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INTERIM REPORT #26 - Analysis of Groundwater Samples STUDY TITLE Analysis of Perfluorooctanoic Acid (PFOA) 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 January 9,2007 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: P0000760 Exygen Study Number: P0000760 Total Pages: 119 Interim Report #26 - Analysis o f Groundwater Samples Exygen Study No.: P0000760 GOOD LABORATORY PRACTICE COMPLIANCE STATEMENT Exygen Study Number P0000760, entitled "Analysis of Perfluorooctanoic Acid (PFOA) 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. Exygen Research Jaisimha ,,, Study Director Weston Solutions, Inc. Michael A. SantorgT Sponsor Representative 3M Company Exygen Research Date Page 2 o f 119 Interim Report #26 - Analysis o f Groundwater Samples Exygen Study No.: P0000760 QUALITY ASSURANCE STATEMENT Exygen Research's Quality Assurance Unit reviewed Exygen Study Number P0000760, entitled, "Analysis of Perfluorooctanoic Acid (PFOA) 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 41. Raw Data & Interim Analytical Report Date Inspected Date Reported to Date Reported to Principal Exygen Date Reported to Investigator Management Study Director 12/28,29/06 01/09/07 01/09/07 01/09/07 'Note: All in-lab inspections will be documented in the QA statement for the final analytical report at the conclusion o f the study. This Q A statement involves only the review of the interim report and associated raw data. Exygen Research Page 3 o f 119 Interim Report #26 - Analysis o f Groundwater Samples Exygen Study No.: P0000760 CERTIFICATION OF AUTHENTICITY This interim report, for Exygen Study Number P0000760, 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: Exygen Research Facility Management: Study Director, Weston Solutions, Inc. K kkX L JaisimhaKesari P.E., DEE Weston Solutions, Inc. Sponsor Representative, 3M Company: Michael A. Santory Director of Regulatory Affairs Exygen Research Date Page 4 o f 119 Interim Report #26 - Analysis o f Groundwater Samples Exygen Study No.: P0000760 STUDY IDENTIFICATION Analysis of Perfluorooctanoic Acid (PFOA) 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: P0000760 EXYGEN STUDY NUMBER: P0000760 TYPE OF STUDY: Residue SAMPLE MATRIX: Ground Water TEST SUBSTANCE: Perfluorooctanoic acid (PFOA) 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: 11/24/06 Interim Analytical Termination Date: 12/06/06 Interim Report Completion Date: 01/09/07 Exygen Research Page 5 o f 119 Interim Report #26 - Analysis o f Groundwater Samples Exygen Study No.: P0000760 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 Chas Simons John Flaherty Karen Risha Amy Sheehan Mark Ammerman Mindy Cressley Ellen Dashem Chrissy Edwards Krista Gallant Title Operations Manager Vice President Laboratory Supervisor Associate Scientist Sample Custodian Technician Technician Technician Technician Exygen Research Page 6 o f 119 Interim Report #26 - Analysis o f Groundwater Samples Exygen Study No.: P0000760 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 TABLES...............................................................................................................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..............................................................................11 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...............................................13 6.3 Chromatography.......................................................................................................14 6.4 Instrument Sensitivity...............................................................................................14 6.5 Description of LC/MS/MS Instrument and Operating Conditions...........................15 6.6 Quantitation and Example Calculation.....................................................................15 7.0 EXPERIMENTAL DESIGN......................................................................................16 8.0 RESULTS...................................................................................................................17 9.0 CONCLUSIONS.........................................................................................................17 10.0 RETENTION OF DATA AND SAMPLES............................................................. 17 Exygen Research Page 7 o f 119 Interim Report #26 - Analysis o f Groundwater Samples Exygen Study No.: P0000760 LIST OF TABLES Page Table I. Summary of PFOA in Groundwater Samples.................................................19 Table II. Matrix Spike Recovery of PFOA in Groundwater Samples..........................20 Table EH. Surrogate Spike Recovery o f 13C PFOA in Groundwater Samples...............21 Exygen Research Page 8 o f 119 Interim Report #26 - Analysis o f Groundwater Samples Exygen Study No.: P0000760 Figure 1. LIST OF FIGURES Page Typical Extracted Calibration Curve for PFOA in Reagent W ater............... 23 Figure 2. Extracted Standards of PFOA in Reagent Water, 0 ng/L and 25 ng/L, Respectively................................................................................................... 24 Figure 3. PFOA in Reagent Water, 50 ng/L Fortified Reagent Spk A, and 500 ng/L Fortified Reagent Spk B, Respectively.......................................... 25 Figure 4. Chromatogram Representing a Groundwater Sample Analyzed for PFOA (Exygen ID: C0222023, Data Set: 112406C)..................................... 26 Figure 5. Typical Extracted Calibration Curve for 13C PFOA in Reagent Water......... 27 Figure 6. Extracted Standards of 13C PFOA in Reagent Water, 0 ng/L and 25 ng/L, Respectively................................................................................................... 28 Figure 7. 13C PFOA in Reagent Water, 50 ng/L Fortified Reagent Spk A, and 500 ng/L Fortified ReagentSpk B, Respectively............................................29 Figure 8. Chromatogram Representing a Groundwater Sample Analyzed for 13C PFOA (Exygen ID: C0222023, Data Set:112406C)................................. 30 Exygen Research Page 9 o f 119 Interim Report #26 - Analysis o f Groundwater Samples Exygen Study No.: P0000760 LIST OF APPENDICES Appendix A Study Protocol P0000760 (Exygen Study No. P0000760) with Analytical Method V0001780: "Method of Analysis for the Determination of Perfluorooctanoic Acid (PFOA) in Water by LC/MS/MS", Protocol Amendments, and Deviations......... Page 31 Exygen Research Page 10 o f 119 Interim Report #26 - Analysis o f Groundwater Samples Exygen Study No.: P0000760 1.0 SUMMARY Exygen Research extracted and analyzed groundwater samples for the determination of perfluorooctanoic acid (PFOA) according to Exygen Method V0001780 (Appendix A). The limit of quantitation (LOQ) for PFOA in ground water was 25 ng/L. Analytical results for the analysis of PFOA in groundwater samples are summarized in Table I. Fortification recoveries for PFOA in the groundwater samples are detailed in Table II. The overall average percent recovery standard deviation for PFOA in groundwater samples was 96 + 22%. Fortification recoveries for the 13C PFOA surrogate spike in the groundwater samples are detailed in Table III. The average percent recovery standard deviation for the 13C PFOA surrogate spike in the groundwater samples was 99 14%. In addition to the groundwater samples, a quality control reagent blank, reagent spike A, and reagent spike B were included in each set. 2.0 OBJECTIVE The objective of the analytical part of this study was to determine levels of perfluorooctanoic acid (PFOA) in groundwater according to Protocol P0000760 (Appendix A). 3.0 INTRODUCTION This report details the results of the analysis for the determination of PFOA in groundwater using the analytical methods 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 November 24, 2006, and the analytical termination date for this interim report was December 6,2006. 4.0 ANALYTICAL TEST SAMPLES Eleven groundwater samples (Exygen ID C0222023 - C0222033) were received on wet ice on November 21,2006 from Tim Frinak at Weston Solutions, Inc. Eight groundwater samples represented two groundwater sampling sites and associated field quality control Exygen Research Page 11 o f 119 Interim Report #26 - Analysis o f Groundwater Samples Exygen Study No.: P0000760 samples. Three water samples represented one trip blank and two trip blank spikes. All 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 standard, PFOA (SP0008065), was purchased from Oakwood Products, Inc. and was received at Exygen on September 8, 2006. The surrogate spiking standard, 13C labeled perfluorooctanoic acid (13C PFOA SP0007767), was received at Exygen on June 13,2006 from the 3M Company. The available information for the reference materials is listed below. PFOA was stored ambient and 13C PFOA was stored frozen. Compound Exygen Inventory No. PFOA SP0008065 13CPFOA SP0007767 Lot# Y16G 3507-277 Purity (%) Expiration Date 98 09/08/08 97.6 07/15/10 The molecular structure of PFOA is given below: PFOA Chemical Name: Perfluorooctanoic acid Molecular Weight: 414 Transitions Monitored: 413 - 369 (for quantification) and 413 -> 219 (for confirmation) Structure: 13C PFOA Chemical Name: 1,2-13C perfluorooctanoic acid Molecular Weight: 416 Transition Monitored: 415-> 370 Structure: Exygen Research Page 12 o f 119 Interim Report #26 - Analysis o f Groundwater Samples Exygen Study No.: P0000760 F F FF F F F F OH 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 the groundwater samples in this study. 6.1. Extraction Procedure A 40 mL aliquot of the water sample was used for the extraction procedure. After fortification of appropriate samples, the samples were loaded onto a Cis 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. 6.2 Preparation of Standards and Fortification Solutions A stock standard solution of PFOA was prepared as specified in Exygen method V 0001780. The stock standard solution w as prepared at a concentration o f 10,000 pg/mL by dissolving 1.0 g of the standard (corrected for purity and salt content, if necessary) in acetonitrile. From this solution, a 1000 pg/mL fortification standard solution was prepared by taking 10 mL of the stock and bringing the volume up to 100 mL with acetonitrile. By taking 10 mL of the 1000 pg/mL fortification standard and bringing the volume up to 100 mL with acetonitrile, a 100 pg/mL fortification standard was prepared. By taking 10 mL of the 100 pg/mL fortification standard and bringing the volume up to 100 mL with acetonitrile, a 10 pg/mL fortification standard was prepared. By taking 10 mL of the 10 pg/mL fortification standard and bringing the volume up to 100 mL with acetonitrile, a 1.0 pg/mL fortification standard was prepared. By taking 10 mL of the 1.0 pg/mL fortification standard and bringing the volume up to 100 mL with acetonitrile, a 0.1 pg/mL fortification standard was prepared. By taking 10 mL of the 0.1 pg/mL fortification standard and bringing the volume up to 100 mL with acetonitrile, a 0.01 pg/mL fortification standard was prepared. Exygen Research Page 13 o f 119 Interim Report #26 - Analysis o f Groundwater Samples Exygen Study No.: P0000760 A stock standard solution of 13C PFOA was also prepared as specified in the raw data. The stock standard solution was prepared at a concentration of 100 pg/mL by dissolving 0.01 g of the standard (corrected for purity and salt content, if necessary) in methanol. From this solution, a 10 pg/mL fortification standard solution was prepared by taking 10 mL of the stock and bringing the volume up to 100 mL with methanol. By taking 10 mL of the 10 pg/mL fortification standard and bringing the volume up to 100 mL with methanol, a 1.0 pg/mL fortification standard was prepared. By taking 10 mL of the 1.0 pg/mL fortification standard and bringing the volume up to 100 mL with methanol, a 0.1 pg/mL fortification standard was prepared. By taking 10 mL of the 0.1 pg/mL fortification standard and bringing the volume up to 100 mL with methanol, a 0.01 pg/mL fortification standard was prepared. A set of standards containing PFOA and 13C PFOA were 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 CL) 00 10 100 10 200 10 400 100 100 100 200 100 400 1of PFOA and 13C PFOA 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 PFOA was accomplished by LC/MS/MS electrospray. The retention time of PFOA was -10.5 min. Peaks above the LOQ were not detected in any of the reagent blank samples corresponding to the analyte retention time. 6.4 Instrument Sensitivity The smallest standard amount injected during the chromatographic run had a concentration of 25 ng/L of PFOA. Exygen Research Page 14 o f 119 Interim Report #26 - Analysis o f Groundwater Samples Exygen Study No.: P0000760 6.5 Description of LC/MS/MS Instrument and Operating Conditions Instrument: Interface: Computer: Software: HPLC: API 4000 Biomolecular Mass Analyzer Turbo Ion Spray Liquid Introduction Interface DELL OptiPlex GX400 Windows NT, Analyst 1.4.1 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 %A 65 65 25 25 65 65 %B 35 35 75 75 35 35 Total run time: ~18 min Flow Rate: 0.3 mL/min Ions monitored: Analvte PFOA PFOA Confirm Ion 13C PFOA Mode negative negative negative Transition Monitored 413 -> 369 413 - 219 415 -- 370 Approximate Retention Time (min) -10.5 min. -10.5 min. -10.5 min. 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. 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. Exygen Research Page 15 o f 119 Interim Report #26 - Analysis o f Groundwater Samples Exygen Study No.: P0000760 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 PFOA and 13C PFOA prior to extraction, Equation 2 calculated the percent recovery. Equation 2: Recovery (%) = (analyte found (ng/L) - analyte in control (ng/Lri xl00% amount added (ng/L) An example of a calculation using an actual sample follows: Water sample Exygen ID C0222023 Spk C (Set: 112406C), fortified at 250 ng/L with PFOA where: peak area intercept slope dilution factor ng/L PFOA added (fort level) amt (ng/L) in corresponding sample = = = = = = 531829 6960 822 1 250 358 (Set: 112406C) From equation 1: Analyte found (ng/L) = r531829 - 69601 x 1 822 From equation 2: % Recovery 639 ng/L (639 - 358 ng/L) x 100% 250 112% NOTE: Numbers may differ slightly from raw data due to rounding. 7.0 EXPERIMENTAL DESIGN 13C PFOA was used as a surrogate for all the samples. 13C PFOA was added to the sample collection bottles in the laboratory before being shipped to the field for sampling. For samples designated as field matrix spikes, PFOA was also 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. Exygen Research Page 16 o f 119 Interim Report #26 - Analysis o f Groundwater Samples Exygen Study No.: P0000760 The samples were extracted in one set. The set included one reagent blank, two reagent blanks fortified at known concentrations. The set also contained two sample sites and the field blank and the field blank spikes collected for the groundwater samples. For each site, a sample, a field duplicate and two-matrix field spikes were collected. For each site, a laboratory duplicate and two laboratory matrix spikes were also extracted. 8.0 RESULTS Analytical results for the analysis of PFOA in the groundwater samples are summarized in Table I. Accuracies were assessed for each sample by reviewing the individual quality control results obtained for each sample site. In most cases, there were two laboratory and two field spike recovery results available for each sample site that were used to assess the accuracy. In instances of failed laboratory or field spikes, recoveries associated with other spikes were used to assess sample accuracy. Fortification recoveries for PFOA in the groundwater samples are detailed in Table II. The average percent recovery standard deviation for PFOA in the groundwater samples was 96 22%. Fortification recoveries for the 13C PFOA surrogate spike in the groundwater samples are detailed in Table III. The average percent recovery standard deviation for the 13C PFOA surrogate spike in the groundwater samples was 99 14%. In addition to the ground water samples, a quality control reagent blank, a low reagent spike A, and a high reagent spike B were included in each set. 9.0 CONCLUSIONS The groundwater samples were successfully extracted and analyzed for PFOA 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 study director. 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 interim 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 119 Interim Report #26 Analysis o f Groundwater Samples Exygen Study No.: P0000760 TABLES Exygen Research Page 18 o f 119 Interim Report #26 - Analysis o f Groundwater Samples Exygen Study No.: P0000760 Table I. Summary of PFOA in Groundwater Samples Exygan ID CQ222023 C0222023 Rep C0222024 C0222027 C0222027 Rep C0222028 C0222031 Cltent S am pli ID DAL-GW-RESW01 -0-061117 DAL-GW-RESW01 -0-061117* DAL-GW-RESW01-DB-061117 DAL-GW-RESW02-0-061117 DAL-GW-RESW02-0-061117* DAL-GW-RESW02-DB-061117 DAL-GW-TRIP-0-061117 Analyte Found (ppt, ng/L) C8 Acid PFOA Perfluorooctanotc Acid 358 364 374 2580 2430 2770 NO Assessed Accuracy (+/- %) 30 30 30 30 30 30 30 'Laboratory duplicate. ND = Notdetected ator above the Limitof Quantitation (LOQ) of 25 ng/L. Exygen Research Page 19 of 119 Interim Report #26 Analysis o f Groundwater Samples Exygen Study No.: P0000760 Table II. Matrix Spike Recovery of PFOA in Groundwater Samples Sample Description DAL-GW-RESW01-0-061117 (C0222023 Spk C, 250 ng/L Lab Spike) DAL-GW-RESW01-0-061117 (C0222023 Spk D, 5000 ng/L Lab Spike) DAL-GW-RESW01-LS-061117 (C0222025,0.25 ppb Field Spike) DAL-GW-RESW01 -HS-061117 (C0222026, 5 ppb Field Spike) DAL-GW-RESW02-0-061117 (C0222027 Spk E, 250 ng/L Lab Spike) DAL-GW-RESW02-0-061117 (C0222027 Spk F, 5000 ng/L Lab Spike) DAL-GW-RESW02-LS-061117 (C0222029,0.25 ppb Field Spike) DAL-GW-RESW02-HS-061117 (C0222030,5 ppb Field Spike) D AL-G W -TR IP-LS-061117 (C0222032,0.25 ppb Field Spike) D AL-G W -TR IP-H S-061117 (C0222033,5 ppb Field Spike) Amount Spiked (ng/L) 250 5000 250 5000 250 5000 250 5000 250 5000 Amt Found in Sample (ng/L) C8 Acid PFOA Amount Recovered (ng/L) 358 638 358 5310 358 667 358 3160 2580 2580 2580 2580 3060 7150 2630 6190 ND 269 ND 5260 Recovery (%) 112 99 124 56 * 91 72 108 105 Average: Standard Deviation: 96 22 ' Sample residue exceeds the spiking level significantly; therefore, an accurate recovery value cannot be calculated ND = Not detected at or above the Limit of Quantitation (LOQ) of 25 ng/L. Note: Since this summary table shows rounded results, recovery values may vary slightly from the values In the raw data. Exygen Research Page 20 o f 119 Interim Report #26 - Analysis o f Groundwater Samples Exygen Study No.: P0000760 Table III. Surrogate Spike Recovery of 13C PFOA in Groundwater Samples Exygen ID C0222023 Spk C C0222023 Spk D C0222023 C0222023 Rep C0222024 C0222025 C0222026 C0222027 Spk E C0222027 Spk F C0222027 C0222027 Rep C0222028 C0222029 C0222030 C0222031 C0222032 C0222033 Sample Description DAL-GW -R E S W 01-0-061117 DAL-GW -R E S W 01-0-061117 DAL-GW-RESW01 -0-061117 DAL-GW-RESW01 -0-061117 DAL-GW-RESW01 -DB-061117 DAL-GW-RESW01-LS-061117 DAL-GW-RESW01 -HS-061117 DAL-GW-RESW02-0-061117 DAL-GW-RESW02-0-061117 DAL-GW-RESW02-0-061117 DAL-GW-RESW02-0-061117 DAL-GW-RESW02-DB-061117 DAL-GW-RESW 02-LS-061117 DAL-GW-RESW02-HS-061117 DAL-GW-TRIP-0-061117 DAL-GW-TRIP-LS-061117 DAL-GW-TRIP-HS-061117 Amount Spiked (ng/L) 750 5500 500 500 500 250 5000 750 5500 500 500 500 250 5000 500 250 5000 13c -p f o a Amount Recovered (ng/L) 811 5860 517 517 475 308 2900 756 5580 485 458 504 244 3930 524 263 5280 Recovery (%) 108 107 103 103 95 123 58 101 101 97 92 101 98 79 105 105 106 Average: Standard Deviation: 99 14 Note: Since this summary table shows rounded results, recovery values may vary slightly from the values in the raw data. Exygen Research Page 21 o f 119 Interim Report #26 - Analysis o f Groundwater Samples Exygen Study No.: P0000760 FIGURES Exygen Research Page 22 o f 119 Interim Report #26 Analysis o f Groundwater Samples Exygen Study No.: P0000760 Figure 1. Typical Extracted Calibration Curve for PFOA in Reagent Water 112406C P760-1131 W jt . i Rts.idb (PFOA): " U n * j f R tg its iio n f l / * KMighting): y 822 x + 6.36+003 ( r 0 .9 M 1 ) Area, counts Exygen Research Page 23 o f 119 Interim Report #26 - Analysis o f Groundwater Samples Exygen Study No.: P0000760 Figure 2. Extracted Standards of PFOA in Reagent Water, 0 ng/L and 25 ng/L, Respectively I XC112406-0 PFOA (Standard) 413.0/369.0 amu - sample 1 of 29 from 11240GCR.wiff (peak not found) XC112404-1-PFOA (Standard) 413.0069.0 am * ta m p h 2 o f 29 from 1124ttC R.w iff A n a : 23275 c o m * Height: 1.60**003 cpo RT: 10.4m i* 10.42 Intensity, cps Intensity, cps Exygen Research Page 24 o f 119 Intensity, cps Interim Report #26 - Analysis o f Groundwater Samples Exygen Study No.: P0000760 Figure 3. PFOA in Reagent Water, 50 ng/L Fortified Reagent Spk A, and 500 ng/L Fortified Reagent Spk B, Respectively R sagsat C ontrol - PFOA (Um* mown) 413.