONCLUSIONS The ground water samples were successfully extracted and analyzed for PFBS, PFHS and PFOS according to analytical method V0001780. 10.0 RETENTION OF DATA AND SAMPLES All original paper data generated by Exygen Research that pertains to this interim report will be shipped to the sponsor. This does not include facility-specific raw data such as instrument or temperature logs. Exact copies of all raw data, as well as a signed copy of the final analytical report and all original facility-specific raw data, will be retained in the Exygen Research archives for the period of time specified in EPA TSCA Good Laboratory Practice Standards 40 CFR 792. Exygen Research Page 17 o f 105 Interim Report #3-Analysis o f Ground Water Samples Exygen Study No. : P0001131 TABLES Exygen Research Page 18 of 105 Interim Report #3-Analysis of Ground Water Samples Exygen Study No.: P0001131 Table I. Summary of PFBS, PFHS and PFOS in Ground Water Samples ExygenID C0058994 C0058994 Rep C0058995 C0058999 C0058999Rep C0059000 C0059004 C0059004 Rep C0059005 C0059009 C0059009Rep C0059010 C0059014 C0059014 Rep C0059015 C0059033 C0059033 Rep C0059037 C0059045 C0059045 Rep C0059046 C0059051 C0059051 Rep C0059052 C0059057 C0059057 Rep C0059058 C0059063 C0059063 Rep C0059064 C0059070 C0059070 Rep C0059071 C0059078 C0059078Rep C0059079 C0059083 C0059083 Rep C0059084 C0059088 C0059088Rep C0059089 C0059093 C0059093 Rep C0059094 C0059098 C0059098 Rep C0059099 Client Sample ID DF09-GW-130L-0-040126 DF09-GW-130L-0-040126* DF09-GW-130L-0-040126 Dup DF09-GW-131L-0-040126 DF09-GW-131L-0-040126* DF09-GW-131L-0-040126 Dup DF06-GW-132L-0-040126 DF06-GW-132L-0-040126* DF06-GW-132L-0-040126 Dup DF06-GW-133L-0-040126 DF06-GW-133L-0-040126* DF06-GW-133L-0-040126 Dup DF8b-GW-134L-0-040126 DF8b-GW-134L-0-040126* DF8b-GW-134L-0-040126 Dup DF09-GW-131R-0-040120 DF09-GW-131R-0-040120* DF09-GW-131R-0-040120 Dup DF09-GW-131S-0-040120 DF09-GW-131S-0-040120* DF09-GW-131S-0-040120 Dup DF06-GW-132R-0-040120 DF06-GW-132R-0-040120* DF06-GW-132R-0-040120 Dup DF06-GW-132S-0-040120 DF06-GW-132S-0-040120* DF06-GW-132S-0-040120 Dup DF06-GW-133R-0-040120 DF06-GW-133R-0-040120* DF06-GW-133R-0-040120 Dup DF06-GW-133S-0-040120 DF06-GW-133S-0-040120* DF06-GW-133S-0-040120 Dup DF8b-GW-134R-0-040121 DF8b-GW-134R-0-040121* DF8b-GW-134R-0-040121 Dup DF8b-GW-134S-0-040121 DF8b-GW-134S-0-040121* DF8b-GW-134S-0-040121 Dup DF8b-GW-135R-0-040121 DF8b-GW-135R-0-040121* DF8b-GW-135R-0-040121 Dup DF8b-GW-135S-0-040121 DF8b-GW-135S-0-040121* DF8b-GW-135S-0-040121 Dup DF8b-GW-135L-0-040121 DF8b-GW-135L-0-040121* DF8b-GW-135L-0-040121 Dup C4 Sulfonate PFBS P e rflu o ro b u ta n e s u ffo n a te 2360 2370 2380 4510 4660 4510 2360 2300 2320 7390 7610 7590 7460 7620 7450 129000 136000 135000 7470 7370 7330 23000 21300 21600 32400 32600 33100 2180 2190 2170 10400 9980 10600 151000 145000 144000 71.0 68.0 66.0 146000 145000 151000 47200 47300 48700 38000 37600 37200 Analyte Found (ppt, ng/L) C6 Sulfonate PFHS Perfluoro haxanasu Ifonate 10500 10300 11400 15700 15500 15200 12200 12100 11900 26000 26000 25900 22200 21600 22100 373000 375000 364000 19300 19600 19400 77900 69200 69600 169000 170000 172000 7470 7420 7590 38000 35100 38000 774000 757000 723000 463 436 452 705000 727000 726000 220000 219000 229000 187000 183000 170000 C8 Sulfonate PFOS P arfluorooctanesu lionate 63800 63900 86900 28600 28300 30200 42800 42900 45000 91400 89400 88400 38600 39500 47800 2340000 2420000 2140000 110000 105000 103000 573000 491000 515000 499000 506000 510000 31800 32500 34800 105000 94600 105000 1320000 1330000 1260000 7930 7410 7190 592000 603000 559000 82700 85600 98500 151000 151000 134000 ` Laboratory Duplicate ND = Not detected at or above 25 ng/L. NQ = Not quantifiable = Measured concentration between 25 ng/L and the Lim it of Quantitation (LO Q ) which is 50 ng/L. NOTE: The assessed accuracy value for each sample can be found In Table II. Exygen Research Page 19 o f 105 Interim Report #3-Analysis o f Ground W ater Samples Exygen Study No.: P0001131 Table I. Summary of PFBS, PFHS and PFOS in Ground Water Samples Continued ExygenID C0059103 C0059103 Rep C0059104 C0059108 C0059108 Rep C0059109 C0059113 C0059113Rep C0059114 C0059118 C0059118 Rep C0059119 C0059123 C0059123Rep C0059124 C0059128 C0059128 Rep C0059129 C0059133 C0059133 Rep C0059134 C0059019 C0059022 Client Sample ID DF09-GW-130R-0-040121 DF09-GW-130R-0-040121* DF09-GW-130R-0-040121 Dup DF09-GW-130S-0-040121 DF09-GW-130S-0-040121* DF09-GW-130S-0-040121 Dup DF14-GW-136R-0-040121 DF14-GW-136R-0-040121 * DF14-GW-136R-0-040121 Dup DF14-GW-136S-0-040121 DF14-GW-136S-0-040121* DF14-GW-136S-0-040121 Dup DF14-GW-136L-0-040121 DF14-GW-136L-0-040121* DF14-GW-136L-0-040121 Dup DF14-GW-137S-0-040121 DF14-GW-137S-0-040121* DF14-GW-137S-0-040121 Dup DF14-GW-137L-0-040121 DF14-GW-137L-0-040121* DF14-GW-137L-0-040121 Dup Field Blank Field Blank C4 Sulfonate PFBS P a rfluo ro b utan e su tfo n a te 22900 23700 23000 13500 14000 13000 204 187 207 3150 3560 3190 5490 5040 5080 101 94.0 102 75.0 83.0 80.0 ND ND Analyte Found (ppt, ng/L) C6 Sulfonate PFHS P a rfluo ro h exa n e sutfo n ata 98600 97500 102000 47700 50300 48300 198 187 211 12600 14700 13400 21100 20400 20900 87.0 84.0 85.0 74.0 81.0 91.0 ND ND C8 Sulfonate PFOS P a rflu o ro o c ta n a s u lfo n a te 440000 434000 466000 62600 66100 61300 1710 1780 2000 89000 101000 92100 135000 132000 141000 339 344 358 1120 1170 1470 ND ND ` Laboratory Duplicate ND = Not detected at or above 25 ng/L. NQ = Not quantifiable = Measured concentration between 25 ng/L and the Lim it of Quantitation (LO Q ) which is 50 ng/L. NOTE: The assessed accuracy value for each sample can be found in Table II. Exygen Research Page 20 o f 105 Interim Report #3-Analysis o f Ground Water Samples Exygen Study No.: P0001131 Table II. Matrix Spike Recovery of PFBS, PFHS and PFOS in Ground Water Samples Sam ple D e scrip tio n DF09-GW -130L-0-040126 (C00MM4Spk C, 5000 r0/t.Spike) D F09-G W -130L-0-040126 (C005SM4Spk D, 50000 ng/LSpike) DF09-GW-130L-0-040126 5 ppb Spk (C005SMS. 5000 ng/LSpike) DF09-GW-130L-0-040126 SO ppb Spk (00056097,50000 ng/LSpike) Amount Spiked (nj .) 5000 50000 5000 50000 C4 Sulfonate PFBS___________________________ C6 Sulfonate PFHS______________________ C8 Sulfonate PFOS Amount Found In Amount Amount Found in Amount Amount Aeeeeeed Found in Amount Aeeeeeed Sample Recovered Ree Accuracy Sample Recovered Ree Accuracy Sample Recovered Ree Accuracy (nfl/L) ("O'1-) (%> (%/%> fngrt.) (ng/L) (%) <%/%> (nan.) (ng/L) (%/-%) 2360 9290 139 100/50 10500 15800 106 30/30 63800 76900 30/30 2360 73700 143 100/50 10500 53100 85 30/30 63800 121000 114 30/30 2360 11500 183 100/50 10500 16300 116 30/30 63800 82500 30/30 2360 81600 158 100/50 10500 61200 101 30/30 63600 159000 190 30/30 DF06-GW-131L-0-040126 (00058696Spk E, 5000 ng/L.Spike) DF09-GW-131 L-0-040126 (C0056666Spk F, 50000 ng/LSpike) DF09-GW-131L-0-040126 5 ppb Spk (C0056001,5000 ng/LSpike) DF06-GW-131L-0-040126 50 ppb Spk (C0059002, 50000 ng/LSpike) 5000 50000 5000 50000 4510 4510 4510 4510 12500 160 100/50 15700 76600 144 100/50 15700 14600 202 100/50 15700 89000 169 100/50 15700 20900 57900 21400 66400 104 30/30 84 30/30 114 30/30 101 30/30 28600 28600 28600 28600 36500 30/30 69300 81 30/30 37000 30/30 85100 113 30/30 D F06-GW -132L-0-040126 (C0096004Spk 0.5000 ng/LSpike) DF06-GW-132L-0-040126 (C0056004Spk H, 50000 ng/L Spike) DF06-GW-132L-0-040126 5 ppb Spk (C0050006,5000 ng/LSpike) DF06-GW-132L-0-040126 50 ppb Spk (C0056007,50000 ng/L Spike) 5000 50000 5000 50000 2360 2360 2360 2360 9460 142 100/50 12200 75600 146 100/50 12200 11700 187 100/50 12200 82000 159 100/50 12200 16300 55300 16200 57700 82 30/30 86 30/30 80 30/30 91 30/30 42800 42800 42800 42800 45000 . 30/30 79500 73 30/30 44500 30/30 77700 70 30/30 DF06-G W -133L-0-040126 (C0056006 Spk 1.5000ng/LSpike) DF06-GW-133L-0-040126 (C0056006Spk J, 50000 ng/l.Spike) DF06-GW-133L-0-040126 5 ppb Spk (C0056011,5000 ng/LSpike) DF06-GW-133L-0-040126 50 ppb Spk (00056012,50000 ng/LSpike) 5000 50000 5000 50000 7390 7390 7390 7390 15100 154 100/50 26000 94100 173 100/50 26000 17400 200 100/50 26000 95600 170 100/50 26000 29800 68500 32600 83100 30/30 85 30/30 * 30/30 114 30/30 91400 91400 91400 91400 83700 . 30/30 130000 77 30/30 117000 . 30/30 158000 133 30/30 D F8b-G W -134L-0-040126 (C0059014 Spk K. 5000ngfl..Spike) DF8b-GW-134L-0-O4O126 (C0056014Spk L. 50000 ng/l. Spike) DF6b-GW-134L-0-040126 5 ppb Spk (C0059016,5000 ng/LSpike) DF8b-GW-134L-0-040126 50 ppb Spk (C0056017,50000 ngfl.Spike) 5000 50000 5000 50000 7460 7460 7460 7460 15700 165 100/50 22200 82200 149 100/50 22200 16800 187 100/50 22200 84200 153 100/50 22200 27300 68100 30400 69600 . 30/30 92 30/30 30/30 95 30/30 38600 38600 38600 38600 54800 30/30 81900 87 30/30 59600 30/30 86800 96 30/30 DF09-GW-131R-0-040120 (00056033 Spk M,50000 ng/LSpHte) 50000 DF09-GW-131R-0-040120 (C0056033Spk N, 500000ngfL Spike) 500000 DF09-GW-131R-0-040120 SO ppb Spk (0005*039,50000 ng/LSpike) 50000 DF09-GW-131R-0-040120 500 ppb Spk (C0056042,500000 ng/LSpike) 500000 129000 129000 129000 129000 214000 170 100/50 373000 908000 156 100/50 373000 226000 194 100/50 373000 972000 169 100/50 373000 437000 . 30/30 2340000 2910000 876000 101 30/30 2340000 3010000 * 441000 30/30 2340000 2710000 . 961000 118 30/30 2340000 3490000 * 100/50 100/50 100/50 100/50 *Sam ple residue exceeds the spiking level significantly; therefore, an accurate recovery value cannot be calculated ND = Not detected at o r above 25 ng/L (ppt). NQ = Not quantifiable = M easured concentration between 25 ng/L (ppt) and the Lim it of Quantitation (LO Q ) which Is 50 ng/L (ppt). Note: Since this summarytable shows rounded results, recovery values may vary slightly from the values in the raw data. Exygen Research Page 21 o f 105 III Interim Report #3-Analysis o f Ground Water Samples Exygen Study No.: P0001131 Table II. Matrix Spike Recovery of PFBS, PFHS and PFOS in Ground Water Samples Continued Sam ple D escrip tio n DF09-GW -131S-0-040120 (C005S0498pk C, 50000ngfLSpike) DF09-GW-131S-0-040120 (C005S045Spk D.500000ng/LSpike) DF09-GW-131S-0-040120 50 ppb Spk (C0050047,50000 ng/LSpike) DF09-GW-131S-0-040120 500 ppb Spk (C0050049,500000 ng/LSpike) Amount Spiked (nan-) 50000 500000 50000 500000 C4 Sulfonate PFBS_____________________CS Sulfonate PFHS_____________________ CS Sulfonate PFOS Amount Amount Amount Found In Amount Found In Amount Found In Amount Sample Recovered Ree Accuracy Sample Recovered Ree Accuracy Sample Recovered Ree (ng/L) (ng/L) (%/-%) (ng/L) (ngfl.) w (+%/-%> (ng/L) <ng/L) 7470 85000 155 100/50 19300 68500 98 30/30 110000 154000 88 30/30 7470 867000 172 100/50 19300 539000 104 30/30 110000 858000 110 30/30 7470 95400 176 100/50 19300 75900 113 30/30 110000 164000 108 30/30 7470 1090000 217 100/50 19300 680000 132 30/30 110000 983000 175 30/30 D F06-GW -132R-0-040120 (C00S50S1 Spk E, 50000 ng/LSpike) DF06-GW-132R-0-040120 (C005S051 Spk F, 500000 ng/LSpike) DF06-GW-132R-0-040120 50 ppb Spk (C00S00S3,50000 ng/LSpike) DF06-GW-132R-0-040120 500 ppb Spk (C005S054,500000 ng/L Spike) 50000 500000 50000 500000 23000 23000 23000 23000 98100 150 100/50 77900 870000 169 100/50 77900 118000 190 100/50 77900 903000 176 100/50 77900 125000 94 30/30 573000 672000 30/30 598000 104 30/30 573000 1190000 123 30/30 137000 118 30/30 573000 722000 30/30 635000 111 30/30 573000 1360000 157 30/30 DF06-GW -132S-0-040120 (C00SS057Spk 0.50000 ng/LSpike) D F06-GW -132S-0-040120 (C005S057 Spk H. 500000 ngfl. Spike) DF06-GW-132S-0-040120 50 ppb Spk (C005S050,50000 ng/L Spike) DF06-GW-132S-0-040120 500 ppb Spk (C005S0S0.500000 ngfl.Spike) 50000 500000 50000 500000 32400 32400 32400 32400 118000 171 100/50 169000 224000 110 30/30 499000 569000 50/50 842000 162 100/50 169000 694000 105 30/30 499000 1270000 154 50/50 129000 193 100/50 169000 232000 126 30/30 499000 672000 50/50 939000 181 100/50 169000 768000 124 30/30 499000 1270000 154 50/50 DF05-GW-133R-0-040120 (C00500S3 Spk 1,50000ng/LSpike) DF06-GW -133R4-040120 (C005S0S5Spk J, 500000 ng/LSpfce) DFOS-GW-133R-0-040120 50 ppb Spk (C00550S5,50000 ngl. Spike) DF06-GW-133R-0-040120 500 ppb Spk (cooseoe*.500000 ngn. spike) 50000 500000 50000 500000 2180 2180 2180 2180 77500 151 100/50 7470 762000 152 100/50 7470 87100 170 100/50 7470 792000 158 100/50 7470 55400 96 30/30 31800 82600 102 30/30 486000 96 30/30 31800 557000 105 30/30 61600 108 30/30 31800 91700 120 30/30 486000 96 30/30 31800 533000 100 30/30 DF06-GW-133S-0-040120 (00050070 Spk K. 50000ngL Spike) DF06-GW-133S-0-040120 (C0050070Spk L. 500000 ng/LSpike) DF0S-GW-133S-0-040120 50 ppb Spk (C005S072,50000 ng/LSpike) DF06-GW-133S-0-040120 500 ppb Spk (C0050073,500000 ng/LSpike) 50000 500000 50000 500000 10400 10400 10400 10400 85200 150 100/50 38000 877000 173 100/50 38000 90000 159 100/50 38000 833000 165 100/50 38000 84500 93 30/30 105000 162000 114 30/30 574000 107 30/30 105000 718000 123 30/30 88900 102 30/30 105000 160000 110 30/30 540000 100 30/30 105000 632000 105 30/30 DF8b-GW-134R-0-040121 (C0050078 Spk M,50000ng/LSpike) DFSb-6W-134R-0-040121 (C005007I Spk N.500000ng/LSpike) DF8b-GW-134R-0-040121 50 ppb Spk (C0050000,50000 ng/LSpike) DF8b-GW-134R-0-040121 500 ppb Spk (C0050061,500000 ng/LSpike) 50000 500000 50000 500000 151000 151000 151000 151000 231000 160 100/50 774000 1290000 228 100/50 774000 231000 160 100/50 774000 1140000 198 100/50 774000 815000 * 30/30 1320000 1240000 100/50 1620000 169 30/30 1320000 2390000 214 100/50 838000 30/30 1320000 1370000 * 100/50 1430000 131 30/30 1320000 2160000 168 100/50 ` Sam ple residue exceeds the spiking level significantly; therefore, an accurate recovery value cannot be calculated NO * Not detected at or above 25 ng/L (ppt). NQ = Not quantifiable = M easured concentration between 25 ng/L (ppt) and the Lim it of Quantitation (LO Q ) which is 50 ng/L (ppt). Note: Since tela summarytable shows rounded results, recovery values mayvary slightly from the values in the raw data. Exygen Research Page 22 o f 105 Interim Report #3-Analysis of Ground Water Samples Exygen Study No.: P0001131 Table II. Matrix Spike Recovery of PFBS, PFHS and PFOS in Ground Water Samples Continued Sam ple D e scrlo tio n DFSb-GW-1 J4S -0-040121 (C009S0S3Spk C, S0000ng/LSpila) DF8b-GW -1J4S-0-040121 (C005SOS3Spk D.500000 ng/LSpila) DF8b-GW-134S-0-040121 50 ppb Spk (C009S0S5,50000 ng/LSpila) DF8b-GW-134S-0-040121 500 ppb Spk (C005S08S,500000 ng/LSpila) Amount Spiked (ng/L) 50000 500000 50000 500000 C4 Sulfonate PFBS_____________________C6 Sulfonate PFHS_____________________ C6 Sulfonate PFOS Amount Amount Amount Found In Sample (ng/L) Amount AM W d Recovered Ree Accuracy <ng/L) ( % /- % ) Found In Sample (ngfl.) Amount Recovered ("O'1-) Ree Accuracy 1*1 (% / -%) Found In Sample (ng/L) Amount Recovered (ng/L) Ai m w d Ree Accuracy w (+%/%) 71.0 74500 149 100/50 463 47400 94 30/30 7930 57400 99 30/30 71.0 751000 150 100/50 463 466000 93 30/30 7930 511000 101 30/30 71.0 81800 163 100/50 463 52200 103 30/30 7930 66400 117 30/30 71.0 785000 157 100/50 463 472000 94 30/30 7930 486000 96 30/30 DF8b-GW-135R-0-040121 (C005S0H Spk E, 50000 ng/LSpila) DF8b-GW-135R-0-040121 (C005S08SSpk F, 500000ng/LSplk) DF8b-GW-135R-0-040121 50 ppb Spk (C009S090,90000 ng/L Spila) DF8b-GW-135R-0-040121 500 ppb Spk (C005S0S1,500000ng/L Spila) 50000 500000 50000 500000 146000 146000 146000 146000 226000 160 100/50 705000 1170000 205 100/50 705000 232000 172 100/50 705000 1220000 215 100/50 705000 746000 30/30 1360000 131 30/30 759000 30/30 1370000 133 30/30 592000 592000 592000 592000 683000 . 30/30 1280000 138 30/30 638000 30/30 1230000 128 30/30 DF8b-GW -135S-0-040121 (C005S0S3Spk 9,50000 ng/LSpila) D F8b-GW -135 S -0-040121 (COOSOOSSspk H,500000ng/LSpila) DF8b-GW-135S-0-040121 50 ppb Spk (C009S0S5,50000 ng/L Spila) DF8b-GW-135S-0-040121 500 ppb Spk (C005S0M,500000 ng/LSplk*) 50000 500000 50000 500000 47200 47200 47200 47200 138000 182 100/50 220000 280000 30/30 82700 818000 154 100/50 220000 729000 102 30/30 82700 142000 190 100/50 220000 269000 30/30 82700 917000 174 100/50 220000 821000 120 30/30 82700 148000 131 30/30 595000 102 30/30 159000 153 30/30 700000 123 30/30 DF8b-GW -135L-0-040121 (cooseoes Spk 1,5000 ng/LSpila) DF8b-GW-135L-0-040121 (COOSOOSSSpk J, 50000 ng/LSpila) DF8b-GW-135L-0-040121 5 ppb Spk (C0090100,5000 ng/LSpila) DF$b-GW-135L-0-040121 50 ppb Spk (C0059101,50000 ng/LSpila) 5000 50000 5000 50000 38000 38000 38000 38000 46200 100/50 187000 199000 30/30 151000 179000 30/30 125000 174 100/50 187000 225000 76 30/30 151000 201000 100 30/30 48600 100/50 187000 203000 30/30 151000 181000 30/30 122000 168 100/50 187000 239000 104 30/30 151000 203000 104 30/30 DF09-GW -130R4-040121 (C005S103Spk K, 50000ng/LSpila) DF09-GW-130R-0-040121 (C0050103 Spk L. 500000 ng/L Spila) DF09-GW-130R-0-040121 50 ppb Spk (C0050105,50000 ng/L Splk) DF09-GW-130R-0-040121 500 ppb Spk (C0050100,500000 ng/LSpila) 50000 500000 50000 500000 22900 22900 22900 22900 97200 149 100/50 98600 791000 154 100/50 98600 115000 184 100/50 98600 840000 163 100/50 96600 148000 99 30/30 440000 452000 30/30 588000 98 30/30 440000 976000 107 30/30 151000 105 30/30 440000 460000 . 30/30 603000 101 30/30 440000 846000 81 30/30 DF08-GW-130S-0-040121 (C005010SSpk M, 50000ng/LSpila) DF09-GW -130S-0-040121 (C0050106Spk N, 500000ng/LSpila) DF09-GW-130S-0-040121 50 ppb Spk (C005S110,50000 ng/LSpila) DF09-GW-130S-0-040121 500 ppb Spk (C0050111,500000 ng/LSpila) 50000 500000 50000 500000 13500 13500 13500 13500 87200 147 100/50 47700 873000 172 100/50 47700 95200 163 100/50 47700 885000 174 100/50 47700 96000 97 30/30 600000 110 30/30 110000 125 30/30 632000 117 30/30 ` Sam ple residue exceeds the striking level significantly; therefore, an accurate recovery value cannot be calculated NO * Not detected at or above 25 ng/L (ppt). NQ = Not quantifiable * M easured concentration between 25 ng/L (ppt) and the Lim it of Quantitation (LO Q ) vrfiich is 50 ng/L (ppt). N ote: S in ce th is sum m ary tab le sh o w s rounded re su lts, reco very va lu e s m ay v a ry slig h tly from the va lu e s In the raw d ata. 62600 62600 62600 62600 123000 121 30/30 616000 111 30/30 130000 135 30/30 427000 73 30/30 Exygen Research Page 23 of 105 Interim Report #3-Analysis o f Ground Water Samples Exygen Study No.: P0001131 Table II. Matrix Spike Recovery of PFBS, PFHS and PFOS in Ground Water Samples Continued Sam ple D e scrip tio n DF14-GW-136R-0-040121 (00059113 Spk C. 5000 ng/LSpike) DF14-GW-136R-0-040121 (C00SS113Spk D, 50000 ng/LSpike) DF14-GW-136R-0-040121 5 ppb Spk (CO0SS11S, 9000 ng/L Spike) DF14-GW-136R-0-040121 50 ppb Spk (C00SS11S,50000 ng/LSpike) li IS s 5000 C4 Sulfonate PFBS_____________________ C6 Sulfonate PFHS_____________________ Ce Sulfonate PFOS A m ount Found in Sim ple (ng/L) Amount Amount Amount Found In Amount Found in Recovered Ree Accuracy Sample Recovered Ree Accuracy Sample <ng/L> l%! (%/%} <ng/L) (ng/L) (%i (%/-%) (ng/L) Amount Recovered (ngA.) Aeeeeeed Ree Accuracy !