Document wq2YXzRxgJan7QB03rVwZd0Kd

INTERIM REPORT #27 - Analysis of Groundwater Samples STUDY TITLE Analysis of Perfluorobutanesulfonate (PFBS), Perfluorohexanesulfonate (PFHS), and Perfluorooctanesulfonate (PFOS) in Water, Soil, Sediment, Fish, Clams, Vegetation, Small Mammal Liver and Small Mammal Serum Using LC/MS/MS for the 3M Decatur Monitoring Program DATA REQUIREMENTS EPA TSCA Good Laboratory Practice Standards 40 CFR 792 STUDY DIRECTOR Jaisimha Kesari P.E., DEE Weston Solutions, Inc. 1400 Weston Way West Chester, PA 19380 Phone: 610-701-3761 INTERIM REPORT COMPLETION DATE February 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: P0001131 Exygen Study Number: P0001131 Total Pages: 119 Interim Report #27 - Analysis o f Groundwater Samples Exygen Study No. : P0001131 GOOD LABORATORY PRACTICE COMPLIANCE STATEMENT Exygen Study Number P0001131, entitled "Analysis of Perfluorobutanesulfonate (PFBS), Perfluorohexanesulfonate (PFHS), and Perfluorooctanesulfonate (PFOS) in Water, Soil, Sediment, Fish, Clams, Vegetation, Small Mammal Liver and Small Mammal Serum Using LC/MS/MS for the 3M Decatur Monitoring Program," conducted for 3M Company, is being performed in compliance with EPA TSCA Good Laboratory Practice Standards 40 CFR 792 by Exygen Research. Chi Prii x >--> Exygen Research f Jaisimha Kesari P.E., DEE Study Director Weston Solutions, Inc. Sponsor Representative 3M Company Exygen Research Date Page 2 o f 119 Interim Report #27 - Analysis o f Groundwater Samples Exygen Study No.: P0001131 QUALITY ASSURANCE STATEMENT Exygen Research's Quality Assurance Unit reviewed Exygen Study Number P0001131, entitled, "Analysis of Perfluorobutanesulfonate (PFBS), Perfluorohexanesulfonate (PFHS), and Perfluorooctanesulfonate (PFOS) in Water, Soil, Sediment, Fish, Clams, Vegetation, Small Mammal Liver and Small Mammal Serum Using LC/MS/MS for the 3M Decatur Monitoring Program". All reviewed phases1 were inspected for conduct according to Exygen Research's Standard Operating Procedures, the Study Protocol, and all applicable Good Laboratory Practice Standards. All findings were reported to the Exygen Principal Investigator and Management and to the Study Director. Phase 48.) Interim Analytical Report and Raw Data Draft review Date Inspected Date Reported to Date Reported to Principal Exygen Date Reported to Investigator Management Study Director 02/07-08/07 02/08/07 02/09/07 02/09/07 'Note: All in-lab inspections will be documented in the QA statement for the final analytical report at the conclusion of the study. This QA statement involves only the review of the interim report and associated raw data. Exygen Research Page 3 of 119 Interim Report #27 - Analysis o f Groundwater Samples Exygen Study No.: P0001131 CERTIFICATION OF AUTHENTICITY This interim report, for Exygen Study Number P0001131, is a true and complete representation of the raw data. Submitted by: Exygen Research 3058 Research Drive State College, PA 16801 (814)272-1039 Principal Investigator, Exygfcn: Chas Simons / j Operations Manager ] Exygen Research _______ -- k ./o 3 Date Exygen Research Facility Management: /f/^ J Ilf. G-k & m Richard A. Grazzini Executive Director Analytical Sciences Exygen Research Date Study Director/lWeston Solutions, Inc. Jaisimha Kesari P.E., DEE Weston Solutions, Inc. Sponsor Representative, 3M Company: Date Michael A. Santoro/ Director of Regulatory Affairs Exygen Research Page 4 of 119 Interim Report #27 - Analysis o f Groundwater Samples Exygen Study No.: P0001131 STUDY IDENTIFICATION Analysis of Perfluorobutanesulfonate (PFBS), Perfluorohexanesulfonate (PFHS), and Perfluorooctanesulfonate (PFOS) in Water, Soil, Sediment, Fish, Clams, Vegetation, Small Mammal Liver and Small Mammal Serum Using LC/MS/MS for the 3M Decatur Monitoring Program PROTOCOL NUMBER: P0001131 EXYGEN STUDY NUMBER: P0001131 TYPE OF STUDY: Residue SAMPLE MATRIX: Ground Water TEST SUBSTANCE: Perfluorobutanesulfonate (PFBS), Perfluorohexanesulfonate (PFHS), and Perfluorooctanesulfonate (PFOS) SPONSOR: 3M Company 3M Building 0236-01-B-10 St. Paul, MN 55144 STUDY DIRECTOR: Jaisimha Kesari P.E., DEE Weston Solutions, Inc. 1400 Weston Way West Chester, PA 19380 STUDY MONITOR: Michael A. Santoro 3M Company 3M Building 0236-01-B-10 St. Paul, MN 55144 PERFORMING LABORATORY: Exygen Research 3058 Research Drive State College, PA 16801 ANALYTICAL PHASE TIMETABLE: Study Initiation Date: 11/05/04 Interim Analytical Start Date: 12/28/06 Interim Analytical Termination Date: 01/16/07 Interim Report Completion Date: 02/09/07 Exygen Research Page 5 of 119 Interim Report #27 - Analysis o f Groundwater Samples Exygen Study No.: P0001131 PROJECT PERSONNEL The Study Director for this project is Jaisimha Kesari at Weston Solutions, Inc. The following personnel from Exygen Research were associated with various phases of this interim portion of the study: Name Chas Simons Karen Risha Mark Ammerman Eric Edwards Chrissy Edwards Krista Gallant Cammy Graybill Title Operations Manager Laboratory Supervisor Sample Custodian Sample Custodian Technician Technician Technician Exygen Research Page 6 o f 119 Interim Report #27 - Analysis o f Groundwater Samples Exygen Study N o.: P0001131 TABLE OF CONTENTS Page TITLE PAGE.......................................................................................................................1 GOOD LABORATORY PRACTICE COMPLIANCE STATEMENT............................. 2 QUALITY ASSURANCE STATEMENT......................................................................... 3 CERTIFICATION OF AUTHENTICITY.......................................................................... 4 STUDY IDENTIFICATION................................................................................................5 PROJECT PERSONNEL.....................................................................................................6 TABLE OF CONTENTS.....................................................................................................7 LIST OF 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...........................14 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 of 119 Interim Report #27 - Analysis o f Groundwater Samples Exygen Study N o.: P0001131 Table I. LIST OF TABLES Page Summary of PFBS, PFHS and PFOS in Groundwater Samples..................... 19 Table II. Matrix Spike Recovery of PFBS, PFHS and PFOS in Groundwater Samples.......................................................................................................... 20 Exygen Research Page 8 of 119 Interim Report #27 - Analysis o f Groundwater Samples Exygen Study No.: P0001131 Figure 1. LIST OF FIGURES Page Typical Extracted Calibration Curve for PFBS in Reagent W ater................23 Figure 2. Extracted Standards of PFBS in Reagent Water, 0 ng/L and 25 ng/L, Respectively....................................................................................................24 Figure 3. PFBS 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 PFBS (Exygen ID: C0221675, Data Set: 122906A)...................................... 26 Figure 5. Typical Extracted Calibration Curve for PFHS in Reagent Water................27 Figure 6. Extracted Standards of PFHS in Reagent Water, 0 ng/L and 25 ng/L, Respectively................................................................................................... 28 Figure 7. PFHS in Reagent Water, 50 ng/L Fortified Reagent Spk A, and 500 ng/L Fortified Reagent Spk B, Respectively.......................................... 29 Figure 8. Chromatogram Representing a Groundwater Sample Analyzed for PFHS (Exygen ID: C0221679, Data Set: 122906A)......................................30 Figure 9. Typical Extracted Calibration Curve for PFOS in Reagent Water............... 31 Figure 10. Extracted Standards of PFOS in Reagent Water, 0 ng/L and 25 ng/L, Respectively....................................................................................................32 Figure 11. PFOS in Reagent Water, 50 ng/L Fortified Reagent Spk A, and 500 ng/L Fortified Reagent Spk B, Respectively...........................................33 Figure 12. Chromatogram Representing a Groundwater Sample Analyzed for PFOS (Exygen ID: C0221658, Data Set: 122806A)......................................34 Exygen Research Page 9 of 119 Interim Report #27 - Analysis o f Groundwater Samples Exygen Study No.: P0001131 LIST OF APPENDICES Page Appendix A Study Protocol P0001131 (Exygen Study No. P0001131) with Analytical Method V0001780, Protocol Amendments, and Deviations................................................................................................. 35 Exygen Research Page 10 of 119 Interim Report #27 - Analysis o f Groundwater Samples Exygen Study No.: P0001131 1.0 SUMMARY Exygen Research extracted and analyzed groundwater samples for the determination of perfluorobutanesulfonate (PFBS), perfluorohexanesulfonate (PFHS), and perfluorooctanesulfonate (PFOS) according to Exygen Method V0001780 (Appendix A). The limit of quantitation (LOQ) for PFBS, PFHS and PFOS in ground water was 25 ng/L. Analytical results and assessed accuracies for the analysis of PFBS, PFHS and PFOS found in groundwater samples are summarized in Table I. Fortification recoveries for PFBS, PFHS and PFOS in the groundwater samples are detailed in Table II. The average percent recoveries standard deviations for PFBS, PFHS and PFOS in the groundwater samples were 103 19%, 99 13% and 118 24%, respectively. Quantitative results were obtained for all samples and analytes except for PFBS for all sample sites except for the trip blank, and one sample site for PFOS, which were not reported (NR) due to quality control failures. 2.0 OBJECTIVE The objective of the analytical part of this study was to determine levels of perfluorobutanesulfonate (PFBS), perfluorohexanesulfonate (PFHS), and perfluorooctanesulfonate (PFOS) in ground water according to Protocol P0001131 (Appendix A). 3.0 INTRODUCTION This report details the results of the analysis for the determination of PFBS, PFHS, and PFOS in 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 December 28, 2006, and the analytical termination date for this interim report was January 16, 2007. 4.0 ANALYTICAL TEST SAMPLES Thirty-five groundwater samples (Exygen ID C0221649 - C0221683) were received on wet ice on November 17, 2006 from Tim Frinak at Weston Solutions, Inc. Thirty-two groundwater samples represented eight groundwater sampling sites and associated field quality control samples. Three water samples represented one trip blank and two trip Exygen Research Page 11 of 119 Interim Report #27 - Analysis of Groundwater Samples Exygen Study No.: P0001131 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 standards, PFBS (SP0008058) and PFHS (SP0008057), were supplied by 3M. PFBS (SP0008058) was received from 3M at Exygen on September 6, 2006. PFHS (SP0008057) was received from 3M at Exygen on September 5, 2006. The analytical standard PFOS (SP0002694) was purchased from Fluka Corporation and was received at Exygen on April 23, 2003. The available information for the reference materials is listed below. PFBS and PFHS were stored frozen and PFOS was stored refrigerated. Compound Exygen Inventory No. Lot # Purity (%) PFBS SP0008058 2 97.3 PFHS SP0008057 NB-120067-69 98.6 PFOS SP0002694 430180-1 101.2 The molecular structures of PFBS, PFHS and PFOS are given below: Expiration Date 01/17/07 10/18/07 10/31/07 PFBS Chemical Name: Perfluorobutanesulfonate Molecular Weight: 338 supplied as the potassium salt (C4F9SO3IC ) Transitions Monitored: 299 -->99 Structure: FF FF F SO 3 FF FF PFHS Chemical Name: Perfluorohexanesulfonate Molecular Weight: 438 supplied as the potassium salt (C6F13SO3TC) Transitions Monitored: 399 -- 80 Structure: FF F F F F F SO3 FFF FFF Exygen Research Page 12 o f 119 Interim Report #27 - Analysis o f Groundwater Samples Exygen Study No.: P0001131 PFOS Chemical Name: Perfluorooctanesulfonate Molecular Weight: 538 supplied as the potassium salt (CgFivSCVK*) Transitions Monitored: 499 -> 80 Structure: FFFF FFFF F SO3 FF FFFF 6.0 DESCRIPTION OF ANALYTICAL METHOD The analytical method "V0001780: Method of Analysis for the Determination of Perfluorooctanoic Acid (PFOA) in Water by LC/MS/MS" was used for 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 Cig 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 mixed stock standard solution of PFBS, PFHS and PFOS was prepared as specified in the raw data. The stock standard solution was prepared at a concentration of 10,000 pg/mL by dissolving 1.0 g of each of the standards (corrected for purity and salt content, if necessary) in methanol. From these solutions, a 1000 pg/mL fortification standard solution was prepared by taking 10 mL of each 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 Exygen Research Page 13 of 119 Interim Report #27 - Analysis o f Groundwater Samples Exygen Study N o.: P0001131 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. A set of standards containing PFBS, PFHS and PFOS 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 (PL) 00 10 100 10 200 10 400 100 100 100 200 100 400 1of PFBS, PFHS and PFOS Volume of Fortified Sample (mL) 40 40 40 40 40 40 40 Final Cone, of Calibration Std. (ng/L) 0 25 50 100 250 500 1000 The stock standard solution and all fortification and calibration standard solutions were stored in a refrigerator (4 2C) when not in use. Documentation of standard preparation is located in the raw data package associated with this interim report. 