$069.9*m b ta m p /t 9 o f 34 from 112496C.wiff A n a : 4409 counts H eight: 1.64*+942 cps RT: 19.5 m in Ara: 40868 oounts Haight: 3.11 003 ops RT: 10.4 min 3000 10.44 2000 1000- 2.47 7.02 0.1 8 . ^ A 89 Tim, min Ragnt Spk B - PFOA(QC)413.0/3G9.0 amu sampl 11 of 3 4 from 112400Cjmff Ara: 421004 counts H a ight 2.80+004 cps RT: 10.4 min 10 10.46 2.04- 1.0*4- 0.0 I I I 12 ...................I- - - --I i ...... 345078 im *. min 13 15 10 17 Intensity, cps Intensity, cps Exygen Research Page 25 o f 119 Interim Report #26 - Analysis o f Groundwater Samples Exygen Study No.: P0000760 Figure 4. Chromatogram Representing a Groundwater Sample Analyzed for PFOA (Exygen ID: C0222023, Data Set: 112406C) I CQ222023 - PFOA (IM txow x) 413.02369.**m * -s tm p lt 1 t o f 34 fro 112406C.wiff A n a : 301SH coaift* H tigXt: 2.*te+004c p t AT: 10.5 mm 10.46 Intensity, cps Exygen Research Page 26 of 119 Interim Report #26 - Analysis o f Groundwater Samples Exygen Study No.: P0000760 Figure 5. Typical Extracted Calibration Curve for 13C PFOA in Reagent Water 112400C P7B0-1131 W i t " Rej.rdb (13C PFOA): 'U n t i l " R ig it r is n C'1 / x " w tigM ing): y - 724 x + 0.00017 ( l - 0.0078) Area, counts Exygen Research Page 27 o f 119 Interim Report #26 - Analysis o f Groundwater Samples Exygen Study No.: P0000760 Figure 6. Extracted Standards of 13C PFOA in Reagent Water, 0 ng/L and 25 ng/L, Respectively I XC11240C-* - 13C PFOA (Standard) 4TSL0/37&O amu -Mampft 1 o f 34from p a a k a o t found) Intensity, cps 10.46 Intensity, cps Exygen Research Page 28 o f 119 Intensity, cps Interim Report #26 - Analysis o f Groundwater Samples Exygen Study No.: P0000760 Figure 7. 13C PFOA in Reagent Water, 50 ng/L Fortified Reagent Spk A, and 500 ng/L Fortified Reagent Spk B, Respectively Roagont Control - 13C PFOA (Unknown) 4T5LA370L0nm u -tu m p f t 9 o f 34 from 11240C.wiff ^ 1 n o t found) 0.06 A rt a: 42406 counts Haight: 3.20e+0Q3 cps RT: 10.4 min 10.46 Area: 373725 counts Height: 2 0 e + 0 0 4 cps RT: 10.9 min 10.46 Intensity, cps Intensity, cps Exygen Research Page 29 o f 119 Interim Report #26 - Analysis o f Groundwater Samples Exygen Study No.: P0000760 Figure 8. Chromatogram Representing a Groundwater Sample Analyzed for 13C PFOA (Exygen ID: C0222023, Data Set: 112406C) I C0222P23 13CPFOA fttokaow K )41S.0370dam n -ta m p /t H a t34from 11240tC.witT A n a : 374225 c o v iftr HaigMt: 3tM n 0 M cp t AT: 10.48 Intensity, cps Exygen Research Page 30 o f 119 Interim Report #26 - Analysis o f Groundwater Samples Exygen Study No.: P0000760 APPENDIX A Study Protocol P0000760 (Exygen Study No. P0000760) with Analytical Method, Protocol Amendments, and Deviations Exygen Research Page 31 o f 119 Interim Report #26 - Analysis o f Groundwater Samples Exygen Study No.: P0000760 Exygen Protocol Number: P0000760 STUDY PROTOCOL Study Title: Analysis of Perfluorooctanoic Acid (PFOA) in Water, 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: P0000760 Performing Laboratory: Exygen Research 3058 Research Drive State College, PA 16801 Phone: (814) 272-1039 Sponsor Representative: Michael A. Santoro Director o f Regulatory Affairs 3M Building 0236-01-B-10 St. Paul, MN 55144 Phone: (651) 733-6374 Page l o f 65 Exygen Research Page 32 o f 119 Interim Report #26 - Analysis o f Groundwater Samples Exygen Study No.: P0000760 Exygen Protocol Number: P0000760 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 33 o f 119 Interim Report #26 Analysis o f Groundwater Samples Exygen Study No.: P0000760 Exygen Protocol Number: P0000760 PROTOCOL APPROVAL Study Title: Analysis of Perfluorooctanoic Acid (PFOA) 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: P0000760 APPROVALS MU JaisimEa Kesari, Study Director Weston Solutions .yQW W Michael A. Sa iro, Sponsor Representative 3M Company A Date Shaffer, Tectinii *gen Research J ___________ l. Quality Assurance Unit Date Page 3 o f 65 Exygen Research Page 34 o f 119 Interim Report #26 - Analysis o f Groundwater Samples Exygen Study No.: P0000760 Exygen Protocol Number: P0000760 TABLE OF CONTENTS TITLE PAGE...................................................................................................................................................... 1 DISTRIBUTION................................................................................................................................................ 2 PROTOCOL APPROVAL................................................................................................................................. 3 TABLE OF CONTENTS................................................................................................................................... 4 INTRODUCTION...............................................................................................................................................5 TEST MATERIAL..............................................................................................................................................5 SURROGATE FIELD SPIKE COMPOUND.................................................................................................... 5 OBJECTIVE.......................................................................................................................................................6 TESTING FACILITY........................................................................................................................................ 6 STUDY DIRECTOR.......................................................................................................................................... 6 SPONSOR REPRESENTATIVE........................................................................................................................7 PRINCIPAL INVESTIGATOR................................................ ........................................................................ 7 PROPOSED EXPERIMENTAL START AND TERMINATION DATES...................................................... 7 IDENTIFICATION AND JUSTIFICATION OF THE TEST SYSTEM.......................................................... 7 SAMPLE PROCUREMENT, RECEIPT AND RETENTION.......................................................................... 8 SAMPLE IDENTIFICATION........................................................................................................................... 8 ANALYTICAL PROCEDURE SUMMARY.......................................................................... .........................9 VERIFICATION OF ANALYTICAL PROCEDURE....................................................................................... 9 METHOD FOR CONTROL OF BIAS............................................................................................................... 11 STATISTICAL METHODS............................................................................................................................... 11 GLP STATEMENT............................................................................................................................................ 11 REPORT............................................................................................................................................................. U SAFETY AND HEALTH...................................................................................................................................12 AMENDMENTS TO PROTOCOL....................................................................................................................13 DATA RECORD KEEPING..............................................................................................................................13 QUALITY ASSURANCE..................................................................................................................................14 RETENTION OF DATA AND ARCHIVING...................................................................................................14 APPENDIX I, ANALYTICAL METHODS.......................................................................................................15 Page 4 o f 65 Exygen Research Page 35 o f 119 Interim Report #26 - Analysis o f Groundwater Samples Exygen Study No.: P0000760 Exygen Protocol Number: P0000760 INTRODUCTION The purpose of this study is to perform analysis for perfluorooctanoic acid (PFOA) 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 MATERIAL The test material is perfluorooctanoic acid (PFOA) and was purchased from Sigma-Aldrich. PFOA Chemical Name: Perfluorooctanoic acid Molecular Weight: 414 Lot Number: 23116HB Purity: 97.64% Transitions Monitored: 413 --> 369 (for quantification) and 413 -->219 (for confirmation) Structure: F SURROGATE FIELD SPIKE COMPOUND The surrogate field spiking compound is UC labeled perfluorooctanoic acid (l3C PFOA) and was purchased by 3M from Perkin-Elmer Life and Analytical Sciences. UC PFOA Chemical Name: 1.2-13C perfluorooctanoic acid Molecular Weight: 416 Lot Number: 3507-195 Purity: 97% Transition Monitored: 415 --370 Page 5 o f 65 Exygen Research Page 36 of 119 Interim Report #26 - Analysis o f Groundwater Samples Exygen Study No.: P0000760 Exygen Protocol Number: P0000760 OBJECTIVE The purpose of this study is to perform analysis for perfluorooctanoic acid (PFOA) 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 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 Page 6 o f 65 Exygen Research Page 37 o f 119 Interim Report #26 - Analysis o f Groundwater Samples Exygen Study No.: P0000760 . Exygen Protocol Number: P0000760 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 john.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 termination dates will be included in the final report. 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 Page 7 o f 65 Exygen Research Page 38 o f 119 Interim Report #26 - Analysis o f Groundwater Samples Exygen Study No.: P0000760 Exygen Protocol Number: P0000760 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 PFOA 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 of 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 die 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 samples will be stored frozen at S -10C. Small mammal whole blood 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 -10"C. The receipt and processing of the samples will be documented in the final report and raw data associated with the study. 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 n um b e r. Sample storage conditions and locations will be documented throughout the study. Page 8 o f 65 Exygen Research Page 39 o f 119 Interim Report #26 - Analysis o f Groundwater Samples Exygen Study No.: P0000760 Exygen Protocol Number: P0000760 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 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" The above methods use analytical conditions capable of separating the isomers of PFOA. The final report will include the isomers summed into total PFOA 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 PFOA sample collection at the testing facility and have been shown to be free of PFOA. All containers used for water sample collection will be shipped to the sample location containing 100 ng of 13C PFOA (equivalent to 500 ng/L in the final sample). 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 spikes will contain PFOA as well as perfluorobutanesulfonate (PFBS), perfluorohexanesulfonate (PFHS), and perfluorooctanesulfonate (PFOS). These compounds are included in the solutions used to spike the samples. The results for PFBS, PFHS and PFOS will not be reported in this study. Exygen will supply one field blank (control water) and two field blank spikes (control water fortified with PFOA at a low Page 9 o f 65 Exygen Research Page 40 o f 119 Interim Report #26 - Analysis o f Groundwater Samples Exygen Study No.: P0000760 Exygen Protocol Number: P0000760 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 PFOA 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 S00 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 PFOA at both a low and high level and processed through the described procedure to determine method accuracy and to check for bias. 13C-PFOA will also be added to each sample in the laboratory prior to extraction at the level specified below. For small mammal liver, Exygen will supply a SO mL polypropylene centrifiige 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 PFOA at both a low and high level and processed through the described procedure to determine method accuracy and to check for bias. >3C-PFOA will also be added to each sample in the laboratory prior to extraction at the level specified below. Low and high spiking levels for each matrix are defined below: Matrix Low PFOA High PFOA "C-PFOA Spiking Level Spiking Level Spiking Level Water 500ng/L 5000 ng/L 500 ng/L Soil 4ng/g 40ng/g 40 ng/g Sediment 4ng/g 40ng/g 40 ng/g Fish lOng/g 100ng/g 100 ng/g Clams lOng/g 100ng/g 100 ng/g Vegetation 10ng/g I00ng/g 100 ng/g Small Mammal Liver 10ng/g 100 ng/g 100 ng/g Small Mammal Serum 10ng/g lOOng/mL 100 ng/mL Recoveries are 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. Prior to sample analysis, a laboratory control spike study will be conducted in water to justify the use of 13C PFOA results to establish precision and accuracy of PFOA samples. The following samples are to be prepared: Page tO o f 65 Exygen Research Page 41 o f 119 Interim Report #26 - Analysis o f Groundwater Samples Exygen Study No.: P0000760 Exygen Protocol Number: P0000760 Two control water blanks Two control water samples fortified at 250 ng/L with l3C PFOA Two control water samples fortified at 250 ng/L with 13C PFOA and at 250 ng/L with PFOA. Two control water samples fortified at 5000 ng/L with 13C PFOA and at 5000 ng/L with PFOA. The above samples are to be extracted and analyzed according to method V0001780 entitled "Method of Analysis for the Determination of Perfluorooctanoic Acid (PFOA) in Water by LC/MS/MS." METHOD FOR CONTROL OF MAS 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: Page I I o f 65 Exygen Research Page 42 o f 119 Interim Report #26 - Analysis o f Groundwater Samples Exygen Study No.: P0000760 Exygen Protocol Number: P0000760 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). 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. SAFETY AND HEALTH Laboratory personnel will 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). P a g e 12 o f 65 Exygen Research Page 43 o f 1 1 9 Interim Report #26 - Analysis o f Groundwater Samples Exygen Study No.: P0000760 Exygen Protocol Number: P0000760 AMENDMENTS TO PROTOCOL All significant changes to the analytical protocol outlined here will be expressed in writing, signed and dated by die 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 die date of the documented change. Copies of 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 of 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, arrow 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 13 o f 65 Exygen Research Page 44 o f 119 Interim Report #26 - Analysis o f Groundwater Samples Exygen Study No.: P0000760 Exygen Protocol Number: P0000760 r * ' 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. All applicable requirements for reporting of study results as per 40 CFR 792.185. 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 14 o f 65 Page 45 o f 119 Interim Report #26 - Analysis o f Groundwater Samples Exygen Study No.: P0000760 Exygen Protocol Number: P0000760 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 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" Exygen Research Page IS o f 65 Page 46 o f 119 Interim Report #26 - Analysis of Groundwater Samples Exygen Study No.: P0000760 Exygen Protocol Number: P0000760 ANALYTICAL METHOD M ethod N um ber V 0001780 M ethod o f A nalysis fo r th e D eterm ination o f P erfluorooctaaolc A cid (PFO A ) in W ater by LC /M S/M S Analytical Testing Facility: Exygen Research 3058 Research Drive State College, PA 16801 Approved By: c-AL____ Paul Connolly ` Technical Leader, LC-M S, Exygen Research D ate / J oJohhnn Flathoerty ' VV iac*e PPirme ciid a li. O p e rin o n e , Exygen R esearch D ate Exygen Research Total Paget: 7 Page 16 o f 65 Page 47 o f 119 Interim Report #26 - Analysis o f Groundwater Samples Exygen Study No.: P0000760 Exygen Protocol Number: P0000760 ExyftaRMMfch Mthod Number VOOOI780 I ANALYTICAL m e t h o d M ethod o f Analysis for the Determ ination o f Perfluorooctanoic A cid (PFO A ) in W ater by LC/M S/M S 1.0 Scope This method is to be employed for the isolation and quantitation o f perfluorooctanoic a d d by H igh Performance Liquid Chrom atography coupled to a tandem M ass Spectrom etnc D e te c t (LC/M S/M S) in water. 2.0 Safety 2.1 Alw ays observe safe laboratory practice!. 2.2 Consult foe appropriate M SDS before handling any chem ical for proper safety precautions. 3.0 Sam ple Requirement 3.1 A t least 40 m L o f test sam ple for extraction. 3.2 N o sample processing is needed for w ater samples. 3.3 Sam ples stored refrigerated should be allow ed to equilibrate to room tem perature. 3.4 All samples m ust be thoroughly m ixed before being sam pled for extraction. 3.5 Any samples w itning particles should be centrifuged at 3000 rpm for 5 minutes and the supernatant used for the extraction. 3.6 Sam ple collection procedures will be specified in foe sam pling plan for this project. 4.0 Reagents and Standards 4.1 W a te r-H P L C grade 4.2 M ethanol - HPLC grade 4.3 Am monium Acetate - A.C.S. R eagent Grade 4.4 Perfluorooctanoic A d d - Sigma-Aldrich 5.0 Instrument and Equipment 5.1 5.2 5.3 5.4 5.5 5.6 5.7 5.8 5.9 5.10 5.11 A high performance liquid chrom atograph capable o f pum ping up to 2 solvents equipped w ith a variable volum e iqjector capable o f injecting 5*200 p L connected to a tandem M ass Spectrom eter (LC/M S/M S). A device to collect raw data for peak integration and quantitation. Analytical balance capable o f reading to 0.00001 g. 50 mL disposable polypropylene centriftige tubes. 15 m L disposable polypropylene centrifoge tubes. Disposable m icropipets (50-100uL, 100-200uL). 125-mL LD PE narrow -m outh bottles. 2 m L clear H PLC vial kit. Disposable pipettes. A utopipettes (100*1000 p L and 10-100 jiL ), w ith disposable tips. W aters Sep Pak Vac 6 cc (Ig ) tC18 SPE cartridges. Page 2 of? Page 7 o f 65 Exygen Research Page 48 o f 119 Interim Report #26 - Analysis o f Groundwater Samples Exygen Study No.: P0000760 Exygen Protocol Number: P0000760 Exygea Reaeaich Method Number V0001780 L ANALYTICAL M ETH OD M ethod o f Analysis for the Determ ination o f Perfluorooctanoic A cid (PFO A ) in W ater by LC/M S/M S 5.12 SPB vacuum manifold. 