%l <%/-%) 204 7080 138 100/50 198 4600 88 30/30 1710 6320 92 30/30 50000 204 67200 134 100/50 198 41700 83 30/30 1710 42600 82 30/30 5000 204 8310 162 100/50 198 5220 100 30/30 1710 6520 96 30/30 50000 204 87100 174 100/50 198 54700 109 30/30 1710 54700 106 30/30 DF14-GW-136S-0-040121 (C009B118Spk E, 5000ng/LSpike) DF14-GW-136S-0-040121 (C00911*Spk F, 50000 ng/LSpike) DF14-GW-136S-0-040121 5 ppb Spk (C009B120,5000 ng/L Spike) DF14-GW-136S-0-040121 50 ppb Spk (C0050121,90000 ngA. Spike) 5000 50000 5000 50000 3150 3150 3150 3150 11700 171 100/50 12600 75100 144 100/50 12600 13000 197 100/50 12600 84300 162 100/50 12600 17500 98 30/30 89000 56800 88 30/30 89000 18600 120 30/30 89000 62500 100 30/30 89000 97800 30/30 136000 94 30/30 94200 30/30 138000 98 30/30 D F14-GW -136L-0-040121 (C0050123Spk 0,5000 ng/LSpike) DF14-GW-136L-0-040121 (C009S123Spk H,50000ng/LSpike) D F14-GW-138L-0-040121 5 ppb Spk (C009B12S,5000 ng/LSpike) DF14-GW-136L-0-040121 50 ppb Spk (C005912S,50000 ngA.Spike) 5000 50000 5000 50000 5490 5490 5490 5490 13800 166 100/50 21100 80900 151 100/50 21100 14400 178 100/50 21100 88400 166 100/50 21100 27800 30/30 135000 158000 30/30 68500 95 30/30 135000 198000 126 30/30 26600 30/30 135000 150000 30/30 71300 100 30/30 135000 184000 98 30/30 DF14-GVV-137S-O-O40121 (C005S12SSpk L 5000 ng/LSpite) DF14-GW-137S-0-040121 (C009S12SSpk J, 50000 ngl. Spike) DF14-GW-137S-0-040121 5 ppb Spk (C009B130,5000 ng/L Spite) DF14-GW-137S-0-040121 50 ppb Spk (C0050131,50000 ngA.Spite) 5000 50000 5000 50000 101 101 101 101 7240 143 100/50 87.0 73100 146 100/50 87.0 8740 173 100/50 87.0 76200 152 100/50 87.0 4800 94 30/30 46500 93 30/30 5820 115 30/30 48300 96 30/30 339 339 339 339 5070 95 30/30 47300 94 30/30 6070 115 30/30 51600 103 30/30 DF14-G W -137L-0-040121 (C0050133Spk K, 9000 ng/LSpite) DF14-GW-137L-0-040121 (C005S133 Spk L. 50000ng/LSpfte) DF14-GW-137L-0-040121 5 ppb Spk (C0098135,5000 ngA. Spite) DF14-GW-137L-0-040121 50 ppb Spk (C005913S, 50000 ngA.Spite) 5000 50000 5000 50000 75.0 75.0 75.0 75.0 7420 147 100/50 74.0 74700 149 100/50 8620 171 100/50 74.0 74.0 82700 165 100/50 74.0 4760 94 30/30 46500 93 30/30 5810 115 30/30 52800 105 30/30 1120 1120 1120 1120 6030 98 30/30 47500 93 30/30 7070 119 30/30 52500 103 30/30 Field Blank 1 Low Spk (C00SS020.100 ngA. Spike) Field Blank 1 High Spk (C005021,10000 ngA.spite) Field Blank 2 Low Spk (C005S023,100ngA. Spite) Field Blank 2 High Spk (C0050024,10000 ngA. Spite) 100 ND 136 136 100/50 ND 103 103 30/30 ND 109 109 30/30 10000 ND 9660 99 100/50 ND 9040 90 30/30 ND 8550 86 30/30 100 ND 108 108 100/50 ND 108 108 30/30 ND 126 126 30/30 10000 ND 11000 110 100/50 Average: 165 Standard Deviation: 21 ND 9690 97 Average: 104 Standard Deviation: 15 30/30 ND 9220 92 Average: 112 Standard Deviation: 28 30/30 ` Sam ple residue exceeds the spiking level significantly; therefore, an accurate recovery value cannot be calculated NO * Not detected at or above 25 ng/L (ppt). NQ * Not quantifiable = M easured concentration between 25 ng/L (ppt) and the Lim it of Quantitation (LO Q ) w hk*i is 50 ng/L (ppt). Note: Since this summary table shows rounded results, recovery values may vary slightly from the values in the raw data. Exygen Research Page 24 of 105 Interim Report #3-Analysis of Ground W ater Samples Exygen Study No.: P0001131 FIGURES Exygen Research Page 25 of 105 Interim Report #3-Analysis o f Ground Water Samples Exygen Study No.: P0001131 Figure 1. Typical Calibration Curve for PFBS in Reagent Water 021806Cidb (P F B S * "lin e a r Regression O / x " weighting): y * 13 x +0.00163 (jr- 0.0071) A rea, counts Exygen Research Page 26 of 105 Interim Report #3-Analysis o f Ground Water Samples Exygen Study No.: P0001131 Figure 2. Extracted Standards of PFBS in Reagent Water, 0 ng/L and 25 ng/L, Respectively XC 0211 0 5-0,0 ng/L Standard PFBS (Standard) 299.0/99.0 am u - aam pta 1 o f 60 from 0216O5C.wiW (poah not found) 0.75 55 T im *, m in I XC021106-I, 25 ng/L Standard PFBS (Standard)299.0/99.0 amu - ampi* 2 of 60 from 02t8O5C.wiff A v a : 5103 counts H ght; 170. ops R T: 4.55 min Exygen Research Page 27 of 105 Interim Report #3-Analysis o f Ground Water Samples Exygen Study No.: P0001131 Figure 3. PFBS in Reagent Water, 500 ng/L Fortified Reagent Water, and 5000 ng/L (DF=10) Fortified Reagent Water, Respectively R a a g a n l Corridoi A P F B S (U n know n) 299.0199.0 am u - sam p )a 9 o f 60 Rom 0 2 1 6 0 5 C .w itf i'paak n o t fou nd) A w : 77001 counts Height: 1700. ops R T: 4.66 min 1900 1000 900 0 0 .8 0 ,,1 .1 4 A4.99 2 .8 8 9 .9 8 7 .4 9 8 .4 1 0 .3 9 1 0 ,4 9 ^ 7 8 9 10 T im e , m in Reagent Spite 8.6000 ng/L. DF10 PFBS (0 0 )2 0 0 8 /0 0 .0 amu sample 11 of 60 from 021806C .iff A e a : 60000 counts Height: 1000. ops R T: 4.62 min 1 1 .9 4 ^ 1 1 .9 4 ,.1 2 .3 0 1 3 .5 9 __________ 1 9 .8 K 1 0 .0 3 11 12 13 1 4 19 19 17 482 & 900 7 .9 7 8.09^ 8.83 1 0 .0 8 8 9 10 T im e , m in ,,1 0 .7 2 11 1 2 .1 8 1 3 .9 9 ^ 14.21 12 13 14 1 9 .1 8 19 1 9 .0 0 ^ 17.31 19 17 Exygen Research Page 28 of 105 Interim Report #3-Analysis of Ground Water Samples Exygen Study No. : P0001131 Figure 4. Chromatogram Representing a Ground Water Sample Analyzed for PFBS (Exygen ID: C0059083, Data Set: 021805C) C00590S3 PFBS fl/aknom rj2 9 9 0 /9 9 0 jm u f i mpio 15 o f 60 from 021805C.m(f Area: 9035 c o u n ts H e ig h t 221. o p s RT: 4.56 min Exygen Research Page 29 of 105 Interim Report #3-Analysis o f Ground Water Samples Exygen Study No.: P0001131 Figure 5. Typical Calibration Curve for PFHS in Reagent Water TM 021806C.rdb (P FH S ): "U noiT Regression O / " weighting): y 621 x + 1.5*-t004(r * 0.9961) Area, counts Exygen Research Page 30 of 105 Interim Report #3-Analysis o f Ground Water Samples Exygen Study No.: P0001131 Figure 6. Extracted Standards of PFHS in Reagent Water, 0 ng/L and 25 ng/L, Respectively XC 02f 105-0, 0 ngfL Standard - PFHS (Standard) 399.0/80.0 ama aampio 1 o f 60 from 021805C.mff Araa: 14906 counts HetgM: 605. ops R 7 :10.8 min 10.75 Intensity, cps A rea: 27333 counts Httght: 1810. ops R T: 10.7 min 10.72 Intensity, cps Exygen Research Page 31 o f 105 Interim Report #3-Analysis of Ground Water Samples Exygen Study No.: P0001131 Figure 7. PFHS in Reagent Water, 500 ng/L Fortified Reagent Water, and 5000 ng/L (DF=10) Fortified Reagent Water, Respectively 60R oagstit C orinti A PFHS (Unknown) 399.0/90.0 am u a m p i o 9 o f on* 021805C.wiff Arsa: 2978 c o u r ts H sig itt 106. o p s RT: 10.8 m in <aoo. 10.75 c T im *, min R*ag*nt Spit* A. 600 ng/L- PFHS (Q C )3090.0 amu sample 10 of 60 from 021806C.wff A * a : 278167 counts Haight: 23300. ops RT: 10.7 min 10.50 &<o 1*** I 1-* 5000.0 OJD 1 2 3 4 5 5 7 8 T im * , min 12 13 14 15 16 17 Exygen Research Page 32 of 105 Interim Report #3-Analysis o f Ground W ater Samples Exygen Study No. : P0001131 Figure 8. Chromatogram Representing a Ground Water Sample Analyzed for PFHS (Exygen ID: C0059083, Data Set: 021805C) C 0 O 5 9 0 6 3 P FH S (Unknow n) 399.0/60.0 u riti ssm p to 15 o f 60 from 0216O5C.wHf A rea : 256031 cou n ts Height: 22000. c p s R T : 10.7 min 2 .1 * 4 2 .0 * 4 1 .9 * 4 1 .8 * 4 1 .7 * 4 1 -6 * 4 1 .5 * 4 - 1 0 .7 0 Intensity, cps 1 .3 * 4 - 1 .2 * 4 1 .1 * 4 1 .0 * 4 9 0 0 0 .0 8 0 0 0 .0 7 0 0 0 .0 6 0 0 0 .0 5 0 0 0 .0 4 0 0 0 .0 3 0 0 0 .0 2000.0 - 1000.0 *1 0 .0 J---- ----- 1---- ----- 1............................................. 12 3 4 5 T im * , m in 12 13 14 15 15 17 Exygen Research Page 33 of 105 Interim Report #3-Analysis of Ground Water Samples Exygen Study No.: P0001131 Figure 9. Typical Calibration Curve for PFOS in Reagent Water 1803CRX (PFOS): "Um a T R*flrwon O / tid in g ): y M2 x + -0.000368 (r 0.9994) Area, counts Exygen Research Page 34 o f 105 Interim Report #3-Analysis of Ground W ater Samples Exygen Study No.: P0001131 Figure 10. Extracted Standards of PFOS in Reagent Water, 0 ng/L and 25 ng/L, Respectively XC022205-0, 0 ngA. Standard - PFOS (Standard) 499.0/60.0 amu sam pl* 1 o f 95 from 021805CF.wff (p*ak not found) 1 2 .2 5 U) Q. C<D rea: 13737 oounts Height: 86Q. ops RT: 12.2 min Exygen Research Page 35 of 105 Interim Report #3-Analysis of Groimd Water Samples Exygen Study No.: P0001131 Figure 11. PFOS in Reagent Water, 500 ng/L Fortified Reagent Water, and 5000 ng/L (DF=10) Fortified Reagent Water, Respectively R gont Cottimi A PFOS (Unhnown) 4 9 9 .OfO.O m u sa m p ln 9 o f 96 from 021805CR.wHf Am a: 943 coarti Hwgtrt 33.9 cp R 7 :12.2 min ouo>. 3 0 - J? m 20 c & 10- 0.67 0a 4.50 i 3.21 4<12 N 5 .2 8 V*1 ^ 0.13 8 -f4 8J1 8?5 ly rr^ i--A'* ^0-. n T im , min Reagent Spite A, 500 ng/L PFOS (Q C) 4WH/80.0 amu sample 10 of 05 from 021805CR.wiff Area: 200541 counts Height: 10300. ops RT: 12.2 min 1 2 .2 4 A/^A ft- *k 16.22 17.55 12.24 Exygen Research Page 36 of 105 Interim Report #3-Analysis of Ground Water Samples Exygen Study No.: P0001131 Figure 12. Chromatogram Representing a Ground Water Sample Analyzed for PFOS, DF=50 (Exygen ID: C00059083, Data Set: 021805CR) C 0 0 59063, OFrnSO - P F O S (U n know n) 499.0*90.0 am u am pio 17 o f 9 5 from 021805CR.W W A roa: 6 7 5 6 5 count H aigh t 4 6 5 0 . op R 7 : 12.3 min 4600* 1 2 .2 5 4000 3500 3000 2500* 11.81 Intensity, cps 2000 1500 1000 500 11 21 31 4 51 60 J ------- --------- ------- --------- ------- --------- -----------------------'-------- ----------------r T im , m in 14 15 16 17 Exygen Research Page 37 of 105 Interim Report #3-Analysis o f Ground Water Samples Exygen Study No.: P0001131 APPENDIX A Study Protocol P0001131 (Exygen Study No. P0001131) with Analytical Method: V0001780:"Method of Analysis for the Determination of Perfluorooctanoic Acid (PFOA) in Water by LC/MS/MS" Exygen Research Page 38 of 105 Interim Report #3-Analysis o f Ground Water Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 STUDY PROTOCOL Study Title: Analysis of Perfluorobutanesulfonate (PFBS), Perfluorohexanesulfonate (PFHS), and Perfluorooctanesulfonate (PFOS) in W ater, Soil, Sediment, Fish, Clams, Vegetation, Small Mammal Liver and Small Mammal Serum Using LC/MS/MS for the 3M Decatur Monitoring Program Exygen Protocol Number: P0001131 Performing Laboratory: Exygen Research 3058 Research Drive State College, PA 16801 Phone: (814) 272-1039 Sponsor Representative: Michael A. Santoro Director of Regulatory Affairs 3M Building 0236-01-B-10 St. Paul, MN 55144 Phone: (651) 733-6374 Page / oj 65 Exygen Research Page 39 o f 105 Interim Report #3-Analysis o f Ground W ater Samples Exygen Study No.: P0001131 Exygen Protocol Number: P 0001131 DISTRIBUTION: 1) Jaisimha Kesari, Study Director, Weston Solutions 2) John M. Flaherty, Principal Investigator, Exygen Research 3) Michael A. Santoro, Sponsor Representative, 3M Company 4) Exygen Research Quality Assurance Unit Exygen Research Page 2 o f 65 Page 40 of 105 M erini Report #3-Analysis of Ground Water Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 PROTOCOL APPROVAL Study Title: Analysis of Perfluorobutanesulfonate (PFBS), Perfluorohexanesulfonate (PFHS), and Perfluorooctanesulfonate (PFOS) in Water, Soil, Sediment, Fish, Clams, Vegetation, Small Mammal Livers and Small Mammal Serum Using LC/MS/MS for the 3M Decatur Monitoring Program Exygen Protocol Number: P0001131 APPROVALS Jaisimhaocesan, Si Weston Solutions Michael A. & toro, Sponsor Representative 3M Compariy Date ^ / Richard A. G Exygen Rese ad, Quality Assurance Unit Date Page 3 o f 65 Exygen Research Page 41 of 105 Interim Report #3-Analysis of Ground Water Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 TABLE OF CONTENTS TITLE P A G E .....................................................................................................................................................................1 D IS T R IB U T IO N ............................................................................................................................................................... 2 PROTOCOL APPROVAL.............................................................................................................................................. 3 TABLE OF CONTENTS................................................................................................................................................ 4 IN T R O D U C T IO N ............................................................................................................................................................. 5 TEST M ATERIA LS.........................................................................................................................................................5 O B JECTIV E......................................................................................................................................................................6 TESTING FACILITY...................................................................................................................................................... 6 STUDY DIRECTOR........................................................................................................................................................ 1 SPONSOR REPRESENTATIVE................................................................................................................................... 7 PRINCIPAL INVESTIGATOR......................................................................................................................................7 PROPOSED EXPERIMENTAL START AND TERMINATION D A TE S............................................................7 IDENTIFICATION AND JUSTIFICATION OF THE TEST SY ST EM ................................................................8 SAMPLE PROCUREMENT, RECEIPT AND RETEN TION ..................................................................................8 SAMPLE IDENTIFICATION........................................................................................................................................9 ANALYTICAL PROCEDURE SUMMARY.............................................................................................................. 9 VERIFICATION OF ANALYTICAL PROCEDURE................................................................................................9 METHOD FOR CONTROL OF B IA S..........................................................................................................................11 STATISTICAL M ETH O D S........................................................................................................................................... 11 GLP STATEM ENT.......................................................................................................................................................... 11 R EPO R T.............................................................................................................................................................................U SAFETY AND H EA LTH ................................................................................................................................................ 12 AMENDMENTS TO PROTOCOL............................................................................................................................... 13 DATA RECORD K EE PIN G .......................................................................................................................................... 13 QUALITY ASSU RA N CE...............................................................................................................................................14 RETENTION OF DATA AND A RCHIVING.............................................................................................................14 APPENDIX I, ANALYTICAL M ETHODS................................................................................................................. 