6.3 Chromatography Quantification of PFBS, PFHS and PFOS was accomplished by LC/MS/MS electrospray. The retention times of PFBS, PFHS and PFOS were ~0.5 mins, -8.4 mins, and -11.1 mins, respectively. 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 PFBS, PFHS and PFOS. 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 Exygen Research Page 14 o f 119 Interim Report #27 - Analysis o f Groundwater Samples Exygen Study No.: P0001131 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 PFBS PFHS PFOS Mode negative negative negative Transition Monitored 299 99 399 80 499 80 Approximate Retention Time (min) -0.5 min. -8.4 min. - 11.1 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 seven concentrations of standards. The concentration was determined from the following equations. Equation 1 calculated the amount of analyte found (in ng/L, based on peak area) using the standard curve (linear regression parameters) generated by the Analyst software program. Equation 1: Analyte found (ng/L) = (Peak area - intercept! x DF slope Where: DF = Dilution Factor, factor by which the final volume was diluted, if necessary. Exygen Research Page 15 o f 119 Interim Report #27 - Analysis o f Groundwater Samples Exygen Study No.: P0001131 For samples fortified with known amounts of PFBS, PFHS and PFOS prior to extraction, Equation 2 was used to calculate the percent recovery. Equation 2: Recovery (%) = (analyte found (ng/L) - analyte in control (ng/L)) xl00% amount added (ng/L) An example of a calculation using an actual sample follows (calculation is for PFOS only): Ground water sample Exygen ID: C0221661 Spk C (Set: 122806C), fortified at 250 ng/L with PFOS where: peak area intercept slope dilution factor ng/L PFHS added (fort level) amt in corresponding sample = = = = = = 172149 628 448 1 250 90.5 (Set:122806C) From equation 1: Analyte found (ng/L) = IT72149 - 6281 x 1 448 = 383 ng/L From equation 2: % Recovery (383 ng/L - 90.5 ng/L) x 100% 250 ng/L 117% NOTE: Numbers may differ slightly from raw data due to rounding. 7.0 EXPERIMENTAL DESIGN For samples designated as field matrix spikes, PFBS, PFHS, and PFOS were added at a known concentration to the bottles in the laboratory before being shipped to the field. The samples were filled to a 200 mL volumetric fill line in the field, with the exception of two samples that were filled to a 100 mL volumetric fill line. Exygen Research Page 16 of 119 Interim Report #27 - Analysis o f Groundwater Samples Exygen Study No. : P0001131 The samples were extracted in three sets. Each set included one reagent blank and two reagent blanks fortified at known concentrations. Two sets contained three sample sites and one set contained two sample sites along with the field blank and the field blank spikes. 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 and assessed accuracies for the analysis of PFBS, PFHS and PFOS found in 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. Quantitative results were obtained for all samples and analytes except for PFBS for all sample sites except for the trip blank, and one sample site for PFOS, which were not reported (NR) due to quality control failures. Fortification recoveries for PFBS, PFHS and PFOS in the groundwater samples are detailed in Table II. The average percent recoveries standard deviations for PFBS, PFHS and PFOS in the groundwater samples were 103 19%, 99 13% and 118 24%, respectively. 9.0 CONCLUSIONS Except as noted above, the groundwater samples were successfully extracted and analyzed for PFBS, PFHS and PFOS according to analytical method V0001780. 10.0 RETENTION OF DATA AND SAMPLES All original paper data generated by Exygen Research that pertains to this interim report will be shipped to the 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 final analytical report and all original facility-specific raw data, will be retained in the Exygen Research archives for the period of time specified in EPA TSCA Good Laboratory Practice Standards 40 CFR 792. Exygen Research Page 17 of 119 Interim Report #27 - Analysis o f Groundwater Samples Exygen Study No.: P0001131 TABLES Exygen Research Page 18 of 119 Interim Report #27 - Analysis o f Groundwater Samples Exygen Study No.: P0001131 Table I. Summary of PFBS, PFHS and PFOS in Groundwater Samples Exygen ID C0221649 CO221649 Rep C0221650 C0221653 C0221653 Rep C0221654 C0221657 CO22 1 6 5 7 Rep C0221658 C0221661 C0221661 Rep C0221662 C0221665 C0221665 Rep C0221666 C0221669 C0221669 Rep C0221670 C0221673 C0221673 Rep C0221674 C0221677 C0221677 Rep C0221678 C0221681 C lie n t S am ple ID G W -327R -Q 4-Y06-C P -0 G W -327R -Q 4-Y 06-C P -0* G W -327R -Q 4-Y06-C P-D B G W -310R -Q 4-Y06-C P -0 G W -310R -Q 4-Y 06-C P -0* G W -310R -Q 4-Y06-C P-D B G W -317L-Q 4-Y06-C P-0 G W -317L-Q 4-Y06-C P-0* G W -317L-Q 4-Y06-C P-D B G W -320L-Q 4-Y06-C P-0 G W -320L-Q 4-Y 06-C P -0* G W -320L-Q 4-Y06-C P-D B G W -226R -Q 4-Y06-L F-0 G W -226R -Q 4-Y 06-L F-0* G W -226R -Q 4-Y06-LF-D B G W -226L-Q 4-Y06-LF-0 G W -226L-Q 4-Y06-LF-0* G W -226L-Q 4-Y06-LF-D B G W -220R -Q 4-Y06-L F-0 G W -220R -Q 4-Y 06-L F-0* G W -220R -Q 4-Y06-LF-D B G W -220L-Q 4-Y06-LF-0 G W -220L-Q 4-Y06-LF-0* G W -220L-Q 4-Y06-LF-D B G W -T R IP -Q 4-Y 06-L F -0 C4 Sulfonate PFBS C6 Sulfonate PFHS C8 Sulfonate PFOS Perfluorobutanesulfonate_______ Perfluorohexanesulfonate_______ Perfluorooctanesulfonate A n a ly te Found (ppb, ng/L) Assessed Accuracy (+/-% ) A n a ly te Found (ppb. ng/L) Assessed Accuracy (+/-% ) A n a ly te Found (ppb, ng/L) Assessed Accuracy < +/-% ) NR 105000 30 357000 50 NR - 88300 30 355000 50 NR - 104000 30 365000 50 NR _ 371000 30 733000 30 NR - 370000 30 744000 30 NR - 342000 30 630000 30 NR . 114 30 157 30 NR - 117 30 158 30 N R - 121 30 160 30 NR . 28.0 30 90.5 40 NR - 26.6 30 91.2 40 NR - ND 30 84.7 40 NR . 2460 30 13300 30 NR - 2560 30 13300 30 NR - 2660 30 16500 30 NR . ND 30 ND 50 NR - ND 30 ND 50 NR - ND 30 ND 50 NR . 38800 30 57500 50 NR - 41300 30 60600 50 NR - 46600 30 72600 50 NR . 53400 30 NR - NR - 55800 30 NR - NR - 60100 30 NR - ND 30 ND 30 ND 30 'Laboratory Duplicate ND = Not detected at or above the Limit of Quantitation (LOQ) of 25 ng/L. NR = Not reported due to quality control failures. Exygen Research Page 19 of 119 Interim Report #27 - Analysis o f Groundwater Samples Exygen Study No.: P0001131 Table II. Matrix Spike Recovery of PFBS, PFHS and PFOS in Groundwater Samples Sam ple D e s c rip tio n GW -327R-Q 4-Y06-CP-0 <00221649 Spk C, 250,000 ng/L Lab Spike) GW -327R-Q 4-Y06-CP-0 (C0221649 Spk P, 5,000,000 ng/L Lab Spike) GW -327R-Q4-Y06-CP-LS (C0221651, 250,000 ng/L Field Spike) GW -327R-Q4-Y06-CP-HS (C0221652, 5,000,000 ng/L Field Spike) GW -310R-Q4-Y06-CP-0 (C0221653 Spk E, 500,000 ng/L Lab Spike) GW -310R-Q4-Y06-CP-0 (C0221653 Spk F, 10,000,000 ng/L Lab Spike) GW -310R-Q4-Y06-CP-LS <00221655, 500,000 ng/L Field Spike) GW -310R-Q4-Y06-CP-HS (C0221656,10,000,000 ng/L Field Spike) GW -317L-Q4-Y06-CP-0 (00221657 Spk 6, 250 ng/L Lab Spike) GW -317L-Q4-Y06-CP-0 (C0221657 Spk H, 5000 ng/L Lab Spike) GW -317L-Q4-Y06-CP-LS (C0221659, 250 ng/L Field Spike) GW -317L-Q4-Y06-CP-HS (C0221660,5000 ng/L Field Spike) GW -320L-Q4-Y06-CP-0 {C0221661 Spk C, 250 ng/L Lab Spike) GW -320L-Q4-Y06-CP-0 (C0221661 Spk D, 5000 ng/L Lab Spike) GW -320L-Q4-Y06-CP-LS (C0221663, 250 ng/L Field Spike) GW -320L-Q4-Y06-CP-HS (C0221664,5000 ng/L Field Spike) GW -226R-Q4-Y06-LF-0 <00221665 Spk E, 5000 ng/L Lab Spike) GW -226R-Q4-Y06-LF-0 (C0221665 Spk F, 100,000 ng/L Lab Spike) GW -226R-Q4-Y06-LF-LS (C0221667, 5000 ng/L Field Spike) GW -226R-Q4-Y06-LF-HS (C0221666,100,000 ng/L Field Spike) Spiked (ng/L) 250000 5000000 250000 5000000 500000 10000000 500000 10000000 250 5000 250 5000 250 5000 250 5000 5000 100000 5000 100000 C 4 S u lfo n a te PFB S __________________ C 6 S u lfo n a te PFH S ___________________C 8 S u lfo n a te P F O S Amount in Sample (no/L) Amount Recovered (ng/L) Recovery (%i Amount in Sample (ng/L) Amount Recovered (ng/L) Recovery <%) Amount in Sample (ng/L) Amount Recovered (ng/L) Recovery (%> NR NR NR 105000 346000 96 357000 622000 106 NR NR NR 105000 5630000 111 357000 6720000 127 NR NR NR 105000 341000 94 357000 726000 148 NR NR NR 105000 6980000 138 357000 8150000 156 NR NR NR 371000 871000 100 733000 1170000 87 NR NR NR 371000 11100000 107 733000 13600000 129 NR NR NR 371000 856000 97 NR NR NR NR NR NR 371000 10300000 99 733000 13700000 130 NR NR NR 114 385 108 157 540 153 NR NR NR 114 4730 92 157 5130 99 NR NR NR 114 388 110 157 473 126 NR NR NR 114 4990 98 157 5440 106 NR NR NR 28.0 286 103 90.5 383 117 NR NR NR 28.0 4570 91 90.5 4710 92 NR NR NR 28.0 300 109 90.5 439 139 NR NR NR 28.0 3590 71 90.5 4280 84 NR NR NR 2460 7210 95 13300 21000 154 NR NR NR 2460 88000 86 13300 104000 91 NR NR NR 2460 8050 112 NR NR NR NR NR NR 2460 88500 86 13300 126000 113 NR ~ Not reported due to quality control failures. Note: Since this summary table shows rounded results, recovery values may vary slightly from the values in the raw data. Exygen Research Page 20 of 119 Interim Report #27 - Analysis o f Groundwater Samples Exygen Study N o.: P0001131 Table II. Matrix Spike Recovery of PFBS, PFHS and PFOS in Groundwater Samples (continued) S am ple D e scrip tio n GW -226L-Q4-Y06-LF-0 (00221669 Spk 0, 250 ng/L Lab Spike) GW -226L-Q4-Y06-LF-0 (C0221669 Spk H. 5000 ng/L Lab Spike) GW -2261-Q4-Y06-LF-LS (C0221671, 250 ng/L Field Spike) GW -226L-Q4-Y06-LF-HS (C0221672, 5000 ng/L Field Spike) GW -220R-Q4-Y06-LF-0 (C0221673 Spk C, 10,000 ng/L Lab Spike) GW -220R-Q4-Y06-LF-0 (C0221673 Spk D, 200,000 ng/L Lab Spike) GW -220R-Q4-Y06-LF-LS (C0221675,10,000 ng/L Field Spike) GW -220R-Q4-Y06-LF-HS (C0221676, 200,000 ng/L Field Spike) GW -220L-Q4-Y06-LF-0 (C0221677 Spk E. 25,000 ng/L Lab Spike) GW -220L-Q4-Y06-LF-0 (C0221677 Spk F, 500,000 ng/L Lab Spike) GW -220L-Q4-Y06-LF-LS (C0221679, 25,000 ng/L Field Spike) GW -220L-Q4-Y06-LF-HS (C0221660. 500,000 ng/L Field Spike) GW -TRIP-Q4-Y06-LF-LS (C0221682, 250 ng/L Field Spike) G W -TRIP-Q4-Y06-LF-HS (C0221683, 100,000 ng/L Field Spike) Amount S p ik e d ("9 C 4 S u lfo n a te PFB S __________________ C 6 S u lfo n a te PFH S ___________________C 8 S u lfo n a te P F O S Amount in Sample (ng/L) Amount Recovered (ng/L) Recovery <*> Amount in Sample (ng/L) Amount Recovered (ng/L) Recovery <*> Amount in Sample (ng/L) Amount Recovered <ng/L) Recovery <*> 250 5000 250 5000 10000 200000 10000 200000 25000 500000 25000 500000 250 100000 NR NR NR ND 250 100 ND 256 102 NR NR NR ND 4550 91 ND 4830 97 NR NR NR ND 315 126 ND 373 149 NR NR NR ND 4800 96 ND 5740 115 NR NR NR 38800 53800 , 57500 82400 . NR NR NR 38800 227000 94 57500 273000 108 NR NR NR 38800 50600 57500 78600 . NR NR NR 38800 247000 104 57500 357000 150 NR NR NR 53400 75000 86 NR NR NR NR 53400 469000 83 NR NR NR NR 53400 79500 104 NR NR NR NR 53400 585000 106 NR NR NR NR NR NR NR NR NR ND 292 117 ND 224 90 ND 89600 90 ND 83800 84 ND 233 93 ND 85100 85 Average: Standard Deviation: 103 19 Average: Standard Deviation: 'S am ple residue exceeds th e spiking level significantly (3x spiking level); therefore, an accurate recovery value cannot be calculated ND = Not detected at or above the Limit o f Quantitation (LOQ) o f2 5 ng/L. NR = Not reported due to quality control failures. Note: Since this summary table shows rounded results, recovery values may vary slightly from the values in the raw data. 99 13 Average: Standard Deviation: 118 24 Exygen Research Page 21 of 119 Interim Report #27 - Analysis o f Groundwater Samples Exygen Study No.: P0001131 FIGURES Exygen Research Page 22 o f 119 Interim Report #27 - Analysis o f Groundwater Samples Exygen Study No.: P0001131 Figure 1. Typical Extracted Calibration Curve for PFBS in Reagent Water 122905A P760-1131 Water, rdb (PF8S): "L in e a r Regression f'1 / x?' weighting): y * 50.2 x + 0.000337 (r * 0.9951) Area, counts Exygen Research Page 23 o f 119 Interim Report #27 - Analysis o f Groundwater Samples Exygen Study No.: P0001131 Figure 2. Extracted Standards of PFBS in Reagent Water, 0 ng/L and 25 ng/L, Respectively XC91M07-Q-PFBS (Standard) 299.W99.0amu -tampin 1 o f 39 from 122996C.wiff $>oa not found) XC010407-1 PFBS (S tandard)299.0/99.0 amu sample 2 of 38 from 122806C.witf Area: 4277 counts Height: 3.37e+002 cps RT: 0.540 min co Q. Exygen Research Page 24 o f 119 Intensity, cps Interim Report #27 - Analysis o f Groundwater Samples Exygen Study No.: P0001131 Figure 3. PFBS in Reagent Water, 50 ng/L Fortified Reagent Spk A, and 500 ng/L