5.13 CentriAige capable o f spinning 50 m L polypropylene tubes at 3000 rpm. 6.0 Chromatographic System 6.1 Analytical Column: Fhiophase R P (K eystone Scientific), 2.1 m m x 50 m m, Sp (P/N: 82505-052130) 6.2 Temperature: 30*C 6.3 M obile Phase (A ) : 2 m M Am m onium Acetate in W ater 6.4 M obile Phase (B) : M ethanol 6.5 Gradient Program: Tim a fminl 0.0 1.0 8.0 20.0 22.5 2tA 65 65 25 25 65 5L 35 35 75 75 35 Flow Rate (m L/m inl 0.3 0.3 0.3 0.3 0.3 6.6 Injection Volum e: 15 pL (can be increased to as m uch as 50 pL). 6.7 Quantitation: Peak A rea - external standard calibration curve. 6.8 R unT im e: 2 3 minutes. The above conditions are intended as a guide and m ay be changed in order to optim ize the HPLC system. 7.0 M S/M S System 7.1 M ode: E lectrospray N egative M R M m ode, m o n ito rin g 413 -* 369 m/z. The above condition* are intended as a guide and m ay be changed in order to optim ize the M SM S system. 8.0 Preparation o f Solutions 8.1 M obile Phase 8.1.1 2 m M am m onium acetate in w a te r is p rep ared b y ad d in g 0 .IS 4 g o f ammonium acetate to 1000 m L o f water. Alternate volumes m ay be prepared. Page .loi Page 18 o f 65 Exygen Research Page 49 o f 119 Interim Report #26 - Analysis o f Groundwater Samples Exygen Study No.: P0000760 Exygen Protocol Number: P0000760 Exygea Research Method Number V00017S0 ANALYTICAL M ETH O D M ethod o f Analysis for die Determination ofPerfluorooctanoic A cid (PFO A ) in W ater by LC/M S/MS 9.0 Standard Preparation 9.1 Standard Stock/Fortification Solution 9.1.1 Prepare a stock so lution o f *100 p g /m L o f P F O A b y w eig h in g t o m g o f analytical standard (corrected for purity) and dilute to 100 mL with m ethanol in a 125-mL LD PE bottle. 9 .1 .2 A 10 p g /m L fo rtification so lu tio n o f P F O A is prep ared b y b ringing 10 m L o f th e 100 p g /m L so lution to a final v o lu m e o f 100 w ith methanol in a 125 m L LO PE bottle. 9.1.3 A 1.0 pg/m L fortification solution o f PFO A is prepared by bringing Io m L o f t h e 10 p g /m L solution to a final v o lu m e o f 100 w ith methanol in a 125 m L LOPE bottle. 9.1.4 A 0 .1 p g /m L fortification so lution o f P F Q A is prepared b y bringing 10 m L o fth e 1.0 pg/m L solution to a final volum e o f 100 w ith methanol in a 125 m L LD PE bottle. 9.1.5 A 0.01 pg/m L fortification solution o f PFO A is prepared by bringing 10 m L o f the 0.1 pg/m L solution to a final volum e o f 100 with methanol in a 125 m L LD PE bottle. 9.1.6 T he stock and fortification solutions are to b e stored in a refrigerator at approximately 4*C and are stable for a m axim um period o f 6 months from the date o f preparation. 9.2 Standard Calibration Solutions 9.2.1 9.2.2 LC/M S/MS calibration standards are prepared in H PLC water. The calibration standards are processed through die extraction procedure, identical to samples. The following is a typical cam ple: additional concentrations m ay be Concentration o f Fortification Solution (nob) 0 10 10 10 100 100 too Fortification Volume of Volume Fortified Control (PL) Sanole (mL) 0 40 100 40 200 40 400 40 100 40 200 40 400 40 Final Concentration of Calibration Standard foot)* 0 25 50 100 250 500 1000 Calibration Standard ID (exaismle) XCmmddyy-Q XCmmddyy-1 XCmmddyy-2 XCmmddyy-3 XCmmddyv-4 XCmmddyy-5 XCmmddw-6 * T he extracted concentration o f the calibration standard is equal to 8x its initial concentration, due to the concentration o f the standard during foe extraction (SPE). X C extracted calibration standard. Page 4 o f Page 19 o f 65 Exygen Research Page 50 o f 119 Interim Report #26 - Analysis o f Groundwater Samples Exygen Study No.: P0000760 Exygen Protocol Number: P0000760 Exygn Research M tftod Number VOOOI7SO ANALYTICAL M ETH O D M ethod o f Analysis for the Determ ination ofPerfluorooctanoic A cid (PFO A ) in W ater by LC/M S/M S 9.2.3 9.2.4 9.2.5 A zero standard solution (reagent blank) m ust be prepared w ith each set o f standards extracted. S tore all extracted calibration standards in 15 -m L polypropylene tubes at 2*C to 6*C, up to two weeks. A lternate volum es and concentrations o f standards m ay b e prepared as needed. 10.0 Batch Set Up 10.1 E ach b atch o f sam ples extracted (typically 2 0 o r leas) m u st include at least one reagent control (method blank using H PLC water) and two reagent controls fortified at known concentrations (lab control spike) to verify procedural recovery for th e batch. 10.2 Requirem ents for field and laboratory duplicates and spikes will be specified in the quality assurance plan for this project. 11.0 Sam ple Extraction 11.1 M easure 40 m L o f sam ple o r a portion o f sam ple diluted to 40 m L w ith w ater into 50 m L polypropylene ccntriflige tubes (fortify as needed, replace lid and m ix well). 11.2 Condition the C u SPE cartridges (1 g, 6 m L ) b y passing 10 m L methanol follow ed b y 5 m L o f H P L C w a te r (* 2 d io p /se c ). D o n o t let co lu m n run dry 11.3 Load sam ple on conditioned C u SPE cartridge. D iscard ehiate. 11.4 E lute w ith *-5 m L 100% m ethanol. C o llect 5 m L o f e lu ate into graduated 15 m L polypropylene centriftige tubes (final volum e 5 m L). 11.5 Analyze sam ples using electrospray LC /M S/M S. 12.0 Chrom atography 12.1 12.2 12.3 12.4 Inject the tam e amount o f each standard, sam ple and fortified sam ple into the L C /M S/M S system . A calibration standard m u st p rec e d e and follow ail analyzed samples. Standards o f PFO A corresponding to at least five o r m ore concentration levels m ust be included in in analytical a c t An entire set o f extracted calibration standards m ust be included at the beginning and at the cod o f a sam ple s e t Extracted standards m ust be interspersed betw een every 5*10 sam ples. A s an alto-native, an entire set of 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 m ust be the first and last injection in a sam ple set. U se linear standard curves for quantitation. U n e a r standard curves a rt generated for the analyte by linear regression using 1/x weighting o f peak area Pag* 5 of 7 Page 20 o f 65 Exygen Research Page 51 o f 119 Interim Report #26 - Analysis o f Groundwater Samples Exygen Study No.: P0000760 Exygen Protocol Number: P0000760 ExyftaR sM ttch M atted Nunbar V0001780 I ANALYTICAL M ETH O D M ethod o f Analysis lor the Determination o f Perfluorooctanoic A cid (PFO A ) in W ater by LC/M S/M S v e n u i calibration standard concentration using M aasLynx 3.3 (or equivalent) software system. 12.3 Sam ple response should not exceed standard responses. A ny sam ples that exceed standard responses should be further diluted and reanalyzed. 13.0 Acceptance Criteria 13.1 13.2 13.3 13.4 13.3 13.6 Chromatogram m ust show a peak o f a daughter ion at 369 am u from a parent o f 413 am u. T he 413 am u parent corresponds to the PF O A anion, while the daughter ion (369 amu) represents the loss o f carbon dioxide. M ethod blanks m ust not contain PFOA at levels greater than the LOQ. if a blank PF O A a t levels g rea te r th an SO ng /L , then a n ew blan k sam ple m ust b e obtained and the entire set m ust b e re-extracted. Recoveries o f control spikes and m atrix spikes m ust be betw een 70-130% o f their know n values. I f a control spike A lls outside the acceptable limits, the entire set o f --TM p w should b e re-extracted. A n y m atrix spike outside 70 130% should be evaluated b y th e analyst to determ ine i f re-extraction is warranted. A ny calibration standard found to be a statistical outlier by using the Huge Error Teat, m ay be excluded from the calculation o f the calibration curve. However, the total num ber o f extracted calibration standards that could be excluded m ust not exceed 20% o f the total num ber o f extracted standards injected. The correlation coefficient (R ) for calibration curves generated m ust be 20.992 (R* 20.983). I f calibration results A ll outside these limits, then appropriate steps m ust be taken to adjust instrum ent operation, and the standards o r the relevant set o f sam ples should be reanalyzed. Retention tim es betw een standards and sam ples m ust not drift m ore than 4 % within an analytical run. I f retention tim e drift exceeds this lim it within an analytical run then the set m utt be reanalyzed. 14.0 Calculations 14.1 U se the follow ing equation to calculate the am ount o f PF O A found (in ng/L, based on peak area) using the standard curve (linear regression parameters) generated b y foe M ass Lynx software program: PFO A found (ng/L) - (Peak area - intercept! x DF slope D F A ctor by which the final volum e w as diluted, i f necessary. Pag 6 of 7 Page 21 o f 65 Exygen Research Page 52 o f 119 Interim Report #26 - Analysis o f Groundwater Samples Exygen Study No.: P0000760 Exygen Protocol Number: P0000760 E x y fn Rcaearck Method Number V0001780 | ANALYTICAL M ETH O D ................ | M ethod o f Analysis for the Determination o f Perfiuocooctanoic A cid (PPO A ) in W ater by LC/M S/M S 14.2 For sam ples fortified w ith know n am ounts o f PFO A 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) Exygen Research Fags 7 o f7 Page 22 o f 65 Page 53 o f 119 Interim Report #26 - Analysis o f Groundwater Samples Exygen Study No.: P0000760 Exygen Protocol Number: P000076 ANALYTICAL METHOD M ethod N um ber V0001781 M ethod o f A nalysis fo r th e Determ loetJoB o f P arfitioroocteaete AcJd (PFO A ) In Soil by LC /M S/M S Analytical Testing Facility: Exygen Research 3058 Research Drive State College, PA 16801 Approved By: C -iL ___ Paul Connolly ' Technical Leader, LC-M S, Exygen Research Date aFlaherty ' Vice President, Operations, Exygen Research D ate Exygen Research Total Pafea: 7 Page 23 o f 65 Page 54 o f 119 Interim Report #26 - Analysis o f Groundwater Samples Exygen Study No.: P0000760 Exygen Protocol Number: P0000760 BxyfttJUMueh MethodNumber VOOOl7g| ANALYTICAL M ETH O D M ethod o f Analysis for the Determination o f Perfluorooctanoic A cid (PFO A ) in Soil by LC/M S/MS 1.0 Scope This m ethod ia to be employed for the isolation and quantitation o f periluorooctanoic acid by High Performance Liquid Chrom atography coupled to a tandem M ass Spectrometric Detector (LC/M S/M S) in soil. 2.0 Safety 2.1 A lw ayi observe safe laboratory practices. 2.2 Consult the appropriate M SDS before handling any chem ical for proper safely precautions. 3.0 Sam ple Requirement 3.1 A t least 15 g o f teat sam ple fo r extraction. 3.2 N o sam ple processing is needed for soil sam ples. 3.3 Sam ples stored refrigerated should be allow ed to equilibrate to room tem perature. 3.4 All samples m ust be thoroughly m ixed before being sam pled for extraction. 3.5 Sam ple collection procedures will be specified in the sam pling plan for this p ro je c t 4.0 Reagents and Standards 4.1 W a te r-H P L C grade 4.2 M eth an o l-H P L C grade 4.3 Ammonium Acetate - A.C.S. Reagent Grade 4.4 Perfluorooctanoic Acid - Sigma-Aldrich 5.0 Instrum ent and Equipment 5.1 5.2 5.3 5.4 5.5 5.6 5.7 5.8 5.9 5.10 5.11 5.12 5.13 A high performance liquid chrom atograph capable o f pum ping up to 2 solvents equipped w ith a variable volum e injector capable o f injecting 5-200 pL connected to a tandem M ass Spectrom eter (LC/M S/M S). A device to collect raw data for peak integration and quantitation. Analytical balance capable o f reading to 0.00001 g. 50 m L disposable polypropylene centrifuge tubes. 15 m L disposable polypropylene centriftige tubes. D isposable m icropipets (50-lOOuL, 100-200uL). 125-m L L D P E narrow -m outh bottles. 2 m L clear HPLC vial Irit Disposable pipettes. Autopipettes (100-1000 pL and 10-100 pL), w ith disposable tips. W aters S ap Pale Vac 6 c c ( l g ) t C l S S P E cartridges. SPE vacuum manifold. U ltrasonic bath. Page 2 o f7 Page 24 o f 65 Exygen Research Page 55 o f 119 Interim Report #26 - Analysis o f Groundwater Samples Exygen Study No.: P0000760 Exygen Protocol Number: P0000760 E xyya Rn n cb Matkod Number V000178I _________________________ A N A L Y T IC A L M E T H O D ____________________________ M ethod o f Analysis for th s D etsnnination o f Perfluorooctanoic A cid (PFO A ) in Soil by LC/M S/MS 5.14 W rist-action shaker. 5.15 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.) m m x 50 mm, 5p (P/N: 82505-052130) 6.2 Temperature: 30*C 6.3 M obile Phase (A ): 2 m M Am m onium Acetate in W ater M obile Phase ( B ) : M ethanol Gradient Program: Tim e fminl 0.0 1.0 8.0 20.0 22.5 2LA 65 65 25 25 65 Flow Rate 2 1 fm L/m inl 35 0.3 35 0.3 75 0.3 75 0.3 35 0.3 6.6 byection V olum e: 15 p L (can be increased to as m uch as 50 pL). 6.7 Quantitation: Peak Area - external standard calibration curve. 6.8 R un Time: - 23 minutes. T he above conditions are inteoded as a guide and m ay b e changed in order to optim ize die HPLC system. 7.0 M S/M S System 7.1 M ode: E lectrospray N egative M R M m ode, m o n ito rin g 41 3 - 369 m /z for PFOA. The above conditions are intended as a guide and m ay be changed in order to optim ize the M SM S system. 8.0 Preparation o f Solutions 8.1 M obile P hase 8.1.1 2 m M am m onium acetate in w ater is prepared b y adding 0.154 g o f ammonium acetate to 1000 m L o f water. Alternate volumes m ay be prepared. Page 3 of? Page 25 o f 65 Exygen Research Page 56 o f 119 Interim Report #26 - Analysis o f Groundwater Samples Exygen Study No.: P0000760 Exygen Protocol Number: P0000760 Exygt Rmarch Method Number V000I7SI ANALYTICAL M ETH O D M ethod o f Analysis for the Determ ination o f Perfluorooctanoic A cid (PFO A ) in Soil by LC/M S/M S 9.0 Standard Preparation 9.1 Standard Stock/Fortification Solution 9.1.1 Prepare a stock solution o f -~100 p g /m L o f P F O A b y w eig h in g 10 m g o f analytical standard (corrected fo r pu rity ) and d ilu te to 100 m L with methanol in a 125-mL LDPB bottle. 9.1.2 A 10 (igtaiL fortification solution o fP F O A is prepared by bringing 10 m L o f th e 100 p g /m L solution to a final v o lu m e o f 100 w ith methanol in a 123 m L LDPE bottle. 9.1.3 A 1.0 pg /m L fortification so lution o f P F O A is p rep ared b y bringing 10 m L o f the 10 p ^ m L solution to a final volum e o f 100 w ith methanol in a 123 m L LD PE bottle. 9.1.4 A 0.1 pg/m L fortification solution o f PFO A is prepared b y bringing 10 m L o f fhe 1.0 pg/raL solution to a final volum e o f 100 w ith methanol in a 123 m L LD PE bottle. 9.1.3 A 0.01 p ^ m L fortification solution o fP F O A is prepared b y bringing 10 m L o f tiie 0.1 p g /ra L solution to s final volum e o f 100 with methanol in a 123 m L L D PE bottle. 9.1.6 T h e stock and fortification so lutions a re to b e sto red in a refrigerator at approxim ately 4*C and are stable for a m axim um period o f 6 months from the date o f preparation. 9.2 Standard Calibration Solutions 9.2.1 9.2.2 LC/M S/M S calibration standards are prepared in HPLC water. The calibration standards are processed through the extraction procedure, identical to samples. D ie following is a typical example: additional concentrations may be prepared 11 needed. Concentration o f Fortification Solution (cob) 0 10 10 10 100 100 100 Fortification Volume o f Volume Fortified Control (PL) Semole (mL) 0 40 100 40 200 40 400 40 100 40 200 40 400 40 Final Concentration of Calibration Standard (not)* 0 25 30 100 250 500 1000 Calibration Standard ID (example) XCmmddyy-0 XCmmddyy-l XCnunddyy-2 XCmmddyy-3 XCminddyy-4 XCnnoddyy-3 XCmnxktW ' * D ie extracted o o a c atratio n o f the calibration standard is equal to 8x its initial concentration, due to the concentration o f the standard during the extraction (SPE). X C extracted calibration standard. Page 4 o f7 Page 26 o f 65 Exygen Research Page 57 o f 119 Interim Report #26 - Analysis o f Groundwater Samples Exygen Study No.: P0000760 Exygen Protocol Number: P0000760 Exygta RMMicb Method Number VOOOI78I ANALYTICAL M ETH O D M ethod o f Analysis for the Determination o f Perfluorooctanoic A cid (PFO A ) in Soil by LC/M S/MS 9.2.3 9.2.4 9.2.3 A zero standard solution (reagent blank) m m t be prepared with each set o f standards extracted. Store all extracted calibration atandards in 15-mL polypropylene tubes at 2*C to 6*C, up to two week. Alternate volumes and concentrations o f standards m ay be prepared as ftffbdi 10.0 Batch Set Up 10.1 F sch b atch o f sam ples extracted (ty p ic ally 2 0 o r less) m u st include at least one reagent control (m ethod blank using 5 m L o f m ethanol) and tw o reagent controls fortified at known concentrations (lab control spike) to verify procedural recovery for the batch. 10.2 Requirem ents for field and laboratory duplicates and spikes will be specified in the quality assurance plan for this project 11.0 Sam ple Extraction 1 u W eigh 5 g o f sam ple into 50 m L polypropylene centrifitge tubes (fortify as needed, replace Ud and m ix well). 11.2 Add 3 m L o f m ethanol and shake on a wrist action shaker for -1 3 minutes. 11.3 Transfer the tubes to an ultrasonic bath and sonicate fo r ~ 1 5 m inutes. 11.4 Bring the volum e up to 40 m L w ith w ater in foe 30 m L polypropylene centrifitge tube. 11.5 Centrifitge fo r- 1 0 m inutes at -3 0 0 0 ipm . 11.6 Condition foe C u SPE cartridges ( l g. 6 m L) by passing 10 m L methanol followed b y 5 m L ofH P L C w ater (~ 2 drop/sec). D o not let colum n run dry 11.7 Load (decant) foe sam ple on the conditioned C u SPE cartridge. Discard eluate. 11.8 Elute w ith - S m L 10034 m ethanol. C ollect 5 m L o f eluate into graduated 15 m L polypropylene centrifitge tubes (final volum e 5 mL). 11.9 Analyze samples using electrospray LC/M S/M S. 12.0 Chrom atography 12.1 Inject th e sam e am ount o f each standard, s am p le an d fo rtified sam ple into the LC /M S/M S system . A calibration standard m u st precede and follow all analyzed samples. 12.2 Standards o f P F O A ---- t o at least five o r m ore concentration levels m ust be included in an analytical set. 12.3 A n entire set o f extracted calibration standards m ust be included at the beginning and at the end o f a sam ple s e t Extracted standards m ust be interspersed betw een every 5-10 samples. A s an alternative, an entire set o f F tp 5 of? Page 27 o f 65 Exygen Research Page 58 o f 119 Interim Report #26 - Analysis o f Groundwater Samples Exygen Study No.: P0000760 Exygen Protocol Number P0000760 Exygei Rncuch Method Numb VOOO171! ANALVTICAL M ETH O D M ethod o f Analysis for the Determination o f Perfluorooctanoic A cid (PFO A ) in Soil by LC/M S/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 m ust be the first and last injection in a sam ple set. 12.4 U se linear standard curves for quantitation. L inear standard curves are generated for the analyte by linear regression using 1/x weighting o f peek area versus calibration standard concentration using M aasLynx 3.3 (or equivalent) software system. 12.5 S im ple response should not exc eed standard responses. A ny sam ples that exceed standard responses should be Anther diluted and reanalyzed. 