15 Page 4 o f 65 Exygen Research Page 42 o f 105 Interim Report #3-Analysis o f Ground Water Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 INTRODUCTION The purpose of this study is to perform analysis for perfluorobutanesulfonate (PFBS), perfluorohexanesulfonate (PFHS) and perfluorooctanesulfonate (PFOS) in water, soil, sediment, fish, clams, vegetation, small mammal livers and small mammal serum using LC/MS/MS for the 3M Decatur Monitoring Program. The study will be audited for compliance with EPA TSCA Good Laboratory Practice Standards 40 CFR 792 by the Quality Assurance Unit of Exygen Research. TEST MATERIALS The test materials are perfluorobutanesulfonate (PFBS), perfluorohexanesulfonate (PFHS) and perfluorooctanesulfonate (PFOS) and are all supplied by 3M. PFBS Chemical Name: Perfluorobutanesulfonate Molecular Weight: 338 supplied as the potassium salt (C FqSCVK*) Lot Number: 101 Purity: 96.7% Transitions Monitored: 299 -> 99 Structure: PFHS Chemical Name: Perfluorohexanesulfonate Molecular Weight: 438 supplied as the potassium salt (CFuSOj'K4) Lot Number: SE036 Purity: 98.6% Transitions Monitored: 399 - * 80 Structure: S03 Page 5 o f 65 Exygen Research Page 43 of 105 Interim Report #3-Analysis of Ground Water Samples Exygen Study No. : P0001131 Exygen Protocol Number: P0001131 PFOS Chemical Name: Perfluorooctanesulfonate Molecular Weight: 538 supplied as the potassium salt (CgF^SOsTC*) Lot Number: 217 Purity: 86.9% Transitions Monitored: 499 -> 99 Structure: OBJECTIVE The purpose of this study is to perform analysis for perfluorobutanesulfonate (PFBS), perfluorohexanesulfonate (PFHS) and perfluorooctanesulfonate (PFOS) in water, soil, sediment, fish, clams, vegetation, small mammal livers and small mammal serum for the 3M Decatur Monitoring Program using the current versions of the following Exygen analytical methods: V0001780: "Method of Analysis for the Determination of Perfluorooctanoic Acid (PFOA) in Water by LC/MS/MS" V0001781: "Method of Analysis for the Determination of Perfluorooctanoic Acid (PFOA) in Soil by LC/MS/MS" V0001782: "Method of Analysis for the Determination of Perfluorooctanoic Acid (PFOA) in Sediment by LC/MS/MS" V0001783: "Method of Analysis for the Determination of Perfluorooctanoic Acid (PFOA) in Fish and Clams by LC/MS/MS" V0001784: "Method of Analysis for the Determination of Perfluorooctanoic Acid (PFOA) in Vegetation by LC/MS/MS" V0001785: "Method of Analysis for the Determination of Perfluorooctanoic Acid (PFOA) in Small Mammal Liver by LC/MS/MS" V0001786: "Method of Analysis for the Determination of Perfluorooctanoic Acid (PFOA) in Small Mammal Serum by LC/MS/MS" TESTING FACILITY Exygen Research 3058 Research Drive State College, PA 16801 Phone:(814)272-1039 Page 6 o f 6S Exygen Research Page 44 of 105 Interim Report #3-Analysis of Ground Water Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 STUDY DIRECTOR Jaisimha Kesari P.E., DEE Weston Solutions, Inc. 1400 Weston Way West Chester, PA 19380 Phone: (610) 701-3761 Fax:(610)701-7401 j.kesari@westonsolutions.com SPONSOR REPRESENTATIVE Michael A. Santoro 3M Company Director of Regulatory Affairs 3M Building 0236-01-B-10 St. Paul, MN 55144 Phone: (651) 733-6374 PRINCIPAL INVESTIGATOR John M. Flaherty Exygen Research 3058 Research Drive State College, PA 16801 Phone: (814) 272-1039 j ohn.flaherty@exygen.com PROPOSED EXPERIMENTAL START AND TERMINATION DATES It is proposed that the analytical portion of this study be conducted from October 01, 2004 to December 31, 2005. The actual experimental start and term ination dates w ill b e included in th e final report. Page 7 o f 65 Exygen Research Page 45 of 105 Interim Report #3-Analysis of Ground Water Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 IDENTIFICATION AND JUSTIFICATION OF THE TEST SYSTEM The following are the test systems for this study: Water (groundwater and surface water) Soil Sediment Fish Clams Vegetation Small Mammal Liver Small Mammal Serum The samples will be collected by Weston Solutions. The control samples will be purchased and prepared by the testing facility. Purchase and processing details for the control samples will be included in the final report associated with this study. The test systems were chosen to access the environmental impact of PFBS, PFHS and PFOS in the Decatur, Alabama area. SAMPLE PROCUREMENT, RECEIPT AND RETENTION Water, soil, sediment, fish, clam, vegetation, small mammal liver and small mammal serum samples will be received at Exygen directly from Weston Solutions. The details o f sample procurement for this study are outlined in the 3M work plan entitled "Phase 2 Work Plan for Sampling Environmental Media." The number and types of samples collected will vary depending availability in the field. The total number of samples received and analyzed for each matrix will be documented in the final report associated with this study. Water, soil, and sediment samples will be used as received without further processing at Exygen. These samples will be stored refrigerated at 2C-8C. Fish, clam, vegetation and small mammal liver samples will be processed according to the appropriate analytical method (see Appendix I). These s a m p le s w ill b e s to re d frozen at -1 0 C . S m a ll m a m m a l w h o le b lo o d samples will be centrifuged in the field at the time of collection and the serum fraction will be used for the study. Small mammal serum will be stored frozen at S -10C. The receipt and processing of the samples will be documented in the final report and raw data associated with the study. Page 8 o f 65 Exygen Research Page 46 of 105 Interim Report #3-Analysis o f Ground Water Samples Exygen Study No. : P0001131 Exygen Protocol Number: P 0001131 SAMPLE IDENTIFICATION Prior to analysis, each sample will be assigned a laboratory sample reference number. The reference number will be unique and will distinguish each laboratory sample that is processed throughout the analytical procedure. Chromatographic data will be identified by the laboratory sample reference number. Sample storage conditions and locations will be documented throughout the study. ANALYTICAL PROCEDURE SUMMARY References: V0001780: "Method of Analysis for the Determination of Perfluorooctanoic Acid (PFOA) in Water by LC/MS/MS" V0001781: "Method of Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Soil by LC/MS/MS" V0001782: "Method of Analysis for the Determination of Perfluorooctanoic Acid (PFOA) in Sediment by LCM SM S" V0001783: "Method of Analysis for the Determination of Perfluorooctanoic Acid (PFOA) in Fish and Clams by LCM SM S" V0001784: "Method of Analysis for the Determination of Perfluorooctanoic Acid (PFOA) in Vegetation by LCM SM S" V0001785: "Method of Analysis for the Determination of Perfluorooctanoic Acid (PFOA) in Small Mammal Liver by LCM SM S" V0001786: "Method of Analysis for the Determination of Perfluorooctanoic Acid (PFOA) in Small Mammal Serum by LCM SM S" The above methods use analytical conditions capable of separating the isomers of PFBS, PFHS and PFOS. The final report will include the isomers summed into total PFBS, total PFHS, and total PFOS found. VERIFICATION OF ANALYTICAL PROCEDURE A laboratory control sample will be used for the preparation of fortified control samples. The test substance will be made into solutions as per the method, and added to the matrices via a micropipette. For water sampling, Exygen will supply one bottle per sample collected. The /*" bottles will be 500 mL precleaned Sci/Spec Premier wide mouth HDPE bottles. These bottles have been routinely used for fluorochemical sample Page 1o f 65 Exygen Research Page 47 of 105 Interim Report #3-Analysis o f Ground Water Samples Exygen Study No. : P0001131 Exygen Protocol Number: P0001131 collection at the testing facility and have been shown to be free of PFBS, PFHS and PFOS. Samples will be added to each container to a volumetric fill line at 200 mL. A field duplicate, a low field spike and a high field spike of each sample will be collected. The low and high field spike bottles will contain PFBS, PFHS and PFOS as well as perfluorooctanoic acid (PFOA) and 1.2-13C perfluorooctanoic acid (l3C PFOA). PFOA and ,3C PFOA are included in the solutions used to spike the samples. The results for PFOA and ,3C PFOA will not be reported in this study. Exygen will supply one field blank (control water) and two field blank spikes (control water fortified with PFBS, PFHS and PFOS at a low and high level) for every twenty samples collected. At the testing facility, each water sample (excluding field duplicates and field spikes) will be extracted in duplicate and will also be fortified at a low and high concentration with PFBS, PFHS and PFOS and processed through the described procedure to determine method accuracy and to check for bias. For soil, sediment, clams, and vegetation, Exygen will supply one SOO mL precleaned Sci/Spec Premier wide mouth HDPE bottle per sample collected or a zip-seal bag. All containers/bags used for sample collection will be shipped to the sample location. Samples will be added to each container or bag in the field. At the testing facility, each sample will be extracted in duplicate and will also be fortified at a known concentration with PFBS, PFHS and PFOS at both a low and high level and processed through the described procedure to determine method accuracy and to check for bias. For small mammal liver, Exygen will supply a SO mL polypropylene centrifuge tube. For small mammal serum, Exygen will supply a collection kit for each sample containing serum separator tubes (red top), vacutainers, needle holders and needles, transfer pipettes, and polypropylene tubes. At the testing facility, each liver and serum sample will be extracted in duplicate and will also be fortified at a known concentration with PFBS, PFHS and PFOS at both a low and high level and processed through the described procedure to determine method accuracy and to check for bias. Low and high spiking levels for each matrix are defined below: Matrix Low Spiking Level High Spiking Level Water 500 ng/L 5000 ng/L Soil 4 ng/g 40 ng/g Sedim ent 4 ng/g 40 ng/g Fish 10 ng/g 100 ng/g Clams 10 ng/g 100 ng/g Vegetation 10 ng/g 100 ng/g Small Mammal Liver 10 ng/g 100 ng/g Small Mammal Serum lOng/mL 100 ng/mL Page 10 o f 65 Exygen Research Page 48 of 105 Interim Report #3-Analysis o f Ground Water Samples Exygen Study No. : P0001131 Exygen Protocol Number: P0001131 Recoveries a anticipated to be between 70% and 130% of the fortified levels; however, the exact precision and accuracy will be determined by the analysis of the quality control samples described above. A statement of accuracy will be included in the final report. METHOD FOR CONTROL OF BIAS Control of bias will be addressed by taking representative sub-samples from a homogeneous mixture of each matrix from untreated control samples, and by analyzing at least two levels of fortifications. STATISTICAL METHODS Statistics will be limited to those specified in the subject methods and to the calculation of average recoveries, as applicable. GLP STATEMENT All aspects of this study shall be performed and reported in compliance with EPA TSCA Good Laboratory Practice Standards 40 CFR 792. The final report or data package (supplied to the Sponsor) shall contain a statement that the study was conducted in compliance with current and applicable GLP standards and will outline any deviations in the study from those standards. This statement will be signed by the Study Director and Sponsor Representative. REPORT A final report will be prepared by the principal investigator or their designee at the conclusion of the study. The report will include, but will not be limited to, the following: The name and address of the Study Director, Sponsor Representative, and of the testing facility. A statement of GLP compliance (any related documentation, such as chain-of-custody records, must be in the study records). Page 11 o f 65 Exygen Research Page 49 o f 105 Interim Report #3-Analysis o f Ground W ater Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 The signed and dated statement by the Exygen Research Quality Assurance Unit regarding dates of study inspections and dates findings were reported to the Study Director and Management. A description of the exact analytical conditions employed in the study. If the subject method was followed exactly, it is necessary to include only a copy of the analytical method. Any modifications to this method will be incorporated into the report. If the method is photo-reduced, the project number and page number must be included on each page. Description of the instrumentation used and operating conditions. All results from all sets analyzed. Control and fortified samples will be identified and the data table will include sample number and fortification level. Representative chromatograms for each analyte in each matrix, including chromatograms of a standard and a control sample, and a chromatogram at a fortification level. The location of the analyte peaks will be clearly identified in all chromatograms. All circumstances that may have affected the quality or integrity of the data will be documented in the report. Locations where raw data and the final report are to be archived. Additions or corrections to the final report shall be in the form of an amendment signed by the Study Director. The amendment shall clearly identify that part of the report that is being altered and the reasons for the alterations. The amendment will be signed and dated by the Study Director and the Sponsor Representative. All applicable requirements for reporting of study results as per 40 CFR 792.185. SAFETY AND HEALTH L aboratory personnel w ill practice good sanitation and health habits. Every reasonable precaution shall be taken to prevent inadvertent exposure of personnel and the environment to the test or reference substance(s). Page 12 o f 65 Exygen Research Page 50 o f 105 Interim Report #3-Analysis o f Ground Water Samples Exygen Study No. : P0001131 Exygen Protocol Number: P0001131 AMENDMENTS TO PROTOCOL All significant changes to the analytical protocol outlined here will be expressed in writing, signed and dated by the Study Director and Sponsor Representative. Amendments usually will be issued prior to initiation of study plan change. However, when a change is required without sufficient time for the issue of a written amendment, that change may be effected verbally with supporting documentation signed and dated by the Study Director and followed with a written amendment as soon as possible. In this case, the effective date of the written amendment will be the date of the documented change. Copies o f the signed amendments will be appended to all distributed study plan copies. The original amendment will be maintained with the original study plan. Any deviations from the study plan or from the analytical method as provided will be documented and reported promptly to the Sponsor Representative. DATA RECORD KEEPING Records to be maintained include the following (as appropriate): Sample tracking sheet(s) Sample receipt records, storage history, and chains o f custody History and preparation of standards (stock, fortification, calibration) Description of any modifications to the method Instrument run sheets, bench-sheets or logs Analytical data tables All chromatographic and instrumental conditions Sample extraction and analysis dates A complete listing of study personnel, signatures and initials Chronological presentation of all study correspondence Any other documentation necessary for the reconstruction of the study Chromatograms- All chromatograms will contain the following: Sample identification, injection date, anew or other indication of the area of interest, and injection number corresponding to the run. Additionally, fortifications will include the amount of analyte added and the sample number of the sample that was fortified. Analytical standard chromatograms will additionally include the concentration (e.g., pg/mL). Page 3 o f 65 Exygen Research Page 51 o f 105 Interim Report #3-Analysis o f Ground Water Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 As part of the documentation the following sheets will be included in each analytical set: a run sheet listing the samples to be run in the set, and an instrument conditions sheet describing the instrument type and operating conditions. QUALITY ASSURANCE The QA Unit of Exygen Research will inspect the study at intervals adequate to assure compliance with GLP's, and will report the findings of audits to the Study Director, Exygen Management, and the Sponsor Representative. RETENTION OF DATA AND ARCHIVING All hard copy raw data, including, but not limited to, the original chromatograms, worksheets, correspondence, and results shall be included with the data package submitted to the Study Director. These will be archived with the original study plan, amendments, final report, and all pertinent information from the Sponsor. The testing facility shall keep all electronic raw data and any instrument, equipment, and storage logs for the period of time specified in 40 CFR 792.195. An exact copy of the materials submitted to the study director will also be kept at Exygen Research. Exygen will obtain permission from the study director before discarding or returning samples. Exygen Research Page 4 o f 65 Page 52 of 105 Interim Report #3-Analysis of Ground Water Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 APPENDIX I ANALYTICAL METHODS V0001780: "Method of Analysis for the Determination of Perfluorooctanoic Acid (PFOA) in Water by LC/MS/MS" V0001781: "Method of Analysis for the Determination of Perfluorooctanoic Acid (PFOA) in Soil by LC/MS/MS" V0001782: "Method of Analysis for the Determination of Perfluorooctanoic Acid (PFOA) in Sediment by LC/MS/MS" V0001783: "Method o f Analysis for the Determination of Perfluorooctanoic Acid (PFOA) in Fish and Clams by LC/MS/MS" V0001784: "Method of Analysis for the Determination of Perfluorooctanoic Acid (PFOA) in Vegetation by LC/MS/MS" V0001785: "Method of Analysis for the Determination of Perfluorooctanoic Acid (PFOA) in Small Mammal Liver by LC/MS/MS" V0001786: "Method of Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Small Mammal Serum by LC/MS/MS" Exygen Research Page 15 o f 65 Page 53 of 105 Interim Report #3-Analysis of Ground Water Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 ANALYTICAL METHOD Method N um ber V0001780 M ethod o f Analysis for the Determ ination o f Perfluorooctanoic Acid (PFOA) in W ater byLC/M S/M S Analytical Totting Facility: Exygen Research 3058 Research Drive State College, PA 16801 Approved By: cU L Paul Connolly 1 Technical Leader, LC-MS, Exygen Research 'John Flaherty / Vice President, Operations, Exygen Research QI tb / a W Date WL Date Exygen Research Total Pages: 7 Page 16 o f 65 Page 54 of 105 Interim Report #3-Analysis of Ground Water Samples Exygen Study No.: P0001131 Exygen Protocol Number: P000113 1 ExygaRutvch Method Number V0001780 ANALYTICAL M ETHOD Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in W ater by L C /M S /M S 1.0 Scope This method is to be employed for foe isolation and quantitation o f perfluorooctanoic acid by High Performance Liquid Chromatography coupled to a tandem Mass Spectromtrie Detector (LC/MS/MS) in water. 2.0 Safety 2.1 Always observe safe laboratory practices. 2.2 Consult foe appropriate MSDS before handling any chemical for proper safety precautions. 