Fortified Reagent Spk B, Respectively Reagent Control PFBS (Unknown) 299.0/99.0<imir -sample 9 o f $4from 1229QSA.wiff freak not found) Reagent Spk A - PFBS (QC) 299.0/99.0 amu sample 10 of 3 4 from 122908A.wiff Area: 2281 counts Height: 1.67 e+002 cps RT: 0.533 min R e agentS pkB - PFBS (Q 0 )299.0/99.0 amu sample 11 of 3 4 from 122908A.wiff Area: 24319 counts Height: 1.79e+003 cps RT: 0.537 min 0.54 Time, min Intensity, cps Intensity, cps Exygen Research Page 25 o f 119 Interim Report #27 - Analysis o f Groundwater Samples Exygen Study No.: P0001131 Figure 4. Chromatogram Representing a Groundwater Sample Analyzed for PFBS (Exygen ID: C0221675, Data Set: 122906A) C0221675 PFBS (QC) 299.0/99.0 am u -sa m p le 19 o f 34 from 122906A.wiff A n a : 19259counts Height: 1.51e*003eps RT: 0.532min 0 .5 3 Intensity, cps Exygen Research Page 26 of 119 Interim Report #27 - Analysis o f Groundwater Samples Exygen Study No.: P0001131 Figure 5. Typical Extracted Calibration Curve for PFHS in Reagent Water 122906A P760-1131 Water.rdb (PFHS): "Lineai*1Recession ("1 /x " weighting): y * 157 x + -0.000643 (r 0.9989) Area, counts Exygen Research Page 27 o f 119 Interim Report #27 - Analysis of Groundwater Samples Exygen Study No.: P0001131 Figure 6. Extracted Standards of PFHS in Reagent Water, 0 ng/L and 25 ng/L, Respectively I XC010407-6-PFHS {Standard)399.0/90.0am u -su m p fe 1 o f 39 from 122S06C.wiff p ea k not found) 13.80 Intensity, cps XC010407-1 . PFHS (Standard) 399.0/80.0 mu - sample 2 of 38 from 122806C.wiff Area: 12632 counts Haight: 5.6'3e+002 cps RT: 3.41 min 8.41 Intensity, cps Exygen Research Page 28 o f 119 Interim Report #27 - Analysis o f Groundwater Samples Exygen Study No.: P0001131 Figure 7. PFHS in Reagent Water, 50 ng/L Fortified Reagent Spk A, and 500 ng/L Fortified Reagent Spk B, Respectively I R ea g en t Control PFHS (Unk n o w n )399.0/80.Oatiw -sa m p le 9 o f 39 from 122906C.wtff freak not found) a. tO I Reagent Spk A - PFHS (QC) 399.0/80.0 amu sample 10 of 38 fiom 122806 C.uuiff Area: 23087 counts Height: 9.74e+002 cps RT: 8.38 min Tim, min 8 .3 8 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 Time, min Reagent Spk B PFHS (QC) 399.0/80.0 amu sample 11 of 38 from 122806C.wiff Area: 256681 counts Height: 1,22e+004 cps R T :8.38 min Exygen Research Page 29 of 119 Interim Report #27 - Analysis o f Groundwater Samples Exygen Study No.: P0001131 Figure 8. Chromatogram Representing a Groundwater Sample Analyzed for PFHS (Exygen ID: C0221679, Data Set: 122906A) I C0221679*PFHS(QC}399.0/90.0am u -sa m p te 26 o f 34 from 122906A.wiff A ria : 124991 c o u n ts Heigtit: &6fte+0O3cp* RT: 9.39 milt Intensity, cps Exygen Research Page 30 of 119 Interim Report #27 - Analysis o f Groundwater Samples Exygen Study N o.: P0001131 Figure 9. Typical Extracted Calibration Curve for PFOS in Reagent Water 122800C P760-1131 Water.rdb (PFOS): "L in e a r R<jfession ("1 / * weighting): y = 448 x + 628 (i = 0.9993) Area, counts Exygen Research Page 31 o f 119 Interim Report #27 - Analysis o f Groundwater Samples Exygen Study N o.: P0001131 Figure 10. Extracted Standards of PFOS in Reagent Water, 0 ng/L and 25 ng/L, Respectively I XC 01M 07-0-PFOS (Standard) 499.W 0.Qamu -sam ptu 1 o f M from 122M6A.wiff $>*at n o t found) V) Gl I XC010407-1 - PFOS (S tandard)499.0/80.0 am u - sample 2 of 38 from 122800A.wiff Area: 9903 counts Height: 0.52e+002 cps RT: 11.0 min Tim e, min 11.04 Tim e, min Exygen Research Page 32 of 119 Interim Report #27 - Analysis o f Groundwater Samples Exygen Study No. : P0001131 Figure 11. PFOS in Reagent Water, 50 ng/L Fortified Reagent Spk A, and 500 ng/L Fortified Reagent Spk B, Respectively Reagent Control PFOS (Unknown) 499.0/B0.0 amu imp/ 9 o f 38from 122d6A.wiff freak not found) | Reagent Spk A - PFOS (QC) 499.0/80.0 amu - sample 10 of 38 from 122806A.wiff Area: 19338 counts Height: 1.25e+003 cps RT: 11.1 min p Reagent Spk B - PFOS (0 0 )4 9 9 .0 /8 0 .0 amu - sample 11 of 38 from 12280GA.wiff Area: 216880 counts Height: 1 ,46e+004 cps RT: 11.1 r Exygen Research Page 33 of 119 Interim Report #27 - Analysis o f Groundwater Samples Exygen Study N o.: P0001131 Figure 12. Chromatogram Representing a Groundwater Sample Analyzed for PFOS (Exygen ID: C0221658, Data Set: 122806A) I C0227658 'PFOS(Unknown) 499.W9&0 amu -sampie 3 3 o f 38 from 122d06A.wiff A n a : 68279 count* Height: 2.95e+003 cps RT: 11.1 min 11.07 Intensity, cps Exygen Research Page 34 o f 119 Interim Report #27 - Analysis o f Groundwater Samples Exygen Study N o.: P0001131 APPENDIX A Study Protocol P0001131 (Exygen Study No. P0001131) with Analytical Methods, Protocol Amendments, and Deviations Exygen Research Page 35 o f 119 Interim Report #27 - Analysis of Groundwater Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 STUDY PROTOCOL Study Title: Analysis of Perfluorobutanesulfonate (PFBS), Perfluorohexanesulfonate (PFHS), and Perfluorooctanesulfonate (PFOS) in Water, Soil, Sediment, Fish, Clams, Vegetation, Small Mammal Liver and Small Mammal Serum Using LC/MS/MS for the 3M Decatur Monitoring Program Exygen Protocol Number: P0001131 Perform ing Laboratory: Exygen R esearch 3058 R esearch D rive State C ollege, PA 16801 Phone: (814) 272-1039 Sponsor R epresentative: M ichael A. Santoro D irector o f R egulatory A ffairs 3M B u ild in g 0 2 3 6 -0 1 -B -10 St. Paul, M N 55144 Phone: (651)733-6374 Page I <>J65 Exygen Research Page 36 o f 119 Interim Report #27 - Analysis o f Groundwater Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 DISTRIBUTION: 1) Jaisim ha K esari, Study D irector, W eston Solutions 2) John M. Flaherty, Principal Investigator, Exygen Research 3) M ichael A. Santoro, Sponsor Representative, 3M Company 4) Exygen Research Quality Assurance Unit Exygen Research Page 2 o f 65 Page 37 of 119 Interim Report #27 - Analysis o f Groundwater Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 PROTOCOL APPROVAL Study T itle: Analysis o f Perfluorobutanesulfonate (PFBS), Perfluorohexanesulfonate (PFHS), and Perfluorooctanesulfonate (PFOS) in W ater, Soil, Sediment, Fish, Clam s, Vegetation, Sm all M am m al Livers and Sm all M am m al Serum U sing LC/M S/M S for the 3M Decatur M onitoring Program E xygen P rotocol N um ber: P 0 0 0 1 131 APPROVALS Jaisim ha W eston Solutions M ichael A. Santoro, Sponsor Representative 3M Compaify bhn M. Flaherty, rincipal Investigator Exygen Research Date Date ead, Quality Assurance Unit Page 3 oj 65 Exygen Research Page 38 of 119 Interim Report #27 - Analysis o f Groundwater Samples Exygen Study No.: P0001131 Exygen Protocol Number: POOO1131 TABLE OF CONTENTS TITLE PA G E .................................................................................................................................................................... 1 D IS T R IB U T IO N ...............................................................................................................................................................2 PROTOCOL APPROVAL..............................................................................................................................................3 TABLE OF CONTENTS................................................................................................................................................4 INTRODUCTION............................................................................................................................................................ 5 TEST M ATERIALS........................................................................................................................................................5 O B IEC TIV E..................................................................................................................................................................... 6 TESTING FACILITY..................................................................................................................................................... 6 STUDY DIRECTOR........................................................................................................................................................7 SPONSOR REPRESENTATIVE...................................................................................................................................7 PRINCIPAL INVESTIGATOR..................................................................................................................................... 7 PROPOSED EXPERIMENTAL START AND TERMINATION D A TE S........................................................... 7 IDENTIFICATION AND JUSTIFICATION OF THE TEST SY ST EM ............................................................... 8 SAMPLE PROCUREMENT. RECEIPT AND RETENTION................................................................................. 8 SAMPLE IDENTIFICATION....................................................................................................................................... 9 ANALYTICAL PROCEDURE SUMMARY..............................................................................................................9 VERIFICATION OF ANALYTICAL PROCEDURE................................................................................................9 METHOD FOR CONTROL OF B IA S......................................................................................................................... 11 STATISTICAL M ETH O D S...........................................................................................................................................11 GLP STATEM ENT......................................................................................................................................................... 11 REPO R T............................................................................................................................................................................ 11 SAFETY AND HEA LTH ............................................................................................................................................... 12 AMENDMENTS TO PROTO CO L...............................................................................................................................13 DATA RECORD K EE PIN G ..........................................................................................................................................13 QUALITY ASSURANCE.............................................................................................................................................. 14 RETENTION OF DATA AND ARCHIVING............................................................................................................ 14 APPENDIX I, ANALYTICAL M ETHODS.................................................................................................................15 Page 4 o f 65 Exygen Research Page 39 o f 119 Interim Report #27 - Analysis o f Groundwater Samples Exygen Study No.: P0001131 Exygen Protoco] Number: P0001131 INTRODUCTION The purpose o f this study is to perform analysis for perfluorobutanesulfonate (PFBS), perfluorohexanesulfonate (PFHS) and perfluorooctanesulfonate (PFOS) in water, soil, sediment, fish, clam s, vegetation, sm all m am m al livers and sm all mam m al serum using LC/M S/M S for the 3M D ecatur M onitoring Program . The study w ill be audited for com pliance with E PA TSC A G ood Laboratory Practice Standards 40 CFR 792 by the Quality A ssurance Unit o f Exygen Research. TEST MATERIALS The test m aterials are perfluorobutanesulfonate (PFBS), perfluorohexanesulfonate (PFHS) and perfluorooctanesulfonate (PFOS) and are all supplied by 3M. PFBS Chemical Name: Perfluorobutanesulfonate M olecular W eight: 338 supplied as the potassium salt (C iFSfV I ) L ot N um ber: 101 Purity: 96.7% Transitions M onitored: 299 -> 99 Structure: PFHS Chemical Name: Perfluorohexanesulfonate M olecular W eight: 438 supplied as the potassium salt (C iFuSC hK *) Lot Number: SE036 Purity: 98.6% T ransitions M onitored: 399 --> 80 Structure: Page 5 o f 65 Exygen Research Page 40 o f 119 Interim Report #27 - Analysis o f Groundwater Samples Exygen Study N o.: P0001131 Exygen Protocol Number: P0001131 PFOS Chemical Name: Perfluorooctanesulfonate M olecular W eight: 538 supplied as the potassium salt (CgFnSCVK") Lot Number: 217 Purity: 86.9% Transitions M onitored: 499 - 99 Structure: OBJECTIVE The purpose o f this study is to perform analysis for perfluorobutanesulfonate (PFBS), perfluorohexanesulfonate (PFHS) and perfluorooctanesulfonate (PFO S) in water, soil, sediment, fish, clam s, vegetation, sm all m am m al livers and sm all m am m al serum for the 3M D ecatur M onitoring Program using the current versions o f the following Exygen analytical methods: V0001780: V0001781: V0001782: V0001783: V0001784: V0001785: V0001786: "M ethod o f Analysis for the Determ ination o f Perfluorooctanoic Acid (PFOA) in W ater by LC/M S/M S" "M ethod o f Analysis for the Determ ination o f Perfluorooctanoic Acid (PFOA) in