13.0 Acceptance Criteria 13.1 13.2 13.3 13.4 13.5 13.6 Chromatogram m ust show a peak o f a daughter ion at 369 am u from a parent o f 413 amu. T he 413 am u parent corresponds to the P F O A anion, w hile the daughter ion (369 amu) represents the loss o f carbon dioxide. M ethod blanks m ust not contain PFO A a t levels greater than the LOQ. If a b lank contains P F O A a t levels greater th an SO ng /L , th en a n e w blan k s im p le m ust be obtained and the entire set m ust be re-extracted. R ecoveries o f control spikes an d m atrix spikes m u st b e betw een 7 0 -130% o f their know n values. I f a control spike foils outside the acceptable lim its, the entire set o f samples should be re-extracted. A ny m atrix spike outside 70 130% should b e evaluated b y th e analyst to determ in e i f re-extrection is warranted. Any calibration standard found to be a statistical outlier b y using the Huge Error Teat, m ay be excluded from the calculation o f the calibration curve. However, the total num ber o f extracted calibration standards that could be excluded m ust not exceed 20% o f the total num ber o f extracted standards injected. The correlation coefficient (R) for calibration curves generated m ust be 20.992 (RJ 20.985). I f calibration results foil outside these lim its, then appropriate steps m ust be taken to adjust instrum ent operation, and the standards o r foe relevant set o f samples should be reanalyzed. Retention tim es betw een standards and sam ples m ust not drift m ore than 4 % w ithin an analytical run. i f retention tim e drift exceeds this lim it within an analytical run then the set m ust be reanalyzed. P*ge6of7 Page 28 o f 65 Exygen Research Page 59 o f 119 Interim Report #26 - Analysis o f Groundwater Samples Exygen Study No.: P0000760 Exygen Protocol Number: P0000760 Exygen lU ltu ch Method Number VOOO1781 I ANALYTICAL M ETH O D M ethod o f Analysis for the Determination o f Perfluorooctanoic A cid (PFO A ) in Soil by L C /M S/M S 14.0 C alculttions 14.1 U n the follow ing equation to calculate the am o u n t o f PF O A found (in ng/L, based a n peak area) using the standard curve (linear regression parameters) generated by foe M ass Lynx software program : PFO A found (ng/L) slope xD F D F factor b y which foe final volum e was diluted, if necessary. 14.2 For samples fortified w ith know n am ounts o f PFO A prior to extraction, use the following equation to calculate foe percent recovery. Recovery (% ) [to tal analyte found (ng/L) - analyte found in control (ng/L)] analyte added (ng/L) 14.3 U se the following equation to convert foe am ount o f PFO A found in ng/L to ng/g(ppb). PFO A found (pnb) (PFOA found fag/L ) x volum e extracted (0.04U1 sample weight (5 g) 14.4 U se the following equation to calculate foe am ount o f PFO A found in ppb baaed on dry w eight PFOA found (ppb) dry weight * PFO A found (ppb) x [100% / total so)ids(%)] Page 7 of ? Page 29 o f 65 Exygen Research Page 60 o f 119 Interim Report #26 - Analysis o f Groundwater Samples Exygen Study No.: P0000760 Exygen Protocol Number: P0000760 ANALYTICAL METHOD M ethod Num ber: V0001782 Method oIA itijnii for the Determination of Perfinorooctanoie Acid (PFOA) in Sediment by LC/MS/MS Analytical Testing Facility: Exygen Research 3058 Research Drive State College, PA 10801 Approved By. V -A (_lL Paul Connolly I Technical Leader, LC-M S, Exygen Research aiJflj / L i / /loloihan F la h o ty f Viicce P resident, O perations, Exygen R esearch __ IQl i b i v i Date D ate Exygen Research Total Pages: 7 Page 30 o f 65 Page 61 o f 119 Interim Report #26 - Analysis o f Groundwater Samples Exygen Study No.: P0000760 Exygen Protocol Number; P0000760 Exygea RMearefc Method Number V0001782 ANALYTICAL M E T H O D M ethod o f Analysis for the Detennination o f Perfluorooctanoic A cid (PFO A ) in Sediment by LC/M S/MS 1.0 Scope Thia m ethod is to be employed for foe isolatimi and quantitation o f perfluorooctanoic acid b y High Performance Liquid Chrom atography coupled to a tandem M ass Spectrometric Detector (LC/M S/M S) in sedim ent. 2.0 Safety 2.1 A lw ays observe safe laboratory practices. 2.2 Consult foe appropriate M SDS before handling any chem ical for proper safety precautions. 3.0 Simple Requirem ent 3.1 A t least 30 g o f te s t sam ple for extraction. 3.2 N o sam ple processing is needed for sedim ent sam ples. 3.3 Sam ples stored refrigerated should be allow ed to equilibrate to room tem perature. 3.4 A ll samples m ust be thoroughly m ixed before being sam pled for extraction. 3.5 Sam ple collection procedures w ill be specified in the sam pling plan for this project. 4.0 Reagents and Standards 4.1 W a te r- HPLC grade 4.2 M ethanol-H P L C grade 4.3 Acetic A cid -R eag en t grade 4.4 Am monium Acetate - A.C.S. R eagent G rade 4.5 Perfluorooctanoic Acid - Sigma-Aldrich 5.0 Instrument and Equipment 5.1 5.2 5.3 5.4 5.5 5.6 5.7 5.8 5.9 5.10 5.11 5.12 A high performance liquid chrom atograph capable o f pum ping up to 2 solvents equipped w ith a variable volum e injector capable o f injecting 5-200 pL connected to a tandem M ass Spectrom eter (LC/M S/M S). A device to collect raw data for peak integration and quantitation. Analytical balance capable o f reading to 0.00001 g. 50 mL disposable polypropylene centrifugo tubes. 15 m L disposable polypropylene centrifoge tubes. D isposable m icropipets (SO-lOOuL, 100-200uL). 125-m L L D P E n arrow -m outh bottles. 2 mL clear HPLC vial k it D p o sa b le pipd tf f A utopipanet (100-1000 pL and 10-100 pL), with diapoaable tipi. W ite n Sep P ik V ac 6 cc (lg ) tC18 SPE cattridgei. SPE vacuum m anifold Page 2 o f7 Page 31 o f 63 Exygen Research Page 62 o f 119 Interim Report #26 - Analysis o f Groundwater Samples Exygen Study No.: P0000760 Exygen Protocol Number: P0000760 E xypa Research Method Number V00017B2 | ANALYTICAL M ETH O D 1 M ethod o f Analysis for die Detennination o f Perfluorooctanoic A cid (PFO A ) in Sediment bv LC/M S/M S 5.13 5.14 5.15 Vortexer. W rist-action ahaker. Centrifuge capable o f spinning 50 m L polypropylene tubes at 3000 rpm. 6.0 Chromatographic System 6.1 A nalytical Colum n: Fluophase R P (K eystone Scientific), 2.1 m m x 50 m m , 5p (P/N: 82505-052130) 6.2 Temperature: 30*C 6.3 M obile Phase (A ): 2 m M Am m onium Acetate in W ater M obile Phase (B ): M ediano! Gradient Program: T h n efarin l 0.0 1.0 8.0 20.0 22.5 &A 65 65 25 25 65 Flow Rate (m L/m ini 35 0.3 35 0.3 75 0.3 75 0.3 35 0.3 6.6 Injection Vohnne: 15 p L (can be increased to as m uch as 50 pL). 6.7 Quantitation: Peak Area - external standard calibration curve. 6.8 R unT im e: - 2 3 minutes. The above conditions are intended as a guide and m ay be changed in order to optim ize the HPLC system. 7.0 M S/M S System 7.1 M ode: Electzospray N egative M R M m ode, m o n ito rin g 413 --36 9 m /z for PFOA. The above conditions are intended ss guide and m ay be changed in order to optim ize the M SM S system. 8.0 Preparation o f Solutions 8.1 M obile Phase 8.1.1 2 m M am m onium acetate in w a te r is p rep ared b y ad d in g 0.154 g o f ammonium acetate to 1000 m L o f water. Page 3 of 7 Page 32 o f 65 Exygen Research Page 63 o f 119 Interim Report #26 - Analysis o f Groundwater Samples Exygen Study No.: P0000760 Exygen Protocol Number: P0000760 ExygsaRcacT b Method Number V0001782 ANALYTICAL M ETH O D I M ethod o f Analytic for the Determ ination o f Periluorooctanoic A cid (PFO A ) in Sediment by LC/M S/MS 8.2 Extraction Solutions 8.2.1 1H acetic acid in w ater ie prepared b y adding 10 m L o f acetic acid to 1000 m L o f water. Alternate volumes m ay be prepared. 9.0 Standard Preparation 9.1 Standard Stock/Fortificatioa Solution 9.1.1 Prepa re a stock solution o f - 1 0 0 p g /m L o f P F O A b y w eig h in g 10 m g o f analytical standard (corrected for purity) and dilute to 100 m L with m ethanol in a 12S-mL LDPB bottle. 9.1.2 A 10 pg /m L fortification so lution o f P F O A is prepared b y bringing 10 m L o f th e 100 p g/m L solution to a final v o lu m e o f 100 w ith methanol in a 125 m L LDPE bottle. 9.1.3 A 1.0 pg /m L fortification so lution o f P F O A is prepared b y bringing 10 m L o fth e 10 pg/m L solution to a final volum e o f 100 w ith methanol in 125 m L LD PE bottle. 9.1.4 A 0.1 p g faiL fortification a o lu tio n o fP F O A is prepared b y b ringing 10 m L o f (he 1.0 p ^ m L solution to a final volum e o f 100 w ith methanol in a 125 m L LD PE bottle. 9.1.5 A 0.01 pg/m L fortification solution o f PFO A is prepared b y bringing 10 m L o f th e 0.1 p g /m L s o lu tio n to a final volum e o f 100 with methanol in a 125 m L LD PE bottle. 9.1.6 T he stock and fortification solutions are to b e stored in a refrigerator at approxim ately 4*C and are stable lo r a m axim um period o f 6 months from the date o f preparation. 9.2 Standard Calibration Solutions 9.2.1 LC/M S/M S calibration standards are prepared in m ethanol via dilution o f foe 0.1 p ^ m L fortification solution. 922 1110 follow ing is a typical exam ple: ad d itio n al concentrations m ay be Concentration o f Fortification Solution fna/inD 100 100 too 10 5 2 Volume (mL) 10 5 2 10 10 10 Diluted to (mL) too 100 100 100 100 100 Final Concentration (n t/m L ) 10.0 5.0 2.0 1.0 O.S 0.2 Page 4 of? Page 33 o f 65 Exygen Research Page 64 o f 119 Interim Report #26 - Analysis o f Groundwater Samples Exygen Study No.: P0000760 Exygen Protocol Number: P0000760 E xyfnRM M rck Method Number V00017I2 \ ANALYTICAL M ETH O D | M ethod o f Analysis for the Determ ination ofPerfluorooctanoic A cid (PFO A ) in Sediment by LC/M S/MS 9.2.3 Store all calibration standard in 125-raL LD PE narrow -m outh bottles at 2*C to 6*C, up to six m onths. 9.2.4 Alternate volum es and concentrations o f standards m ay be prepared as 10.0 Batch Set Up 10.1 batch o f cam ples extracted (typically 20 o r less) m ust include at least one untreated control and tw o untreated controls fortified at known concentrations (lab control qpike) to v e rity pro ced u ral reco v ery few the batch. 10.2 Requirem ents fbr field and laboratory duplicates and spikes will be specified in tee quality assurance plan fbr this project 11.0 Sam ple Extraction 11.1 W eigh 5 g o f sam ple into SO m L poly p ro p y len e c e n trifu g e tubes (fortify as needed, replace lid and m ix well). 11.2 A dd 35 m L o f 1% acetic acid, cap, vortex and shake on a wrist action shaker for - 4 0 minutes, 113 Centrifuge the tubes at -3 0 0 0 rpm for -2 0 minutes. 11.4 Condition the C u SPE cartridges (1 g, 6 m L) b y passing 10 m L methanol followed b y 20 m L o f HPLC w ater (~ 2 drop/sec). D o not let colum n run dry 11.5 Load (decant) the sam ple on the conditioned C u SPE cartridge. Discard eluate. 11.6 A dd 2 0 m L o f m ethanol to th e sedim ent le ft in th e b o tto m o f th e SO m L centrifoge tube. Cap, vortex and shake on a w rist action shaker for -3 0 11.7 11.8 11.9 11.10 11.11 11.12 11.13 Centrifuge tee tubes at -3 0 0 0 rpm for -2 0 minutes. D ecant the m ethanol onto the sam e SPE cartridge. C ollect the eluate. W aah tee colum n w ith 4 m L o f m ethanol. C ollect tee eluate and add it to the eluate collected in step 11.8. Condition a second C u SPE cartridge (1 g, 6 m L ) by passing 10 m L methanol followed by 20 m L o f HPLC w ater ( - 2 drop/sec). D o not let colum n run dry Add tee m ethanol to -2 0 0 m L o f w ater and load on tee second conditioned SPE cartridge. Elute w ith - 5 m L 100% m ethanol. C ollect S m L o f eluate into graduated 15 m L polypropylene centrifoge tubes (final volum e * S m L). A nalyze sam ples using electrospray LC/M S/M S. Page 5 o f7 Page 34 o f 65 Exygen Research Page 65 o f 119 Interim Report #26 - Analysis o f Groundwater Samples Exygen Study No.: P0000760 Exygen Protocol Number: P0000760 BxyusuIUs --i c h Method N u o tar VOOOI7I2 1 ANALYTICAL M ETH O D | M ethod o f Analyste for the Determination o f Perfluorooctanoic A cid (PFO A ) in Sediment by LC/M S/M S 12.0 Chrom atography 12.1 Iqject the sam e am ount o f each standard, cam ple a n d fortified sam ple into the L C /M S/M S lyatem . A calibration standard m u st p rec e d e an d follow all analysed samples. 12.2 Standards o f PFO A corresponding to at least five or m ore concentration levels m ust be included in an analytical set. 12.3 A n entire aet o f extracted calibration standards m ust b e included t i the beginning and at foe end o f a sam ple s e t Standards m ust be interspersed betw een every 5-10 sim ples. A s an alternative, an entire set o f calibration standards m ay be injected at the beginning o f a set followed by calibration standards interspersed every 5-10 sim ples (to account for a second set o f standards). In either esse, calibration standards m ust be foe first and last injection in a sample se t 12.4 U se linear standard curvet for quantitatioa 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. 12.5 Sam ple response should not exceed standard responses. A ny sam ples that exceed standard responses should be forfoer diluted and reanalyzed. 13.0 Acceptance Criteria 13.1 13.2 13.3 13.4 13.5 Chromatogram m ust show a peak o f a daughter ion at 369 am u from a parent o f 413 amu. T he 413 am u parent corresponds to th e PFO A anion, w hile foe ^fvgM ^r ion (369 am u) icpreecnts foe lo st o f carbon dioxide. M ethod blades m ust not contain PFO A at levels greater than the LOQ. If a blank contain PFO A a t levels greater than 0.2 ng/m L, then a new blank sample must be obtained and the entire set m ust be re-extracted. Recoveries o f control spikes and m atrix spikes m ust be betw een 70-130% o f their known values. I f a oootrol qpike falls outside foe acceptable lim its, foe entire set o f should b e re-extracted. A ny m atrix spike outside 70 130% should b e evaluated b y foe analyst to de te rm in e i f re-extraction is warranted. A ny calibration standard found to be a statistical outlier by using die Huge Error Test, m ay be excluded from the calculation o f foe calibration curve. However, the total num ber o f extracted calibration standards that could be excluded m ust not exceed 20% o f foe total num ber o f extracted standards iO)a c te d . The correlation coefficient (R ) for calibration curves generated m ust be 20.992 (R* 20.985). I f calibration results fall outside these lim its, then appropriate stapa m ust b e taken to adjust instrum ent operation, and the standards o r foe relevant set o f samples should be reanalyzed. Pag 6 o f? Page 35 o f 65 Exygen Research Page 66 o f 119 Interim Report #26 - Analysis o f Groundwater Samples Exygen Study No.: P0000760 Exygen Protocol Number: P0000760 BxyyaRjMirefc Method N u o t a V000J7I2 1 a n a l y t ic a l m e t h o d I M ethod o f A nalysis for the D ettnm nation o f Perfluorooctanoic A cid (PFO A ) in Sediment by LC/M S/MS 13.6 R etention tim ea betw een etandanh and sam ples m ust not drift m ore than 4 % within an analytical nm . I f retention tim e drift exceeds this lim it within an analytical run then the set m ust b e reanalyzed. 14.0 C a lc u la tio n s 14.1 U se the follow ing equation to calculate th e am o u n t o f P F O A found (in ng/m L. baaed on peak area) using the standard curve (linear regression parameters) generated b y th e M am Lynx softw are p rogram : PFOA found (ngftnL) slope xD F D F - factor b y w hich the final volume waa diluted, if necessary. 14.2 For sam ples fortified w ith know n am ounts o f PFO A prior to extraction, use die following equation to calculate die percent recovery. Recovery (% ) " f total analyte found (ng/mL) - analyte found in control (ng/m L)] ^ analyte added (ng/mL) 14.3 U se die following equation to convert die am ount o f PFO A found in ng/m L to n * /f (ppb). PP O A found fpyiM - fPFO A found fay /m L l * final v o lu m e (5 mL)1 sample weight (3 g) 14.4 U se the following equation ( if necessary) to calculate the am ount o f PFOA found in ppb based on dry weight. PFO A found (ppb) dry weight - PFO A found (ppb) x [100% / total solida(%)) Pig* 7 o f 7 Page 36 o f 65 Exygen Research Page 67 o f 119 Interim Report #26 - Analysis o f Groundwater Samples Exygen Study No.: P0000760 Exygen Protocol Number: P0000760 ANALYTICAL METHOD M ethod N um ber V0001783 M ethod of A nalysis fo r th e D eterm ination o f F erflaorooctanotc A d d (FFO A ) In Fish ta d C lam s by LC /M S/M S Analytical Testing Facility: Exygen Research 30S8 Research Drive State College. PA 16801 Approved By. Paul Connolly Technical Leader, LC-M S, Exygen Research D ate >/ohn Flaherty ' Vic*e President, O perations, Exygen Research j A 4 r D ate Exygen Research Total Pages: 8 Page 37 o f 65 Page 68 o f 119 Interim Report #26 - Analysis o f Groundwater Samples Exygen Study No.: P0000760 Exygen Protocol Number: P0000760 Exygen Research Method Number VOOOI783 I ANALYTICAL m e t h o d I M ethod o f Analysis for die Determination o f Perftuorooctanoic A cid (PFO A ) in Fish and Clams by LC/M S/M S 1.0 Scope This m ethod is to be employed for the isolation and quantitation o f periluorooctanoic acid b y High Performance Liquid Chrom atography coupled to a tandem M ass Spectromtrie Detector (LC/M S/M S) in fish and clam s. 2.0 Safety 2.1 Alw ays observe safe laboratory practices. 2.2 Consult the appropriate M SDS before handling any chem ical for proper safely precautions. 3.0 Sam ple Requirement 3.1 A t least 20 g o f test sam ple for extraction. 32 Sam ples should b e processed before extraction. P lace th e frozen sam ple in a food ptoces io r and homogenize w ith dry ice. P lace the sam ples in containers and leave open in frozen storage overnight to allow for carbon dioxide piMimatinti Seal and place foe sam ples in frozen storage until tim e o f analysis. 3.3 S im ple collection procedures w ill be specified in foe sam pling plan for this p ro je c t 4.0 Reagents and Standards 4.1 4.2 4.3 4.4 4.5 4.6 4.7 4.8 4.9 4.10 4.11 4.12 4.13 W ater-H P L C grade Acetonitrile - HPLC grade Carbon (120-400 m esh) - Reagent grade M ethanol-H P L C grade Silica gel (60*200 m esh) - Reagent grade Florisil (60*100 m esh) - Reagent grade Superclean LC-N Hj - Reagent grade 1-Octanol - HPLC grade L*A scorbic acid - R eagent grade DimethyldichlorosUane - Reagent grade Toluene - Reagent grade Ammonium Acetate - A.C.S. Reagent Grade Periluorooctanoic Acid - Sigma-Aldrich 5.0 Instrument and Equipment 5.1 A hig h perform ance liquid c hrom atograph c a p ab le o f pu m p in g up to 2 solvents equipped w ith a variable volum e iqjector capable o f injecting S-200 jiL connected to a tandem Maas Spectrom eter (LC/M S/M S). 5.2 A device to collect raw data for peak integration and quantitation. 5.3 A nalytical balance capable o f read in g to 0.00001 g. Page 2 ufS Page 38 o f 63 Exygen Research Page 69 o f 119 Interim Report #26 - Analysis o f Groundwater Samples Exygen Study No.: P0000760 Exygen Protocol Number: P0000760 Exygen R aiifch Method Number V0001783 ANALYTICAL M ETH O D M ethod o f Analyst for die Determ ination o f Perfluorooctanoic A cid (PFO A ) in Fish and Clam s b y LC/M S/M S 5.4 5.5 5.6 5.7 5.8 5.9 5.10 5.11 5.12 5.13 5.14 5.15 5.16 Rotary evaporator. T iasum izer. 125 m L pear-shaped flasks. 50 m L disposable polypropylene centrifoge tubes. 15 m L disposable polypropylene centrifoge tubes. Disposable micropipeta (50-100uL, 100-200uL). 125-m LLD PE narrow -m outh bottles. 2 m L clear HPLC vial k it Disposable pipettea. Autopipettes (100-1000 p L and 10-100 jiL), w ith dispoaable tips. SPE tubes (20mL) (Supelco c a t no. N 057177). W rist action shaker. Centrifoge capable o f spinning 50 m L polypropylene tubes at 2000 rpm. 6.0 Chromatographic Syriern 6.1 Analytical Colum n: Fluophase R P (K eystone Scientific), 2.1 m m x 50 m m. 5p (P/N: 82505-052130) 6.2 Temperature: 30*C 6.3 M obile Phase (A ) : 2 m M Am m onium Acetate in W ater M obile Phase (B ) : M ethanol Gradient Program: Tim e/m int 0.0 1.0 8.0 20.0 22.5 S iA 65 65 25 25 65 Flow Rate & 35 0.3 35 0.3 75 0.3 75 0.3 35 0.3 6.6 Injection Volum e: 15 pL (can be increased to as m uch as 50 pL). 6.7 Quantitation: Peak A re a -e x te rn a l standard calibration curve. 6.8 R unT im e: - 2 3 minutes. The above conditions are intended as a guide and m ay be changed in ord er to optim ize the HPLC system. Page 3 of 8 Page 39 o f 65 Exygen Research Page 70 o f 119 Interim Report #26 - Analysis o f Groundwater Samples Exygen Study No.: P0000760 Exygen Protocol Number: P0000760 Exyfea Research Method N u o t a V0001783 ANALYTICAL M ETH O D M ethod o f Analysis for foe Determ ination ofPcrfluoroocUDOic A cid (PFO A ) in F iih and C lam i by LC/M S/M S 7.0 M S/M S System 7.1 M ode: B lectiospray N egative M R M m ode, m o n ito rin g 413 369 m /z for PFOA. T he above conditions are intended as a guide and m ay be changed in order to optim ize the M SM S system. 