3.0 Sim ple Requirement 3.1 At least 40 m L o f test sample for extraction. 3.2 No sample processing is needed for water samples. 3.3 Samples stored refrigerated should be allowed to equilibrate to room temperature. 3.4 All samples must be thoroughly mixed before being sampled for extraction 3.5 Any samples containing particles should be centrifuged at -3 0 0 0 rpm for -5 minutes and foe supernatant used for the extraction. 3.6 Sample collection procedures will be specified in foe sampling plan for this project 4.0 Reagents and Standards 4.1 W ater-H P L C grade 4.2 Methanol - HPLC grade 4.3 Ammonium Acetate - A.C.S. Reagent Grade 4.4 Perfluorooctanoic Acid - Sigma-Aldrich 5.0 Instrument and Equipment 5.1 A high performance liquid chromatograph capable o f pumping up to 2 solvents equipped with a variable volume injector capable o f injecting 5-200 pL connected to a tandem Mass Spectrometer (LC/MS/MS). 5.2 A device to collect raw data for peak integration and quantitation. 5.3 Analytical balance capable o f reading to 0.00001 g. 5.4 50 mL disposable polypropylene ccntrifogc tubes. 5.5 IS mL disposable polypropylene centrifuge tubes. 5.6 Disposable micropipets (50-100uL, 100-200uL). 5.7 125-mL LDPE narrow-mouth bottles. 5.8 2 m L clear HPLC vial kit. 5.9 Disposable pipettes. 5.10 Autopipettes (100*1000 pL and 10-100 pL), with disposable tips. 5.11 Waters Sep Pak Vac 6 cc (Ig ) tC18 SPE cartridges. Page 2 o f ? Page 17 o f 65 Exygen Research Page 55 of 105 Interim Report #3-Analysis o f Ground Water Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 Exygsn Research Method Number V0001780 a n a l y t ic a l m eth o d Method o f Analysis for the Deterraituton ofPerfluorooctanoic Acid (PFOA) in Water by L C /M S/M S 3.12 SPB vacuum manifold. 3.13 Centrifuge capable o f spinning 30 m L polypropylene tubes at 3000 rpm. 6.0 Chromatographic System 6.1 Analytical Column: Fluophaie RP (Keystone Scientific), 2.1 mm x 50 mm. 5m (P/N: 82305-052130) 6.2 Temperature: 30C 6.3 Mobile Phase (A) : 2 mM Ammonium Acetate in W ater 6.4 Mobile Phase (B) : Methanol 6.5 Gradient Program: Time (mint 0.0 1.0 8.0 20.0 22.5 65 63 23 25 63 Flow Rate % B (mL/mjnl 35 0.3 35 0.3 75 0.3 75 0.3 35 0.3 6.6 Injection Volume: 15 jiL (can b e increased to as much as SO pL). 6.7 Quantitation: Peak Area - external standard calibration curve. 6.8 Run Time: - 23 minutes. The above conditions are intended as a guide and may be changed in order to optimize the HPLC system. 7.0 MS/MS System 7.1 Mode: Electrospray Negative M R M m ode,m onitoring4 1 3 - 3 6 9 m/z. The above conditions are intended as a guide end m ay be changed in order to optimize the MSMS system. 8.0 Preparation o f Solutions 8.1 Mobile Phase 8.1.1 2 mM ammonium acetate in water is prepared b y adding 0.154 g o f ammonium acetate to 1000 mL o f water. Alternate volumes m ay be prepared. Page3ol ' Page 18 o f 65 Exygen Research Page 56 of 105 Interim Report #3-Analysis o f Ground W ater Samples Exygen Study No.: P0001131 Exygen Protocol Number: P000U31 Exygca Rctetrch Method Number VOW1780 ANALYTICAL METHOD Method o f Analysis for the Determination o f Periluorooctanoic Acid (PFOA) in W ater by L C /M S/M S 9.0 Standard Preparation 9.1 Standard Stock/Fortification Solution 9.1.1 Prepare a stock solution o f **100 pg/mL o f PFOA by weighing 10 mg o f analytical standard (corrected for purity) and dilute to 100 mL with methanol in a 125-mL LDPE bottle. 9.1.2 A 10 pg/intL fortification solution o f PFOA is prepared by bringing 10 m L o f the 100 pg/mL solution to a final volume o f 100 with methanol in a 125 m L LDPE bottle. 9.1.3 A 1.0 p ^ m L fortification solution o f PFOA is prepared by bringing 10 m L o f the 10 pg/mL solution to a final volume o f 100 with methanol in a 125 mL LDPE bottle. 9.1.4 A 0.1 pg/raL fortification solution o fPFO A is prepared by bringing 10 mL o f the 1.0 pg/mL solution to a final volume o f 100 with methanol in a 125 m L LDPE bottle. 9.1.5 A 0.01 pg/mL fortification solution o fP F O A is prepared by bringing 10 mL o f the 0.1 pg/mL solution to a final volume o f 100 with methanol in a 125 m L LDPE bottle. 9.1.6 The stock and fortification solutions are to be stored in a refrigerator at approximately 4C and are stable for a maximum period o f 6 months from foe date o f preparation. 9.2 Standard Calibration Solutions 9.2.1 9.2.2 LC/MS/MS calibration standards are prepared in HPLC water. The calibration standards are processed through the extraction procedure, identicsl to samples. The following is a typical example: additional concentrations may be prepared as needed. Final Concentration Fortification Voltane o f Concentration o f Calibration ofFortificition Volume Fortified Control Calibration Standard ID Solution (oob) 0 10 10 10 100 100 100 (PL) 0 100 200 400 100 200 400 Semole (m Ll 40 40 40 40 40 40 40 Standard (not)* 0 25 50 100 2S0 500 1000 (examole) XCmmddyy-0 XCmmddyy-1 XCmmddyy-2 XCnunddyyO XCmmddyy-4 XCmmddyy-5 XCmmddw-6 * The extracted concentration o f foe calibration standard is equal to 8x its initial concentration, due to the concentration o f the standard during foe extraction (SPE). XC " extracted calibration standard. Plgc4 o f' Page 19 o f 65 Exygen Research Page 57 o f 105 Interim Report #3-Analysis o f Ground Water Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 Exygea Retasrch Mfltbod Number VOOO1780 | ANALYTICAL M ETHOD | Method o f Analyst for the Determination o f Periluorooctanoic Acid (PFOA) in Water by L C /M S /M S 9.2.3 9.2.4 9.2.5 A zero standard solutioa (reagent blank) m ust be prepared with each set o f standards extracted. Store all extracted calibration standards in 15-mL polypropylene tubes at 2*C to 6 'C , up to two weeks. Alternate volumes and concentrations o f standards m ay be prepared as needed. 10.0 Batch Set Up 10.1 Each batch o f samples extracted (typically 20 or less) must include at least one reagent control (method blank using HPLC water) and (wo reagent controls fortified at known concentrations (lab control spike) to verify procedural recovery for the batch. 10.2 Requirements for field and laboratory duplicates and spikes will be specified in tfie quality assurance plan for this project 11.0 Sample Extraction 11.1 Measure 40 m L o f sim ple o r a portion o f sample diluted to 40 m L with water mto 50 m L polypropylene eentrifoge tubes (fortify as needed, replace lid and mix well). 11.2 Condition the C u SPE cartridges (1 g, 6 mL) by passing 10 mL methanol followed b y 5 m L o f HPLC water ( - 2 drop/sec). Do not let column run dry 11.3 Load sample on conditioned C u SPE cartridge. Discard eluate. 11.4 Elute with - 5 m L 100% m ethanol Collect 5 m L o f eluate into graduated 15 m L polypropylene centrifiige tubes (final volume 5 mL). 11.5 Analyze samples using electrospray LC/MS/MS. 12.0 Chromatography 12.1 Inject the same amount o f each standard, sample and fortified sample into the LC/MS/MS system. A calibration standard m ust precede and follow all analyzed samples. 12.2 Standards o f PFOA corresponding to at least five o r m ore concentration levels must be included in an analytical set. 12.3 An entire act o f extracted calibration standards m ust be included at the beginning and at the end o f a sample set. Extracted standards must be interspersed between every 5-10 samples. As an alternative, an entire set o f extracted calibration standards may be ityected at the beginning o f a set followed by extracted calibration standards interspersed every 5-10 samples (to account for a second set o f extracted standards). In either esse, extracted calibration standards must be the first and last injection in s sample set. 12.4 Use linear standard curves for quantitation. Linear standard curves are generated for die analyte by linear regression using 1/x weighting o f peak area Page 20 o f 65 Exygen Research Page 58 of 105 Interim Report #3-Analysis of Ground Water Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 ExygtaK m arch Method Number V0001780 ANALYTICAL M ETHOD Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Water by L C /M S/M S versus calibration standard concentration using MasaLynx 3.3 (or equivalent) soAware system. 12.5 Sample response should not exceed standard responses. Any samples that exceed standard responses should b e farther diluted and reanalyzed. 13.0 Acceptance Criteria 13.1 Chromatogram must show a peak o f a daughter ion at 369 amu Aom a parent o f 413 amu. The 413 amu parent corresponds to the PFOA anion, while the daughter ion (369 amu) represents the loss o f carbon dioxide. 13.2 Method blanks must not contain PFOA at levels greater than the LOQ. If a blank contains PFOA at levels greater than SO ng/L, then a new blank sample must be obtained and the entire set must be re-extracted. 133 Recoveries o f control spikes and matrix spikes must be between 70-130% of their known values. I f a control spike foils outside the acceptable limits, the entire set o f samples should be re-extracted. Any matrix spike outside 70 130% should be evaluated by the analyst to determine if re-extraction is warranted. 13.4 Any calibration standard found to be a statistical outlier by using the Huge Error Teat, may be excluded from the calculation o f the calibration curve. However, the total number o f extracted calibration standards that could be excluded must not exceed 20% o f the total num ber o f extracted standards injected. 13.S The correlation coefficient (R) for calibration curves generated must be 0.992 (R* 0.985). I f calibration results fall outride these limits, then appropriate steps must be taken to adjust instrument operation, and the standards or the relevant set o f samples should b e reanalyzed. 13.6 Retention times between standards and samples must not drift more than 4 % within an analytical run. I f retention tim e drift exceeds this limit within an analytical run then the set must be reanalyzed. 14.0 Calculations 14.1 Use the following equation to calculate the amount o f PFOA found (in ng/L. baaed on peak area) using the standard curve (linear regression parameters) generated by the Mass Lynx software program: PFOA found (ng/L) - fP elk m m - intercept) x DF slope DF * factor by which the final volume was diluted, if necessary. Page 6 of 7 Page 21 o f 65 Exygen Research Page 59 of 105 Interim Report #3-Analysis of Ground Water Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 ExyfM RuMrch Mstbod Number VOOO1780 | ANALYTICAL M ETHOD ~| Method o f Analysis for die Determination ofPerfluorooctanoic Acid (PFOA) in Water by L C /M S /M S \A 2 For samples fortified with known amounts o f PFOA prior to extraction, use the following equation to calculate the percent recovery. Recovery (%) f total analyte found (ng/L) - analyte found in control (ng/L)] ^ analyte added (ng/L) Exygen Research Page 7of7 Page 22 o f 65 Page 60 of 105 Interim Report #3-Analysis o f Ground Water Samples Exygen Study No.: P0001131 Exygen Protocol Number: POOO1131 ANALYTICAL METHOD Method N um ber V000178J M ethod o f Analysis for the D eterm iaatioo o f P e rflu o ro o c tu o k Acid (PFOA) In Soil by LC /M S/M S Analytical Testing Facility: Exygen Research 3058 Research Drive State College, PA 16801 Approved By: r i u , c - J i,____ Paul Connolly ' Technical Leader, LC-MS, Exygen Research ohn Flaherty ' Vice Prsidait, Operations, Exygen Research Date **r Date Exygen Research Total Pager 7 Page 23 o f 65 Page 61 o f 105 Interim Report #3-Analysis o f Ground Water Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 ExygraRnevch Method Number V000178J I ANALYTICAL M ETHOD Method o f Analysis for the Determination ofperfluorooctanoic Acid (PFOA) in Soil by L C /M S/M S 1.0 Scope This method is to be employed for the isolation and quantitation ofperfluorooctanoic a d d by High Performance Liquid Chromatography coupled to a tandem Mass Spectromtrie Detector (LC/MS/MS) in soil. 2.0 Safety 2.1 Always observe safe laboratory practices. 2.2 Consult the appropriate MSDS before handling any chemical for proper safety precautions. 3.0 Sample Requirement 3.) At least 15 g o f test sample for tractio n . 3.2 No sample processing is needed for soil samples. 3.3 Samples stored refrigerated should be allowed to equilibrate to room temperature. 3.4 All samples must be thoroughly mixed before being sampled for traction. 3.5 Sample collection procedures will be specified in the sampling plan for this project. 4.0 Reagents and Standards 4.1 W ater-H P L C grade 4.2 Methanol - HPLC grade 4.3 Ammonium Acetate - A.C.S. Reagent Grade 4.4 Perfluorooctanoic Acid - Sigma*Aldrich 5.0 Instrument sod Equipment 5.1 A high performance liquid chromatograph capable o f pumping up to 2 solvents equipped with variable volume injector capable o f injecting 5-200 pL connected to a tandem Maas Spectrometer (LC/MS/MS). 5.2 A device to collect raw data for peak integration and quantitation. 5.3 Analytical balance capable o f reading to 0.00001 g. 5.4 50 mL disposable polypropylene centrifuge tubes. 5.5 15 mL disposable polypropylene centriftjge tubes. 5.6 Disposable micropipets (S0-t00uL, 100-200uL). 5.7 125-mL LDPE narrow-mouth bottles. 5.8 2 m L clear HPLC vial kit. 5.9 Disposable pipettes. 5.10 Autopipettes (100-1000 pL and 10-100 pL), w ith disposable tips. 5.11 Waters Sep Pak Vac 6 cc (Ig ) tC18 SPE cartridges. 5.12 SPE vacuum manifold. 5.13 Ultrasonic bath. Page 2 of ? Page 24 o f 65 Exygen Research Page 62 of 105 Interim Report #3-Analysis o f Ground Water Samples Exygen Study No.: P0001131 Exygen Protocol Number: POOOi 131 Exygea Research Mtthod Number VOO1781 ANALYTICAL METHOD LC/MS/MSMethod o f Analytic for the Determination o f Perfluorooctanoic Acid (PFOA) in Soil by 5.14 Wrist-action shaker. 5.1 S Centrifuge capable o f spinning 50 m L polypropylene tubes at 5000 rpm. 6.0 Chromatographic System 6.1 Analytical Column: Fluophase RP (Keystone Scientific), 2 .1 mm x 50 mm. 5p (P/N: 82505-052130) 6.2 Temperature: 30*C 6.3 Mobile Phase (A) : 2 mM Ammonium Acetate in Water 6.4 Mobile Phase (B) : Methanol 6.5 Gradient Program: 0.0Time fmint 1.0 8.0 20.0 22.5 s a 65 65 25 25 65 Flow Rate % B ImL/min3 35 0.3 35 0.3 75 0.3 75 0.3 35 0.3 6.6 Injection Volume: 15 pL (can be increased to as m uch as 50 pL). 6.7 Quantitation: Peak Area - external standard calibration curve. 6.8 Run Time: - 23 minutes. The above conditions are intended as a guide and m ay b e changed in order (o optimize the HPLC system. 7.0 MS/MS System PFOA.7.1 Mode: Electrospray Negative MRM mode m onitoring 413 - 369 m/z for The above conditions are intended as a guide and m ay be changed in order to optimize die MSMS system. 8.0 Preparation o f Solutions 8.1 Mobile Phase 8.1.1 2 mM ammonium acetate in water is prepared by adding 0.154 g o f ammonium acetate to 1000 m L o f water. Alternate volume may be prepared. Page 3 of 7 Page 25 o f 65 Exygen Research Page 63 o f 105 Interim Report #3-Analysis of Ground Water Samples Exygen Study No.: P0001131 Exygen Protocol Number: POOOl 131 Exygea Rewrch Method Number VOOOI79I ANALYTICAL M ETHOD | Method o f Analysis for foe Determination o f Perfluorooctanoic Acid (PFOA) in Soil by L C /M S/M S 9.0 Standard Preparation 9.1 Standard Stock/Fortification Solution 9.1.1 Prepare a stock solution o f -1 0 0 pg/raL o f PFOA by weighing 10 mg o f analytical standard (corrected for purity) and dilute to 100 mL with 10methanol in a 125-mL LDPE bottle. 9.1.2 A 10 pg/mL fortification solution o f PFOA is prepared by bringing mL o f the 100 pg/mL solution to a final volume o f 100 with methanol in a 125 mL LDPE bottle. 9.1.3 A 1.0 pg/mL fortification solution o f PFOA is prepared by bringing 10 m L o ftb e 10 pg/mL solution to a final volume o f 100 with methanol in a 125 mL LDPE bottle. 9.1.4 A 0.1 pg/mL fortification solution o f PFOA la prepared by bringing 10 m L o f foe 1.0 pg/mL solution to a final volume o f 100 with methanol in a 125 mL LDPE bottle. 9.1.5 A 0.01 pg/mL fortification solution o f PFOA is prepared by bringing 10 m L o f foe 0.1 pg/m L solution to a firm! volume o f 100 with methanol in a 125 m L LDPE bottle. 9.1.6 The stock and fortification solutions am to be stored in a refrigerator at approximately 4*C and are stable for a maximum period o f 6 months from foe date o f preparation. 9 2 Standard Calibration Solutions 9.2.1 9.2.2 LC/MS/MS calibration standards are prepared in HPLC water. The calibration standards are processed through the extraction procedure, identical to samples. The following is a typical example: additional concentrations may be prepared a* needed. Final Conceaffatioa Fortification Volume of Concentration of Calibration ofFoctifioation Volume Fortified Control Calibration Standard ID Solution (nob) (ML) Santole (mL) Standard foot)* fexamole) 0 0 40 0 XCmmddyy-0 10 100 10 200 10 400 100 100 100 200 100 400 40 40 40 40 40 40 25 XCmmddyy-l SO XCmmddyy-2 100 XCmmddyyO 250 XCmmddyy-4 500 XCmmddyy-5 1000 XCmmddw-6 * The extracted concentration o f foe calibration standard is equal to 8x its initial concentration, due to the concentration o f the standard during foe extraction (SPE). XC extracted calibration standard. Page 4 of 7 Page 26 o f 65 Exygen Research Page 64 of 105 Interim Report #3-Analysis o f Ground W ater Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 Exygta Rnearck Method Number VOOOl 78] I ANALYTICAL METHOD Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Soil by LC/MS/MS 9.2.3 9.2.4 9.2.5 A zero standard solution (reagent blank) must be prepared with each set o f standards extracted. Store all extracted calibration standards in 15-mL polypropylene lubes at 2*C to 6C, up to two weeks. Alternate volumes and concentrations o f standards may be prepared as needed. 10.0 Batch Set Up lO.t Each batch o f samples extracted (typically 20 o r less) must include at least one reagent control (method blank using 5 raL o f methanol) and two reagent controls fortified at known concentrations (lab control spike) to verify procedural recovery for the batch. 10.2 Requirements for field and laboratory duplicates and spikes will be specified in the quality assurance plan for this project. 