Soil by LC/M S/M S" "M ethod o f Analysis for the Determ ination o f Perfluorooctanoic Acid (PFOA) in Sedim ent by LC/M S/M S" "M ethod o f Analysis for the Determ ination o f Perfluorooctanoic Acid (PFOA) in Fish and Clams by LC/M S/M S" "M ethod o f Analysis for the Determ ination o f Perfluorooctanoic Acid (PFOA) in Vegetation by LC /M S/M S" "M ethod o f Analysis for the Determ ination o f Perfluorooctanoic Acid (PFOA) in Small M am mal Liver by LC/M S/M S" "M ethod o f Analysis for the Determ ination o f Perfluorooctanoic Acid (PFOA) in Small M am mal Serum by LC/M S/M S" TESTING FACILITY Exygen Research 3058 Research Drive State College, PA 16801 Phone: (814) 272-1039 Page 6 o f 65 Exygen Research Page 41 of 119 Interim Report #27 - Analysis o f Groundwater Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 STUDY DIRECTOR Jaisim ha Kesari P.E., DEE W eston Solutions, Inc. 1400 W eston W ay W est Chester, PA 19380 Phone: (610) 701-3761 Fax: (610) 701-7401 j .kesari@ w estonsolutions.com SPONSOR REPRESENTATIVE M ichael A. Santoro 3M Company Director o f Regulatory Affairs 3M Building 0236-01-B-10 St. Paul, M N 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 o f this study be conducted from O ctober 01, 2004 to D ecem ber 31, 2005. The actual experim ental start and term ination dates w ill be included in the final report. Page 7 o f 65 Exygen Research Page 42 of 119 Interim Report #27 - Analysis o f Groundwater Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 IDENTIFICATION AND JUSTIFICATION OF THE TEST SYSTEM The following are the test systems for this study: W ater (groundwater and surface water) Soil Sediment Fish Clams Vegetation Sm all M am mal Liver Small M am mal Serum The sam ples w ill be collected by W eston Solutions. The control sam ples will be purchased and prepared by the testing facility. Purchase and processing details for the control sam ples w ill be included in the final report associated w ith this study. The test systems were chosen to access the environm ental impact o f PFBS, PFHS and PFOS in the Decatur, A labam a area. SAMPLE PROCUREMENT, RECEIPT AND RETENTION W ater, soil, sediment, fish, clam, vegetation, sm all m am m al liver and small m am m al serum samples w ill be received at Exygen directly from W eston Solutions. The details o f sam ple procurem ent for this study are outlined in the 3M w ork plan entitled "Phase 2 W ork Plan for Sam pling Environm ental M edia." The num ber and types o f samples collected w ill vary depending availability in the field. The total num ber o f sam ples received and analyzed for each m atrix will be documented in the final report associated with this study. W ater, soil, and sedim ent sam ples w ill be used as received without further processing at Exygen. These samples will be stored refrigerated at 2C-8C. Fish, clam , vegetation and sm all m am m al liver sam ples w ill be processed according to the appropriate analytical m ethod (see A ppendix I). These sam ples w ill be stored frozen at < -10C. Sm all m am m al w hole blood samples will be centrifuged in the field at the tim e o f collection and the serum fraction w ill be used for the study. Sm all m am m al serum will be stored frozen at -10C. The receipt and processing o f the sam ples will be docum ented in the final report and raw data associated with the study. Page 8 o f 65 Exygen Research Page 43 of 119 Interim Report #27 - Analysis o f Groundwater Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 SAMPLE IDENTIFICATION Prior to analysis, each sam ple will be assigned a laboratory sam ple reference number. The reference num ber will be unique and will distinguish each laboratory sample that is processed throughout the analytical procedure. Chrom atographic data will be identified by the laboratory sam ple reference num ber. Sam ple storage conditions and locations w ill be docum ented throughout the study. ANALYTICAL PROCEDURE SUMMARY R eferences: V0001780: "M ethod o f Analysis for the Determ ination o f Perfluorooctanoic Acid (PFOA) in W ater by LC/M S/M S" V0001781: "M ethod o f Analysis for the Determ ination o f Perfluorooctanoic Acid (PFOA) in Soil by LC/M S/M S" V0001782: "M ethod o f Analysis for the Determ ination o f Perfluorooctanoic Acid (PFOA) in Sediment by LC/M S/M S" V0001783: "M ethod o f Analysis for the Determ ination o f Perfluorooctanoic A cid (PFOA) in Fish and Clam s b y LC/M S/M S" V0001784: "M ethod o f Analysis for the Determ ination o f Perfluorooctanoic A cid (PFOA) in Vegetation by LC /M S/M S" V0001785: "M ethod o f Analysis for the Determ ination o f Perfluorooctanoic A cid (PFOA) in Small M am mal Liver by LC/M S/M S" V0001786: "M ethod o f Analysis for the Determ ination o f Perfluorooctanoic A cid (PFOA) in Small M am mal Serum by LC /M S/M S" The above m ethods use analytical conditions capable o f separating the isom ers o f PFBS, PFHS and PFOS. The final report will include the isomers sum m ed into total PFBS, total PFHS, and total PFO S found. VERIFICATION OF ANALYTICAL PROCEDURE A laboratory control sam ple will be used for the preparation o f fortified control samples. The test substance w ill be m ade into solutions as per the m ethod, and added to the m atrices via a m icropipette. For w ater sampling, Exygen will supply one bottle per sam ple collected. The /**'- bottles w ill be 500 m L p recleaned Sci/S pec P re m ie r w id e m o u th H D P E bottles. These bottles have been routinely used for fluorochem ical sample Page 9 o f 65 Exygen Research Page 44 o f 119 Interim Report #27 - Analysis o f Groundwater Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 collection at the testing facility and have been show n to be free o f PFBS, P F H S and P F O S . Sam ples w ill b e ad ded to each co n tain er to a v olum etric fill line at 200 mL. A field duplicate, a low field spike and a high field spike o f each sam ple w ill be collected. T he low and high field spike bottles will contain PFBS, PFHS and PFOS as well as perfluorooctanoic acid (PFOA) and 1.2-13C perfluorooctanoic acid ( l3C PF O A ). P F O A and l3C P F O A are included in the solutions used to spike the sam ples. T he results for PFOA and l3C P F O A w ill not b e reported in th is study. E x y g en w ill su p p ly on e field blank (control water) and two field blank spikes (control w ater fortified with PFBS, PFHS and PFOS at a low and high level) for every tw enty samples collected. At the testing facility, each w ater sam ple (excluding field duplicates and field spikes) w ill be extracted in duplicate and w ill also be fortified at a low and high concentration with PFBS, PFH S and PFOS and processed through the described procedure to determ ine m ethod accuracy and to check for bias. For soil, sediment, clam s, and vegetation, Exygen will supply one 500 mL precleaned Sci/Spec Prem ier wide m outh HD PE bottle per sam ple collected or a zip-seal bag. All containers/bags used for sam ple collection will be shipped to the sam ple location. Sam ples will be added to each container or bag in the field. At the testing facility, each sam ple w ill be extracted in duplicate and will also be fortified at a know n concentration w ith PFBS, PFHS and PFOS at both a low and high level and processed through the described procedure to determ ine m ethod accuracy and to check for bias. For sm all m am m al liver, Exygen w ill supply a 50 m L polypropylene centrifuge tube. For sm all m am m al serum, Exygen w ill supply a collection kit for each sam ple 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 w ill be extracted in duplicate and will also be fortified at a know n concentration with PFBS, PFH S and PFOS at both a low and high level and processed through the described procedure to determ ine m ethod accuracy and to check for bias. Low and high spiking levels for each m atrix are defined below: M atrix Low Spiking Level H igh Spiking Level W ater 500 ng/L 5000 ng/L Soil 4 ng/g 40 ng/g S edim ent 4 ng/g 40 ng/g Fish 10 ng/g 100 ng/g C lam s 10 ng/g 100 ng/g V e g eta tio n 10 ng/g 100 ng/g Small M am mal Liver 10 ng/g 100 ng/g Small M am mal Serum 10 ng/m L 100 ng/mL Page 10 o f 65 Exygen Research Page 45 o f 119 Interim Report #27 - Analysis o f Groundwater Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 Recoveries are anticipated to be betw een 70% and 130% o f the fortified levels; however, the exact precision and accuracy w ill be determ ined by the analysis o f the quality control sam ples described above. A statem ent o f accuracy will be included in the final report. METHOD FOR CONTROL OF BIAS Control o f bias will be addressed by taking representative sub-sam ples from a hom ogeneous m ixture o f each m atrix from untreated control sam ples, and by analyzing at least two levels o f fortifications. STATISTICAL METHODS Statistics will be limited to those specified in the subject m ethods and to the calculation o f average recoveries, as applicable. GLP STATEMENT A ll aspects o f this study shall be perform ed and reported in com pliance with EPA TSC A G ood L aboratory Practice Standards 40 C FR 792. The final report or data package (supplied to the Sponsor) shall contain a statem ent that the study was conducted in com pliance with current and applicable GLP standards and w ill outline any deviations in the study from those standards. This statem ent will be signed by the Study Director and Sponsor Representative. REPORT A final report w ill be prepared by the principal investigator or their designee at the conclusion o f the study. The report w ill include, but w ill not be limited to, the following: The nam e and address o f the Study Director, Sponsor Representative, and o f the testing facility. A statem ent o f GLP com pliance (any related docum entation, such as chain-of-custody records, m ust be in the study records). Page 11 o f 65 Exygen Research Page 46 of 119 Interim Report #27 - Analysis of Groundwater Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 The signed and dated statem ent by the Exygen Research Quality A ssurance Unit regarding dates o f study inspections and dates findings were reported to the Study Director and M anagem ent. A description o f the exact analytical conditions em ployed in the study. If the subject m ethod was followed exactly, it is necessary to include only a copy o f the analytical method. A ny m odifications to this m ethod w ill be incorporated into the report. I f the m ethod is photo-reduced, the project num ber and page num ber must be included on each page. Description o f the instrumentation used and operating conditions. All results from all sets analyzed. Control and fortified sam ples will be identified and the data table will include sam ple num ber and fortification level. Representative chrom atogram s for each analyte in each m atrix, including chrom atogram s o f a standard and a control sam ple, and a chrom atogram at a fortification level. The location o f the analyte peaks w ill be clearly identified in all chromatograms. A ll circumstances that m ay have affected the quality or integrity o f the data w ill be documented in the report. Locations w here raw data and the final report are to be archived. A dditions or corrections to the final report shall be in the form o f an am endm ent signed by the Study Director. The am endm ent shall clearly identify that part o f the report that is being altered and the reasons for the alterations. The amendm ent w ill be signed and dated by the Study D irector and the Sponsor Representative. All applicable requirements for reporting o f study results as per 40 CFR 792.185. SAFETY AND HEALTH Laboratory personnel w ill practice good sanitation and health habits. Every reasonable precaution shall be taken to prevent inadvertent exposure o f personnel and the environm ent to the test or reference substance(s). Page 12 o f 65 Exygen Research Page 47 o f 119 Interim Report #27 - Analysis o f Groundwater Samples Exygen Study No. : P0001131 Exygen Protocol Number: POOO1131 AMENDMENTS TO PROTOCOL All significant changes to the analytical protocol outlined here will be expressed in writing, signed and dated by the Study Director and Sponsor Representative. Amendments usually will be issued prior to initiation o f study plan change. However, w hen a change is required w ithout sufficient tim e for the issue o f a written amendment, that change m ay be effected verbally with supporting documentation signed and dated by the Study Director and follow ed with a written am endm ent as soon as possible. In this case, the effective date o f the w ritten am endm ent w ill be the date o f the documented change. C opies o f the signed am endm ents w ill b e appended to all distributed study plan copies. T he original amendm ent will be m aintained with the original study plan. A ny deviations from the study plan or from the analytical m ethod as provided will be documented and reported prom ptly to the Sponsor R epresentative. DATA RECORD KEEPING Records to be