8.0 Preparation o f Solutions 8.1 M obile Phase 8.1.1 2 m M am m onium acetate in w ater is prepared b y adding 0.154 g o f ammonium acetate to 1000 m L o f water. 8.2 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% Dimefoyldichloioailane in toluene i t prepared by bringing 3 mL o f dimefoyldichloroailane to a final volum e o f 10 m L w ith toluene. Alternate volumes m ay be prepared. 9.0 Standard Preparation 9.1 Standard Slodc/F crtification S olution 9.1.1 9.1.2 9.1.3 9.1.4 9.1.$ Prepare a stock solution o f ~100 pg/m L o f P F O A b y w eighing 10 mg o f analytical standard (corrected for purity) and dilute to 100 mL with methanol in a 125-mL LD PE bottle. A 1.0 pg/m L fortification solution o f PFO A is prepared by bringing 1 m L o f foe 100 p g /m L so lution to a final v o lu m e o f 100 w ith methanol in e 12$ m L LDPE bottle. A 0 .1 pg/m L fortification solution o f P F O A is prepared b y bringing 10 m L o f foe 1.0 p g/m L so lution to a final v o lu m e o f 100 w ith methanol in e 12$ m L LD PE bottle. A 0.01 p ^ m L fortification solution o f PFO A is prepared by bringing 10 m L o f th e 0.1 pgfm L s o lu tio n to a final volum e o f 100 w ith methanol in n 12$ m L LD PE bottle. T he stock and fortification solutions a re to b e sto re d in a refrigerator at approxim ately 4C and are stable for a m axim um period o f 6 months from foe date o f preparation. Page 4 ofS Page 40 o f 65 Exygen Research Page 71 o f 119 Interim Report #26 - Analysis o f Groundwater Samples Exygen Study No.: P0000760 Exygen Protocol Number: P0000760 Exygea Research Method Number V00017I3 I ANALYTICAL M E T H O D ...... M ethod o f A nalysis for the Determ ination ofPerfluorooctanoic A cid (PFO A ) in Fish and Clams by LC/M S/M S 9.2 Standard Calibration Solutions 9.2.1 LC/M S/M S calibration standards are prepared in m ethanol via dilution o f the 1.0 pg/m L fortification solution. 9.2.2 The following is a typical exam ple: additional concentrations m ay be Coooeotration Final o f Fortification Volume Solution (ux/mLl (mL) Diluted to (mL) Concentration (ligftnL) 1.0 3.0 100 1.0 2.5 too 1.0 1.0 100 0.05 0.025 0.01 0.03 10 100 0.005 0.025 10 100 0.002S 0.1 10 100 0.001 0.003 10 100 0.0005 9.2.3 Store all calibration standards in 125-m L L D PE narrow -m outh bottles at 2*C to 6*C, up to six m onths. 9.2.4 Alternate volum es and concentrations o f standards m ay be prepared as 10.0 Batch Set Up 10.1 Bach batch o f sam ples extracted (typically 2 0 o r l e u ) m ust include at least one untreated control and tw o untreated controls fortified at known concentrations (lab control spike) to verify procedural recovery for the batch. 10.2 Requirem ents for field and laboratory duplicates and spikes will be specified in the quality assurance plan for this project. 11.0 Sim ple Extraction 11.1 11.2 11.3 11.4 11.3 11.6 W eigh 5 g o f frozen sam ple into 30 m L polypropylene centrifuge tubes (fortify u needed, replace lid and m ix well). A dd 30 m L o f acetonitrile and shake on a w rist action shaker fo r-1 5 minutes. Place the tubes in a freezer for ~ l hour. Pack and condition the SPE tubea and silanize the pear-shaped flasks. Pack foe 20 m L SPE tubes in sequence w ith 2 g florisit, 2 g silica gel. 2 g carbon, and 1 g LC-NHj. Condition the colum ns w ith 20 m L o f methanol, then 20 m L o f acetonitrile. D iscard all w ashes. D o n o t allow the colum n to dry. Silanize the 123 m L pear-shaped flasks by rinsing with the 30% dimethyidichlorosilane in toluene solution. R inse the flask w ith toluene once, followed b y m ethanol (three tim es). D ry the flasks com pletely before use. either b y air-drying o r w ith a stream o f nitrogen. PSe 5 of K Page 4! o f 65 Exygen Research Page 72 o f 119 Interim Report #26 - Analysis o f Groundwater Samples Exygen Study No.: P0000760 Exygen Protocol Number: P0000760 Exygea Rm arck Matted Number V000178J [ ANALYTICAL M ETH OD M ethod o f Analysis for the Determ ination ofPerfluorooctanoic A cid (PFO A ) in Fish and Clam s by LC/M S/M S 11.7 Centrifoge the 50 m L polypropylene tubes containing sam ple at 2000 rpm for 10 minutes. 11.8 Decant the extract on to a conditioned SPE colum n fitted inside the m outh o f the pear-shaped flask. C ollect the eluate in the 125 m L silanized pear-shape flask. 11.9 A dd 10 m L o f acetonitrile to the cam ple in th e 50 m L centrifuge tube. Hom ogenize the frozen fat phase using s tisium izer for 30 seconds and rinse foe tiasumizer w ith 10 m L o f acetonitrile into the tube. 11.10 Shake the sam ple again for 10 m inutes on a w rist-action shaker. 11.11 Place the tubes in a freezer for 1 hour m ore. 11.12 Ccntri& ge the 50 m L polypropylene tubes containing sam ple at 2000 rpm for 10 minutes. . 11.13 Decant foe extract onto foe sam e S PE colum n. C ollect the eluate into foe sam e pear-shaped flask and com bine w ith the eluent from foe initial extraction. 11.14 P ais 20 m L o f acetonitrile through the S P E colum n and com bine the eluate in the tam e pear-shaped flask. 11.15 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). 11.16 M ake the final volum e, b y adding 2 m L o f 2% ascorbic acid in methanol to the pear-shaped flask and swirl to mix/diasolve. 11.17 Transfer the extracts to HPLC vials using disposable pipets. 11.18 Analyze samples using electrospray LC/M S/M S. 12.0 Chrom atography 12.1 12.2 12.3 12.4 Inject the sam e amount o f each standard, sim p le and fortified sam ple into the LC /M S/M S system . A calibration standard m ust precede and follow all analyzed samples. Standards o f P F O A co rre q x md in g to at least five o r m o re concentration levels m ust be included in an analytical set. A n entire set o f calibration standards m ust b e included at the beginning and at the end o f a sample set. Standards m ust be interspersed betw een every 5-10 samples. A s an alternative, an entire set o f calibration standards m ay be iiyocted at the beginning o f a set followed b y calibration standards interspersed ev e ry 5-10 s im p le s (to a cco u n t fo r a s e co n d set o f standards). In either case, calibration standards m ust be tits first and last injection in a sample se t U se linear standard curves for quantitation. Linear standard curves are generated for the analyte by linear regression using 1/x weighting o f peak area v enue calibration standard concentration using M aasLynx 3.3 (or equivalent) software system. Pag*6 of8 Page 42 o f 65 Exygen Research Page 73 o f 119 Interim Report #26 - Analysis o f Groundwater Samples Exygen Study No.: P0000760 Exygen Protocol Number: P0000760 ExypsRm uch Method Number VOOOI7S3 | ANALYTICAL M ETH O D I M ethod o f Analysis for the Determination o f Perfhiorooctanoic A cid (PFO A ) in Fish and C laim byLC /M S/M S 12.S Sam ple response should not exceed standard responses. A ny sam ples that exceed standard responses should be further diluted and reanalyzed. 13.0 Acceptance Criteria 13.1 C hrom atogram m ust show a p eak o f a d au g h ter io n at 36 9 am u from s parent o f 413 amu. The 413 am u parent corresponds to the PFO A anion, while the daughter ion (369 am u) represents the lo ts o f carbon dioxide. 13.2 M ethod blanks m ust not contain PFO A at levels greater than die LOQ. If a blank contains PFOA at levels greater than 0.3 ppb. then a new blank sample m ust be obtained and the entire set m ust be re-extracted. 13.3 R ecoveries o f control spikes and m atrix spikes m ust b e betw een 70*130% o f their know n values. I f a control spike falls outside the acceptable limits, the entire set o f samples should be re-extracted. 13.4 A ny calibration standard found to be a statistical outlier b y using the Huge Error Test, m ay be excluded from die calculation o f the calibration curve. However, die total num ber o f calibration standards that could be excluded m ust not exceed 20% o f the total num ber o f standards injected. 13.5 T he correlation coefficient (R ) fo r calibration curves generated m ust be 20.992 (R3 20.98$). I f calibration results fall outside these lim its, then appropriate steps m ust be taken to adjust instrum ent operation, and the standards or die relevant set o f sam ples should be reanalyzed. R etention tim es betw een standards and sam ples m ust not drift m ore than 1 4 % within an analytical run. I f retention tim e drift exceeds this limit within an analytical run then the set m ust be reanalyzed. 14.0 Calculations 14.1 U se th e follow ing equation to calculate the am o u n t o f P F O A found (in ng/m L, baaed o n peak area) using die standard curve (linear regression parameters) generated by the Maas Lynx software program: PFOA found fag/m Lt - (Peak area - intercept) slope 14.2 U ae the following equation to convert th e am ount o f PFO A found in ng/m L to ng/g(ppb). P F O A found (ppb) * fP F O A fou n d (ifM iLi * final v o lu m e fm L ) x DF1 sample weight (g) DF factor b y which the final volum e w as diluted, if necessary. tFase 7 of Page 43 o f 65 Exygen Research Page 74 o f 119 Interim Report #26 - Analysis o f Groundwater Samples Exygen Study No.: P0000760 Exygen Protocol Number: P0000760 Elften Research Method Number V0001783 L ANALYTICAL M ETH O D M ethod o f Analysis for the Determ ination ofPerfluorooctanoic A cid (PFO A ) in Fish and Clam s by LC/M S/M S 14.3 For sam ples fortified w ith know n am ounts o f PFO A prior to extraction, use the following equation to calculate the percent recovery. Recovery (H )* [to tal analyte found (ng/g) - analyte found in control (ng/g)] analyte added (ng/g) qq Exygen Research h |i8 o ft Page 44 o f 65 Page 75 o f 119 Interim Report #26 - Analysis o f Groundwater Samples Exygen Study No.: P0000760 Exygen Protocol Number: P0000760 ANALYTICAL METHOD M ethod Number: V 00017M M ethod o f A nalysis fo r th e D eterm in atio n o f P e rilu o ro o ctan o ic A cht (PFO A ) In V egetation by LC /M S/M S Analytical Testing Facility: Exygen Research 3058 Research Drive State College, P A 16401 Approved By: T t - l C JiL __ Paul Connolly ' Technical Leader, LC-M S, Exygen Research Date q /z n /iU / >/6hn Flaherty ' Vice President, Operations, Exygen Research Date Exygen Research Total Pages: 7 Page 45 o f 65 Page 76 o f 119 Interim Report #26 - Analysis o f Groundwater Samples Exygen Study No.: P0000760 Exygen Protocol Number: P0000760 fccyieo Rwciir h Mwhod Number V0001784 I ANALYTICAL M ETH O D M ethod o f A n a ly st for the Determination o f Perfluorooctanoic A cid (PFO A ) in Vegetation by LC/M S/M S 1.0 Scope This method it to be employed for die isolation and quantitation o f perfluorooctanoic acid b y High Performance Liquid Chrom atography coupled to a tandem M att Spectrom etric D etector (LC/M S/M S) in vegetation. 2.0 Safety 2.1 A lw a y i observe sa fe laboratory practices. 2.2 Consult the appropriate M SDS before handling any chemical for proper safety precautions. 3.0 Sam ple Requirement 3.1 A t least 2 0 g o f test sam ple t o extraction. 32 Sam ples should be processed before extraction. P lace th e frozen sam ple in a food procesaor and hom ogenize w ith dry ice. P lace the sam ples in containers and leave open in frozen storage overnight to allow for carbon dioxide sublimation. Seal and place die sam ples in frozen storage until tim e o f analysis. 3 3 Sam ple collection procedures w ill b e specified in th e sam pling plan for this project. 4.0 Reagents rod Standards 4.1 4.2 4.3 4.4 4.5 4.6 4.7 4.8 4.9 4.10 4.11 4.12 4.13 W ater-H P L C grade Acetonitrile - HPLC grade Carbon (120-400 m esh) - Reagent grade M ethanol - HPLC grade Silica gel (60-200 mesh) - Reagent grade Flonsil (60-100 mesh) - Reagent grade Superclean LC-NHj - Reagent grade 1-O ctanol-H PL C grade L-Ascorbic acid - R eagent grade D im ethyidkhlorosilane - Reagent grade T oluene-R eagent grade Am m onium Acetate - A.C.S. Reagent Grade Perfluorooctanoic Acid - Sigma-Aldrich 5.0 Instrument ro d Equipment 5.1 A hig h p c rto m a n c e liquid c hrom atograph c a p ab le o f pu m p in g up to 2 solvents equipped w ith a variable volum e injector capable o f injecting 5-200 p L connected to a tandem M ass Spectrom eter (LC/M S/M S). 52 A device to collect raw data t o peak integration and quantitation. 5.3 Analytical balance e n a b le o f reading to 0.00001 g. Pag 2 o f7 Page 46 o f 65 Exygen Research Page 77 o f 119 Interim Report #26 - Analysis o f Groundwater Samples Exygen Study No.: P0000760 Exygen Protocol Number: P0000760 Exygea Research Method Number VOOOI784 ANALYTICAL M ETH O D M ethod o f Analysis for die Determination o f Perfluorooctanoic A cid (PFO A ) in Vegetation by LC/M S/M S 5.4 5.5 5.6 5.7 5.8 5.9 5.10 5.11 5.12 5.13 5.14 5.15 R otary evaporator. 125 m L pear-shaped flasks. SO m L disposable p olypropylene c e n tr if lif e ttb e s . 15 m L disposable polypropylene centrifuge tubes. D isposable m icropipets (50>1 OOuL, 100-200uL). 125-mL LDPE narrow -m outh bottles. 2 m L d e a r H P L C v ial Idt. Dispoaable pipettes. Autopipettes (100-1000 pL and 10-100 pL ), w ith disposable tips. SPE tubes (20mL) (Supelco cat. no. N0S7177). W rist action shaker. CentriiUge capable o f spinning 50 m L polypropylene tubes st 2000 rpm. 6.0 Chromatographic System 6.1 A nalytical Column: Fluophase R P (K eystone Scientiflc), 2.1 m m x 50 mm, 5m (P/N: 82505-052130) 62 Tem perature: 30"C 6.3 M obile Phase (A ) : 2 m M Am m onium Acetate in W ater 6.4 M obile M use (B ) : Methanol 6.5 Gradient Program: 11100 rimiri 0.0 1.0 8.0 20.0 22.5 SLA 65 65 25 2$ 65 SLB 35 35 75 75 35 Flow Rate fm L/m inl 0.3 0.3 0.3 0.3 0.3 6 .6 In jectio n V olum e: 15 mL (ca n b e in creased to a s m u ch as 5 0 p L ). 6.7 Quantitation: Peak A rea - external standard calibration curve. 6.8 R u n T im e: 2 3 m inutes. The above conditions are intended as a guide and m ay be changed in order to optim ize the HPLC system. 7.0 M S/M S System 7.1 M ode: Electrosprmy N egative M R M m o d e, m o n ito rin g 41 3 - 3 6 9 m /z for PFOA. Pagt 3 of 7 Page 47 o f 65 Exygen Research Page 78 o f 119 Interim Report #26 - Analysis o f Groundwater Samples Exygen Study No.: P0000760 Exygen Protocol Number: P0000760 ExygenlUmreb Method Number V0001784 | ANALYTICAL m e t h o d M ethod o f Analyst* for the Detennination o f Periluorooctanoic A cid (PFO A ) in Vegetation byLC /M S/M S The above conditions are intended as a guide and m ay be changed in order to optim ize foe M SM S system. 8.0 Preparation o f Solutions 8.1 M obile Phase 8.1.1 2 n M am m onium acetate in w a te r is p rep ared b y ad d in g 0.154 g o f M M M ulim acetate to 1000 m L o f water. 8.2 Extraction Solutions 8.2.1 8.2.2 2% iso o rb ic acid in m ediano! is p rep ared b y d isso lv in g 2 g o f ascorbic a d d in 100 m L o f m ethanol. 30% DimethyldichlorosUaDe in toluene is prepared b y bringing 3 m L o f dimethyldichlorosilane to a final volum e o f 10 m L w ith toluene. Alternate volumes m ay be prepared. 9.0 Standard Preparation 9.1 Standard Stock/Fortification Solution 9.1.1 9.1.2 9.1.4 9.1.5 P repare a stock solution o f 1 0 0 p g faiL o f P F O A b y w e ig h in g 10 m g o f analytical standard (corrected for purity) and dilute to 100 m L with methanol in a 125-mL LD PE bottle. A 1.0 pgfaiL fortification solution o f PFO A is prepared by bringing I m L o f th e 100 p g /m L solution to a final v o lu m e o f 100 w ith methanol in a 125 m L LDPE bottle. A 0.1 p g/m L fortification so lu tio n o f P F O A is prepared b y bringing 10 m L o fth e 1.0 pg/m L solution to a final volum e o f 100 w ith methanol in a 125 m L LD PE bottle. A 0.01 pg/m L fortification solution o f PFO A is prepared b y bringing 10 m L o f the 0.1 pg/m L solution to a final volum e o f 100 with methanol in a 125 m L L D PE bottle. T h e d o c k and fortification solutions w e to b e sto red in e refrig erato r at approximately 4*C and are stable for e m axim um period o f 6 months from the date o f preparation. 9.2 Standard Calibration Solutions 9.2.1 LC /M S/M S calibration standards are prepare d in m ethanol via ditution o f the 1.0 pgfaiL fortification solution. Page 4 of ? Page 48 o f 65 Exygen Research Page 79 of 119 Interim Report #26 - Analysis o f Groundwater Samples Exygen Study No.: P0000760 Exygen Protocol Number: P0000760 Exygen R--etich Method Number VOOOI784 ANALYTICAL m e t h o d M ethod o f Analysis for the Determ ination o f Perfluoiooctanoic A d d (PFO A ) in Vegetation by LC/M S/M S 9.22 The following ie a typical example: additional concentration* may be prepared n eed ed . Concamradoo o f Fortification Solution fus/mL) Volume (mL) D iluted (mL) Final Concentration (ugftnL) 1.0 1.0 1.0 0.05 0.025 0.1 0.005 5.0 2.5 1.0 10 10 10 10 100 100 100 100 100 100 100 0.05 0.025 0.01 0.005 0.0025 0.001 0.0005 9.2.3 Store all calibration etandarda in 125-m L L D PE narrow -m outh bottles at 2*C to 6*C, up to six months. 9.2.4 Alternate volum es and concentrations o f standards m ay be prepared as 10.0 Batch Set U p 10.1 Each batch o f sam ples extracted (ty p ic d ly 20 o r less) m u st include at least one untreated control end two untreated controls fortified at known concentrations (lab control spike) to verity procedural recovery for the batch. 10.2 Requirem ents for field and laboratory duplicates and spikes will be specified in the quality assurance plan for this project- 11.0 Sam ple Extraction 11.1 11.2 11.3 11.4 11.5 11.6 11.7 W eigh 5 g o f frozen sam ple info 5 0 m L polypropylene centrifuge tubes (fortify as needed, replace lid and m ix well). A dd 30 m L o f acetonitrile and shake o n a w rist action shaker for -1 5 minutes. Centrifiige the 50 m L polypropylm e tubes containing sam ple at -2 0 0 0 rpm fo r- 1 0 minutes. Pack and condition the SPE tubes and silanize the pear-shaped flasks. Pack the 20 m L SPE tubes in sequence w ith 2 g florisil, 2 g silica gel. 2 g carbon, and 1 g LC-NH*. Condition the colum ns w ith 20 m L o f methanol, then 20 m L o f acetonitrile. D iscard all w ashes. D o not allow the column to diy. Silanize foe 125 m L pceM haped flasks b y rinsing w ith the 30% dimethyldichlorofilane in toluene solution. R inse the flask with toluene once, followed b y m ethanol ( ti n e tim es). D ry the flasks com pletely before use, either b y air-drying o r w ith a stream o f nitrogen. Decent the extract on to a conditioned SPE colum n fitted inside the m outh o f the pear-shaped flask. Collect the eluate in foe 125 m L silanized pear-shape flask. Page 5 oi'? Page 49 o f 65 Exygen Research Page 80 o f 119 Interim Report #26 - Analysis o f Groundwater Samples Exygen Study No.: P0000760 Exygen Protocol Number: P0000760 Exygen Research Method Number V0001784 | ANALYTICAL M ETH OD M ethod o f Analysis for the Determination o f Perfluorooctanoic A cid (PFO A ) in Vegetation by LC/M S/M S 11.8 A dd 20 m L o f acetonitrile to the sam ple in the 50 m LcentriA ige tube. 11.9 Shake the eample again fo r 10 minute on a w riat-action shaker. 11.10 O f-- if t g e th e SO m L polypropylene tu b es co n tain in g sa m p le at -2 0 0 0 rpm fo r- 5 minutes. 11.11 Decam foe extract onto the sam e S P E colum n. C ollect the eluate into the sam e pear-shaped flask and com bine w ith foe eluent from foe initial extraction. 11.12 R epeatsteps 11.8through 11.11 again. 11.13 A dd 3-4 drops o f l*octanol to foe extract in the pear-shaped flask and evaporate at reduced pressure using a rotary evaporator (at < 40*C). 11.14 M aim the final volume, b y adding 2 m L o f 2% ascorbic acid in methanol to the pear-shaped flask and sw irl to imx/dieeolve, 11.15 T ran sftr the extracts to H P LC vials using dispoiable pipets. 