11.0 Sample Extraction 1 U W eigh 5 g o f sample into 50 m L polypropylene centrifuge tubes (fortify as needed, replace lid and mix well). 1U Add 5 m L o f methanol and shake on a wrist action shaker for 15 minutes 1 U Transfer the tubes to an ultrasonic bath and sonicate for 15 minutes. 11.4 Bring the volume up to 40 m L with water in the 50 m L polypropylene centrifoge tube. 11.5 Centrifuge fo r- 1 0 minutes at -3 0 0 0 rpm. 11.6 Condition the C SPE cartridges (1 g. 6 mL) by passing 10 mL methanol followed by 5 m L o f HPLC water ( - 2 drop/sec). Do not let column run dry 11.7 Load (decant) the sample on the conditioned C u SPE cartridge. Discard eluate. 11.8 Elute with - 5 mL 100% methanol. Collect 5 m L o f eluate into graduated 15 m L polypropylene centrifoge tubes (final volume - 5 mL). 11.9 Analyze samples using eleotrospray LC/MS/MS. 12.0 Chromatography 12.1 Inject the same amount o f each standard, sample and fortified sample into the LC/MS/MS system. A calibration standard m ust precede and follow all analyzed samples. 12.2 Standards o f PFOA corresponding to at least five or m ore concentration levels must be included in an analytical set. 12.3 A n entire sec o f extracted calibration standards must be included at the beginning and at the end o f a sample s e t Extracted standards must be interspersed between every 5-10 samples. As an alternative, an entire set o f Page 3 of 7 Page 27 o f 65 Exygen Research Page 65 of 105 Interim Report #3-Analysis o f Ground Water Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 Exygea Research Method Number V000i 781 I ANALYTICAL M E T H O D ...... Method o f Analysis for the Determination o f Perfluorooctaaoic Acid (PFOA) in Soil by LC/MS/MS extracted calibration standards m ay be injected at the beginning o f a set followed by extracted calibration standards interspersed every 5*10 samples (to account for a second set o f extracted standards). In either case, extracted calibration standards must be the tin t and last injection in a sample set. 12.4 Use linear standard curves for quantitation. Linear standard curves are generated for foe analyte by linear regression using i/x w eighting o f peak area versus calibration standard concentration using MassLynx 3.3 (or equivalent) software system. 12.5 Sample response should not exceed standard responses. Any samples that exceed standard responses should be further diluted and reanalyzed. 13.0 Acceptance Criteria 13.1 13.2 13.3 13.4 13.5 13.6 Chromatogram must show a peak o f a daughter ion at 369 amu from a parent o f 413 amu. The 413 amu parent corresponds to the PFOA anion, while the daughter ion (369 amu) represents the loss o f carbon dioxide. Method blanks must not contain PFOA at levels greater than the LOQ If a blank contains PFOA at levels greeter than SO ng/L, then a new blank sample must be obtained and the entire set must be re-extracted. Recoveries o f control spikes and matrix spikes must be between 70-130% of their known values. I f a control apike falls outside the acceptable limits, the entire set o f samples should be re-extracted. Any matrix spike outside 70 130% should be evaluated by foe analyst to determine if re-extraction is warranted. Any calibration standard found to be a statistical outlier by using the Huge Error Test, may be excluded from the calculation o f foe calibration curve. However, the total number o f extracted calibration standards that could be excluded must not exceed 20% o f the total number o f extracted standards iryected The correlation coefficient (R) for calibration curves generated must be 20.992 (R] 20.985). If calibration results fall outside these limits, then appropriate steps must be taken to adjust instrument operation, and the standards or the relevant set o f samples should be reanalyzed Retention tim et between standards and samples m ust not drift more than 4 % within an analytical run. I f retention tim e drift exceeds this limit within an analytical run then foe set must be reanalyzed. Page 6 o f 7 Page 28 o f 6} Exygen Research Page 66 o f 105 Interim Report #3-Analysis o f Ground W ater Samples Exygen Study No.: P0001131 Exygcn Protocol Number: P0001131 Exyfco RMMicb Method Number V0001781 ANALYTICAL M ETH O D __________________________ Method o f Analytic for the Determination o f Periluorooctanoic Acid (PFOA) in Soil by L C /M S/M S 14.0 Calculations 14.1 Use the following equation to calculate foe amount o f PFOA found (in ng/L, based on peak area) using the standard curve (linear regression parameters) generated by foe Mass Lynx software program: PFOA found (ng/L) - (Peak area - intercept) x DF slope DF factor by which foe final volume was diluted, i f necessary. 14.2 For samples fortified with known amounts o f PFOA prior to extraction, use the following equation to calculate foe percent recovery. Recovery (% ) [total analyte found (ng/L) - analyte found in control (ng/L)] analyte added (ng/L) 14.3 Use foe following equation to convert the amount o f PFOA found in ng/L to ng/g(ppb). PFOA found (ppb) - fPFQA found fnc/L) x volume extracted (0.Q4L11 sample weight (5 g) 14.4 Uae foe following equation to calculate the amount o f PFOA found in ppb based on dry weight PFOA found (ppb) dry weight PFOA found (ppb) x [100% / total sotids(%)] Pag*7 o f7 P age 29 o j 65 Exygen Research Page 67 of 105 Interim Report #3-Analysis of Ground Water Samples Exygen Study No. : P0001131 Exygen Protocol Number: POOO1131 ANALYTICAL METHOD Method Number: V0001782 M ethod o f Analysis fo r the D eterm ination o f P erllaorooctanoic Acid (PFO A ) to Sediment by LC/MS/MS Analytical Testing Facility: Exygen Research 3058 Research Drive State College, PA 16801 Approved By: C JL Paul Connolly [ Technical Leader, LC-MS, Exygen Research io k .b h '1 Date Z Zj'if i ? j j j y ydiobhin Flaherty f Viicc<e President, Operations, Exygen Research Date Exygen Research Total Paget: 7 Page 30 o f 65 Page 68 of 105 Interim Report #3-Analysis o f Ground Water Samples Exygen Study No. : P0001131 Exygen Protocol Number: POOO1131 E x y p s Rcacuvb Method Number V0001782 ANALYTICAL M ETHOD Method o f Analysis for the Determination o f Periluorooctanoic Acid (PFOA) in Sediment by L C /M S/M S 1.0 Scope T hii method i t to b e employed for the eolation and quantitation o f periluorooctanoic acid by High Performance Liquid Chromatography coupled to a tandem M ass Spectrametric Detector (LC/MS/MS) in sediment. 2.0 Safety 2.1 Always observe safe laboratory practices. 2.2 Consult the appropriate MSDS before handling any chemical for proper safety precautions. 3.0 Sample Requirement 3.1 At least 30 g o f te s t sample for extraction. 3.2 No sample processing is needed for sediment samples. 3.3 Samples stored refrigerated should b e allowed to equilibrate to room temperature. 3.4 All samples must be thoroughly m ixed before being sampled for extraction. 3.5 Sample collection procedures will be specified in the sampling plan for this project 4.0 Reagents and Standards 4.1 W ater - HPLC grade 4.2 Methanol - HPLC grade 4.3 Acetic A d d - Reagent grade 4.4 Ammonium Acetate - A.C.S. Reagent Grade 4.5 Periluorooctanoic Acid - Sigma-Aldrich 5.0 Instrument and Equipment 5.1 A high performance liquid chromatograph capable o f pumping up to 2 solvents equipped with a variable volume injector capable o f injecting 5-200 pL connected to a tandem Mass Spectrometer (LC/MS/MS). 5.2 A device to collect raw data for peak integration and quantitation. 5.3 Analytical balance capable o f reading to 0.00001 g. 5.4 50 mL disposable polypropylene ccntrifiige tubes. ' $.5 15 m L disposable polypropylene centrifuge tubes. 5.6 Disposable micropipets (50-100uL, !00-200uL). 5.7 125-mL LDPE narrow-mouth bottles. 5.8 2 mL clear HPLC vial kit. 5.9 Disposable pipettes. 5.10 Autopipettes (100-1000 pL and 10-100 pL), with disposable tips. 5.11 Waters Ssp Pak Vac 6 c c ( l g ) t C l 6 SPE cartridges. 5.12 SPE vacuum manifold. Pag* 2 of 7 Page 31 o f 65 Exygen Research Page 69 of 105 Interim Report #3-Analysis o f Ground Water Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 Exygea Research Method Number V0001782 I ANALYTICAL M ETHOD Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Sediment bv L C /M S /M S ' 5.13 Vortexer. 5.14 Wrist-actioo shaker. 5.15 Centrifiige capable o f pinning 50 xnL polypropylene tubes at 3000 rpm. 6.0 Chromatographic System 6 .1 Analytical Column: Fluophase RP (Keystone Scientific), 2.1 mm x 50 mm. 5p (P/K: 82505*052130) 6.2 Temperature: 30*C 6.3 Mobile Phase (A) : 2 m M Ammonium Acetate in W ater 6.4 Mobile Phase (B) : Methanol 6.5 Gradient Program: Tima fminl 0.0 1.0 8.0 20.0 22.5 2LA 6$ 65 25 25 65 Flow Rate 5LB (mL/minl 35 0.3 35 0.3 75 0.3 75 0.3 3$ 0.3 6.6 Injection Volume: 15 p L (can be increased to aa much as 50 pL). 6.7 Quantitation: Peak Area - external standard calibration curve. 6.8 RunTim e: - 2 3 minute*. The above conditions are intended as a guide and m ay be changed in order to optimize the HPLC system. 7.0 MS/MS System 7.1 Mode: HlectrOspray Negative MRM mode, monitoring 413 369 m/z for PFOA. The above conditions are intended aa a guide and m ay b e changed in order to optimize tite MSMS system. 8.0 Preparation o f Solutions 8.1 Mobile Phase 8.1.1 2 mM ammonium acetate in water is prepared by adding 0.IS4 g o f ammonium acetate to 1000 m L o f water. Page 3 of 7 Page 32 o f 65 Exygen Research Page 70 o f 105 Interim Report #3-Analysis o f Ground W ater Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 Exygen Raiaareh Method Number VOOO1782 I ANALYTICAL METHOD "] Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Sediment by L C /M S/M S 8.2 Extraction Solutions 8.2.1 1% acetic acid in water is prepared by adding 10 m L o f acetic acid to 1000 m L o f water. Alternate volumes m ay b e prepared. 9.0 Standard Preparation 9.1 Standard Stock/Fortification Solution 9.1.1 Prepare a stock solution o f -100 pg/m L o f PFOA by weighing 10 mg o f analytical standard (corrected for purity) and dilute to 100 mL with methanol in a 125-mL LDPE bottle. 9.1.2 A 10 pg/mL fortification solution o f PFOA i t prepared by bringing 10 m L o f the 100 pg/mL solution to a final volume o f 100 with methanol in a 125 mL LDPE bottle. 9.1.3 A 1.0 pg/mL fortificatimi solution o f PFOA is prepared by bringing 10 m L o f die 10 pg/mL solution to a final volume o f 100 with methanol in a 123 m L LDPE bottle. 9.1.4 A 0.1 pg/mL fortification solution o f PFOA is prepared by bringing 10 m L o f die 1.0 jtg/mL solution to a final volume o f 100 with methanol in a 125 mL LDPE bottle. 9.1.5 A 0.01 figrinL fortification solution o f PFOA is prepared by bringing 10 mL o f the 0.1 pg/inL solution to a final volume o f 100 with methanol in a 125 mL LDPE bottle. 9.1.6 H ie stock and fortification solutions are to be stored in a refrigerator at approximately 4*C and are stable for a maximum period o f 6 months from die date o f preparation. 9.2 Standard Calibration Solutions 9.2.1 LC/MS/MS calibration standards are prepared in methanol via dilution o f die 0.1 pg/mL fortification solution. 9.2.2 The following i t a typical example: additional concentrations may be Concentration o f Fortification Solution fna/mL) 100 100 too 10 5 2 Volume (mL) 10 5 2 10 10 10 . Diluted to (mL) 100 100 100 100 100 100 Final Concentration (ng/mL) 10.0 so 20 1.0 OS 0.2 Page 4 of 7 Page 33 o f 65 Exygen Research Page 71 o f 105 Interim Report #3-Analysis o f Ground Water Samples Exygen Study No. : P0001131 Exygen Protocol Number: P0001131 Exyfcs&Mearcb Method Nimber V00017S2 ANALYTICAL M ETHOD | Method o f Anilysis for the Determination ofPerfluorooctsnolc Acid (PFOA) in Sediment by L C /M S/M S 9.2.3 9.2.4 Store all calibration standards in 125-mL LDPE narrow-mouth bottles at 2C to 6*C, up to fix months. Alternate volumes and concentrations o f standards may be prepared as pfwjftd 10.0 Batch Set Up 10.1 Each batch o f samples extracted (typically 20 o r leas) must include at least one untreated control and two untreated controls fortified at known concentrations (lab control spike) to verify procedural recovery for the batch. 10.2 Requirements for field end laboratory duplicates and spikes will be specified in the quality assurance plan for this project 11.0 Sample Extraction 1l .l Weigh 5 g o f sample into 50 mL polypropylene centriftige tubes (fortify as needed, replace lid and mix well). 11.2 Add 35 m L o f 1% acetic acid, cap, vortex and shake on a wrist action shaker for - 6 0 minutes. 11.3 Centriftige the tubes at ~3000 (pm for - 2 0 minutes. 11.4 Condition the C n SPE cartridges (1 g, 6 m L) by passing 10 mL methanol followed by 20 m L ofH PL C water (~ 2 drop/sec). D o not let column run dry 11.5 Load (decant) the sample on the conditioned Cis SPE cartridge. Discard chute. 11.6 Add 20 m L o f methanol to the sediment left in the bottom o f the 50 mL centriftige tube. Cap, vortex and shake on a wrist action shaker for *-30 minutes. 11.7 Centriftige the tubes i t ~3000 rpm for ~20 minutes. 11.8 Decant the methanol onto the same SPE cartridge. Collect the eluate. 11.9 Wash the column with 4 m L o f methanol. Collect the eluate and add it to the ehiite collected in step 11.8. 11.10 Condition a second C u SPE cartridge (1 g, 6 mL) by passing 10 mL methanol followed by 20 m L ofH PL C water (~ 2 drop/sec). Do not let column run dry 11.11 Add the methanol to - 2 0 0 m L o f water and load on the second conditioned SPE cartridge. 11.12 Elute with - 5 mL 100% m ethanol Collect 5 mL o f eluate into graduated 15 mL polypropylene centriftige tubes (final volume 5 mL). 11.13 Analyze samples using electrospray LC/MS/MS. P>S* 3 of 7 Page 34 o f 65 Exygen Research Page 72 of 105 Interim Report #3-Analysis of Ground Water Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 Exyge&Rmarcb Method Number V00017S2 ANALYTICAL M ETHOD 1 Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Sediment by L C /M S /M S 12.0 Chromatography 12. i Inject the same amount o f each standard, sample and fortified sample into the LC/MS/MS system, A calibration standard m ust precede and follow all analyzed samples. - 12.2 Standards o f PFOA corresponding to t least five o r more concentration levels must be included in an analytical set. 12.3 A n t ir e set o f extracted calibration standards must be included at the beginning and at the end o f a sample set. Standards must be interspersed between every 3-10 samples. A s an alternative, an entire set o f calibration standards may be injected at die beginning o f a set followed by calibration standards interspersed every 3-10 samples (to account for a second set of standards). In s itte r case, calibration standards must be the first and last injection in a ample set. 12.4 Use linear standard curves for quantitation. Linear standard curves arc generated for the analyte by lin e regression using 1/x weighting o f peak area versus calibration standard concentration using M assLynx 3.3 (or equivalent! software system. 12.3 Sim ple response should not exceed standard responses. Any samples that exceed standard responses should be fla tte r diluted end reanalyzed. 13.0 Acceptance Criteria 13.1 Chromatogram must show peak o f t daughter ion at 369 amu from a parent o f 413 amu. The 413 am u parent corresponds to the PFOA anion, while the daughter ion (369 amu) represents the loss o f carbon dioxide. 13.2 Method blanks must not contain PFOA t levels greater than the LOQ. If a blank contains PFOA t levels greater than 0.2 ng/mL, then a new blank sample m ust be obtained and the tr e set m ust be re-extracted. 13.3 Recoveries o f control spikes and matrix spikes m ust be between 70 130% o f their known values. I f a control spike M s outside the acceptable limits, the entire set o f samples should be re-extracted. Any matrix spike outside 70 130% should be evaluated by the analyst to determine i f re-extraction is warranted. 13.4 Any calibration standard found to be a statistical outlier by using the Huge Error Test, may b e excluded from the calculation o f the calibration curve. However the total number o f extracted calibration standards that could be excluded must not exceed 20% o f the total number o f extracted standards iqjectod. 13.3 The correlation coefficient (R) for calibration curves generated must be 0.992 (R2 0.985). I f calibration resulte fall outside these limits, then appropriate steps must be taken to adjust instrument operation, and the standards or the relevant set o f samples should be reanalyzed. Page 6 of 7 Page 35 o f 65 Exygen Research Page 73 of 105 Interim Report #3-Analysis o f Ground Water Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 Exygn&MOich Method Number V0001782 | ANALYTICAL METHOD | Method o f AnalycU for the Detennination ofPerfluorooctanoic Acid (PFOA) in Sediment by L C /M S /M S 13.6 Retention times between standards and samples must not drift more than 4 % within an analytical run. I f retention tim e drift exceeds this limit within an analytical ran then the set must be reanalyzed. 14.0 Calculations 14.1 Use the following equation to calculate the amount o f PFOA found (in ng/mL. baaed on peak area) using the standard curve (linear regression parameters) generated by the Mass Lynx software program: PPOA found (ng/mL) - (Peak are* - intercept) x OF slope DF " factor by which the final volume was diluted, if necessary. 