m aintained include the following (as appropriate): Sam ple tracking sheet(s) Sam ple receipt records, storage history, and chains o f custody History and preparation o f standards (stock, fortification, calibration) Description o f any modifications to the m ethod Instrum ent run sheets, bench-sheets or logs Analytical data tables All chromatographic and instrumental conditions Sam ple extraction and analysis dates A com plete listing o f study personnel, signatures and initials Chronological presentation o f all study correspondence A ny other documentation necessary for the reconstruction o f the study Chromatograms- A ll chrom atogram s w ill contain the follow ing: Sam ple identification, injection date, arrow or other indication o f the area o f interest, and injection num ber corresponding to the run. Additionally, fortifications w ill include the am ount o f analyte added and the sam ple num ber o f the sam ple that w as fortified. A nalytical standard chrom atogram s will additionally include the concentration (e.g., pg/mL). Page 13 o f 65 Exygen Research Page 48 o f 119 Interim Report #27 - Analysis o f Groundwater Samples Exygen Study No. : P0001131 Exygen Protocol Number: P000H31 A s part o f the docum entation the follow ing sheets w ill be included in each analytical set: a run sheet listing the sam ples to be run in the set, and an instrument conditions sheet describing the instrum ent type and operating conditions. QUALITY ASSURANCE The QA Unit o f Exygen Research w ill inspect the study at intervals adequate to assu re com pliance w ith G L P 's, and w ill report the findings o f audits to the Study Director, Exygen M anagement, and the Sponsor Representative. RETENTION OF DATA AND ARCHIVING All hard copy raw data, including, but not lim ited to, the original chromatograms, worksheets, correspondence, and results shall be included with the data package subm itted to the Study Director. These will be archived w ith the original study plan, amendm ents, 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 o f tim e specified in 40 CFR 792.195. A n exact copy o f the m aterials subm itted to the study director will also be kept at Exygen Research. Exygen w ill obtain perm ission from the study director before discarding or returning samples. Exygen Research Page 4 o f 65 Page 49 of 119 Interim Report #27 - Analysis o f Groundwater Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 APPENDIX I ANALYTICAL METHODS V0001780: V0001781: V0001782: V0001783: V0001784: V0001785: V0001786: "M ethod o f A nalysis for the D eterm ination o f Perfluorooctanoic Acid (PFOA) in W ater by LC/M S/M S" "M ethod o f Analysis for the Determ ination o f Perfluorooctanoic Acid (PFOA) in Soil by LC/M S/M S" "M ethod o f Analysis for the D eterm ination o f Perfluorooctanoic A cid (PFO A ) in Sedim ent by L C /M S/M S" "M ethod o f Analysis for the Determ ination o f Perfluorooctanoic Acid (PFOA) in Fish and Clams by LC/M S/M S" `M e th o d o f A nalysis fo r the D eterm in atio n o f P erflu orooctanoic Acid (PFOA) in Vegetation by LC/M S/M S" "M ethod o f Analysis for the Determ ination o f Perfluorooctanoic Acid (PFOA) in Sm all M am mal Liver by LC/M S/M S" "M ethod o f Analysis for the Determ ination o f Perfluorooctanoic Acid (PFOA) in Sm all M am mal Serum b y L C /M S/M S" Exygen Research Page 15 o f 65 Page 50 of 119 Interim Report #27 - Analysis o f Groundwater Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 ANALYTICAL METHOD Method Number: V000J760 Method of Analysis for the Determination of Periloorooctanoic Acid (PFOA) in Water by LC/MS/MS Analytical Testing Facility: Exygen Research 3058 Research Drive State College, PA 16801 Approved By: "vLi ______ Paul Connolly ' Technical Leader, LC-MS, Exygen Research 2. John Flaherty / Vice President, Operations, Exygen Research Date Date Exygen Research Total Pages: 7 Page 16 o f 65 Page 51 o f 119 Interim Report #27 - Analysis o f Groundwater Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 Exygen Rcjearch Method Number VO0OP8O [ ANALYTICAL METHOD Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Water by LC/MS/MS 1.0 Scope This method is to be employed for the isolation and quantitation o f perfluorooctanoic acid by High Performance Liquid Chromatography coupled to a tandem Mass Spectrometric Detector (LC/MS/MS) in water. 2.0 Safety 2.1 Always observe safe laboratory practices. 2.2 Consult the appropriate MSDS before handling any chemical for proper safety precautions. 3.0 Sample Requirement 3. L At least 40 mL o f test sample for extraction. 3.2 No sample processing is needed for water samples. 3.3 Samples stored refrigerated should be allowed to equilibrate to room temperature. 3.4 All samples must be thoroughly mixed before being sampled for extraction 3.5 Any samples containing particles should be centrifuged at ~3000 rpm for -5 minutes and the supernatant used for the extraction. 3.6 Sample collection procedures will be specified in the sampling plan for this project. 4.0 Reagents and Standards 4.1 W ater-H P L C grade 4.2 Methanol - HPLC grade 4.3 Ammonium Acetate - A.C.S. Reagent Grade 4.4 Perfluorooctanoic Acid - Sigma-Aldrich 5.0 Instrument and Equipment 5.1 A high performance liquid chromatograph capable o f pumping up to 2 solvents equipped with a variable volume injector capable o f injecting 5-200 pL connected to a tandem Mass Spectrometer (LC/MS/MS). 5.2 A device to collect raw data for peak integration and quantitation. 5.3 Analytical balance capable o f reading to 0.00001 g. 5.4 50 mL disposable polypropylene centrifuge tubes. 5.5 15 mL disposable polypropylene centrifuge tubes. 5.6 Disposable micropipets (50-100uL, 100-200uL). 5.7 125-raL LDPE narrow-mouth bottles. 5.8 2 m L clear HPLC vial kit. 5.9 Disposable pipettes. 5.10 Autopipettes (100-1000 pL and 10-100 pL), with disposable tips. 5.11 Waters Sep Pak Vac 6 cc (lg ) tC l8 SPE cartridges. Page 2 of ? Page 7 o f 65 Exygen Research Page 52 o f 119 Interim Report #27 - Analysis o f Groundwater Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 Exygen Reward) Method Number V0001780 ANALYTICAL M ETHOD Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Water by LC/MS/MS 5.12 SPE vacuum manifold. 5.13 Centrifuge capable o f spinning 50 mL polypropylene tubes at 3000 rpm. 6.0 Chromatographic System 6 .1 Analytical Column: Fluophase RP (Keystone Scientific), 2.1 mm x 50 mm, 5p (P/N: 82505-052130) 6.2 Temperature: 30C 6.3 Mobile Phase (A) : 2 mM Ammonium Acetate in Water 6.4 Mobile Phase (B) : Methanol 6.5 Gradient Program: Time iminl 0.0 1.0 8.0 20.0 22.5 %A 65 65 25 25 65 Flow Rate m imL/minl 35 0.3 35 0.3 75 0.3 75 0.3 35 0.3 6.6 Injection Volume: 15 p L (can be increased to as m uch as 50 jiL). 6.7 Quantitation: Peak Area - external standard calibration curve. 6.8 RunTim e: - 2 3 minutes. The above conditions are intended as a guide and m ay b e changed in order to optimize the HPLC system. 7.0 MS/MS System 7.1 Mode: Electrospray Negative MRM mode, monitoring 413 -* 369 nvz. The above conditions are intended as a guide and may be changed m order to optimize the MSMS system. 8.0 Preparation o f Solutions 8.1 Mobile Phase 8.1.1 2 mM ammonium acetate in water is prepared by adding 0.154 g of ammonium acetate to 1000 mL o f water. Alternate volumes m ay be prepared. Page 3 ol ` Page /8 o f 65 Exygen Research Page 53 of 119 Interim Report #27 - Analysis o f Groundwater Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 Exygen Research Method Number VQQQ\ 780 ANALYTICAL METHOD Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Water by LC/MS/MS 9.0 Standard Preparation 9.1 Standard Stock/Fortification Solution 9.1.1 Prepare a stock solution o f -1 0 0 pg/mL o f PFOA by weighing 10 mg o f analytical standard (corrected for purity) and dilute to 100 mL with methanol in a 125-mL LDPE bottle. 9.1.2 A 10 pg/mL fortification solution o f PFOA is prepared by bringing 10 m L o f the 100 pg/mL solution to a final volume o f 100 with methanol in a 125 mL LDPE bottle. 9.1.3 A 1.0 pg/mL fortification solution o f PFOA is prepared by bringing 10 m L o fth c 10 pg/mL solution to a final volume o f 100 with methanol in a 125 mL LDPE bottle. 9.1.4 A 0.1 pg/mL fortification solution o f PFOA is prepared by bringing 10 m L o fth e 1.0 pg/raL solution to a final volume o f 100 with methanol in a 125 mL LDPE bottle. 9.1.5 A 0.01 pg/mL fortification solution o f PFOA is prepared by bringing 10 mL o f the 0.1 pg/mL solution to a final volume o f 100 with methanol in a 125 mL LDPE bottle. 9.1.6 The stock and fortification solutions are to be stored in a refrigerator at approximately 4C and are stable for a maximum period o f 6 months from the date o f preparation. 9.2 Standard Calibration Solutions 9.2.1 9.2.2 LC/MS/MS calibration standards are prepared in HPLC water. The calibration standards are processed through the extraction procedure, identical to samples. The following is a typical example: additional concentrations may be prepared as needed. Final Concentration Fortification V olum e o f Concentration o f Calibration o f Fortification Volume Fortified Control C a lib r a tio n Standard ID Solution (ppb) 0 (PL) .... Sample (m L) 0 40 Standard (ppt)* 0 (exam ple) X C m m ddyy-0 10 100 40 25 XCmmddyy-1 10 200 40 50 XCmmddyy-2 10 400 40 100 XCmmddyyO 100 100 40 250 XCmmddyv-4 100 200 40 500 XCmmddyy-5 100 400 40 1000 XCm m ddw-6 * The extracted concentration o f the calibration standard is equal to 8x its initial concentration, due to the concentration o f the standard during the extraction (SPE) XC - extracted calibration standard. Page A of ^ Page 19 o f 65 Exygen Research Page 54 of 119 Interim Report #27 - Analysis o f Groundwater Samples Exygen Study No.: P0001131 Exygen Protocol Number: POOO1131 Exygen Research Method Number V0001780 I ANALYTICAL METHOD ~ Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Water by LC/MS/MS 9.2.3 9.2.4 9.2.5 A zero standard solution (reagent blank) must be prepared with each set o f standards extracted. Store all extracted calibration standards in 15-mL polypropylene tubes at 2C to 6*C, up to two weeks. Alternate volumes and concentrations o f standards may be prepared as needed. 10.0 Batch Set Up 10.1 Each batch o f samples extracted (typically 20 or less) must include at least one reagent control (method blank using HPLC water) and two reagent controls fortified at known concentrations (lab control spike) to verily procedural recovery for the batch. 10.2 Requirements for field and laboratory duplicates and spikes will be specified in the quality assurance plan for this project. 11.0 Sample Extraction 11.1 Measure 40 mL o f sample or a portion o f sample diluted to 40 mL with water into 50 mL polypropylene centriftige tubes (fortify as needed, replace lid and mix well). 11.2 Condition the C n SPE cartridges (1 g, 6 mL) by passing 10 mL methanol followed by 5 m L o f HPLC water (*- 2 drop/sec). Do not let column run dry 11.3 Load sample on conditioned Ci %SPE cartridge. Discard eluate. 11.4 Elute with ~5 mL 100% methanol. Collect 5 mL o f eluate into graduated 15 m L polypropylene centriftige tubes (final volume " 5 mL). 11.5 Analyze samples using electrospray LC/MS/MS. 12.0 Chromatography 12.1 Inject the same amount o f each standard, sample and fortified sample into the LC/MS/MS system. A calibration standard must precede and follow all analyzed samples. 12.2 Standards o f PFOA corresponding to at least five or m ore concentration levels must be included in an analytical set. 12.3 An entire set o f extracted calibration standards must be included at the beginning and at the end o f a sample set. Extracted standards must he interspersed between every 5*10 samples. As an alternative, an entire set of extracted calibration standards may be injected at the beginning o f a set followed by extracted calibration standards interspersed every 5*10 samples (to account for a second set o f extracted standards). In either case, extracted calibration standards must be the 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 Pages of7 Page 20 o f 65 Exygen Research Page 55 o f 119 Interim Report #27 - Analysis o f Groundwater Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 Exyges Resetreh Method Number V0001780 ANALYTICAL METHOD____________________ Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Water by LC/MS/MS versus calibration standard concentration using M assLynx 3.3 (or equivalent) software system. 12.5 Sample response should not exceed standard responses. Any samples that exceed standard responses should be further diluted and reanalyzed. 13.0 Acceptance Criteria 13.1 Chromatogram must show a peak o f a daughter ion at 369 amu from a parent o f 413 amu. The 413 amu parent corresponds to the PFOA anion, while the daughter ion (369 amu) represents the loss o f carbon dioxide. 13.2 Method blanks must not contain PFOA at levels greater than the LOQ. If a blank contains PFOA at levels greater than 50 ng/L, then a new blank sample must be obtained and the entire set must be re-extracted. 