11.16 Analyze samples using electroipray LC/M S/M S. 12.0 Chrom atography 12.1 12.2 12.3 12.4 12.3 Inject foe came am ount o f each standard, sam ple and fortified sam ple into the LC /M S/M S system. A calibration standard m ust precede and follow all analyzed samples. Standards o f PFO A corresponding to at least five o r m ore concentration levels m ust be included in an analytical s e t Aji entire set o f extracted calibration standards m ust be included at the beginning and at foe end o f a sample set. Extracted standards m ust be interspersed betw een every 3-10 sam ples. A s an alternative, an entire set o f extracted calibration standard m ay b e injected at foe beginning o f a set followed b y extracted calibration standards interspersed every 3-10 samples (to account for a second set o f extracted standards). In o th e r case, extracted calibration standards m ust b e the first end last injection in a sam ple set. U se linear standard curves for quantitation, lin e a r standard curves are generated fo r foe analyte b y linear regression using lAr w eighting o f peak area versus calibration standard concentration using M assLynx 3.3 (or equivalent) software system. Sam ple response should no t exceed standard responses. A ny sam ples that exceed standard responses should be further diluted and reanalyzed. 13.0 Acceptance Criteria 13.1 C hrom atogram m ust abow a peak o f a daughter io n at 369 am u from a parent o f 413 amu. The 413 am u parent corresponds to the PFO A tnion, while the daughter ion (369 am u) represents the lose o f carbon dioxide. Page 6 of 7 Page 50 o f 65 Exygen Research Page 81 o f 119 Interim Report #26 - Analysis o f Groundwater Samples Exygen Study No.: P0000760 Exygen Protocol Number: P0000760 Exygoa RwMrch M eted Nwabtr VOOOI7I4 I AXALVnCALM ETHOP M ethod o f A n a ly st for the Determination o f Perfluocooctanoic A cid (PFO A ) in Vegetation by LC/M S/MS 13.2 13.3 13.4 13.5 13.6 M ethod blanki m ust not contain PFOA at levels greater than the LOQ. If a blank contain! PFOA at levels greater than 0.5 ppb. then a new blank sample m ust be obtained and die entire set m ust b e re-extracted. Recoveries o f control spikes and m atrix spikes m ust be betw een 70-130% o f their known values. If a control spike falls outside the acceptable limits, the entire eat o f samples should be re-extracted. A ny calibration standard found to be a statistical outlier by using the Huge Error Test, m ay be excluded from the calculation o f the calibration curve. However, die total num ber o f calibration standards that could be excluded m ust not exceed 20% o f die total num ber o f standards injected. The correlation coefficient (R) for calibration curves generated m ust be 20.992 (R3 20.985). I f calibration results foil outside these lim its, then appropriate steps m ust be taken to adjust instrum ent operation, and the standards o r the relevant set o f sam ples should be reanalyzed. Retention tim es betw een standards and sam ples m ust not drift m ore than 1 4 % w ithin an analytical run. I f retention tim e drift exceeds this limit within an analytical run then the set m ust be reanalyzed. 14.0 Calculations 14.1 U se th e follow ing equation to calculate th e am o u n t o f P F O A found (in ng/m L, based on peak area) using the standard curve (linear regression parameters) generated by the M aas Lynx software program: PFO A found (ng/m L) - fPeok m m - intercept) slope 14.2 U se the following equation to convert the am ount o f PFO A found in ng/mL to ntfgippb). PFO A found (poM - fPFO A found fng/m L) linel volum e ( m L i x DF1 sample weight (g) DF * lector b y w hich the final volum e w as diluted, if necessary. 14.3 For sam ples fortified w ith know n am ounts o f PFO A p rior to extraction, use the following equation to calculate foe percent recovery. Recovery (% ) - [ total analyte found (ng/g) - analyte found in control (ng/g)] analyte added (ng/g) Page 7 oH Page 51 o f 65 Exygen Research Page 82 of 119 Interim Report #26 - Analysis o f Groundwater Samples Exygen Study No.: P0000760 Exygen Protocol Number: P0000760 ANALYTICAL METHOD M ethod N um ber V0001785 M ethod o f A nalysis for th e D eterm ination o f P erfluorooctaaoic A cid (PFO A ) la Sm all M am m al U v e r by LC /M S/M S A nalytical Testing Facility: Exygen Research 3058 Research D rive State College, PA 16801 Approved By: _C--iLj___ Paul Connolly { Technical Leader, LC-M S, Exygen Research g /M d -4 ^ ________ J6 h n Flaherty ' Vice President, Operations, Exygen Research D ue D ate Exygen Research Total Pages: 7 Page 52 o f 65 Page 83 o f 119 Interim Report #26 - Analysis o f Groundwater Samples Exygen Study No.: P0000760 Exygen Protocol Number: P0000760 Exygea Research Matbod Number VOOOI78S | ANALYTICAL M ETH O D M ethod o f Analysis for the Determ ination o f Perfluorooctanoic A cid (PFO A ) in Small M ammal Liver b y LC/M S/M S 1.0 Scope This m ethod is to be employed for foe isolation and quantitation o f perfluorooctanoic acid by High Perform ance Liquid Chrom atography coupled to a tandem Mass Spectrom etric D etector (LC/M S/M S) in sm all m am m al liver. 2.0 Safety 2.1 Alw ays obeerve aafe laboratory practices. 2.2 Consult foe appropriate M SDS before handling any chem ical for proper safety precautions. 2.0 Sam ple Requirement 3.1 A t least 3 g o f test sam ple for extraction. 3.2 Sam ples should be processed before extraction. P lace foe frozen sam ple in a food processor and hom ogenize w ith dry ice. P lace the sam ples in containers and leave open in frozen storage overnight to allow for carbon dioxide sublimation. Seal and place foe sam ples in frozen storage until tim e o f analysis. Alternately, i f there is an insufficient am ount o f sam ple (-less than 5 g), then no processing is necessary and foe sam ple can be used as supplied. 3.3 Sam ple collection procedures w ill be specified in foe sam pling plan for this p ro je c t 4.0 R eagans and Standards 4.1 W a te r-H P L C grade 4.2 M ethanol - HPLC p a d s 4.3 A cetonitrile-H P L C grade 4.4 Ammonium Acetate -A .C .S . R eagent G rade 4.3 Perfluorooctanoic A d d - Sigma-Aldrich 3.0 Instrument and Equipment 3.1 A high perform ance liquid chrom atograph capable o f pum ping up to 2 solvents equipped w ith a variable volum e iqjector capable o f injecting 5*200 p L connected to a tandem M aas Spectrom eter (LC/M S/M S). 3.2 A device to collect raw data for peak integration and quantitation. 3.3 A nalytical balance capable o f reading to 0.00001 g. 3.4 30 m L disposable polypropylene centrifuge tubes. 3.3 15 m L disposable polypropylene centrifuge tubes. 3.6 D isposable m icropipets (SO-lOOuL, 100-200uL). 3.7 123*mL LDPE narrow -m outh bottles. 3.5 2 m L clear HPLC vial kit. P fe2 o n Page 53 o f 65 Exygen Research Page 84 o f 119 Interim Report #26 - Analysis o f Groundwater Samples Exygen Study No.: P0000760 Exygen Protocol Number: P0000760 Bxygea Research Method Number VOOOI785 I ANALYTICAL M ETH O D M ethod o f Analysis for the Determ ination o f Perfluorooctanoic A cid (PFO A ) in Smell M ammal Liver b y LC/M S/M S 5.9 5.10 $.11 $.12 $.13 $.14 $.1$ D isposable pipette*. Autopipette* (100*1000 pL and 10*100 pL ), w ith disposable tips. W aters Sep Pak Vac 6 cc (lg ) tC18 SPE cartridges. SPE vacuum manifold. T issuem izer. Wrist*aetk>n shaker. Centriftige capable o f spinning 1$ m L polypropylene tubes at 3000 rpm. 6.0 Chromatographic System 6.1 Analytical Colum n; Fluopbase R P (K eystone Scientific), 2.1 m m x 50 m m, 5p (P/N: 82505*052130) 6.2 Temperature: 30*C 6.3 M obile Phssc (A ): 2 m M Am m onium A cetate in W ater M obile Phase (B ) : Methanol G n d ien t Program: T im * fwitnl 0.0 1.0 8.0 20.0 22.5 65 65 2$ 2$ 65 5LB 35 35 75 75 35 Flow Rate fm L/m inl 0.3 0.3 0.3 0.3 0.3 6.6 Injection Volum e: 1$ pL (can be increased to as m uch as 50 pL). 6.7 Quantitation: Peak Area - external standard calibration curve. 6.8 R unT im e: ~23m inutes. The above conditions are intended as a guide and m ay b e changed in order to optim ize the HPLC system. 7.0 M S/M S System 7.1 M ode: E le ctro ap n y N egative M R M m o d e, m o n ito rin g 41 3 -* 369 m /z for PFOA. The above conditions are intended as a guide and m ay be changed in order to optim ize the M SM S system. Page 3 of7 Page 54 o f 65 Exygen Research Page 85 o f 119 In te r im Report #26 - Analysis o f Groundwater Samples Exygen Study No.: P0000760 Exygen Protocol Number: P000760 ExypaXaNKCt Method Number VOM7 I A N a L Y T I C A L M E T H O P ____________________________ M ethod o f Analysis for A e Determ ination o f Perfiuorooctanoic A cid (PFO A ) in Small M ammal Liver by LC/M S/M S 8.0 Preparation o f Solutiona 8.1 M obile P hase 8.1.1 2 m M am m onium acetate in w a te r is prepared b y ad d in g 0.154 g o f ammonium acetate to 1000 m L o f water. Alternate volumes m ay be prepared. 9.0 Standard Preparation 9.1 Standard Stock/Fortification Solution 9.1.1 P repare a s to c k solution o f - 1 0 0 p g /m L o f P F O A b y w eig h in g 10 m g o f analytical standard (corrected fo r pu rity ) a n d d ilu te to 100 m L with methanol in a 125-mL LD PE bottle. 9.1.2 A 1.0 pg/raL fortification solution o f PFO A is prepared by bringing I m L o f th e 100 p g /m L s o lu tio n to a final v o lu m e o f 100 w ith methanol in a 125 m L LDPE bottle. 9 . 1 J A 0.1 p g /m L fortification s o lu tio n o f P F O A is p rep ared b y bringing 10 m L o f the 1.0 pg/m L solution to a final volum e o f 100 w ith methanol in a 125 m L LD PE bottle. 9.1.4 The stock and fortification aohitions are to be stored in a refrigerator at approximately 4C and are table for a m axim um period o f 6 months from the date o f preparation. 9.2 Standard Calibration Solutions 9.2.1 9.2.2 LC/M S/M S calibration standards are prepared in m ethanol via dilution o fth e 0.1 pg/m L fortification solution. The following is a typical exam ple: additional concentrations may be prepared as needed. Concentration o f Fortification Volume Diluted to Final Concentration Solution (nafaiL) 100 (mL) 5.0 (mL) too ina/m L ) S.0 100 2.0 100 100 1.0 too 2.0 1.0 5.0 10 100 2.0 10 100 1.0 10 100 0.5 02 0.) 9.2.3 Store all calibration standards in 125-m L LD PE nanow -m outh bottles at 2*C to 6*C, up to six months. 9.2.4 A lternate volum es and concentrations o f sta n d a rd s m t y b e prepared as Pge4of7 Page 55 o f 65 Exygen Research Page 86 o f 119 Interim Report #26 - Analysis o f Groundwater Samples Exygen Study No.: P0000760 Exygen Protocol Number: P0000760 Exy*eo Research Method Number VOOOI783 I ANALYTICAL M ETH OD M ethod o f Analysis for the Determ ination o f Perfluorooctanoic A cid (PFO A ) in Small M ammal Liver b y LC/M S/M S 10.0 Batch Set Up 10.1 E ach batch o f f a n c ie s extracted (typically 20 o r less) m ust include at least one untreated control and tw o untreated controls fortified at known concentrations (lab control spike) to verify procedural recovery for the batch. 10.2 Requirem ents for field and laboratory duplicates and spikes will be specified in the quality assurance plan for this project. 11.0 Sam ple Extraction 11.1 W rig h 1 g o f sam ple into a SO m L poly p ro p y len e c e n trifu g e tubes (fortify as needed, replace lid and m ix well). N ote that alternate w eights o f liver m ay be m easured depending on the sam ple size available for use. 11.2 A dd w ater to the sam ple for a final volum e o f lO m L . 11J Hom ogenize sam ple using a tiatuem izer for -1 m inute. 11.4 T ransfer 1 m L o f th e sam ple u s in g a d isp o sa b le p ip ette into a 15 m L disposable centrifbge tube. 11.5 A dd 5 m L o f acetonitrile and shake for - 2 0 m inutes on a wrist-action shaker. 11.6 Centrifbge foe tubes at -3 0 0 0 rpm for - 5 m inutes. 11.7 D ecant th e supernatant into a SO m L d isp o sab le c e n trifu g e tu b e and add 35 m L o f water. 11.8 Condition foe C tt S P 6 cartridges (1 g , 6 m L) by passing 10 m L methanol followed b y $ m L o f HPLC w ater (~ 2 drop/sec). D o not let colum n run dry 11.9 Load foe sam ple on conditioned C SPB cartridge. D iscard eluate. 11.10 Elute w ith - 2 m L o f m ethanol. C ollect 2 m L o f eluate into a graduated 15 m L polypropylene centrifbge tube (final volum e - 2 m L). 11.11 Analyze sam ples using electrospray LC /M S/M S. 12.0 Chrom atography 12.1 12.2 12J 12.4 Inject the same o f each standard, sam ple and fortified sam ple into the LC /M S/M S system . A calibration stan d ard m u st p rec e d e and follow ell analyzed samples. Standards o f PFO A corresponding to at least five o r m ore concentration levels m ust be included in an analytical act. A n entire set o f calibration standards m ust be included at foe beginning and at foe end o f a sample set. Standards m ust be interspersed betw een every 5-10 sam ples. A s an alternative, an entire set o f calibration standards m ay be injected a t the beginning o f a set followed by calibration standards in terspers ed ev ery 5-10 sam ples (to account fo r a se co n d set o f standards). In either case, calibration standards m ust be the first and last injection in a sam ple set. Use linear standard curves for quantitation. L inear standard curves are g enerated for foe analyte b y lin ear reg ressio n u s in g 1/ x w eig h tin g o f p eak area Pais 5 o f7 Page 56 o f 65 Exygen Research Page 87 o f 119 Interim Report #26 - Analysis o f Groundwater Samples Exygen Study No.: P0000760 Exygen Protocol Number: P0000760 BxygnRMMrch Method NvuatoerV00017I3 I ANALYTICAL M E T H O D M ethod o f Analysis for the D eterm ination o f Perfiuorooctanoic A cid (PFO A ) in Small M amm al Liver b y LC/M S/M S venue calibration standard concentration using M aesLynx 3.3 (or equivalent) software system. 12.5 Sam ple response should not exceed standard responses. A ny sam ples that exceed standard responses should be Auther diluted and reanalyzed. 13.0 Acceptance Criteria 13.2 13.3 13.6 Chrom atogran m ust show a peak o f a daughter ion at 369 am u from a parent o f 413 amu. The 413 am u parent corresponds to foe PFO A anion, while the daughter ion (369 am u) represents the loss o f carbon dioxide. M ethod blanks m ust not contain PFO A at levels greater than the LOQ. If a blank contains PFO A at levels greater than 10 ng/g, then a new blank sample must be obtained and the entire set m ust be re-extracted. Recoveries o f control spikes and m atrix spikes m ust be betw een 70-130% o f their known values. I f a control spike foils outside the acceptable lim its, the entire set o f samples should be re-extracted. A ny m atrix spike outside 70 130% should b e evaluated b y d ie analyst to determ ine i f re-extraction is warranted. A ny calibration standard found to be a statistical outlier by using the Huge E rror Test, m ay be excluded from die calculation o f the calibration curve. However, die total num ber o f calibration standards that could be excluded m ust not exceed 20% o f the total num ber o f standards injected. The correlation coefficient (R ) for calibration curves generated m ust be 0.992 (R : 0.985). I f calibration results foil outside these lim its, then appropriate steps m ust b e taken to adjust instrum ent operation, and the standards or the relevant set o f samples should be reanalyzed. Retention times between standards and samples m ust not drift more than 4 % within an analytical run. If retention tim e drift exceeds this limit within in analytical run then the set m ust be reanalyzed. 14.0 Calculations 14.1 U se the follow ing equation to calculate th e am ount o f PF O A found (in ng/mL. baaed on peak area) using the standard curve (linear regression parameters) generated by die M ass Lynx software program : PFO A found (ng/mL) * fPeak area - intercept) x D F x aliquot factor slope D F factor b y which the final volum e w u diluted, if necessary. A liquot factor 10 Page 6 of 7 Page 57 o f 65 Exygen Research Page 88 o f 119 Interim Report #26 - Analysis o f Groundwater Samples Exygen Study No.: P0000760 Exygen Protocol Nuzriber: P0000760 Exy|en lUMareh Me*od Number V00017I5 | ANALYTICAL M ETH O D M etbod o f Analysis for the D eterm ination o f Perfiuorooctanoic A cid (PFO A ) in Small M ammal Liver by LC/M S/M S 14.2 For sam ples fortified w ith know n am ounts o f PFO A prior to extraction, use foe following equation to calculate foe percent recovery. Recovery (%) [ total analyte found (n^m L ) analyte found in control (ng/m L)] ^ analyte added (ng/m L) 14.3 U ae foe follow ing equation to convert foe am o u n t o f P F O A found in ng/m L to ngfg(ppb). PFO A found fpnbl - fPFOA found fna/m L l x final volum e fm Lll sample weight (g) Exygen Research Pag. 7 of 7 Page 58 o f 65 Page 89 o f 119 Interim Report #26 - Analysis o f Groundwater Samples Exygen Study No.: P0000760 Exygen Protocol Number: P0000760 ANALYTICAL METHOD M ethod Number: V0001786 M ethod o f A nalysis fo r th e D eterm ination o f F erflnorooctanolc A d d (PFO A ) la Sm all M am m al S eram by LC /M S/M S Analytical Teeting Facility: Exygen Research 3058 Research Drive State College, P A 16801 Approved By: __ C-- Paul Connolly I Technical Leader, LC*MS, Exygen Research D ale S l//7 ) / / J o b n F Ia h e rty / V Viice President, O perations, Exygen Research D ale Exygen Research Total Pegee: 7 Page 59 o f 65 Page 90 o f 119 Interim Report #26 - Analysis o f Groundwater Samples Exygen Study No.: P0000760 Exygen Protocol Number: P0000760 jliygM R w n d i Method Nunber V0001786 I ANALYTICAL M ETH O D M ethod o f Analysis for the Determ ination o f Perfluorooctanoic A cid (PFO A ) in Small M ammal Serum by LC/M S/M S 1.0 Scope This m ethod is to be employed for the isolation and quantitation o f perfluorooctanoic acid by High Performance Liquid Chrom atography coupled to a tandem M u s Spectromtrie D etector (LC/M S/M S) in amall m am m al serum . 2.0 Safety 2.1 Alw ays observe aafo laboratory practice. 2.2 Consult foe appropriate M SDS before handling any chem ical for proper safety 3.0 Sam ple Requirement 3.1 A t least 1 m L o f tes t sam ple fo r extraction. 3.2 N o sam ple processing is needed for serum sam ples. H ow ever, frozen serum sem ples m ust to allowed to com pletely thaw to room tem perature before use. 3.3 Sam ple collection procedures w ill be specified in die sam pling plan for this p ro je c t 4.0 Reagents and Standards 4.1 W a tc r-H P L C grade 42 M ethanol - HPLC grade 43 Acetonitrile - HPLC grade 4.4 Am m onium Acetate - A.C.S. R eagent Grade 4.5 Perfluorooctanoic A cid - Sigma*Aldrich 5.0 Instrument and Equipment 5.1 5.2 5.3 5.4 5.5 5.6 5.7 5.8 5.9 5.10 5.11 5.12 5.13 A high performance liquid chrom atograph capable o f pum ping up to 2 solvents equipped w ith a variable volum e injector cepable o f injecting 5-200 pL connected to a tandem M ass Spectrom eter (LC/M S/M S). A device to collect raw data for peak integration and quantitation. Analytical balance capable o f reading to 0.00001 g. 50 m L disposable polypropylene centrifuge tubes. 15 m L disposable polypropylene eentrifoge tubes. Diqaoeable m icropipets (SO-lOOuL, 100*200uL). 123>mL LDPE narrow -m outh bottles. 2 mL clear HPLC vial k it Disposable pipettes. Autopipettes (100*1000 pL and 10*100 pL ), w ith disposable tipi. W aters Sep Pak Vac 6 cc ( lg ) tC l 8 SP E cartridges. SPE vacuum manifold. Vortexer. Page 2 o f7 Page 60 o f 65 Exygen Research Page 91 o f 119 Interim Report #26 - Analysis o f Groundwater Samples Exygen Study No.: P0000760 E xy g en P ro to co l N um ber: P 00 0 0 7 6 0 Exygea Remrch Mtthod Number VOOQI786 I A1VALVT1CAL m e t h o d M ethod o f Analysis for the Determ ination o f Perfluorooctanoic A cid (PFO A ) in Small M ammal Seram by LC/M S/M S 5.14 W rist-action shaker. 