14.2 For samples fortified with known amounts o f PFOA prior to extraction, use the following equation to calculate the percent recovery. Recovery ( % ) - f total analyte found (ng/mL) - analyte found in oontrol (ng/mL)] ] ^ analyte added (ng/mL) 14.3 Use the following equation to convert the amount o f PFOA found in ng/mL to ng/g (ppb). PFOA found (ppb) * fPFQA found foe/m Ll x final volume (3 mL)1 sample weight (3 g) 14.4 Use foe following equation (if necesasry) to calculate the amount o f PFOA found in ppb based on dry weight. PFOA found (ppb) dry weight PFOA found (ppb) x [1 0 0 % / total solids(%)] Page 7 of 7 Page 36 o f 65 Exygen Research Page 74 of 105 Interim Report #3-Analysis o f Ground W ater Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 ANALYTICAL METHOD Method N um ber V0001783 M ethod of Analysis for the Determ ination o f Perflvorooctaoolc Acid (PFOA) to Fish and Clame by LC/MS/MS Analytical Testing Facility: Exygen Research 305$ Research Drive State College, PA 16801 Approved By. rj L Paul Connolly 1 Technical Leader LC-MS, Exygen Research 4 2 f,/ 07 lohn Flaherty * Viccee President, Operations, Exygen Research Date Date Exygen Research Total Pages: 8 Page 37 o f 65 Page 75 of 105 Interim Report #3-Analysis o f Ground Water Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 Exygen Research Method Number VOOO1783 ANALYTICAL m e t h o d Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Fish and Clams by LC/MS/MS 1.0 Scope This method is to be employed for the isolation and quantitation o f perfluorooctanoic a d d by High Performance Liquid Chromatography coupled to a tandem Mass Spectrometric Detector (LC/MS/MS) in Ash and clams. 2.0 Safety 2.1 Always observe safe laboratory practices. 2.2 Consult the appropriate MSDS before handling any chemical for proper safely precautions. 3.0 Sample Requirement 3.1 At least 20 g o f test sample for extraction 3.2 Sample should be processed before extraction. Place the frozen sample in a food processor and homogenize with dry ice. Place foe samples in containers and leave open in frozen storage overnight to allow for carbon dioxide sublimation. Seal and place the samples in frozen storage until time o f analysis. 3.3 Sample collection procedures will be specified in the sampling plan for this project 4.0 Reagents and Standards 4.1 W ater-H P L C grade 4.2 Acetonitrile - HPLC grade 4.3 Carbon (120-400 mesh) - Reagent grade 4.4 Methanol - HPLC grade 4.3 Silica gel (60-200 mesh) - Reagent grade 4.6 FlorUil (60-100 mesh) - Reagent grade 4.7 Superclean LC-NHj - Reagent grade 4.8 1-Octanol - HPLC grade 4.9 L-Ascotbic a d d - Reagent grade 4.10 Dimethyldichloroetiaiie - Reagent grade 4.11 Toluene - Reagent grade 4.12 Ammonium Acetate - A.C.S. Reagent Grade 4.13 Perfluorooctanoic Acid - Sigma-Aldrich 3.0 Instrument and Equipment 5.1 A high performance liquid chromatograph capable o f pumping up to 2 solvents equipped with a variable volume injector capable o f injecting 5-200 pL connected to a tandem M ast Spectrometer (LC/MS/MS). 5.2 A device (o collect raw data for peak integration and quantitation. 3.3 Analytical balance capable o f reading to 0.00001 g. Page 2 of 8 Page 38 o f 65 Exygen Research Page 76 of 105 Interim Report #3-Analysis o f Ground Water Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 Exygett Rmarch Method Number V0001783 ANALYTICAL m e t h o d Method o f Analytic for the Determination o f Perfluorooctanoic Acid (PFOA) in Fish and Clim e by LC/MS/MS 5.4 Rotary evaporator. 5.5 Tiasumizer. 5.6 125 mL pear-shaped flasks. 5.7 50 mL disposable polypropylene centrifuge tubes. 5.8 15 mL disposable polypropylene centrifuge tubes. 5.9 Disposable micropipets (50-100uL, 100*200uL). 5.10 12S-mL LDPE narrow-mouth bottles. 5.11 2 mL clear HPLC vial Id t 5.12 Disposable pipettes. 5.13 Autopipettes (100*1000 pL and 10-100 pL), with disposable tips. 5.14 SPE tubes (20mL) (Supelco cat. no. N057I77). 5.15 Wrist action shaker. 5.16 Centrifuge capable o f spuming 50 m L polypropylene tubes at 2000 rpm. 6.0 Chromatographic System 6.1 Analytical Column: Fluophase RP (Keystone Scientific), 2.1 mm x 50 mm. 5p (P/N: 82505*052130) 6.2 Temperature: 30C 6.3 Mobile Phase (A) : 2 mM Ammonium Acetate in Water 6.4 Mobile Phase (B ): Methanol 6.5 Gradient Program: T h w a/m nit 0.0 1.0 8.0 20.0 22.5 5kA 65 65 25 25 65 Flow Rate % B fmL/miril 35 0.3 35 0.3 75 0.3 75 0.3 35 0.3 6.6 Injection Volume: 15 pL (can be increased to as much as 50 pL). 6.7 Quantitation: Peak Area - external standard calibration curve. 6.8 RunTim e: - 2 3 minutes. The above conditions are intended as s guide and may be changed in order to optimize the HPLC system. Page 3 of8 Page 39 o f 65 Exygen Research Page 77 of 105 Interim Report #3-Analysis of Ground Water Samples Exygen Study No.: P0001131 Z "' Exygen Protocol Number: P0001131 Exygea Rneveh Method Number V0001783 ANALYTICAL METHOD Method o f Analysis for the Determination o f Perfhiorooctsnotc Acid (PFOA) in Fish and Clams by LC/MS/MS 7 0 MS/MS System 7.1 Mode: Electrospray Negative MRM mode, monitoring 413 - 369 m/z for PFOA. The above conditions are intended as a guide sod m ay be changed in order to optimize the MSMS system. 8.0 Preparation o f Solutions 8.1 Mobile Phase 8.1.1 2 mM ammonium acetate in water is prepared by adding 0.IS4 g o f ammonium acetate to 1000 mL o f water. 82 Extraction Solutions 8.2.1 8.2.2 2% ascorbic acid in methanol is prepared by dissolving 2 g o f ascorbic a d d in 100 m L o f methanol. 30% DimethyMichlorotilane in toluene is prepared by bringing 3 mL o f dimethyldichlorosilane to s final volume o f 10 mL with toluene. Alternate volumes may be prepared. 9.0 Standard Preparation 9.1 Standard StoettFortification Solution 9.1.1 9.1.2 9.1.3 9.1.4 9.1.5 Prepare a node solution o f ~10G pg/m L o f PFOA by weighing 10 mg o f analytical standard (corrected for purity) and dilute to 100 mL with methanol in a 125-mL LDPE bottle. A 1.0 pg/mL fortification solution o f PFOA is prepared by bringing 1 m L o f the 100 pg/mL solution to a final volume o f 100 with methanol in a 123 m L LDPE bottle. A 0.1 pgfatL fortification solution o f PFOA is prepared by bringing 10 m L o f foe 1.0 p g ta L solution to a final volume o f 100 with methanol in a 125 mL LDPE bottle. A 0.01 pgfmL fortification solution o f PFOA is prepared by bringing 10 m L o f the 0.1 pgfmL solution to a final volume o f 100 with methanol in a 125 raL LDPE bottle. The stock and fortification solutions are to b e stored in a refrigerator at approximately 4*C and are stable for a maximum period o f 6 months from the date o f preparation. Page 4 of 8 Page 40 o f 65 Exygen Research Page 78 of 105 Interim Report #3-Analysis o f Ground W ater Samples Exygen Study No.: P0001131 Exygen Protocol Number: POOO1131 Exygea Research Method Number V0001783 1 ANALYTICAL M ETHOD | Method o f Analysis for the Determination o f Pcrftuorooctanoic Acid (PFOA) in Fish and Clams by LC/MS/MS 9.2 Standard Calibration Solutions 9.2.1 922 LC/MS/MS calibration standards are prepared in methanol via dilution o f the 1.0 pg/mL fortification solution. The following is a typical example: additional concentrations may be prepared aa needed. Concentration o f Fortification Solution fue/raL) 1.0 1.0 1.0 0.05 0.025 0.1 0.005 Volume (mL) $.0 2.5 1.0 10 10 10 10 Diluted to (mL) 100 100 100 100 100 100 100 Final Concentration fua/mU 0.05 0.025 0.01 0.005 0.0025 0.001 0.0005 9.2.3 Store all calibration standards in 125-mL LDPE narrow-mouth bottles at 2C to 6*C, up to six months. 9 2 .4 Alternate volumes and concentrations o f standards m ay be prepared as needed. 10.0 Batch Set Up 10.1 Each batch o f samples extracted (typically 20 o r lees) m ust include at least one untreated control and two untreated controls fortified at known concentrations (lab control spike) to verify procedural recovery for foe batch. 10.2 Requirements for field and Laboratory duplicates and spikes will be specified in the quality assurance plan for this project. 11.0 Sample Extraction 11.1 Weigh 5 $ o f frozen sample into 50 m L polypropylene centrifuge tubes (fortify aa needed, replace lid and mix well). 11.2 Add 30 m L o f acetonitrile and shake on a wrist action shaker for - I S minutes. 11.3 Place the tubes in a freezer for ~1 hour. 11.4 Pack and condition the SPE tubes and silanize the pear-shaped flasks. 11.5 Pack the 20 m L SPE tubes in sequence w ith 2 g flonril, 2 g silica gel, 2 g carbon, and t g LC-NHj. Condition the columns with 20 m L o f methanol, then 20 m L o f acetonitrile. Discard all washes. D o not allow foe column to dry. 11.6 Silanize foe 125 m L pear-shaped flasks b y rinsing with foe 30% diraefoykiichlorosiUne in toluene solution. Rinse foe flask with toluene once, followed by methanol (three times). Dry the flasks completely before use, either by air-drying o r with a stream o f nitrogen. Page S of a P age 4 / o f 65 Exygen Research Page 79 o f 105 Interim Report #3-Analysis o f Ground W ater Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 Exygen RetMich Method Number V000I7&3 ANALYTICAL METHOD Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Fish and Chuns by LC/MS/MS 11.7 Centrifoge the 50 mL polypropylene tubes containing sample at -2 0 0 0 rpm fo r- 1 0 minutes. 11.8 Decant the extract on to a conditioned SPE column fitted inside the mouth o f the pear-shaped flask. Collect the eluaie in the 125 m L silanized pear-shape flask. 11.9 Add 10 mL o f acetonitrile to the sample in the SO m L centrifiige tube. Homogenize the frozen fat phase using a H fin w i r for - 3 0 seconds and rinse the tiaaumizer with - 1 0 m L o f acetonitrile into the tube. 11.10 Shake die sample again fo r- 1 0 minute cm a wrist-action shaker. 11.11 Place die tubes in a freezer for - 1 hour more. 11.12 Centrifuge the 50 m L polypropylene tubes containing sample at -2 0 0 0 rpm fo r-1 0 minutes. 11.13 Decant die extract onto die same SPE column. Collect the eluate into the tam e pear-shaped flask and combine with die eluent from the initial extraction. 11.14 Pass 20 mL o f acetonitrile through the SPE colum n and combine the eluate in the same pear-chaped flask. 11.15 Add 3-4 drops o f 1-octsnol to the extract in the pear-shaped flask and evaporate at reduced pressure using a rotary evaporator (at < 40C). 11.16 Make the final volume, by adding 2 m L o f 2% ascorbic acid in methanol to the pear-shaped flask and swirl to mix/dissolve. 11.17 Transfer the extracts to HPLC vials using disposable pipets. 11.18 Analyze samples using electrospray LC/MS/MS. 12.0 Chromatography 12.1 Irgoct the same amount o f each standard, sample and fortified sample into the LC/MS/MS system. A calibration standard m ust precede and follow all analyzed samples. 12.2 Standards o f PFOA corresponding to at least five o r m ore concentration levels must be included in an analytical set 12.3 An a ttire set o f calibration standards m ust be included at the beginning and at the end o f a sample set. Standards must be interspersed between every 5 -10 samples. As an alternative, an entire set o f calibration standards may be injected at the beginning o f a set followed by calibration standards interspersed every 5-10 samples (to account for a second set o f standards). In either ease, calibration standards must be the first and last injection in a sample set 12.4 Use linear standard curvet for quantitation. Linear standard curves are generated for the analyte by linear regression using 1/x weighting o f peak area versus calibration standard concentration using M assLynx 3.3 (or equivalent) software system. Page 6 of 8 P age 42 o f 65 Exygen Research Page 80 of 105 Interim Report #3-Analysis of Ground Water Samples Exygen Study No. : P0001131 Exygen Protocol Number: P0001131 ExygtaRMMrcfa 0001Method Number V 783 ANALYTICAL M ETHOD Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Fish and Clams by LC/MS/MS 12.5 Sample response should not exceed standard responses. Any samples that exceed standard responses should be Anther diluted and reanalyzed. 13.0 Acceptance Criteria 13.1 Chromatogram m ust show a peak o f a daughter ion at 369 amu from a parent o f 413 amu. The 413 amu parent corresponds to the PFOA anion, while the daughter ion (369 amu) represents the loes o f carbon dioxide. 13.2 Method blanks must not contain PFOA at levels greater than (he LOQ. I f a blank contains PFOA at levels greater than 0.5 ppb, then a new blank sample must be obtained and the entire set must b e re-extracted. 13.3 Recoveries o f control spikes and matrix spikes must be between 70-130% o f their known values. I f a control spike Adis outside the acceptable limits, the entire set o f samples should be re-extracted. 13.4 Any calibration standard found to be a statistical outlier by using the Huge Error Test, may be excluded from the calculation o f the calibration curve. However, the total number o f calibration standards that could be excluded must not exceed 20% o f the total number o f standards injected. 13.5 The correlation coefficient (R) for calibration curves generated must be 20.9% (R2 20.985). I f calibration results foil outside these limits, then appropriate steps must be taken to adjust instrument operation, and the standards o r the relevant set o f samples should b e reanalyzed. 13.6 Retention times between standards and samples must not drift more than 4 % within an analytical run. I f retention time drift exceeds this limit within an analytical run then the set must be reanalyzed. 14.0 Calculations 14.1 Use the following equation to calculate the amount o f PFOA found (in ng/mL. based on peak area) using the standard curve (linear regression parameters) generated by die M ew Lynx software program: PFOA found (ng/raL) - (Peak area - intercept) slope 14.2 Use the following equation to convert the amount o f PFOA found in ng/mL to ng/g (R>b). PFOA found fapb) - (PFOA found fn e/m D x final volume fmLl x DF1 sample weight (g) DF * factor by which the final volume was diluted, if necessary. Page 7 of 8 Page 43 o f 65 Exygen Research Page 81 o f 105 Interim Report #3-Analysis o f Ground Water Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 Exygen Research Method Number VOOOi 783 ANALYTICAL m e t h o d Method o f Analysis for the Determination ofPerfluorooctanoic Acid (PFOA) in Fish and Clams by LC/MS/MS 14.3 For samples fortified with known amounts o f PFOA prior to extraction, use the following equation to calculate the percent recovery. Recovery (% ) [ total analyte found (n g /g ) analyte found in control (ng/g)) ^ analyte added (ng/g) Exygen Research Page 8 ofS Page 44 o f 65 Page 82 of 105 Interim Report #3-Analysis of Ground Water Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 ANALYTICAL METHOD Method Number: V001784 M ethod o f Analysis for th e D eterm ination o f Perflnorooetaaolc A d d (PFO A ) in Vegetation by LC/MS/MS Analytical Testing Facility: Exygen Research 3058 Research Drive State College, P A 16801 Approved By: TtA cJiL Paul Connolly Technical Leader, LC-MS, Exygen Research n /m fiL C /___________ J o h n Flaherty * ^ Vice President, Operations, Exygen Research -- la jw H Date Exygen Research Total Pages: 7 Page 45 o f 65 Page 83 o f 105 Interim Report #3-Analysis o f Ground Water Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 Bxygen Reseeidi M ethod Number VOQUI 7S4 ANALVTiCAL M ETHOD Method o f Analysis for the Determinedon o f Periluorooctanoic Acid (PFOA) in Vegetation by LC/MS/MS 1.0 Scope This method is to be employed for the isolation end quantitation o f perfluorooctanoic acid b y High Performance Liquid Chromatography coupled to a tandem Mass Spectromtrie Detector (LC/MS/MS) in vegetation. 2.0 Safety 2.1 Always observe safe laboratory practices. 2.2 Consult foe appropriate MSDS before handling any chemical for proper safety precautions. 3.0 Sample Requirement 3.1 At leest 20 g o f test sample for extraction. 3.2 Samples should be processed before extraction Place the frozen sample in a food processor and homogenize with dry ice. Place foe samples in containers and leave open in frozen storage overnight to allow for carbon dioxide sublimation. Seal and place foe samples in frozen storage until time o f analysis. 3.3 Sample collection procedures will b e specified in the sampling plan for this project 4.0 Reagents and Standards 4.1 W tte r-H P L C grade 4.2 Acetonitrile - HPLC grade 4.3 Carbon (120*400 mesh) - Reagent grade 4.4 Methanol - HPLC grade 4.3 Silica gel (60-200 mesh) - Reagent grade 4.6 Floriail (60-100 mesh) - Reagent grade 4.7 Superclean LC-NHj - Reagent grade 4.8 1-Octanol - HPLC grade 4.9 L-Ascorbic a d d - Reagent grade 4.10 Dimethyklichlorosilane - Reagent grade 4.11 Toluene - Reagent grade 4.12 Ammonium Acetate - A.C.S. Reagent Grade 4.13 Perfluorooctanoic Acid - Sigma-Aldrich 3.0 Inatnm ent and Equipment 3.1 A high performance liquid chromatograph capable o f pumping up to 2 solvents equipped w ith a variable volume injector capable o f injecting 3-200 pL connected to a tandem Mass Spectrometer (LC/MS/MS). 3.2 A device to collect raw data for peak integration and quantitation. 3.3 Analytical balance capable o f reading to 0.00001 g. Page 2 ut' 7 Page 46 o f 65 Exygen Research Page 84 of 105 Interim Report #3-Analysis o f Ground Water Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 Exygen Research Method Number VOOO1784 ANALYTICAL METHOD Method o f Analysis for the Detenunation o f Pcrfluorooctanoic Acid (PFOA) in Vegetation by LC/MS/MS $.4 Rotary evaporator. 5.5 125 mL pear-shaped flasks. 5.6 50 mL disposable polypropylene centrifuge tubes. 5.7 15 m L disposable polypropylene centrifuge tubes, 5.8 Disposable micropipets (50*1 OOuL, 100-200uL). 5.9 125-mL LDPE narrow-mouth bottles. 5.10 2 mL clear HPLC vial k it 5.11 Disposable pipettes. 5.12 Autopipettes (100-1000 jiL and 10-100 pL), w ith disposable tips. 5.13 SPE tubes (20mL) (Supelco cat. no. N057177). 5.14 Wrist action shaker. 5.15 Centrifiige capable o f spinning 50 m L polypropylene tubes at 2000 ipm. 6.0 Chromatographic System 6.1 Analytical Column: FhiophaaeRP (Keystone Scientific), 2.1 mm x SO mm, 5p (P/N: 82505-052130) 6.2 Temperature: 30*C 6.3 Mobile Phase (A ): 2 mM Ammonium Acetate in W ater Mobile P h aae(B ): Methanol Gradient Program: T in fmin) 0.0 1.0 8.0 20.0 22.5 && 65 65 25 25 65 Flow Rate 3LB fmL/min) 35 0.3 35 0.3 75 0.3 75 0.3 35 0.3 6.6 Injection Volume: 15 jiL (can b e increased to as much as 50 pL). 6.7 Quantitation: Peak A rea-ex tern al standard calibration curve. 6.8 RunTim e: - 2 3 minutes. The above conditions are intended as t guide id m ay b e changed in order to optimize the HPLC system. 