13.3 Recoveries o f control spikes and matrix spikes must be between 70-130% o f their known values. I f a control spike falls outside the acceptable limits, the entire set o f samples should be re-extracted. Any matrix spike outside 70 130% should be evaluated by the analyst to determine if rc-extraction is warranted. 13.4 Any calibration standard found to be a statistical outlier by using the Huge Error Test, may be excluded from the calculation o f the calibration curve However, the total number o f extracted calibration standards that could be excluded must not exceed 20% o f the total number o f extracted standards injected. 13.5 The correlation coefficient (R) for calibration curves generated must be 0.992 (R2 0.985). If calibration results fall outside these limits, then appropriate steps must be taken to adjust instrument operation, and the standards or the relevant set o f samples should be reanalyzed. 13.6 Retention times between standards and samples must not drift more than 4 % within an analytical run. I f retention time drift exceeds this hmit within an analytical run then the set must be reanalyzed. 14.0 Calculations 14.1 Use the following equation to calculate the amount o f PFOA found (in ng/L. based on peak area) using the standard curve (linear regression parameters) generated by the Mass Lynx software program: PFOA found (ng/L) (Peak area - intercept) x DF slope DF * factor by which the final volume was diluted, if necessary. Page 6 of 7 Page 21 o f 65 Exygen Research Page 56 o f 119 Interim Report #27 - Analysis o f Groundwater Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 Exygea Research Method Number V0001780 | ..... ANALYTICAL METHOD Method o f Analysis for the Determination ofPerfluorooctanoic Acid (PFOA) in Water by L C /M S/M S 14.2 For samples fortified with known amounts o f PFOA prior to extraction, use the following equation to calculate the percent recovery. Recovery (%) [ total analyte found (ng/L) - analyte found in control (ng/L)] ^ ^ analyte added (ng/L) Exygen Research P ge70f7 Page 22 o f 65 Page 57 of 119 Interim Report #27 - Analysis o f Groundwater Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 ANALYTICAL METHOD Method Num ber V0001781 Method of Analysis for the Determination of Perfluorooctanoic Acid (PFOA) In Soil by LC/MS/MS Analytical Testing Facility: Exygen Research 3058 Research Drive State College, PA 16801 Approved By: c jL PPaaul Connollyv > Technical Leader, LC-MS, Exygen Research 7 //n d l/ ihn Flaherty Vice President, Operations, Exygen Research Date s*t Date Exygen Research Total Pages: 7 Page 23 o f 65 Page 58 o f 119 Interim Report #27 - Analysis o f Groundwater Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 Exygen Roearcfc Method Number V0Q01781 I ANALYTICAL M ETHOD Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Soil by LC/MS/MS 1.0 Scope This method is to be employed for the isolation and quantitation o f perfluorooctanoic acid by High Performance Liquid Chromatography coupled to a tandem Mass Spectrometric Detector (LC/MS/MS) in soil. 2.0 Safety 2.1 Always observe safe laboratory practices. 2.2 Consult the appropriate MSDS before handling any chemical for proper safety precautions. 3 .0 Sample Requirement 3.1 At least 15 g o f test sample for extraction. 3.2 No sample processing is needed for soil samples. 3.3 Samples stored refrigerated should be allowed to equilibrate to room temperature. 3.4 All samples must be thoroughly mixed before being sampled for extraction. 3.5 Sample collection procedures will be specified in the sampling plan for this project. Reagents and Standards 4.1 Water -H P L C grade 4.2 M ethanol-H PL C grade 4.3 Ammonium Acetate - A.C.S. Reagent Grade 4.4 Perfluorooctanoic Acid - Sigma*Aldrich 5.0 Instrument and Equipment 5.1 A high performance liquid chromatograph capable o f pumping up to 2 solvents equipped with a variable volume injector capable o f injecting 5-200 pL connected to a tandem Mass Spectrometer (LC/MS/MS). 5.2 A device to collect raw data for peak integration and quantitation. 5.3 Analytical balance capable ofreading to 0.00001 g. 5.4 50 m L disposable polypropylene centrifuge tubes. 5.5 15 mL disposable polypropylene centrifuge tubes. 5.6 Disposable micropipets (50-100uL, 10Q-200uL). 5.7 125-mL LDPE nairow-mouth bottles. 5.8 2 m L clear HPLC vial kit. 5.9 Disposable pipettes. 5.10 Autopipettes (100-1000 pL and 10-100 pL), with disposable tips. 5.11 Waters Sep Pak Vac 6 cc (lg ) tC18 SPE cartridges. 5.12 SPE vacuum manifold. 5.13 Ultrasonic bath. ">Pa$e 2 of Page 24 o f 65 Exygen Research Page 59 o f 119 Interim Report #27 - Analysis o f Groundwater Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 Exygen Reeetrcb Method Number V 0001781 I ANALYTICAL M ETHOD Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Soil by LC/MS/MS 5.14 Wrist-action shaker. 5.15 Centrifuge capable o f spinning 50 mL polypropylene tubes at 5000 rpm. 6.0 Chromatographic System 6.1 Analytical Column: FluophaseRP (Keystone Scientific), 2,1 mm x 50 mm. 5^ (P/N: 82505-052130) 6.2 Temperature. 30C 6.3 Mobile Phase (A) : 2 mM Ammonium Acetate in Water 6.4 Mobile Phase (B) : Methanol 6.5 Gradient Program: Time fminl 0.0 1.0 8.0 20.0 22.5 %A 65 65 25 25 65 Flow Rate % B (mL/min) 35 0.3 35 0.3 75 0.3 75 0.3 35 0.3 6.6 Injection Volume: 15 pL (can be increased to as m uch as 50 pL). 6.7 Quantitation: Peak Area - external standard calibration curve. 6.8 RunTime: - 2 3 minutes. The above conditions axe intended as a guide and may be changed in order to optimize the HPLC system. 7.0 MS/MS System 7.1 Mode: Electrospray Negative MRM mode, monitoring 4 1 3 - 369 m/'z for PFOA. The above conditions are intended as a guide and m ay be changed in order to optimize the MSMS system. 8.0 Preparation o f Solutions 8.1 Mobile Phase 8.1.1 2 mM ammonium acetate in water is prepared by adding 0.154 g of ammonium acetate to 1000 m L o f water. Alternate volumes may be prepared. Page 3 o f 7 Page 25 o f 65 Exygen Research Page 60 of 119 Interim Report #27 - Analysis o f Groundwater Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 ExygenResearch Method Number VOGO1781 ANALYTICAL METHOD Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Soil by LC/MS/MS 9.0 Standard Preparation 9.1 Standard Stock/Fortification Solution 9.1.1 Prepare a stock solution o f -1 0 0 pg/raL o f PFOA by weighing 10 mg o f analytical standard (corrected for purity) and dilute to 100 mL with methanol in a 125-mL LDPE bottle. 9.1.2 A 10 pg/mL fortification solution o f PFOA is prepared by bringing 10 mL o f the 100 pg/mL solution to a final volume o f 100 with methanol in a 125 mL LDPE bottle. 9.1.3 A 1.0 pg/mL fortification solution o f PFOA is prepared by bringing 10 mL o f the 10 pg/mL solution to a final volume o f 100 with methanol in a 125 mL LDPE bottle. 9.1.4 A 0.1 pg/mL fortification solution o f PFOA is prepared by bringing 10 mL o f (he 1.0 pg/mL solution to a final volume o f 100 with methanol in a 125 mL LDPE bottle. 9.1.5 A 0.01 pg/mL fortification solution o f PFOA is prepared by bringing 10 m L o f die 0.1 pg/mL solution to a final volume o f 100 with methanol in a 125 mL LDPE bottle. 9.1.6 The stock and fortification solutions are to be stored in a refrigerator at approximately 4C and are stable for a maximum period o f 6 months from the date o f preparation. 9.2 Standard Calibration Solutions 9.2.1 9.2.2 LC/MS/MS calibration standards are prepared in HPLC water The calibration standards are processed through the extraction procedure, identical to samples. The following is a typical example: additional concentrations may be prepared as needed. Final Concentration Fortification Volume o f Concentration o f C a li b r a tio n o f Fortification Volume Fortified Control Calibration Standard ID Solution DDb) (PL) Samole (mL) Standard Coot)* (exam p le) 0 0 40 0 XCmmddyy-0 10 100 40 25 XC m m ddyy-1 10 200 40 50 XCmmddyy-2 10 400 40 100 XCmmddyy-3 100 100 40 250 XCmmddyy-4 100 200 40 500 XCmmddyy-5 100 400 40 1000 XCm m ddw-6 * The extracted concentration o f the calibration standard is equal to 8x its initial concentration, due to the concentration o f the standard during the extraction (SPE). XC * extracted calibration standard. Page 4 o f7 Page 26 o f 65 Exygen Research Page 61 o f 119 Interim Report #27 - Analysis o f Groundwater Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 Exygen Resetreh Method Number V0001781 ___________________ ANALYTICAL METHOD____________________ Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Soil by LC/MS/MS 9.2.3 9.2.4 9.2.5 A zero standard solution (reagent blank) must be prepared with each set o f standards extracted. Store all attracted calibration standards in 15-mi, polypropylene lubes at 2C to 6C, up to two weeks. Alternate volumes and concentrations o f standards may be prepared as needed. 10.0 Batch Set Up 10.1 Each batch o f samples extracted (typically 20 o r less) must include at least one reagent control (method blank using 5 mL o f methanol) and two reagent controls fortified at known concentrations (lab control spike) to verify procedural recovery for the batch. 10.2 Requirements for field and laboratory duplicates and spikes will be specified in the quality assurance plan for this project. 11.0 Sample Extraction 11.1 Weigh 5 g o f sample into 50 m L polypropylene centrifuge tubes (fortify as needed, replace lid and mix well). 11.2 Add 5 mL o f methanol and shake on a wrist action shaker for -1 5 minutes 11.3 Transfer the tubes to an ultrasonic bath and sonicate f o r -1 5 minutes. 11.4 Bring the volume up to 40 mL with water in the 50 mL polypropylene centrifuge tube. 11.5 Centrifuge for -1 0 minutes at -3 0 0 0 rpm. 11.6 Condition the C|g SPE cartridges (1 g, 6 mL) by passing 10 mL methanol followed by 5 mL o f HPLC water ( - 2 drop/sec). Do not let column run dry 11.7 Load (decant) the sample on the conditioned C n SPE cartridge. Discard eluate. Elute with - 5 mL 100% methanol. Collect 5 mL o f eluate into graduated IS mL polypropylene centrifuge tubes (final volume " 5 mL). 11.9 Analyze samples using electrospray LC/MS/MS. 12.0 Chromatography 12.1 Inject the same amount o f each standard, sample and fortified sample into the LC/MS/MS system. A calibration standard must precede and follow all analyzed samples. 12.2 Standards o f PFOA corresponding to at least five or more concentration levels must be included in an analytical set. 12.3 An entire set o f extracted calibration standards must be included at the beginning and at the end o f a sample set. Extracted standards must be interspersed between every 5*10 samples. As an alternative, an entire set of Page S o f 7 Page 27 o f 65 Exygen Research Page 62 o f 119 Interim Report #27 - Analysis o f Groundwater Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 Exygeo Research________________________________________________ Method Number V0001781 I ANALYTICAL METHOD Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Soil by LC/MS/MS extracted calibration standards may be injected at the beginning o f a set followed by extracted calibration standards interspersed every 5-10 samples (to account for a second set o f extracted standards). In either case, extracted calibration standards must be the 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 MassLynx 3 3 (or equivalent) software system. 12.5 Sample response should not exceed standard responses. Any samples that exceed standard responses should be further diluted and reanalyzed. 13.0 Acceptance Criteria 13.1 Chromatogram must show a peak o f a daughter ion at 369 amu from a parent o f 413 amu. The 413 amu parent corresponds to the PFOA anion, while the daughter ion (369 amu) represents the loss o f carbon dioxide. 13.2 Method blanks must not contain PFOA at levels greater than the LOQ. If a blank contains PFOA at levels greater than 50 ng/L, then a new blank sample must be obtained and the entire set must be re-extracted. 13.3 Recoveries o f control spikes and matrix spikes must be between 70-130% of their known values. If a control spike falls outside the acceptable limits, the entire set o f samples should be re-extracted. Any matrix spike outside 70 130% should be evaluated by the analyst to determine if re-extraction is warranted. 13.4 Any calibratimi standard found to be a statistical outlier by using the Huge Error Test, may be excluded from the calculation o f the calibration curve. However, the total number o f extracted calibration standards that could be excluded must not exceed 20% o f the total number o f extracted standards injected 13.5 The correlation coefficient (R) for calibration curves generated must be 20.992 (R3 20.985). If calibration results fall outside these limns, then appropriate steps must be taken to adjust instrument operation, and the standards or the relevant set o f samples should be reanalyzed. 