5.15 Centrtftige capable o f spinning 15 m L polypropylene tubes at 3000 rpm. 6.0 Chromatographic System 6.1 A nalytical Column: Fluophase R P (K eystone Scientific), 2 .1 m m x 5 0 m m , 5p (P/N: 82505-052130) 6.2 Temperature: 30*C 6.3 M obile Phase (A ) : 2 noM Am m onium Acetate in W ater 6.4 M obile Phase (B ): M ethanol 6.5 Gradient Program: Time fminl 0.0 1.0 8.0 20.0 22.5 2U 65 6$ 25 25 65 Flow Rate (m L/m inl 35 0.3 35 0.3 75 0.3 75 0.3 3$ 0.3 6.6 Injection Volum e: 15 pL (c a n b e increased to as m uch as 50 pL). 6.7 Quantitation: Peak A re a -e x te rn a l standard calibration curve. 6.8 R un Time: - 23 minutes. The above conditions are intended as a guide and m ay be changed in order to optim ize the HPLC system. 7.0 M S/M S System 7.1 M ode: Electroepray N egative M R M m o d e, m o n ito rin g 413 --36 9 m /z for PFOA. T he above conditions are intended as a guide and m ay be changed in order to optim ize die M SM S system. 8.0 Preparation o f Solutions 8.1 M obile Phase 8.1.1 2 m M am m onium acetate in w a te r is p rep ared b y ad d in g 0.154 g o f ammonium acetate to 1000 m L o f water. Alternate volum es m ay b e prepared. p*g3on Page 6i o f 65 Exygen Research Page 92 o f 119 Interim Report #26 - Analysis o f Groundwater Samples Exygen Study No.: P0000760 Exygen Protocol Number: P0000760 Exygea RcMuch Method Number V0001786 I ANALYTICAL M ETH O D M ethod o f Analysis for the Determ ination ofPerfluorooctanoic A cid (PFO A ) in Small M amm al Sw um by LC/M S/M S 9.0 Standard Preparation 9.1 S tandard Stock/Fortificatioii Solution 9.1.1 Prepare a stock aolution o f ~ 100 pg/n>L o f PFO A by w eighing 10 mg o f analytical standard (corrected for purity) and dilute to 100 m L with m ethanol in a 125-mL LD PE bottle. 9.1.2 A 1.0 pg/m L fortification solution o f PFO A if prepared by bringing I m L o f th e 100 p g /m L solution to a final v o lu m e o f 100 w ith methanol in a 125 m L LDPE bottle. 9.1.3 A 0 .1 p g/m L fortification so lution o f P F O A is prepared b y bringing 10 m L o f th e 1.0 pg /m L so lution to a final vo lu m e o f 100 w ith methanol in a 125 m L LDPE bottle. 9.1.4 The stock and fortification solutions are to be stored in a refrigerator at approximately 4*C and are stable for a m axim um period o f 6 months from the date o f preparation. 9.2 Standard Calibration Solutions 9.2.1 9.2.2 LC/M S/M S calibration standards are prepared in m ethanol via dilution o f the 0.1 pg/m L fortification solution. The following is typical example: additional concentrations m ay be prepared aa needed. Concentration o f Fortification Solution fna/taL) Volume (mL) Diluted to (mL) Final Concentration (nx/mL) 100 5.0 100 100 2.0 100 100 1.0 too 5.0 2.0 1.0 5.0 10 100 0.5 2.0 10 100 0.2 1.0 10 100 0.1 9.2.3 Store all calibration standards in 125-mL LD PE narrow -m outh bottles at 2'C to 6*C, up to six m onths. 9.2.4 Alternate volumes and concentrations o f standards m ay be prepared as needed. 10.0 Batch Set Up 10.1 E ach batch o f sam ples extracted (typically 2 0 o r le u ) m ust include at least one untreated control and tw o untreated controls fortified at known concentrations (lab control spike) to verify procedural recovery for the batch 10.2 Requirem ent! for field and laboratory duplicates and spikes will be specified in the quality assurance plan for this project. Page 4 of 7 Page 62 o f 65 Exygen Research Page 93 o f 119 Interim Report # 2 6 - Analysis o f Groundwater Samples Exygen Study No.: P0000760 Exygen Protocol Number: P0000760 Exygen Reieich Method Number VOOOI786 ANALYTICAL M ETH O D M ethod o f A nalysis for the Determ ination o f Perfluorooctanoic A cid (PFO A ) in Small M ammal Serum b y LC/M S/M S 1 11.0 Sam ple Extraction ' tt.l 11.2 11.3 11.4 11.5 11.6 11.7 11.8 11.9 11.10 11.11 M easure 1 m L o f sam ple into a SO m L polyp ro p y len e c e n trifu g e tu b er (fortify as Deeded, replace lid and m ix w ell). N ote that alternate volum es o f serum m ay be measured depending on the sam ple size available for use. A dd water to the sample for a final volum e o f 20 m L. C ap tightly Vortex fo r-1 minute. T ransfer 1 m L o f th e sam ple u sin g d isp o sab le p ip ette into a 15 m L disposable ccntrifiige tube. Add 5 raL o f acetonitrile and ahake for - 2 0 m inutes on a wrist-action shaker. Cetftrifcge the tubes at -3 0 0 0 rpm for - 5 minutes. D ecant th e supernatant into a SO m L d isp o sab le centrifU ge tu b e and add 33 m L o f water. Condition the C SPE cartridges (1 g. 6 m L) b y passing 10 m L methanol followed b y 5 m L o f HPLC w ater ( - 2 drop/sec). D o not let colum n run dry Load the sample on conditioned C n SPE cartridge. D iscard eluate. Elute with - 2 m L o f m ethanol. C ollect 2 m L o f eluate into a graduated 15 m L polypropylene centriflige tube (final volum e 2 m L). Analyze samples using electrospny LC/M S/M S. 12.0 Chrom atography 12.1 Iqject the sam e am ount o f each standard, sam ple and fortified sam ple into the LC /M S/M S system . A calibration standard m u st precede and follow all analyzed samples. 12.2 Standards o f PFO A corresponding to at least five o r m ore concentration levels m ust be included in an analytical set. 12.3 A n entire set o f calibration standards m ust be included at the beginning and at the end o f sample s e t Standards m ust be interspersed between every 5-10 samples. A s an alternative, an entire set o f calibration standards m ay be injected at the beginning o f a set followed by calibration standards interspersed ev ery 5 -10 sam ples (to account fo r a seco n d s e t o f standards), (n 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 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. 12.5 Sam ple response should not exceed standard responses. A ny sim ples that exceed standard responses should be further diluted and reanalyzed. Page $ or 7 Page 63 o f 65 Exygen Research Page 94 o f 119 Interim Report #26 - Analysis o f Groundwater Samples Exygen Study No.: P0000760 Exygen Protocol Number: P0000760 BxygnRssauch Method Number VOOO1786 I ANALYTICAL M ETH O D M ethod o f Analysis for the Determ ination o f Perfluorooctanoic A cid (PFO A ) in Small M ammal Serum by LC/M S/M S 13.0 Acceptance Criteria 13.1 13.2 13.3 13.4 13.5 13.6 Chrom atogram m ust show a peak o f a daughter ion at 369 am u from a parent o f 413 amu. The 413 am u parent corresponds to the PFO A anion, w hile the daughter ion (369 amu) represent! die loss o f carbon dioxide. M ethod blanks m ust not contain PFOA at levels greater than the LOQ. If a blank contains PFO A at levels greater than 10 ng/m L, then a new blank sample m ust be obtained and die entire set m ust be re-extracted. Recoveries o f control spikes and m atrix spikes m ust be betw een 70-130% o f their known values. If a control spike falls outside the acceptable limits, the entire set o f samples should be re-extracted. A ny m atrix spike outside 70 130% should b e evaluated b y th e analyst to de te rm in e i f re-extraction is warranted. A ny calibration standard found to be a statistical outlier by using the Huge E rror Test, m ay be excluded from the calculation o f the calibration curve. However, die total num ber o f calibration standards that could be excluded m ust not exceed 20% o f the total num ber o f standards injected. The correlation coefficient (R ) for calibration curves generated m ust be 2 0 .9 9 2 (R* 20.985). I f calibration resu lts fall ou tsid e these lim its, then appropriate steps m ust be taken to adjust instrum ent operation, and the standards or die relevant set o f sam ples should be reanalyzed. Retention tim es between standards and sam ples m ust not drift m ore than 1 4 % within an analytical run. I f retention tim e drift exceeds this limit within an analytical run then the set m ust be reanalyzed. 14.0 Calculations 14.1 U se die follow ing equation to calcu late th e am o u n t o f P F O A found (in ng/m L. based on peak area) using the standard curve (linear regression parameters) generated by the M ass Lynx software program: PFOA found (ng/mL) - (Peak area - intercept! x D F x aliquot factor slope D F ffcctor b y w h ich the final volum e w a s diluted, i f necessary. Aliquot factor - 20 14.2 For sam ples fortified w ith known am ounts o f PFO A prior to extraction, use the following equation to calculate the percent recovery. Recovery (% ) [ total analyte found (ng/mL) - analyte found in control (ng/m L)] analyte added (ng/mL) Page 6 of7 Page 64 o f 65 Exygen Research Page 95 o f 119 Interim Report #26 - Analysis o f Groundwater Samples Exygen Study No.: P0000760 Exygen Protocol Number: P0000760 Exygen Remrch Method Number VOOO1786 I A N A lV nC A lT M ETHOD ... M ethod o f Analysis for the Determ ination o f Perfluorooctanoic A cid (PFO A ) in Small M ammal Serum by LC/M S/M S 14.3 U se the following equation to convert the am ount o f PFO A found in ng/m L to ppb. PFO A found (ppb) - [PFO A found fau/m L ) x fin] volum e (m L)l sample volum e (mL) Exygen Research Page 7 of ? Page 65 o f 65 Page 96 o f 119 Interim Report #26 - Analysis o f Groundwater Samples Exygen Study No.: P0000760 3058 Research Drive Phone: 814-272-1039 State College, PA 16801 Fax: 814-231-1580 PROTOCOL AMENDMENT Amendment Number_J__ Effective Date: 01/19/05 Exygen Study Number: P0000760_______ Client Study Number Page 1 of 1 DESCRIPTION OF AMENDED SECTION 1) Analytical Procedure Summary V0001780:Section 9.1 2) Verification of Analytical Procedure None AMENDED TO 1) Add to Section 9.1: Section 9.1.6, Alternate weights of standards may be used to prepare alternate concentrations of stock solutions as necessary. Alternate levels of fortification solutions may also be prepared. 2) Low and high spiking levels of the analytes for each matrix may be altered depending on sample size available for extraction and/or to cover analyte concentrations expected in the samples. RATIONALE 1) Higher concentrations of standards need to be prepared in order to spike the sample bottles at higher levels. 2) The sample size available for small mammal liver and serum was smaller than expected. Spiking at the pre-determined levels in the protocol puts the spiked ' concentration lower than the detection limit. Also, the analyte levels in the ground water samples are expected to greatly exceed the pre-determined spiking levels listed in the protocol. When the levels in the samples greatly exceed the spiking levels, an accurate recovery value cannot be calculated for the QC sample. Higher spiking levels In the bottles will cover the analyte concentrations expected in the water samples. IMPACT ON STUDY The LOQ is 100 ng/g for a 0.1 g sample of small mammal liver and is 1000 ng/mL for a 0.01 mL sample of small mammal serum. Higher levels of spiking for the water samples will ensure that more QC recovery data can be used. LIBRARY IO: W 0001226-6 Exygen QAU Review U iS n ilti/n r ADM INISTRATIVE FORM Exygen Research Page 97 o f 119 Interim Report #26 - Analysis o f Groundwater Samples Exygen Study No.: P0000760 E RESEARCH 3058 Research Drive Phone: 814-272-1039 State College, PA 16801 Fax: 814-231-1580 Am endm ent Number: Effective Date: Exygen Study Number PROTOCOL AMENDMENT 2 03/07/05 ' P0000760 Client Study Number: Page 1 of 1 None DESCRIPTION OF AMENDED SECTION Report, page 11 of 65 AMENDED TO Instead of one final report, interim reports will be issued. RATIONALE Due to the excessive sizes of the data sets, interim reports will be issued to allow the client to receive data in a timelier manor. e s jt*,lc s - IMPACT ON STUDY The client will be able to receive and review the data more quickly. Study DirectorSignature / al Pfitidpal Investigator Signatire 's k h Study Director Management Signature -------- Exygen Manageibepif)nature Sponsor Signa)tfre (if required) 3 /q I oB Dater z /v rf Date Date Date i/ta /o S Dat 1 Exvaen QAU Review L O S O il liS LIBRARY ID: V0001226-8 ADM INISTRATIVE FORM Exygen Research Page 98 o f 119 Interim Report #26 - Analysis o f Groundwater Samples Exygen Study No.: P0000760 06-02-2005 OS:11ai Froa-MESTON SOLUTIONS T-027 P.002/002 F-066 305 (testardi Drive Phono: 814-272-1030 State College, PA 16801 Fax: 814-231-1580 Amendment Number: Effective Darts: Exygen Study Number PROTOCOL. AMENDMENT 3 04/13/05 . P760 Client Study Number: Pao*1 * * 1 NA_ _ Analytical Procedure Summaiy: VOOOi780^Mettnd ofAnalysis for the Determination of Perfluoroooctanoic Add (PFOA) in Water by LG/MS/M5.' Sections 9.2 and 11.0 of tee method. amended to Section 0.2. Non-wdracted calibration standards may be prepared In order to quantitate samples directly injected onto the LCMSMS system. Section 11,0. Samples may be analysed without going through the extraction procedure. Instead, they may be diluted, if appropriate, and directly injected onto the LCMSMS system for analysis. ' ----- ---------- ------------- BB5SS1------------------- : " Some axtracted results between labomtoiy end field spflws were not In agreement end/br were unusable. Directly injecting tne extracts may add some Insight into the matrix fleets on the data results. ' im p a c t o n snJbV More usable data results may be obtained. U9RAKriD-V000122Sa RECEIVED Tire JUN. 2. 9:29011 PRINT TIME adm inistrative form JUN. 2. 9:300(1 Exygen Research Page 99 of 119 Interim Report #26 - Analysis of Groundwater Samples Exygen Study No.: P0000760 J U L .Z 5 .2 0 0 6 8:561*1 EXYGEN RESEARCH NO.7 7 4 P .4 3058 Research Drive Phone: 814-272-1039 State College, PA 16801 Fax; 814 231-1580 Amendment Number. Effective Date; Exygen Study Number PROTOCOL AMENDMENT 4. 07/18/05 P780 Client Study Number p*8 *1 11 NA DESCRIPTION OF AMENDED SECTiN~ Verification of Analytical Procedure, page 10 of protocol. AM iNBliUS ^ The field duplicate can be used-for the laboratory pikes and replicate when the primary sample volume la limited. . fVfflSMftlE " The sample size for a water sample Is 200 m L If a sample site requires re-extraction for any reason, there would not be enough of the primary sample to repeat two laboratory spices and a replicate. The field duplicate Is technically the same sample as the primary sample and therefore, can be used for laboratory spikes and replicates as nesded. IMPACT ON-STUDY No negative impact on the study. Using the duplicate sample allows for the tufi QC of the sample site to be completed. Study '/ /n //a _ PrjiJpa`iInvestigatorSlgnafcff k y / B osen M i ftto iiJ , d Sponioc S fcnaturyf rauirad) b a le Oats X -JU I-6 S Dais Exygen QAU Review ftn .n v LIBRARYID:V000122W ADMtNtSTRATVHFORM Exygen Research Page 100 o f 119 Interim Report #26 - Analysis of Groundwater Samples Exygen Study No.: P0000760 3058 Research Drive Phone: 814-272-1039 State College, PA 16801 Fax: 814-231-1580 Amendment Number: Effective Date: Exygen Study Number PROTOCOL AMENDMENT ______ 5______ 0 9 /1 3 /0 5 P760 Client Study N um ber Pa01 of 1 ______ N A DESCRIPTION OF AMENDED SECTION Analytical Procedure Summary: V0001780:"Method of Analysis for the Determination of Perfluoroooctanoic Add (PFOA) in Water by LC/MS/MS." AMENDED TO The reanalysis of two water samples, DF09-GW-131R-0-040126 and DFB-GW-135L-0040121 will be performed using the following procedure: An aliquot of the water sample is spiked with the surrogate standard and/or other matrix spike compounds as appropriate. Add methanol to the fortified aqueous sample. Mix and aliquot for LC/MS/MS analysis. The LC conditions indude the use of a Betasil C IS, 2.1 x 50 mm column, a gradient of 30% B to 100% B in 10 minutes, where A=2mM ammonium acetate in water and B*Methanol (6 min equilibration). Use a 5 pL injection volume and a 0.3 mL/min flow rate. LC conditions may be further modified as appropriate for the system being used. An interim report will be issued that will indude the exact details of the reanalysis. R A T IO N A LE DF09-GW-131R-0-040126 and DF8b-GW-135L-0-040121 could not be reported quantitatively in interim report #3 due to quality control sample failures. The study director and sponsor have requested reanalysis using the above conditions. IM P A C T O N S TU D Y No negative impact on the study. More usable data may be obtained. Study Director Slanature principal InvestigatorSignature A Study Director Maoag lent Signature Exygen Management Signature Sponsor Signature (if required) Date Date Date Date Date Exygen QAU Review ? // i/ o S ' LIBRARY ID: V0001226-6 ADM INISTRATIVE FORM Exygen Research Page 101 o f 119 Interim Report #26 - Analysis o f Groundwater Samples Exygen Study No.: P0000760 -. .,-5-2003 oe : 19 FROM:31 ENU. LOB 651 778 6176 n P .13.2005 l ! 5lPM EXYGEN RESEARCH TO:*08142311580 NO.294 P .2 P :2 '2 3038 Research Drive Phone: 814-272-1039 State College, PA 16801 Fax: 814-7.31-1580 Amendment Number Effective Date: Exygen Study Number PROTOCOL AMENDMENT 5 09/13/05 P760 Client Study Number Page 1 of 1 NA DESCRIPTION OF AMENDED SECTION Analytical Procedura Summary: V0001780;"Method of Analysis for the Determination of Perfluoroooctanofc Add (PFOA) In Water by LC/MS/MS.' AMENDED TO The reanalysis of two water samples, DF09-GW-131R-0-040128 and DFB-GW-135L-D040121 will be performed using the fblowing procedure: An aliquot of the water sample is spiked with the surrogate standard and/or other matrix spike compounds as appropriate. Add methanol to the fortified aqueous sample. MU and aliquot for LC/MS/MS analysis. The LC conditions indude the use of a Betasil C18, 2.1 x 50 mm column, a gradient of 30% B to 100% B in 10 minutes, where A*2mM ammonium acetate in water and B=Methanol (6 min equlibration). Use a 5 pL injection volume and a 0.3 m l/min flow rate. LC conditions may be further modified as appropriate for the system being used. An interim report will be issued that will include the exact details of tne reanalysis. ------------------------------------------------------------------------------ R a t i o n a l e ------------------------------------------------------------------------------ DF09-GW-131R-0-040126 and DF8b-GW-135L-0-040121 could not be reported quantitatively in interim report #3 due to quaRy control sample tenures. The study director and sponsor have requested reanalysis using the above conditions. IMPACT ON STUDY No negativa impact on the study. More usable data may be obtained. Study Oil IkK L Principal Invoatigalor Signature Study Director Management Signature * Evygan M anagam ant Signature Sponsor Signature (if required) Date Date Dale Dais Exygen QAU Review LIBRARY ID : V0O1226-8 RECEIVED TIME SEP.14. 10:37fiM ADM INISTRATIVe FORM PRINT TIME SEP.14. 10:381 Exygen Research Page 102 o f 119 Interim Report #26 - Analysis o f Groundwater Samples Exygen Study No.: P0000760 CHEM EHSR 236 IB ,E P .13 .8 0 0 5 1 ! 51PPI 651 733 1958 EXYtSEN RESEARCH 0 9 /1 4 '0 5 0 9 :2 7 NO.915 0 2 /0 2 IX T .& 9 r .* Exysen S30ta5t8eRCeosleleagrceh, MPrivl6o801 PhFonwec: 814-272*103* 814-231-1380 % 7 ^ I rtsea rch AEmffeecntidvme eDnattaN:umber. . Exygen Study Number PROTOCOL AMENDMENT 5 09P/1736/0 5 ClientStudyNumber Pagai et1 NA APanrafllyutoicraoloPorcotachedoluerAedSdum(PimsPfaOcrMAy:)dIVnt a0Wi0R0a1tge7Hr8bQayJ:'SMLOPejtMghoSadf.MoXfS5A'KnaSlyi sisterthe DetemlnsBon of aatva2a0AThp.pmno4nh1efl0pdKuema1rexmealor2xoilqpe1caSineqaoruOcinwiuumotaaamotdimtnlptetloeydmfobaftfaooauoetirarsihneclpLstotedoeha0lCosfwru.leffe/tme3MoawmseI~rtynnoeSmmesa,r/wtwMLpemeasdap/maamSatrgtmueeonalrrsbnrpapsniaenlrdslayi(nieagniallseodmyiigtmnsswesB.t.puisespbsls.rfeAMeafaosATdcdlt.3lseeMh.do0Dtd.ehw%EmARawLiNnnJneLiCBtWgDtohCihnlcEpaSttto(roDhenWcr3oreoaoTci1nmn-lfes01Otiuddfti08niorruio%1teernritqRpooehsuong:-Bei0Hrasnt-tfIhd0woebm4urnsit0lda1tirlfe1y0etbiine2otedmdbBnhiw)eeais.anqsudnufuUuudsteeereesndOtoashdo,uPtaeffhwsroBa5arsh-tmGaBoevwmtraWohleitldpeeti-Antr1sliien3je*me.c6d2'CclamLutMtila-dorM8Cbinesx,tdqDiuErae0nc5tt-oiCtraWatinv!