7.0 MS/MS System 7.1 Mode: Electroepny Negative MRM m ode, monitoring 413 --369 m /z for PFOA. Page 3 o f7 Page 47 o f 65 Exygen Research Page 85 of 105 Interim Report #3-Analysis of Ground Water Samples Exygen Study No. : P0001131 Exygen Protocol Number: P0001131 Exygsa Research Method Number V0001784 ANALYTICAL M ETHOD Method o f Analysis for foe Determination o f Perfluoiooctanoic Acid (PFOA) in Vegetation by LC/MS/MS The above conditions are intended as a guide and m ay be changed in order to optimize foe MSMS system. 8.0 Preparation o f Solutions 8.1 Mobile Phase 8.1.1 2 mM ammonium acetate in water is prepared by adding 0.154 g o f ammonium acetate to 1000 mL o f water. 8.2 Extraction Solutions 8.2.1 8.2.2 2% ascorbic acid in methanol is p repared b y dissolving 2 g o f ascorbic a d d in 100 m L o f mefoaaoL 30% Dimethyidichlorosilane in toluene is prepared by bringing 3 mL o f dimefoyktiehlorotilane to a final volume o f 10 m L with toluene. Alternate volumes may be prepared. 9.0 Standard Preparation 9.1 Standard Stock/Fortification Solution 9.1.1 9.1.2 9.1.3 9.1.4 9.1.5 Prepare a stock solution o f -100 pg/mL o f PFOA by weighing 10 mg o f analytical standard (corrected for purity) and dilute to 100 mL with methanol in a 125-mL LDPE bottle. A 1.0 pg/mL fortification solution o f PFOA is prepared by bringing I mL o f foe 100 pg/raL solution to a final volume o f 100 with methanol in a 125 m L LDPE bottle. A 0.1 pg/mL fortification solution o f PFOA is prepared by bringing 10 m L o f die 1.0 pg/mL solution to a final volume o f 100 with methanol in a 125 m L LDPE bottle. A 0.01 pg/mL fortification solution o f PFOA is prepared by bringing 10 mL o f the 0.1 pg/raL solution to a final volume o f 100 with methanol in a 125 m L LDPE bottle. The stock and fortification solutions are to b e stored in a refrigerator at approximately 4C and are stable for a maximum period o f 6 months from tiie date o f preparation. 9.2 Standard Calibration Solutions 9.2.1 LC/MS/MS calibration standards are prepared in methanol via dilution o f the 1.0 pg/mL fortification solution. Page 4 ut P age 48 o f 65 Exygen Research Page 86 of 105 Interim Report #3-Analysis of Ground Water Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 Exygen Rm arch Method Number VOOO1784 ANa IYU CAJL M ETHOD Method o f Analysi* for the Determination o f Perfluorooctanoic Acid (PFOA) in Vegetation by LC/MS/MS 9.2.2 The following it a typical example: additional concentrations may be prepared a t needed. Concentration ofFortification Volume Diluted to Final Concentration Solutioniue/mL) (mL) 1.0 S.O (mL) 100 (us/mU 0.05 1.0 2.5 100 1.0 1.0 100 0.0S 10 too 0.025 0.01 0.005 0.025 0.1 0.005 10 10 10 100 100 100 0.0025 0.001 0.0005 9.2.3 Store all calibration standards in 125-mL LDPE narrow-mouth bottles at 2*C to 6*C, up to six months. 9.2.4 Alternate volume* and concentrations o f standards m ay be prepared as needed. 10.0 Batch Set Up 10.1 Each batch o f samples extracted (typically 20 o r less) must include at least one untreated control and two untreated controls fortified at known concentrations (lab control spike) to verify procedural recovery for the batch 10.2 Requirements for field and laboratory duplicates and spikes will be specified in the quality assurance plan for this project. 11.0 Sample Extraction 11.1 Weigh 5 g o f frozen sample into $0 m L polypropylene centrifuge tubes (fortify as needed, replace lid and mix well). 11.2 Add 30 m L o f acetonitrile and shake on a wrist action shaker for - 1 S minuies. 11.3 Centrifuge the SO mL polypropylene tubes containing sample at ~2000 rpm for ~10 minutes. 11.4 Pack and condition the SP6 tubes and siLanize the pear-shaped flasks. 11.$ Pack the 20 mL SPE tubes in sequence with 2 g florisil, 2 g silica get. 2 g carbon, end 1 g LC-NHj. Condition the columns with 20 m L o f methanol, then 20 mL o f acetonitrile. Discard all washes. D o not allow the column to dry. 11.6 Silanize the 12$ mL pear-shaped flasks by nosing with the 30% dimethyldichlorosUane in toluene solution. Rinse the flask with toluene once, followed by methanol (three times). D ry the flasks completely before use. either by air-drying or with a stream o f nitrogen. 11.7 Decant the extract on to a conditioned SPE colum n fitted inside the mouth o f the pear-shaped flask. Collect the eluate in the 125 m L silanized pear-shape flask. P*ge 5 of - Page 49 o f 65 Exygen Research Page 87 of 105 Interim Report #3-Analysis o f Ground W ater Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 Exygso Research Method Number VOQO1784 | A iU L V nC A l. METHOD .. " Method o f Analysis for the Determination o f Perfluorooctanoic A d d (PFOA) in Vegetation by LC/MS/MS 11.8 Add 20 m L o f acetonitrile to the sample in the 50 m L centrifuge tube. 11.9 Shake the sample again for ~10 minutes on a wrist-action shaker. 11.10 Centri&ige the SO m L polypropylene tubes containing sample at -2 0 0 0 rpm for - 5 minutes. 11.11 Decant the extract onto the same SPE column. Collect the eluate into the seme pear-shaped flask and combine with the eluent from the initial extraction. 11.12 Repeatstepa 11.8th ro u g h ll. 11 again. 11.13 Add 3*4 drops o f 1-octanol to the extract in the pear-shaped flask and evaporate at reduced pressure using a rotary evaporator (at < 40C). 11.14 M ake the final volume, by adding 2 m L o f 2% ascorbic acid in methanol to the pear-shaped flask and swirl to mix/dissolve. 11.15 Transfer the extracts to HPLC vials using disposable pipets. 11.16 Analyze samples using electrospray LC/MS/MS. Chromatography 12.1 Inject the same amount o f each standard, sample and fortified sample into the LC/MS/MS system. A calibration standard must precede and follow all analyzed samples. 12.2 Standards o f PFOA corresponding to at least five o r m ore concentration levels must be included in an analytical set. 12.3 An entire set o f extracted calibration standards must be included at the beginning and at the end o f a sample set. Extracted standards must be interspersed between every 5-10 samples. As an alternative, an entire set o f extracted calibration standards m ay b e injected at the beginning o f a set followed b y extracted calibration standards interspersed every 5-10 samples (to account for a second set o f extracted standards). In either case, extracted calibration standards must be the first and last injection in a sample set. 12.4 U se linear standard curves for quantitation. Linear standard curves we generated for the analyte by linear regression using l/x weighting o f peak area versus calibration standard concentration using M assLynx 3.3 (or equivalent! software system. 12.5 Sample response should not exceed standard responses. Any samples that exceed standard response* should be further diluted and reanalyzed. 13.0 Acceptance Criteria 13.1 Chromatogram must show a peak o f a daughter ion at 369 amu from a parent o f 413 amu. The 413 amu parent corresponds to the PFOA anion, while the daughter ion (369 amu) represents the loss o f carbon dioxide. Pag*6 of 7 Page 50 o f 65 Exygen Research Page 88 o f 105 Interim Report #3-Analysis o f Ground Water Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 Exyje&Reicarch Method Number V0001784 a n a l y t ic a l m e t h o d I Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Vegetation by LC/MS/MS 13.2 Method blanks most not contain PFOA at levels greater than the LOQ. If a blank contains PFOA at levels greater than 0.5 ppb, then a new blank sample most be obtained and the entire set must be re-extracted. 13.3 Recoveries o f control spikes and matrix spikes m ust be between 70>130*A o f tbeir known values. I f a control spike falls outside the acceptable limits, the entire set o f samples should b e re-extracted. 13.4 Any calibration standard found to be a statistical outlier by using the Huge Error Teat, may b e excluded from the calculation o f the calibration curve. However, the total number o f calibration standards that could be excluded must not exceed 20% o f the total number o f standards injected. 13.5 The correlation coefficient (R) for calibration curves generated must be 20.992 (R* 20.985). I f calibration results fall outside these limits, then appropriate steps must be taken to adjust instrument operation, and the standards or die relevant set o f samples should be reanalyzed. 13.6 Retention times between standards and samples must not drift more than 4 % within an analytical run. I f retention tim e drift exceeds this limit within an analytical nm then the set must be reanalyzed. 14.0 Calculations 14.1 Use the following equation to calculate the amount o f PFOA found (in ng/mL, based on peak area) using the standard curve (linear regression parameters) generated by the Mass Lynx software program: PFOA found (ng/mL) (Ptffr I*" --intercept) slope 14.2 Use the following equation to convert the amount o f PFOA found in ng/mL to ng/g (ppb). PFOA found fpphl - iPFOA found fn a/m U x flm l volume (m L ) DF1 sample weight (g) DF factor by which the final volume waa diluted, if necessary. 14.3 For samples fortified with known amounts o f PFOA prior to extraction, use die following equation to calculate the percent recovery. Recovery (%) | total analyte found (n ^ g ) - analyte found in control (ng/g)j analyte added (ng/g) Page 7 ol 7 Page S I o f 65 Exygen Research Page 89 of 105 Interim Report #3-Analysis o f Ground Water Samples Exygen Study No.: P0001131 Exygen Protocol Number: POOOi 131 ANALYTICAL METHOD Method Number: V0001785 M ethod o f Analysis for die D eterm ination o f P e rfln o ro o ctaio tc Acid (PFO A ) in Smell M am m al Liver by LC/MS/MS Analytical Testing Facility: Exygen Research 3058 Research Drive State College, PA 16801 Approved By: C -- iX j______ Paul Connolly t Technical Leader, LC-MS, Exygen Research ml Jfochhn Flaeherty / ' Vice Presiicdent, Operations, Exygen Research Date Date Exygen Research Total Pages: 7 Page 52 o f 65 Page 90 of 105 Interim Report #3-Analysis of Ground Water Samples Exygen Study No.: P0001131 Exygen Protocol Number: POOO1131 ExygttiUMtrcfc Method Number VQ001785 I AlNALYTICAL METHOD Method o f Analysis fbr tbe Determination o f Perfluorooctanoic Acid (PFOA) in Small Mammal Liver by LC/MS/MS 1.0 Scope This method is to be employed for the isolation and quantitation o f perfluorooctanoic acid by High Performance Liquid Chromatography coupled to a tandem Mass Spectrometric Detector (LC/MS/MS) in small mammal liver. 2.0 Safety 2.1 Always observe safe laboratory practices. 2.2 Consult the appropriate MSDS before handling any chemical for proper safety precautions. 3.0 Sample Requirement 3.1 At least 5 g o f test sample for extraction. 3.2 Samples should be processed before extraction. Place the frozen sample in a food prooeteor and homogenize with dry ice. Place the samples in containers and leave open in frozen storage overnight to allow for carbon dioxide sublimation. Seal and place the samples in frozen storage until time of analysis. Alternately, i f there is an insufficient amount o f sample (-less than 5 g). then no processing is necessary and the sample can be used as supplied 3.3 Sample collection procedures will be specified in the sampling plan for this project 4.0 Reagents and Standards 4.1 W ater - HPLC grade 4.2 Meritano]-H P L C grade 4.3 A cetonitrile-HPLC grade 4.4 Ammonium Acetate - A.C.S. Reagent Grade 4.5 Perfluorooctanoic A r i d - Sigma*Aldrich 5.0 Instrument and Equipment 5.1 A high performance liquid chromatograph capable o f pumping up to 2 solvents equipped with a variable volume injector capable o f injecting 5*200 yL connected to a tandem Mass Spectrometer (LC/MS/MS). $.2 A device to co lled raw data for peak integration and quantitation. 5.3 Analytical balance capable o f reading to 0.00001 g. 5.4 50 mL disposable polypropylene centrifoge tubes. 5.5 15 tnL disposable polypropylene centrifoge tubes. 5.6 Disposable micropipet (30*1OOuL, 100-200uL). 5.7 125*mL LDPE narrow-mouth bottles. 5.8 2 m L clear HPLC vial k it Ptge 2 of7 Page 53 o f 65 Exygen Research Page 91 o f 105 Interim Report #3-Analysis o f Ground Water Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 Exyjca Research Method Number V0001785 I ANALYTICAL METHOD Method o f Aoitysis for the Determiniti] o f Perfluorooctanoic Acid (PFOA) in Smal I Mammal Liver by LC/MS/MS 5.9 Diiposable pipettes. 5.10 Autopipettef (100-1000 p L and 10*100 pL), with disposable tips. 5.11 W stert Sep Pak Vac 6 cc (lg ) tC I8 SPE cartridges. 5.12 SPE vacuum manifold. 5.13 Tiasuemizer. 5.14 Wrist-action shaker. 5.15 Centrifoge capable o f spinning 15 m i, polypropylene tubes at 3000 rpm. 6.0 Chromatographic System 6.1 Analytical Column; Fluophaae RP (Keystone Scientific), 2.1 mm x $0 mm. 5m (P/N: 82505*052130) 6.2 Temperature: 30*C 6.3 Mobile Phase (A) : 2 mM Ammonium Acetate in Water 6.4 Mobile Phase (B) : Methanol 6.5 Gradient Program: Time (mini 0.0 1.0 8.0 20.0 22.5 StA 65 65 25 25 65 Flow Rate 5L (mL/miri) 35 0 3 35 0.3 75 0.3 75 0.3 35 0.3 6.6 Injection Volume: 15 p L (can b e increased to as m uch as 5 0 iL). 6.7 Quantitation: Peak A rea-ex tern al standard calibration curve. 6.8 RunTim e: - 2 3 minutes. The above conditions are intended a t a guide and m ay b e changed in order to optimize the HPLC system. 7.0 MS/MS System 7.1 Mode: Electiospray Negative MRM mode, monitoring 413 - 369 m/z for PFOA. The above conditions are intended as a guide and m ay be changed in order to optimize the MSMS system. P *|<3oP Page 54 o f 65 Exygen Research Page 92 of 105 Interim Report #3-Analysis o f Ground Water Samples Exygen Study No.: P0001131 Exygen Protocol Number: POOO1131 Exygen Raseileh Meihod Number VOQOI ' 85 | ANALYTICAL M ETHOD Method o f A nalyse for the Determination o f Perftuorooctanoic Acid (PFOA) in Small Mammal Liver by LC/MS/MS 8.0 Preparation o f Solutions 8.1 Mobile Phase 8.1.1 2 mM ammonium acetate in water ia prepared by adding 0.154 g of ammonium acetate to 1000 m L o f water. Alternate volumes may be (pared. 9.0 Standard Preparation 9.1 Standard Stock/Fortification Solution 9.1.1 Prepare a stock solution o f -1 0 0 pg/raL o f PFOA by weighing 10 mg o f analytical standard (corrected l or purity) and dilute to 100 mL with methanol in a 125-mL LDPE bottle. 9 .1 2 A 1.0 figfrnL fortification solution o f PFOA is prepared by bringing I m L o f the 100 pg/mL solution to a final volume o f 100 with methanol in a 125 m L LDPE bottle. 9.1.3 A 0.1 pg/mL fortification solution o f PFOA is prepared by bringing 10 m L o f foe 1.0 iigftnL solution to a final volume o f 100 with methanol in a 12S m L LDPE bottle. 9.1.4 The stock and fortification solutions are to b e stored in a refrigerator at approximately 4*C and are stable for a maximum period o f 6 months from the date o f preparation. 9.2 Staodaid Calibration Solutions 9.2.1 922 LC/MS/MS calibration standards are prepared in methanol via dilution o f the 0.1 pg/mL fortification solution. The following is a typical example: additional concentrations may be prepared as needed. Concentration of Fortification Volume Solution fac/mL) (mL) Diluted to (mL) Final Concentration (ng/mL) 100 5.0 100 100 2.0 100 100 1.0 100 5.0 to 100 2.0 10 100 1.0 10 100 5.0 2.0 1.0 0.5 0.2 0.1 9.2.3 Store all calibration standards in 125>mL LDPE narrow-mouth bottles at 2C to 6*C, up to six months. 9.2.4 Alternate volumes and concentrations o f standards may be prepared as needed. Page 4 of 7 Page 55 o f 65 Exygen Research Page 93 of 105 Interim Report #3-Analysis of Ground Water Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 Exyfca RakmA Method Number V0001785 | ..... ANALYTICAL METHOD Method o f Analyais for the Determination o f Perfluorooctanoic Acid (PFOA) in Small Mammal Liver by LC/MS/MS 10.0 Batch Set Up 10.1 Each batch o f samples extracted (typically 20 o r less) must include at least one untreated control and two untreated controls fortified at known concentrations (lab control spike) to verify procedural recovery for the batch. 10.2 Requirements for field and laboratory duplicates and spikes will be specified in the quality assurance plan for this project. 11.0 Sample Extraction 11.1 Weigh 1 g o f sample into a 50 m L polypropylene centrifuge tubes (fortify as needed, replace lid and mix well). Note that alternate weights o f liver may be measured depending on the sample size available for use. 11.2 Add water to the sample for a final volume o f 10 mL. 11.3 Homogenize sample using a tissuemizer for- 1minute. 11.4 Transfer t mL o f die sample using a disposable pipette into a IS mL disposable centrifuge tube. 11.$ Add 5 mL o f acetonitrile and shake for -20 m inutes on a wrist-action shaker. 11.6 Centrifuge the tubes at -3 0 0 0 rpm for -S minutes. 11.7 Decant the supernatant into a SO m L disposable centrifiige cube and add 3S m L o f water. 11.8 Condition the C SPE cartridges (1 g, 6 mL) by passing 10 mL methanol followed by S m L o f HPLC water ( - 2 drop/sec). Do not let column run dry 11.9 Load tire sample on conditioned C u SPE cartridge. Discard eluate. 11.10 Elute with - 2 m L o f methanol. Collect 2 m L o f eluate into a graduated 15 mL polypropylene centrifUge tube (final volume 2 mL). 11.11 Analyze samples using electrospray LC/MS/MS. 12.0 Chromatography 12.1 Inject the tam e amount o f each standard, sample and fortified sample into the LC/MS/MS system. A calibration standard m ust precede and follow all analyzed samples. 12.2 Standards o f PFOA corresponding to at least five or m ore concentration levels must be included in an analytical s e t 12.3 An entire set o f calibration standards m ust be included at the beginning and at the end o f a sample set. Standards m utt be interspersed between every 5-iU samples. As an alternative, an entire set o f calibration standards may be injected at the beginning o f sec followed by calibration standards interspersed every 5-10 samples (to account for a second set o f standards). In either case, calibration standards m ust be the first and last injection in a sample se t 12.4 Use linear standard curves for quantitation. Linear standard curves are generated for die analyte by linear regression using 1/x weighting o f peak area Page 5 of 7 Page 56 o f 65 Exygen Research Page 94 o f 105 Interim Report #3-Analysis o f Ground Water Samples Exygen Study No. : P0001131 Exygen Protocol Number: P0001131 ExyfM&Mcuch Method Number V000178$ I ANALYTICAL METHOD Method o f Analysis for the Determination ofPerfluorooctanoic Acid (PFOA) in Small Mammal Liver by LC/MS/MS | venue calibratioo standard concentration using MaesLynx 3.3 (o r equivalent) software system. 