13.6 Retention times between standards and samples must not drift more than 4 % within an analytical run. I f retention time drift exceeds this limit within an analytical run then the set must be reanalyzed. Page 6 n Page 28 o f 65 Exygen Research Page 63 o f 119 Interim Report #27 - Analysis o f Groundwater Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 Exygen Research Method Number VOQO1781 _______________________ ANALYTICAL METHOD________________________ Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Soil by LC/MS/MS 14.0 Calculations 14.1 Use the following equation to calculate the amount o f PFOA found (in ng/L, baaed on peak area) using the standard curve (linear regression parameters) generated by the Mass Lynx software program: PFOA found (ngO.) - (Peak area - imercepi) x DF slope DF ~ factor by which the final volume was diluted, if necessary. 14.2 For samples fortified with known amounts o f PFOA prior to extraction, use the following equation to calculate the percent recovery. Recovery (%) * [total analyte found (ng/L) - analyte found in control (ng/L)] ^ ^ analyte added (ng/L) 14.3 Use the following equation to convert the amount o f PFOA found in ng/L to ng/g (ppb). PFOA found (ppb) - IFFOA found (ng/L) x volume extracted (0.04L)1 sample weight (5 g) 14.4 Use the following equation to calculate the amount o f PFOA found in ppb based on dry weight. PFOA found (ppb) dry weight - PFOA found (ppb) x [ 100% / total solids(%)] Page 7 of 7 Page 29 o f 65 Exygen Research Page 64 o f 119 Interim Report #27 - Analysis o f Groundwater Samples Exygen Study No. : P0001131 Exygen Protocol Number: P0001131 ANALYTICAL METHOD Method Number. V0001782 Method of Analysis for the Determinttioo of Perfluorooctanoic Acid (PFOA) in Sediment by LC/MS/MS Analytical Testing Facility: Exygen Research 3058 Research Drive State College, PA 16801 Approved By: c j tL Paul Connolly ( Technical Leader, LC-MS, Exygen Research 'J/n r f U / lohn Flaherty Vice President, Operations, Exygen Research ____ l O l l - b M Date Date Exygen Research Total Pages: 7 Page 30 o f 65 Page 65 o f 119 Interim Report #27 - Analysis o f Groundwater Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 Exygen Research Method Number V00017B2 ANALYTICAL M ETHOD Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Sediment by L C /M S/M S ' 1.0 Scope This method is to be employed for the isolation and quantitation o f perfluorooctanoic acid by High Performance Liquid Chromatography coupled to a tandem Mass Spectrometrie Detector (LC/MS/MS) in sediment. 2.0 Safety 2.1 Always observe safe laboratory practices. 2.2 Consult the appropriate MSDS before handling any chemical for proper safety precautions. 3.0 Sample Requirement 3.1 At least 30 g o f test sample for extraction. 3.2 No sample processing is needed for sediment samples. 3.3 Samples stored refrigerated should be allowed to equilibrate to room temperature. 3.4 AH samples must be thoroughly m ixed before being sampled for extraction. 3.5 Sample collection procedures will be specified in the sampling plan for this project 4.0 Reagents and Standards 4.1 W a te r- HPLC grade 4.2 M ethanol-H PL C grade 4.3 Acetic Acid - Reagent grade 4.4 Ammonium Acetate - A.C.S. Reagent Grade 4.5 Perfluorooctanoic Acid - Sigma-Aldrich 5.0 Instrument and Equipment 5.1 A high performance liquid chromatograph capable o f pumping up to 2 solvents equipped with a variable volume injector capable o f injecting 5-200 pL connected to a tandem Mass Spectrometer (LC/MS/MS). 5.2 A device to collect raw data for peak integration and quantitation. 5.3 Analytical balance capable o f reading to 0.00001 g. 5.4 50 mL disposable polypropylene centrifuge tubes. 5.5 15 mL disposable polypropylene centrifuge tubes. 5.6 Disposable micropipets (50-100uL, 100-200uL). 5.7 125-mL LDPE narrow-mouth bottles. 5.8 2 mL clear HPLC vial kit. 5.9 Disposable pipettes. 5.10 Autopipettes (100-1000 pL and 10-100 pL). with disposable tips. S.l 1 Waters Sep Pak Vac 6 cc (Ig ) tC18 SPE cartridges. 5.12 SPE vacuum manifold. Page 2 o f 7 Page 31 o f 65 Exygen Research Page 66 of 119 Interim Report #27 - Analysis o f Groundwater Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 Exygen ReKvch Method Number V0001782 ANALYTICAL METHOD Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Sediment bv L C /M S/M S ' 5.13 Vortexer, 5.14 Wrist-action shaker. 5.15 Centrifhge capable o f spinning 50 m L polypropylene tubes at 3000 rpm. 6.0 Chromatographic System 6. t Analytical Column: Fiuophase RP (Keystone Scientific), 2.1 mm x 50 mm, (P/N: 82505-052130) 6.2 Temperature: 30C 6.3 Mobile Phase (A) : 2 mM Ammonium Acetate in W ater Mobile Phase (B) : Methanol Gradient Program: Time (min) 0.0 1.0 8.0 20.0 22.5 %A 65 65 25 25 65 Flow Rate MB (mUminl 35 0.3 35 0.3 75 0.3 75 0.3 3S 0.3 6.6 Injection Volume: 15 pL (can be increased to as much as 50 pL). 6.7 Quantitation: Peak Area - external standard calibration curve. 6.8 Run Time: - 23 minutes. The above conditions are intended as a guide and m ay be changed in order to optimize the HPLC system. 7.0 MS/MS System 7.1 Mode: Electrospray Negative MRM mode, monitoring 413 369 rcJz for PFOA. The above conditions are intended as a guide and may be changed in order to optimize the MSMS system. 8.0 Preparation o f Solutions 8.1 Mobile Phase 8.1.1 2 mM ammonium acetate in water is prepared by adding 0.154 g of ammonium acetate to 1000 mL o f water. Pige 3 of 7 Page 32 o f 63 Exygen Research Page 67 of 119 Interim Report #27 - Analysis o f Groundwater Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 Exygen Research Method Number VQ001 782 | ANALYTICAL METHOD ( Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Sediment by LC/MS/MS 8.2 Extraction Solutions 8.2.1 \% acetic acid in water is prepared by adding 10 m L o f acetic acid to 1000 mL o f water. Alternate volumes may be prepared. 9.0 Standard Preparation 9.1 Standard Stock/Fortification Solution 9.1.1 Prepare a stock solution o f -1 0 0 pg/mL o f PFOA by weighing 10 mg o f analytical standard (corrected for purity) and dilute to 100 mL with methanol in a 125-mL LDPE bottle. 9.1.2 A 10 pg/mL fortification solution o f PFOA is prepared by bringing 10 m L o f the 100 pg/raL solution to a final volume o f 100 with methanol in a 125 mL LDPE bottle. 9.1.3 A 1.0 pg/mL fortification solution o f PFOA is prepared by bringing 10 mL o f the 10 pg/mL solution to a final volume o f 100 with methanol in a 125 mL LDPE bottle. 9.1.4 A 0.1 pg/mL fortification solution o fPFO A is prepared by bringing 10 m L o f the 1.0 pg/mL solution to a final volume o f 100 with methanol in a 125 mL LDPE bottle. 9.1.5 A 0.01 pg/mL fortification solution o f PFOA is prepared by bringing 10 mL o f the 0.1 pg/mL solution to a final volume o f 100 with methanol in a 125 mL LDPE bottle. 9.1.6 The stock and fortification solutions are to be stored in a refrigerator at approximately 4C and are stable for a maximum period o f 6 months from the date o f preparation. 9.2 Standard Calibration Solutions 9.2.1 LC/MS/MS calibration standards are prepared in methanol via dilution o f the 0.1 pg/mL fortification solution. 9.2.2 The following is a typical example: additional concentrations may be _______ prepared as needed.________________________________ Concentration Final of Fortification Volume Diluted to Concentration Solution (na/mL) (mL) (mL) fng/mL) 100 10 100 10.0 to o 5 100 too 2 100 10 10 100 5.0 20 1.0 5 10 to o 0.5 2 10 100 0.2 Page 4 a f 7 Page 33 o f 65 Exygen Research Page 68 of 119 Interim Report #27 - Analysis o f Groundwater Samples Exygen Study N o.: P0001131 Exygen Protocol Number: POOO1131 Exygen Research Method Number V0001782 | ANALYTICAL M ETHOD ~ Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Sediment by LC/MS/MS 9.2.3 Store alt calibration standards in 125-mL LDPE narrow-mouth bottles at 2C to 6C, up to six months. 9.2.4 Alternate volumes and concentrations o f standards may be prepared as 10.0 Batch Set Up 10.1 Each batch o f samples extracted (typically 20 or less) must include at least one untreated control and two untreated controls fortified at known concentrations (lab control spike) to verify procedural recovery for the batch. 10.2 Requirements for field and laboratory duplicates and spikes will be specified in the quality assurance plan for this project. 11.0 Sample Extraction 11.1 Weigh 5 g o f sample into 50 mL polypropylene centrifuge tubes (fortify as needed, replace lid and mix well). 11.2 Add 35 mL o f \% acetic acid, cap, vortex and shake on a wrist action shaker for -60 minutes. 11.3 Centrifuge the tubes at -3000 rpm for - 2 0 minutes. 11.4 Condition the C u SPE cartridges ( l g, 6 mL) by passing 10 mL methanol followed by 20 mL o f HPLC water ( - 2 drop/sec). Do not let column run dry 11.5 Load (decant) file sample on the conditioned C it SPE cartridge. Discard eluate. 11.6 Add 20 m L o f methanol to the sediment left in the bottom o f the 50 mL centrifuge tube. Cap, vortex and shake on a wrist action shaker for -3 0 minutes. 11.7 Centrifuge the tubes at -3 0 0 0 rpm for - 2 0 minutes. 11.8 Decant the methanol onto the same SPE cartridge. Collect the eluate. 11.9 Wash the column with 4 mL o f methanol. Collect the eluate and add it to the eluate collected in step 11.8. 11.10 Condition a second C u SPE cartridge (1 g, 6 mL) by passing 10 mL methanol followed by 20 m L o f HPLC water ( - 2 drop/sec). Do not let column run dry 11.11 Add the methanol to -2 0 0 mL o f water and load on the second conditioned SPE cartridge. 11.12 Elute with - 3 mL 100% methanol. Collect 5 mL o f eluate into graduated 15 mL polypropylene centrifuge tubes (final volume * 5 mL). 11.13 Analyze samples using electrospray LC/MS/MS. Page 5 of 7 Page 34 o f 65 Exygen Research Page 69 of 119 Interim Report #27 - Analysis o f Groundwater Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 Exygen Reicercb Method Number V00017B2 1 ANALYTICAL M E T H O D ........... Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Sediment bv L C /M S/M S ' 12.0 Chromatography 12.1 Inject the same amount o f each standard, sample and fortified sample into the LC/MS/MS system. A calibration standard must precede and follow all analyzed samples. 12.2 Standards o f PFOA corresponding to at least five o r m ore concentration levels must be included in an analytical set. 12.3 An entire set o f extracted calibration standards must be included at the beginning and at the end o f a sample set. Standards must be interspersed between every 5-10 samples. As an alternative, an entire set o f calibration standards may be injected at the beginning o f a set followed by calibration standards interspersed every 5-10 samples (to account for a second set of standards). In either case, calibration standards must be the first and last injection in a sample set. 12.4 Use linear standard curves for quantitation. Linear standard curves arc generated for the analyte by linear regression using 1/x weighting o f peak area versus calibration standard concentration using MassLynx 3 3 (or equivalent) software system. 12.5 Sample response should not exceed standard responses. Any samples that exceed standard responses should be Author diluted and reanalyzed. 13.0 Acceptance Criteria 13.1 Chromatogram must show a peak o f a daughter ion at 369 amu from a parent o f 413 amu. The 413 amu parent corresponds to the PFOA anion, while the daughter ion (369 amu) represents the loss o f carbon dioxide. 13.2 Method blanks must not contain PFOA at levels greater than the LOQ. If a blank contains PFOA at levels greater than 0.2 ng/mL, then a new blank sample must be obtained and the entire set must be re-extracted. 13.3 Recoveries o f control spikes and matrix spikes must be between 70-130% o f their known values. I f a control spike falls outside the acceptable limits, the entire set o f samples should be re-extracted. Any matrix spike outside 70 130% should be evaluated by the analyst to determine if re-extraction is warranted. 13.4 Any calibration standard found to be a statistical outlier by using the Huge Error Test, may be excluded from the calculation o f the calibration curve However, the total number o f extracted calibration standards that could be excluded must not exceed 20% o f the total number o f extracted standards injected. 13.5 The correlation coefficient (R) for calibration curves generated must be 0.992 (R2 0.985). I f calibration results fall outside these limits, then appropriate steps must be taken to adjust instrument operation, and the standards or the relevant set o f samples should be reanalyzed. Page 6 of 1 Page 35 o f 65 Exygen Research Page 70 of 119 Interim Report #27 - Analysis of Groundwater Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 Exygen Research Method Number V0001782 1 ANALYTICAL M ETHOD | Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Sediment by L C /M S/M S 13.6 Retention times between standards and samples must not drift more than 4 % within an analytical run. If retention tim e drift exceeds this limit within an analytical run then the set must be reanalyzed. 