-ed1ly3sp1IRonn-0Isn-ot0re4rhi0ma1v2er8erpeoaqrnutde#s3teDddFur5ReeKAatnTo3aIWOlqyN-us1iaAs3lL6iutKEysiWncgoMnthtr0eo1la2b1saomvcepoculoelndtdeintniuoorntess..beThreepsotrutdeyd Nonegative impacionthestudy. IMMoPrAeCuTs1aOblNe dSartiaibmraybeobtained. Study tX rw ro r S o n s tu m " PrhdM InvostbetorSisnabie Study Director Manajumor* Signature Data SST Ja k-- Exvobti QAU Review (7t>.w, U B R A R riD :V tX X M 2S M RECEIVED TIME SEP. 14. 10:43! AtTM M lSTIWTTVe PORM PRINT TIME SEP. 14. 10:441 Exygen Research Page 103 o f 119 Interim Report #26 - Analysis o f Groundwater Samples Exygen Study No.: P0000760 3058 Research Drive Phone: 814-272-1039 State College, PA 16801 Fax: 814-231-1580 Amendment Number Effective Date: Exygen Study Number PROTOCOL AMENDMENT 6 09/20/05 P760 Client Study Number: Pa98 1 f 1 NA DESCRIPTION OF AMENDED SECTION The identification of the two water samples listed in protocol amendment No 5 are incorrect. AMENDED TO The two water samples for reanalysis should be DF09-GW -131R-0-040120 and DF8D-GW-135R-0-040121. RATIONALE The correct samples are identified for reanalysis. IMPACT ON STUDY No negative impact on the study. Study Director Signature. y a //} ? / / - Y ^principal Investigator Siglature UA Study Director Management Signature ----------- Exygen MarggmegLS^nature Sponsor Signature (if required) Date Date Date 2 S -JQ P -0 S Dale Date Exygen QAU Review /Tv. '' ^ / x o l o ^ LIBRARY ID : V0001226-8 ADM INISTRATIVE FORM Exygen Research Page 104 o f 119 Interim Report #26 - Analysis of Groundwater Samples Exygen Study No.: P0000760 M fiY -l-2 0 0 3 06:37 FROM: 31 EhW. Lfl 651 770 6176 > SEP-20 -2005 2'29PM EXYGEN RESEARCH TO:*86107017401 NO.380 P :2 / 2 P.Z 3058 Research Drive Phon; 814-272-1039 State Cotte PA 16801 Fax: 514-231-1580 Amendment Number: Effective Date: Exygen Study Number PROTOCOL AMENDMENT 6 OS/ZO/OS~ P760 Client Study Number Pagai o fi NA PES *A M E N P E ft-S E C fjo fr The dentification of the two water samples listed in protocol amendment No S ere incorrect " AMENDED TO The two water samples for reanalysis should be DF0&-GW-131R-WM0120 and DF8b-GW-135R-0-040121. " ' ~ RATIONALE The correctsamples are Identified for reanalysis. im p a c t o n a r u p T No negative impact on the study. Study MSSbr Signatum Print** In v w tjjiio r Stntemi tL StIunddy nrraaccttoorrMl anagumemSignature Exygin MsragcmsntSignature Sponsor Stonature (if lequSredT" 3W E Dare Dato Dit Dite Exygen QAU Review f i * . * ' 9/xo(oS ` LIBRARY ID: V0OO1226-8 A D M N K T R A T M IFO R M Exygen Research Page 105 o f 119 Interim Report #26 - Analysis of Groundwater Samples Exygen Study No.: P0000760 CHEM EHSR 2 3 6 1 B 651 733 1958 0 9 /2 0 '0 5 1 3 :4 6 .N O .9 2 0 0 2 /0 2 S C P .Z 0 .2 0 0 S 229PC1 EXYGEN RESEARCH NO-360 P.2 E\ygSn RESEARCH 3058 Research Drive Phon: 814-272-1039 State College, PA 16801 Fax: B14-23MS80 Amendment Number Effective Date: Exygen Study Number PROTOCOLAMENDMENT 6 09/20/05 P760 Client Study Number fa 1 o f1 NA DESCRIPTION OFi w The IdermfioBon of the two water samples Retail in protocol amendment No S ere incorrect. The two water amples tor reanalysb should be DF09-GW-131R-0-OW12 and DF8b-QW-13SR-0>040121. The correctsamples ere identified torreanalysis. INTACTPMSTVBT No negative Impact on the study. StudyOlreeOrSismeni- Pimele kweMgebrSIginim- sMtSSSSrr !M m o g e n e n t S g i w a n E q sa n M m a p H n a n l H i (Ah A SponsorSii f i n n n r c(rrfrioqutneO ) Me l/hiS Data Exvoan Q A U w aview A < ^ 9 /* * lo ? LIBRARY IO: V D 0O 122S4 RECEIVED TIME SEP.20. 3:01PM AOWNISTRATTVE PORM PRINT TIME .SEP.20. 3:02PM Exygen Research Page 106 o f 119 Interim Report #26 - Analysis of Groundwater Samples Exygen Study No.: P0000760 1I-J3-20C5 M-.Um FraHUSTON SOLUTIONS NOV.2 2 .2 0 0 5 4 !57PM EXYGEN RESEARCH T-S7T P m /0 0 3 F-TI3 NO.914 r . e n RESEARCH 3058 ResearchDrive Phone: 14-272-1039 Stete College, PA 16801 Fav 814*231-1580 Amendment Number Effective Date: Exygen Study Number PROTOCOL AMENDMENT 3# Client Study Number: Paga l o f i NA DESCRIPTION OF AMENDED-SECTCN Analytical Procedure Summary. VW17W.,Meilifld ofAnalyse for the De lamini lion of PeffluoraeoctanolcAdd (PFOA) in Water by LC/MS/MS." Secon 11.0 ofthe method. Section 11.0. Samples may be A T iQ N A L e 7 If a 40 mL portion or sample wDI not load onto the Cu SPE cartridge, a pre-dilution can be prepared and extracted. - MPdfNsrosy No negative impacton the study. More usable data may be obtained so n a ta S p r n ia r 3 ta rw m ft tqulfa) 5S" Data Ms ~ Gtvnmnfla il Revtew /X -H V ilf m / o Z ' LIBRARYID;V000122S-E RECEIVED TIME NOV.23. 5=40PM ADMINISTRATIVEFORM PRINT TIME NOV.23. 5:42PM Exygen Research Page 107 o f 119 Interim Report #26- Analysis of Groundwater Samples Exygen Study No.: P0000760 CHEM EHSR 236 IB 651 733 1958 o s i l e r r o j r i i o n e r w . l k b 1 <(a o i r o 11/23 '05 14:11 NO.979 02/03 P * ..i'4 N O V .aa.20W 4:57PM EXYGEN RESEPRCH NO.914 p .2 n research 3058 Reieerch Drive pfwnt: 814*272-1039 S U CoUefe, PA18801 Fax: 814-231-1580 Amendment Number: Effective Date: Exyflfin Study Number PROTOCOL AMENDMENT 8 11/22/0$ P7TO cneot Study Number Page 1 of i NA DESCRIPTION OFaMeNOED SEtfllON Analytical Pnocadum Summary: VDOOlTBOfMaOiod ofAnemia fro Pitarmtnaflon of PaifluoroooctanoicAdd (PFOA) in WbU tby LC/MS/MS." Section 11.0 ofha method. ------------------------------- ~&MEMPebW----------------------------- Section 11.0. Sample may be diluted beforegoing throughthe euttiacBcn procedure. -------------------------------m m ik If a 40 mL portion of sample wfll not load onto the Cw SPE cartridge, a pre-dilution can be prepared and extracted. I No negative impacton trie study. More usable data my te obtained, Studybhectorforurti ess----------,M r S on* **-u i -e y use---------- aw,' 2rt>S B a *..... ........... ExvnenQAU Review 'tfa /o J Z t LIBRARY ttX V 000122B -S RECEIVED TIME NOV.23. 3:32PM . ADMINISTRATIVE PORM PRINT TIME NOV.23. 3:33PM Exygen Research Page 108 o f 119 Interim Report #26 - Analysis o f Groundwater Samples Exygen Study No.: P0000760 EHS OPNS ENVIRONMENTAL LAB . ff.651 778 2 6 07/11/06 11:46 0 :05/07 NO:681 RESEARCH 3058 Research Drive Phone:. 14-272-1039. State College, PA 16801 Fax: 814-23M580 Amendment Number. Effective Date: Exygen Study Number PROTOCOL AMENDMENT ; S. ;' : 6/2B/06 P760 Client Study.Number :... . ' ' NA PEMIPTON OP AMENDED 9 HEMKT-i Reference V00017B1: "Method of Analysis tor the Determination of PerfjuprobCtanolc Add (PFOA) in Sol by LC/MS/MS we* usedtonalyze aludjje aa well. . '. . ' ; . ftMENDEEBr The purpose of this study Is to perform analysis for perfluoropOtenoJc add (PFOA) in w*tor, soli, sediment, sludge, fish, clams, vegetation, smsl mammal livers, and small mammal serum using LC/MS/MS for the 3M Decatur Monitoring Program . . . . . . . : ~~ rationale"^ ^ ; ; ~ ' [ Sludge Is constituted of soil with si greater percentage of water. " '." ' ' b r a c to n a m e s Using a known method produced viable data applicable to this study. Exvban QU inttJDate td(L 1 iJiL, ' LIBRARY ID : V 0 0 0 1 2 2 M ' -" A ru w K iifrr T ivp i c n n u Exygen Research Page 109 o f 119 Interim Report #26 - Analysis of Groundwater Samples Exygen Study No.: P0000760 01-08-2007 03:5 Frw-KSTON SOLUTIONS + . T-021 P-003/005 f-053 3058 Research Drive Phone: 814-272-1039 I T .State College, PA 16801 Fa 814-23M580 RESEARCH Amendment Number Effective Date: Exygen Study Number PROTOCOL AMENDMENT 12 12/21106 P0000760 Client Study Number F a g e ia fi P0000760 'QEScagnON OF AMENPgb SECTION" JohnProtocol Dbtribulton Section: 2) M. Flaherty, Princfeal Investigator, BorgenResearch ! AMENDBITO 2} Charles Simons, Principal Investigator, Exygen Research RATIONALE John Flaherty retired and Chas Simons has taken over John's rale as Principal Investigatorforthe study. No negative Im p act IMPACTANffTVg" BMjyDMGrBignstx Rgnatm S r^ M g S iS n r < *8. m l 5 sta * *--------- ExygenOAUIniL/Dsts U & /o fle R lfT ^ j^ LIBRARY ;V 0 0 0 1 2 2 M RECEIVED TIME! JflN. 8. 4:02PM - ADMINISTRATIVE FORM PRINT TIME JPN. 8. 4:04PM Exygen Research Page 110 o f 119 Interim Report #26 - Analysis o f Groundwater Samples Exygen Study No.: P0000760 SEP. 20.2005 1:52PM EXYGEN RESEBRCH NO.377 P .2 3058 Research Drive Phone: 814-272-1039 State College, PA 16801 Fax: 814-231-1580 DEVIATION FORM General: X Project Specific Deviation ____Facility Deviation Date of Occurrence: 03/18/05 Exygen Project # : P760/P1131 Deviation # : 1/1 Client Prcfleot#: NA Reference#: 05-122 Regulatory Driver: ____ X ____ ____ 0MP GLP Other None Sample Description: Deviaflon Tuna: (Include W fo r m ethods and SOPs) _____ Protocol _____ M ethod X SOP Vfc 0001858-3 Notebook reference: NA ' Login#: ______ NA______ Container#: _______NA______L o t# :_________ NA Summary of Deviation: , This deviation pertains to all soil and sediment samples analyzed for percent soffde before 07/07/05. a. No blanks or duplicates were run as required bysection 9.3. b. Some sample weights exceed the allowable range (> 10g). Cause: ___ Preparation____ Analysis____ Instrument____ Client Request X Other There has bean no negative impact on the study. All ofthe percent solid values that were determined during the time period in question are considered valid, although the SOP was not fottowed. In the newly revised version of Vie SOP blanks and duplicates are no longer required. Also, in the new SOP, the alowable amount of sample to be used is < 20 g. All ofthe samples in question Inthis deviation weighed less than 20 g. The technician analyzing the samples for percent solids was following the new procedure before it was formallyapproved. Corrective Actions: A new version ofthe SOP has bean Issued and approved (V0000427-3). Signatures: . a / /n /z tA tf* Principal lmfistlgator / l/ j/ t s / /y j. 0 9 ` r Exygen Majfegepfont ASc/EM-dT Date ^ fc O A l Study DirlfotSr " ( f if e Sponsor Representative Data t V ^ - -v J *-- - t i l fa S Quality Assurance at N ft Sponsor Management Dal LIBRARY ID : V0001640-6 ADM INISTRATIVE FORM Exygen Research Page 111 o f 119 Interim Report #26 - Analysis o f Groundwater Samples Exygen Study No.: P0000760 SEP.20.2005 152PM EXYGEN RESEARCH NO.377 P .3 3058 Research Drive Phone: 814-272-1039 State College, PA 16801 Fax: 814-23MS80 DEVIATION FORM ____________________________ ______________________ ___________________Pane 1 of 1 G eneral: X Project Specific Deviation ___ Facility Deviation Date of Occurrence: 06/29/05 Exygen Project#: P760/P1131 Deviation#: 2/2 Client Project#: NA Reference#: Regulatory Driver: Deviation T w a: (Include V# fo r m ethods a nd SOPs) ____ X _____ ____ GMP GLP Obrer None Semole D escription: X Protocol ____ Method V # NA Notebook reference: NA "! ____ SOP ' Login#: L4264/L4256 Conteiner#: C0056480-86 Lot#: ________ NA________ Summfv of Deviation: " The protocol states that control and fortified control samples of each matrixwill be analyzed; however. LIBRARY ID : V000164Q-6 Exygen Research ADM INISTRATIVE FORM Page 112 o f 119 Interim Report #26 - Analysis of Groundwater Samples Exygen Study No.: P0000760 SEP. 30.2005 1.-52PM EXYGEN RESEARCH NO.377 P. 4 nr RESEARCH 358 Research Drive Phone: 814*272*1039 State College, PA 16801 Fax: 814-231*1580 General: X Project Specific Deviation DEVIATION FORM Facility Deviation P a o e lo fl Date of Occurrence: 12/27/04 Exygen Project # : P760/P1131 Deviation # : 3/3 Client Project#: NA Reference # : o S '- f ^ f Regulatory Driver: Deviation Tvne: (Indude V# fo r m ethods and SOPs) GMP GLP Other None Semole Description: _____ Protocol V#: 202-20 Section 5.2.3 Notebook reference: NA Method X SOP Login#: ______ NA Container# : NA Lot#: NA Summary of Deviation: SL2114 (30% Qlmethyldichlorosilane in Toluene) was given the expiration date of 02/13/05, but RE544 (Toluene) used to make SL2114 expired on 03/28/04. SL2114 was used to silanlzed glassware prepared for the fish extraction from 12/27/04 through 01/07/05. Cauta: Preparation Analysis Instrument Client Request X Other Impact: No negative impacton the study. Toluene was used onlyas a solvent for the glassware preparation. Dimethyldlchlorosilane, which is the coating agent, was not expired. Corrective Actions: Deviation Issued. S la n a tu ra ; s ______/Parimln(ciap9j|nvdestigatior / k Date OM ; Study Director (m m 'Dataf , S i __ -- Exygen ^anjgement l Sponsor Representative Quality Assurance Date Sponsor Management A>*rt M S Date Date Date LIBRARY ID: V0001640-6 ADMINISTRATIVE FORM Exygen Research Page 113 o f 119 Interim Report #26 - Analysis o f Groundwater Samples Exygen Study No.: P0000760 3058 Research Orive Phone: 814-272-1039 State College, PA 16801 Fax: 814-231-1580 DEVIATION FORM Ganaral: X Project Spedile Deviation ____FaeSty Deviation Pa> lp t2 Date of Occurrence: 04/26/06 Exygen Prelect#: P7601P1131 Deviation#: ____ 6 CIieri Project # : NA Reference#: 084)76 Regulatory Driver Deviation Type: (In clud e VU fo r m ethods e nd SO Ps) _____ X _____ _____ GMP GLP Other None Smele Description: X Protocol _____Method V#:NA Notebook reference: NA ____ SOP Login#: U0008191 Container#: NA L ot#: _________NA Summary o f Deviation: The three sediment samples in L8191 (C0172892 - C0172894) were originally extracted using the sediment method V0001782. Poor recoveries were obtained for PFOS, PFOA and UC PFOA Because of this, the study sponsor requested the use of an alternative extraction for these compounds, as follows: Direct Injection Method: Before the samples were weighed for the extraction, they were mixed thoroughly by vigorously shaking the container. Aone-gram portion of sediment was weighed into a 15-miHiKter centrifuge tube for the extraction. Ten milliliters of 1% acetic acid In methanol was added to each sample. The samples were then shaken by hand, vortexed, and sonicated for thirty minutes. The samples were then centrifuged for -10 minutes at -3000 rpm. Each sample was analyzed by LC/MS/M5 etectrospray. Using this method acceptable data was obtainedfor PFOS. but the recoveries for PFOA and '*C PFOA were still poor. Another alternative method was then used for PFOA and ,JC PFOA, as follows: Alternative SPE Method: The samples that were prepared in 1% acetic add for the direct injection method ware used for this extraction. Five mfliiiters of each sample was aliquoted into a 50-mL polypropylene centrifuge tube and the volume was taken to 40 mL with water. The samples were then centrifuged for -10 minutes at -3000 rpm. The supernatant was then loaded onto a C ,, SPE cartridge conditioned with 10 mL of methanol and 5 mL of water. The eluale was discarded. Approximately five milliters of methanol was added to the cartridge. Five milliters of eluate was collected into a graduated 1S mL polypropylene centrifuge tube. Each sample was analyzed by LC/MS/MS electrospray. Cause: ___ Preparation_____Analysis____ Instrument____ Client Request X Other Impact: More usable data was obtained. LIBRARYID: V0001B40-6 ADMINISTRATIVE FORM Exygen Research Page 114 o f 119 Interim Report #26 - Analysis of Groundwater Samples Exygen Study No.: P0000760 CHEH EHS 236 1B 651 733 1958 OMMM 01:11 FrnrtHTBl SOLUTIOHS 0+5/01 '06 13:46 NT04.H106F.OM03/O/O0I3 H U 1| l>. JOM ta N r tt Orfvt Phorir It+ a T flO T SMtColtofl, M U m stauch w w im v a M iM N * !' .< % '* *' > .. ' '" j->f ? f " ' l i . ? .- '. v . :i s' - ' 1 Exygen Research Page 115 o f 119 Interim Report #26 - Analysis o f Groundwater Samples Exygen Study No.: P0000760 EHS OPNS ENVIRONMENTAL LAB 651 778 4226 07/11/06 11:46 0:04/07 NO:681 RESEARCH 3Q58 Research Prive Phone: 814-272-1039. State College, PA 16801 Fax: 814-23M580 OBVIATION FORM onerai; '\ , X Project SpecificDeviation ' FeclltyDeviation liti. Date'ofOccurrence: 0*888)6 BtHjen Project#: P760/P1131 Dviation#: ' . fc "; . ClientProject# : Na - 'Rejitanoe#: ".'.'bfmO'. RlBUHtWY Prtyar GWP OLP Other None Dvistion fyoe: /Induri W fo r matheXI* and SOP) ____ ' Protocol ' X Method ' ___ ' :P '; V # V 00427-3 Notebook reference: NA h m r i f D M c la iU m Login#: NA Container:- C0015M 46 Lot#: JfiL SummaryofDevtetten: On# soNsample (C00158446) was weighed at ~Bgfor percent eoHdanalysis, retherthan et -20g whleh lastated In th method. ' ' " ' Causa: ___ Preparation____ Analysis____ Instrument _j___dient Request "X- Olhisr Imnacfr ; ... : . No negative Impact An accuratepercentsolidnumber was obtained b slightlyalteringth calculation used. . ' ' ' ... ' . '. Correttiva Actions; Deviation Issued. PflhcipeUbivestlgator ' / 'm i t o .______ SSJdyCCflrsctor . Dale 4k Oeta / Exygen(^ntgemeat Sppoonnssoorr RIapmaantatfve. ' ... Bat . l.Bal Dale Sponsor Management T5 T Exygen Research Page 116 o f 119 Interim Report #26 - Analysis o f Groundwater Samples Exygen Study No.: P0000760 EHS OPNS ENVIRONMENTAL LAB '651 778 4226 07/11/06 11:46 0 :06/07 N0:681 ^RESEARCH 3058 Remrch Drive Phone: 814-272-T039 State College, P16801 Fax: 814^31-1580 , > '0 V IA T lO N FORM isti X . Protect Specific Deviation __ _ Facility Deviation Data ofOccurrence: 66/28W6 EmmenProtect ; P7BQ/P1131 ; ' ' Deviation#: ' W -"-. ' Client Project # : NA . Referanoe# : RwuliWrr Priva r Deviation TVne: (Includa V# formethod* and SOPa) ____ X ____ . __. ' OMP SLP Other None _____ Protocol ____ _ Method VRtjM O Notebook reference: - X - Q F ' n ie P treteilen; loom#: NA Confelher#: NA Lot#:' JL innrirgfPtvHitlsn; 'aer review of raw data wee not documented per SOP V1800 prior ie 6/28X56. Caiiee; ___ Preparation____ ^ Adele__ _ Irxtrument___ Cllant Raquea! V" Other :____ :____ TZ.y-- ___ ' . ' . -TT _ ' Rewdate wll be more thoroughly evaluated and reviewed before Q Inepectlon. ' fiarm flyt Actlpni; A note to fBeWn be hiud to a i subiequent reporte i tetina that overall tummarlec have bben peer reviewed. SponsorManagement Exygen Research Page 117 o f 119 Interim Report #26 - Analysis of Groundwater Samples Exygen Study No.: P0000760 EHS OPNS ENVIRONMENTAL LAB '651 778 4226 07/11/06 11:46 0 :07/07 N0:681 n RESEARCH 3058 Research Drive Phone: 814^72-1039 State College, PA 16801 Fax: 814-231-1580 . .. DEVIATION Fffol P e n e re i: . . . ' . X Project Specific D e v ia tio n ___ Facility Deviatori Peteof Occurrenos: 06g8it. Exygen Project#: P7B/P1131 Oevlatlon#: M. Client Prcjact # : ______ NA Niflalarv Driver: pwtaHenVene: (Include v to r m ethodsa n d S O P tl ___^ . X... ___' QMP OLP Other None 8 awptiOeacrtetlof>: X -Protocol _____Method VA N A '. Notebook reference: __ _ SOP Login#: L0OO8121 L008051 Container#: .. P tflijlg n i ! Samples were Wad to 250 mL Inataad of 200 mL. C06R347 C0169354 C17188 : L u i# : NA Cause: X Preparation _^-AnjWyais Ihetrument ___ ClientRequest Impact: .: .: Sampiee could not be extracted according to the protocol. Other ttE S tta o x Spiking!evalewereadjustedtoaccommodate thealternativevolume. pal Invaetlgator / %khk. Study.Oli lu e ^ A se u rin c e -4 * 6 -? M JtiMk Date Sponsor Re^nentattve- ilL Sponsor Management -Dele Exygen Research Page 118 o f 119 Interim Report #26 - Analysis o f Groundwater Samples Exygen Study No.: P0000760 01-01-2007 03:51m Froi-KSTCM SOLUTIONS T-021 P .004/005 F-051 IT RESEARCH 3058 Research Orfvs , Mime: 814-272-1039 State College, PA 16801 Fax: 814-231-1580 DEVIATION FORM 1 of 1 X Prefect SpacllcDavMkm FacilityDeviation Dale of Occorrane* Nov. 2006Jan.gffl7. B w enPitjsct#: Cdant Project#: 000760 DevW oni: Rotarne#: ifl'OOS lBvB55tTCp!7 S S j* VftrmemoriandSOPri ** GMP GLP Other V#: V0001780 Notebook roference: X Method ____ SOP L o g in # : U010162 LOO101B3 Container# : la t# : Stockstandard andfsrtifieation standards uwd h dieextractiontertesa loginswarapraparod In acelonM Initaad ofmathanol. Coma: X Prop*raion___ Analysis____ Instrument____ COantRequest___ ,, Other Nonegativa impact. Compoundstre complexlysolubleinneetonftfe astheyare in methanol. ComaaBvaAction*" Davtstkm issued. B ra u a n tstn M a S C f No actiontaken. LIBRARY IO ; vo om 840-7 RECEIVED TIME JflN. B. 4:02PM AD M M SntATlVENO RM PRINT TIME JAN. 8. 4:04PM Exygen Research Page 119 o f 119