12.5 Sample response should not exceed standard responses. Any samples that exceed standard responses should be Anther diluted and reanalyzed. 13.0 Acceptance Criteria 13.1 Chromatogram must show a peak o f a daughter ion at 369 amu from a parent o f 413 amu. The 413 amu parent corresponds to the PFOA anion, while the daughter ion (369 amu) represents die loss o f carbon dioxide. 13.2 Method blanks must not contain PFOA at levels greater than the LOQ. If a blank contains PFOA at levels greater than 10 ng/g, then a new blank sample must be obtained and the entire set must be re-extracted. 13.3 Recoveries o f control spikes and matrix spikes must be between 70-130% of their known values. I f a control spike falls outside the acceptable limits, the entire set o f samples should be re-extracted. Any matrix spike outside 70 130% should be evaluated by the analyst to determine if re-extraction is warranted. 13.4 Any calibration standard found to be a statistical outlier by using the Huge Error Test, may be excluded from the calculation o f the calibration curve. However, the total number o f calibration standards that could be excluded must not exceed 20% o f the total number o f standards injected. 13.3 The correlation coefficient (R) for calibration curves generated must be 20.992 (R] 20.985). I f calibration results fall outside these limits, then appropriate steps must be taken to adjust instrument operation, and the standards or the relevant set o f samples should be reanalyzed. 13.6 Retention times between standards and samples must not drift more than 4 % within an analytical run. I f retention tim e drift exceeds this limit within an analytical run then the set must be reanalyzed. 14.0 Calculations 14.1 Use the following equation to calculate the amount o f PFOA found (in ng/mL. based on peak area) using the standard curve (linear regression parameters) generated by die Mass Lynx software program: PFOA found (ng/mL) - fffgfr fiTTI - intercept) x O F x aliquot factor slope DF - factor by which the final volume was diluted, if necessary. Aliquot facto r* 10 Page 6 of 7 Page 57 o f 65 Exygen Research Page 95 of 105 Interim Report #3-Analysis of Ground Water Samples Exygen Study No.: P0001131 Exygen Protocol Number: POOOI131 ExygeaResearch Method Number V000l78i | ANALYTICAL m e t h o d Method o f A nalytii for the Determination o f Perfluorooctanoic Acid (PFOA) in Small Mammal Liver by LC/MS/MS 14 2 For samples fortified with known amounts o f PFOA prior to extraction, use the following equation to calculate the percent recovery. Recovery (%) - [ total analyte found (ngftnL) - analyte found in control (ng/mL)] analyte added (ng/mL) 14.3 Use the following equation to convert the amount o f PFOA found in ng/mL to ng/g(ppb). PFOA found fppb) - fPFOA found in g /m D x final volume fmLII sample weight (g) Exygen Research Page 7 o f 7 Page 58 o f 65 Page 96 of 105 Interim Report #3-Analysis of Ground Water Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 ANALYTICAL METHOD Method Number: V0001786 Method o f Analysis for the D eterm ination of Perfluorooctanolc Acid (PFOA) io Smalt M amm al Serum by LC/MS/MS Analytical Testing Facility: Exygen Research 3058 Research Drive State College. PA 16801 Approved By: c j UL Paul Connolly I Technical Leader, LC-MS, Exygen Research Jo h n Flaherty / Vice Presidesn!t, Operation*, Exygen Research Date a/4 ^ - Due Exygen Research Total Pages: 7 Page 59 o f 65 Page 97 of 105 Interim Report #3-Analysis o f Ground Water Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 Exygea Roicarch Method Number V0001786 | AM A LiTICAL M ETHOD M ethod o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Small Mammal Serum by LC/MS/MS 1.0 Scope This method is to be employed for die isolation and quantitation o f perfluorooctanoic acid by High Performance Liquid Chromatography coupled to a tandem Mass Spectrometric Detector (LC/MS/MS) in small mammal serum. 2.0 Safety 2.1 Always observe safe laboratory practices. 2.2 Consult the appropriate MSDS before handling any chemical for proper safety precautions. 3.0 Sample Requirement 3.1 At least 1 m L o f test sample for extraction. 3.2 No sample processing is needed for serum samples. However, frozen serum samples must to allowed to completely thaw to room temperature before use. 3.3 Sample collection procedures will be specified in the sampling plan for this project 4.0 Reagents and Standards 4.1 W ater-H P L C grade 4.2 Methanol - HPLC grade 4.3 Acetonitrile >H PLC grade 4.4 Ammonium Acetate - A.C.S. Reagent Grade 4.3 Perfluorooctanoic Acid - Sigma-Aldrich 3.0 Instrument and Equipment 5.1 A high performance liquid chromatograph capable o f pumping up to 2 solvents equipped with a variable volume injector capable o f injecting 3*200 pL connected to a tandem Mass Spectrometer (LC/MS/MS). 5.2 A device to collect raw data for peak integration and quantitation. 5.3 Analytical balance capable o f reading to 0.00001 g. 5.4 50 m L disposable polypropylene centrifoge tube*. 5.5 15 m L disposable polypropylene centrifiige tubes. 5.6 Disposable micropipets (50-lOOuL, 10Q-200uL). 5.7 125-mL LDPE narrow-mouth bottles. 5.8 2 m L clear HPLC vial k it 5.9 Disposable pipettes. 5.10 Autopipettes (100*1000 p L and 10-100 pL), with disposable tips. 5.11 Wster* S cpPak Vac 6 c c (!g )tC 1 8 S P E cartridges. 5.12 SPE vacuum manifold. 5.13 V ortex. Page 2 of? Page 60 o f 65 Exygen Research Page 98 of 105 Interim Report #3-Analysis of Ground Water Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 Exygcs Rcaearch Method Number V00017S6 | A lW V nC A L METHOD Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Small Mammal Serum by LC/MS/MS $.14 Wrist-action shaker. 5.15 Centrifuge capable o f spinning 15 raL polypropylene tubes at 3000 rpm. 6.0 Chromatographic System 6.1 Analytical Column: Pluophaae RP (Keystone Scientific), 2.1 mm x 50 mm. 5p (P/N: 82505-052130) 6.2 Temperature: 30*C 6.3 Mobile Phase (A) : 2 mM Ammonium Acetate in W ater 6.4 Mobile Phase (B) : Methanol 6.5 Gradient Program: Time (mini 0.0 1.0 8.0 20.0 22.5 SLA 65 65 25 25 65 Flow Rate % B fmL/minl 35 0.3 35 0.3 75 0.3 75 0.3 35 0.3 6.6 Infection Volume: 15 pL (can be increased to as much as 50 pL). 6.7 Quantitation: Peak Area - external standard calibration curve. 6.8 RunTim e: - 2 3 minutes. The above conditions are intended as a guide and m ay be changed in order to optimize the HPLC system. 7.0 MS/MS System 7.1 Mode: Electrospray Negative MRM m ode, monitoring 4 1 3 - 369 m/z for PFOA. The above conditions are intended as a guide and may be changed in order to optimize the MSMS system. 8.0 Preparation o f Solutions 8.1 Mobile Phase 8.1.1 2 mM ammonium acetate in water is prepared by adding 0.1S4 g o f ammonium acetate to 1000 m L o f water. Alternate volumes may be prepared. Page 3 of 7 Page 61 o f 65 Exygen Research Page 99 of 105 Interim Report #3-Analysis o f Ground W ater Samples Exygen Study No.: P0001131 E x y g e n P ro to c o l N u m b e r: POOO1131 ExygtnFidWfCh Method Number V00017S6 ANALYTICAL METHOD Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Small Mammal Serum by LC/MS/MS ] 9.0 Standard Preparation 9.1 Standard Stock/Fortificstion Solution 9.1.1 Prepare stock solution o f *-100 ng/mL o f PFOA by weighing 10 mg o f analytical atandard (corrected for purity) and dilute to 100 mL with methanol in a 125-mL LDPE bottle. 9.1.2 A 1.0 pg/mL fortification solution o f PFOA is prepared by bringing l m L o f the 100 pg/raL solution to a final volume o f 100 with methanol in 125 mL LDPE bottle. 9.1.3 A 0.1 pg/mL fortification solution o f PFOA is prepared by bringing 10 m L o f foe 1.0 pg/mL solution to a final volume o f 100 with methanol in 125 mL LDPE bottle. 9.1.4 The stock and fortification solutions are to b e stored in a refrigerator at approximately 4C and are stable for maximum period o f 6 months from foe date o f preparation. 9.2 Standard Calibration Solutions 9.2.1 92.2 LC/MS/MS calibration standards are prepared in methanol via dilution o f foe 0.1 pg/mL fortification solution. The following is a typical example: additional concentrations may be prepared as needed. Concentration o f Fortification Volume Diluted to Final Concentration Solution fna/mL) (mL) (mL) (na/mL) 100 5.0 100 100 2.0 100 100 1.0 100 5.0 2.0 1.0 5.0 10 100 2.0 10 100 1.0 10 100 0.5 0.2 0.1 9.2.3 Store all calibration standards in 125-mL LDPE narrow-mouth bottles st 2*C to 6*C, up to six months. 9.2.4 Alternate volumes and concentrations o f standards may be prepared as needed. 10.0 Batch Set Up 10.1 Each batch o f samples extracted (typically 20 o r less) m ust include at least one untreated control and two untreated controls fortified at known concentrations (lab control spike) to verify procedural recovery for the batch. 10.2 Requirements for field and laboratory duplicates and spikes will be specified in the quality assurance plan for this project. Page 4 o f 7 Page 62 o f 65 Exygen Research Page 100 of 105 Interim Report #3-Analysis of Ground Water Samples Exygen Study No. : P0001131 Exygen Protocol Number? POOOU31 Exygio Research Method Number V0001786 I ANALYTICAL METHOD Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Small Mammal Serum by LC/MS/MS 11.0 Sample Extraction 11.1 Measure 1 m L o f sample into a SO mL polypropylene centrifuge tubes (fortify as needed, replace lid and mix well). Note that alternate volumes o f serum m ay be measured depending on the sample size available for use. 11.2 Add water to the sample for a final volume o f 20 m L Cap tightly 11.3 Vortex fo r-1 minute. 11.4 Transfer 1 m L o f the sample using a disposable pipette into a IS mL disposable oentriflige tube. 11.5 Add 5 mL o f acetonitrile and shake for - 2 0 minutes on a wrist-action shaker. 11.6 Centrifuge the tubes at -3 0 0 0 ipm for - S minutes. 11.7 Decant the supernatant into t SO m L disposable centrifuge tube and add 35 m L o f water. 11.8 Condition the C u SPE cartridges (1 g, 6 mL) by passing 10 mL methanol followed b y 5 m L o f HPLC water ( - 2 drop/sec). Do not let column run dry 11.9 Load the sample on conditioned C u SPE cartridge. Discard eluatc. 11.10 Elute with - 2 mL o f methanol. Collect 2 m L o f cluate into a graduated IS m L polypropylene centrifuge tube (final volume 2 mL). 11.11 Analyze samples using electroepray LC/MS/MS. 12.0 Chromatography 12.1 Iqject the same amount o f each standard, sample and fortified sample into the LC/MS/MS system. A calibration standard must precede and follow all analyzed samples. 12.2 Standards o f PFOA corresponding to at least five o r more concentration levels must be included in an analytical set. 12.3 An entire set o f calibration standards m ust be included at the beginning and at the end o f a sample set. Standards must be interspersed between every 5 -1o samples. As an alternative, an entire set o f calibration standards may be injected at die beginning o f a set followed by calibration standards interspersed every 5*10 samples (to account for a second set o f standards). In either case, calibration standards m ust be the first and last injection in a sample set 12.4 Use linear standard curves for quantitation. Linear standard curves are generated forth analyte by linear regression using 1/x weighting o f peak area versus calibration standard concentration using M assLynx 3.3 (or equivalent) software system. 12.5 Sample response should not exceed standard responses. Any samples that exceed standard responses should be further diluted and reanalyzed. Page 5 o f " Page 63 o f 65 Exygen Research Page 101 o f 105 Interim Report #3-Analysis o f Ground W ater Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 Exygen Reaeaich M ethod N um ber VOOOl 786 a n a l y t ic a l m ic t h o d Method o f Analysis fix-the Detennination o f Perfluorooctanoic Acid (PFOA) in Small Mammal Serum by LC/MS/MS ] 13.0 Acceptance Criteria 13.1 Chromatogram m ust show a peak o f a daughter ion at 369 amu from a parent o f 413 amu. The 413 amu parent correaponds to the PFOA anion, while the daughter ion (369 amu) represents the loss o f carbon dioxide. 13.2 Method blanks must not contain PFOA at levels greater than the LOQ. if a blank contains PFOA at levels greater than 10 ng/mL, then a new blank sample must be obtained and foe entire set must be re-extracted. 13.3 Recoveries o f control spikes and matrix spikes must be between 70-130% o f their known values. I f a control spike falls outside the acceptable limits, the entire set o f samples should be re-extracted. Any matrix spike outside 70 130% should be evaluated by die analyst to determine if re-extraction is warranted. 13.4 Any calibration standard found to be a statistical outlier by using the Huge Error Test, may be excluded from the calculation o f the calibration curve However, the total number o f calibration standards that could be excluded must not exceed 20% o f the total number o f standards injected. 13.5 The correlation coefficient (R) for calibration curves generated must be &0.992 (R* 0.985). I f calibration results fall outside these limits, then appropriate steps must be taken to adjust instrument operation, and the standards o r the relevant set o f samples should be reanalyzed. 13.6 Retention times between standards and samples must not drift more than 1 4 % within an analytical run. I f retention time drift exceeds this limit within an analytical run then the set must be reanalyzed. 14.0 Calculations 14.1 Use the following equation to calculate the amount o f PFOA found (in ng/mL. based on peak area) using foe standard curve (linear regression parameters) generated by the Mass Lynx software program: PFOA found (ng/m L) (Peak area -intercept) x DF x aliquot factor slope DF - factor by which foe final volume w as diluted, if necessary. Aliquot factor- 2 0 14.2 For samples fortified with known amounts o f PFOA prior to extraction, use the following equation to calculate the percent recovery. Recovery (%) - [total analyte found (ng/mL) - analyte found in control (ng/mL)] ][10Q analyte added (ng/mL) Page 6 o f7 Page 64 o f 65 Exygen Research Page 102 of 105 Interim Report #3-Analysis o f Ground W ater Samples Exygen Study No. : P0001131 Exygen Protocol Number: POOO1131 E xyya RMtrch Method Number VOOO17S6 I .... "Na L vT IC A L M E T H O D ... Method o f Analysii for die Determination ofPerfluorooctanoic Acid (PFOA) in Small Mammal Serum by LC/MS/MS 14.3 Use the following equation to convert the amount o f PFOA found in ng/mL to ppb. PFOA found (ppb) - fPFOA found frffrn M * final volume fmU ) ample volume (mL) Exygen Research Page 7 of 7 Page 65 o f 65 Page 103 of 105 Interim Report #3-Analysis of Ground Water Samples Exygen Study No.: P0001131 3058 Research Drive Phone: 814-272-1039 State College, PA 16801 Fax: 814-231-1580 P R O TO C O L AM EN DM EN T Am endm ent Number. 1 Effective D ate: 01/19/05 Exygen Study Number P0001131 Client Study N u m b er. Page 1 of 1 None D ESC R IP T IO N O F AM EN D ED SEC T IO N 1) Analytical Procedure Sum m ary V 0001780:Section 9.1 2) Verification of Analytical Procedure AM ENDED TO 1) Add to Section 9 .1 : Section 9 .1 .6, Alternate w eights of standards m ay be used to prepare alternate concentrations of stock solutions a s n ecessary. Alternate levels of fortification solutions m ay also be prepared. 2 ) Low and high spiking levels of the analytes for e ach matrix m ay be altered depending on sam ple size available for extraction and/or to cover analyte concentrations expected in the sam p les. R A TIO N A LE 1) Higher concentrations of standards need to be prepared in order to spike the sam ple bottles at higher levels. 2) Th e sam ple size available for sm all mammal liver and serum w as sm aller than expected. Spiking at the pre-determined levels in the protocol puts the spiked concentration lower than the detection lim it A lso, the analyte levels in the ground w ater sam p les a re expected to greatly exceed the pre-determined spiking levels listed in the protocol. W hen the levels in the sam ples greatly exceed the spiking levels, an accurate recovery value cannot be calculated for the Q C sam ple. Higher spiking levels in the bottles will cover the analyte concentrations expected in the w ater sam p les. IM PA CT ON S T U D Y T h e LO Q is 100 n g /g fb ra0.1 g sam ple of sm all mammal liver and is 1000 ng/mL for a 0.01 m L sam ple of sm all mammal serum . Higher levels of spiking for the w ater sam p les will ensure that more Q C recovery data can be used. LIBRARY ID: W0001226*6` /... ' ADMINISTRATIVE FORM Exygen Research Page 104 of 105 Interim Report #3-Analysis o f Ground Water Samples Exygen Study No. : P0001131 3058 Research Drive Phone: 814-272-1039 State College, PA 16801 Fax: 814-231-1580 Amendment Number Effective Date: Exygen Study Number PRO TO CO L AMENDMENT 2 03/07/05 P0001131 Client Study Number Page 1 of 1 None D ESC R IP T IO N O F AM EN D ED SEC T IO N 1) Report, page 11 of 65 2) T e st M aterials, page 6 of 65: P F O S transition monitored 499 -> 99. AM ENDED TO 1) Instead of one final report, interim reports will be issu ed . 2) P F O S transition monitored m ay also be 499 *> 80. RA TIO N A LE 1) D ue to the e x cessive size s of the data se ts, interim reports will be issu ed to allow the d ien t to receive data in a tim elier m anoR e%faits- 2) Th e A P I 4000 LC/M S/M S system s detect the 4 99 -> 80 P F O S transition with greater sensitivity than the 4 99 -> 99 transition. IM PA CT ON S T U D Y 1) T h e d ien t will be able to receive and review the data more quickly. 2) T h e 499 -> 80 transition can be detected with greater sensitivity; therefore, giving better chrom atography. LIBRARY ID: VD00122M- - Exygen Q AU R eview . ADMINISTRATIVE FORM Exygen Research Page 105 o f 105