14.0 Calculations 14.1 Use the following equation to calculate the amount o f PFOA found (in ng/mL. based on peak area) using the standard curve (linear regression parameters) generated by the Mass Lynx software program: PFOA found (ng/mL) (Peak area - intercept) x DF slope DF " factor by which the final volume was diluted, if necessary. 14.2 For samples fortified with known amounts o f PFOA prior to extraction, use the following equation to calculate the percent recovery. Recovery (%) - [ total analyte found (ng/mL) - analyte found in control (ng/mL)] ^ ^qq analyte added (ng/mL) 14.3 Use the following equation to convert the amount o f PFOA found in ng/mL to ng/g (ppb). p f o a found (ppb) - [PFOA found. (qft'gU,) x final w l a n s (i. m ill sample weight (3 g) 14.4 Use the following equation (if necessary) to calculate the amount o f PFOA found in ppb based on dry weight. PFOA found (ppb) dry weight PFOA found (ppb) x [100% / total solids(%>] Page 7 of 7 Pag e 36 o f 65 Exygen Research Page 71 o f 119 Interim Report #27 - Analysis o f Groundwater Samples Exygen Study No.: P0001131 Exygen Protocol Number; POQQ1131 ANALYTICAL METHOD Method Number: V001783 M ethod o f Analysis for the D eterm ination o f Perfluorooctaoolc Acid (PFOA) in Fish and Clami by LC/MS/MS Analytical Testing Facility: Exygen Research 3058 Research Drive State College, PA 16801 Approved By: Paul Connolly Technical Leader, LC-MS, Exygen Research '/07 /b /S fohn Flaherty Vice President, Operations, Exygen Research Date Date Exygen Research Total Pages: 8 Page 37 o f 65 Page 72 of 119 Interim Report #27 - Analysis o f Groundwater Samples Exygen Study No. : P0001131 Exygen Protocol Number: P0001131 Exygen Research Method Number VOOO1783 ANALYTICAL m e t h o d __________________________ Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Fish and Clams by LC/MS/MS 1.0 Scope This method is to be employed for the isolation and quantitation o f perfluorooctanoic acid by High Performance Liquid Chromatography coupled to a tandem Mass Spectrometric Detector (LC/MS/MS) in fish and clams. 2.0 Safety 2.1 Always observe safe laboratory practices. 2.2 Consult the appropriate MSDS before handling any chemical for proper safety precautions. 3.0 Sample Requirement 3.1 At least 20 g o fte s t sample for extraction. 3.2 Samples should be processed before extraction. Place the frozen sample in a food processor and homogenize w ith dry ice. Place the samples in containers and leave open in frozen storage overnight to allow for carbon dioxide sublimation. Seal and place the samples in frozen storage until time o f analysis. 3.3 Sample collection procedures will be specified in the sampling plan for this project. Reagents and Standards 4.1 Water - HPLC grade 4.2 Acetonitrile - HPLC grade 4.3 Carbon (120-400 mesh) - Reagent grade 4.4 Methanol - HPLC grade 4.5 Silica gel (60-200 mesh) - Reagent grade 4.6 Floruil (60-100 mesh) - Reagent grade 4.7 Superclean LC-NHj - Reagent grade 4.8 1-Octanol - HPLC grade 4.9 L-Ascorbic acid - Reagent grade 4.10 Dimethyldichlorosilane - Reagent grade 4.11 Toluene - Reagent grade 4.12 Ammonium Acetate - A.C.S. Reagent Grade 4.13 Perfluorooctanoic Acid - Sigma-Aldrich 5.0 Instrument and Equipment 5.1 A high performance liquid chromatograph capable o f pumping up to 2 solvents equipped with a variable volume injector capable o f injecting 5 -2 0 0 pL connected to a tandem Mass Spectrometer (LC/MS/MS). 5.2 A device to collect raw data for peak integration and quantitation. 5.3 Analytical balance capable o f reading to 0.00001 g. Page 2 o f 8 Page 38 of 65 Exygen Research Page 73 o f 119 Interim Report #27 - Analysis of Groundwater Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 Exygen Reietrch Method Number V0001783 ANALYTICAL M ETHOD Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Fish and Clams by LC/MS/MS 5.4 Rotary evaporator. 5.5 Tissumizer. 5.6 125 mL pear-shaped flasks. 5.7 50 m L disposable polypropylene centrifuge tubes. 5.8 15 mL disposable polypropylene centrifuge tubes. 5.9 Disposable micropipets (50-lOOuL, 100-200uL). 5.10 125-mL LDPE narrow-mouth bottles. 5.11 2 mL clear HPLC vial kit. 5.12 Disposable pipettes. 5.13 Autopipettes (100*1000 pL and 10-100 pL), with disposable tips. 5.14 SPE tubes (20mL) (Supelco cat. no. N057177). 5.15 Wrist action shaker. 5.16 Centrifuge capable o f spinning 50 m L polypropylene tubes at 2000 rpm. 6.0 Chromatographic System 6.1 Analytical Column: Fluopbase RP (Keystone Scientific). 2.1 mm x 50 mm. 5m (P/N: 82505-052130) 6.2 Temperature: 30*C 6.3 Mobile Phase (A) : 2 mM Ammonium Acetate in W ater 6.4 Mobile Phase (B) : Methanol 6.5 Gradient Program: T im t (mill) 0.0 1.0 8.0 20.0 22.5 %A 65 65 25 25 65 Flow Rate % B ImL/minl 35 0.3 35 0.3 75 0.3 75 0.3 35 0.3 6.6 Injection Volume: 15 pL (can be increased to as much as 50 pL). 6.7 Quantitation: Peak Area - external standard calibration curve. 6.8 Run Time: - 23 minutes. The above conditions are intended as a guide and may be changed in order to optimize the HPLC system. Page 3 o f8 Page 39 of 65 Exygen Research Page 74 o f 119 Interim Report #27 - Analysis o f Groundwater Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 Exygen Research . Method Number VQ001783 I ANALYTICAL M ETHOD Method o f Analyst for the Detennination o f Perfluorooctanoic Acid (PFOA) in Fish and Clams by LC/MS/MS 7.0 MS/MS System 7.1 Mode: Electrospray Negative MRM mode, monitoring 413 -- 369 m/z for PFOA. The above conditions are intended as a guide and may be changed in order to optimize the MSMS system. 8.0 Preparation o f Solutions 8.1 Mobile Phase 8.1.1 2 mM ammonium acetate in water is prepared by adding 0.154 g of ammonium acetate to 1000 m L o f water. 8.2 Extraction Solutions 8.2.1 5.2.2 2% ascorbic acid in methanol is prepared by dissolving 2 g o f ascorbic acid in 100 mL o f methanol. 30% Dimethyldichlorositane in toluene is prepared by bringing 3 ml. o f dimethyldichloroailane to a final volume o f 10 mL with toluene. Alternate volumes may be prepared. 9.0 Standard Preparation 9.1 Standard Stock/Fortification Solution 9.1.1 9.1.2 9.1.3 9.1.4 9.1.5 Prepare a stock solution o f -1 0 0 pg/mL o f PFOA by weighing 10 mg o f analytical standard (conected for purity) and dilute to 100 mL with methanol in a 125-raL LDPE bottle. A 1.0 p ^ m L fortification solution o f PFOA is prepared by bringing 1 mL o f the 100 pg/mL solution to a final volume o f 100 with methanol in a 125 m L LDPE bottle. A 0.1 pgfaiL fortification solution o f PFOA is prepared by bringing 10 mL o f the 1.0 pg/mL solution lo a final volume o f 100 with methanol in a 125 mL LDPE bottle. A 0.01 pg/mL fortification solution o f PFOA is prepared by bringing 10 mL o f the 0.1 pg/mL solution to a final volume o f 100 with methanol in a 125 mL LDPE bottle. The stock and fortification solutions are to be stored in a refrigerator at approximately 4C and are stable for a maximum period o f 6 months from the date o f preparation. Page 4 or'8 Page 40 o f 65 Exygen Research Page 75 of 119 Interim Report #27 - Analysis o f Groundwater Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 Exygen Research Method Number VOOO1783 I ANALYTICAL METHOD Method o f Analysis for the Determination o f Pcrfluorooctanoic Acid (PFOA) in Fish and Clams by LC/MS/MS 9.2 Standard Calibration Solutions 9.2.1 9.2.2 LC/MS/MS calibration, standards are prepared in methanol via dilution o f the l .0 pg/mL fortification solution. The following is a typical example: additional concentrations may be prepared as needed. Concentration o f Fortification Volume Solution (uttfaiL) (mL) Diluted to (mL) Final Concentration (ua/mL) 1.0 5.0 100 0.05 1.0 2.5 100 1.0 1.0 100 0.05 10 too 0.025 0.01 0.005 0.025 10 100 0.0025 0.1 0.005 10 10 100 100 0.001 0.0005 9.2.3 Store all calibration standards in 125-mL LDPE narrow-mouth bottles at 2CC to 6C, up to six months. 9.2.4 Alternate volumes and concentrations o f standards may be prepared as needed. 10.0 Batch Set Up 10.1 Each batch o f samples extracted (typically 20 or less) must include at least one untreated control and two untreated controls fortified at known concentrations (lab control spike) to verity procedural recovery for the batch. 10.2 Requirements for field and laboratory duplicates and spikes will be specified in the quality assurance plan for this project. 11.0 Sample Extraction 11.1 Weigh 5 g o f frozen sample into 50 mL polypropylene centrifuge tubes (fortify as needed, replace lid and mix well). 11.2 Add 30 mL o f acetonitrile and shake on a wrist action shaker for -15 minutes 11.3 Place the tubes in a freezer for- 1 hour. 11.4 Pack and condition the SPE tubes and silanize the pear-shaped flasks. 11.5 Pack the 20 mL SPE tubes in sequence with 2 g florisil, 2 g silica gel, 2 g carbon, and I g LC-NHj. Condition the columns with 20 mL o f methanol, then 20 mL o f acetonitrile. Discard all washes. Do not allow the column to dry. 11.6 Silanize the 125 m L pear-shaped flasks by rinsing with the 30% dimethyldicblorosilane in toluene solution. Rinse the flask with toluene once, followed by methanol (three times). Dry the flasks completely before use. either by air-drying or with a stream o f nitrogen. Page 5 ofX Page 4! o f 65 Exygen Research Page 76 o f 119 Interim Report #27 - Analysis o f Groundwater Samples Exygen Study N o.: P0001131 Exygen Protocol Number: P0001131 Exygen Reiearch Method Number VOOOPS3 ANALYTICAL M ETHOD 1 Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Fish and Clams by LC/MS/MS 11.7 Centrifuge the 50 mL polypropylene tubes containing sample at -2000 rpm fo r-1 0 minutes. 11.8 Decant the extract on to a conditioned SPE column fitted inside the mouth o f the pear-shaped flask. Collect the eluate in the 125 m L siianized pear-shape flask. 11.9 Add 10 mL o f acetonitrile to the sample in the 50 mL centrifuge tube. Homogenize the frozen fat phase using a tissumizer for - 3 0 seconds and rinse the tissumizer with -1 0 m L o f acetonitrile into the tube. 11.10 Shake die sample again fo r-1 0 minutes on a wrist-action shaker. 11.11 Place the tubes in a freezer for - 1 hour more. 11.12 Centrifuge the 50 mL polypropylene tubes containing sample at -2000 rpm fo r-1 0 minutes. 11.13 Decant the extract onto the same SPE column. Collect the eluate into the same pear-shaped flask and combine with the eluent from the initial extraction. I t. 14 Pass 20 mL o f acetonitrile through the SPE column and combine the eluate in the same pear-shaped flask. 11.15 Add 3-4 drops o f l-octanol to the extract in the pear-shaped flask and evaporate at reduced pressure using a rotary evaporator (at < 40C). 11.16 Make the final volume, by adding 2 m L o f 2% ascorbic acid in methanol to the pear-shaped flask and swirl to mix/dissolve. 11.17 Transfer the extracts to HPLC vials using disposable pipets. 11.18 Analyze samples using electrospray LC/MS/MS. 12.0 Chromatography 12.1 Inject die same amount o f each standard, sample and fortified sample into the LC/MS/MS system. A calibration standard must precede and follow all analyzed samples. 12.2 Standards o f PFOA corresponding to at least five or more concentration levels must be included in an analytical set. 12.3 An entire set o f calibration standards must be included at the beginning and at the end o f a sample set. Standards must be interspersed between every 5-10 samples. As an alternative, an entire set o f calibration standards may be injected at the beginning o f a set followed by calibration standards interspersed every 5-10 samples (to account for a second set o f standards). In either case, calibration standards must 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 MassLynx 3-3 (or equivalent) software system. Page 6 of 8 Page 42 o f 65 Exygen Research Page 77 of 119 Interim Report #27 - Analysis o f Groundwater Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 Exygen Reswch_______________________ Method Number V0001783 I ANALYTICAL m e t h o d Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Fish and Clams by LC/MS/MS 12.S Sample response should not exceed standard responses. Any samples that exceed standard responses should be Anther diluted and reanalyzed. 13.0 Acceptance Criteria 13.1 Chromatogram must show a peak o f a daughter ion at 369 amu from a parent o f 413 amu. The 413 amu parent corresponds to the PFOA anion, while the daughter ion (369 amu) represents the loss o f carbon dioxide. 13.2 Method blanks must not contain PFOA at levels greater than the LOQ. If a blank contains PFOA at levels greater than 0.5 ppb, then a new blank sample must be obtained and the entire set must be re-extracted. 13.3 Recoveries o f control spikes and matrix spikes must be between 70-130% of their known values. I f a control spike falls outside the acceptable limits, the entire set o f samples should b e re-extracted. 13.4 Any calibration standard found to be a statistical outlier by using the Huge Error Test, may be excluded from the calculation o f the calibration curve. However, the total number o f calibration standards that could be excluded must not exceed 20% o f the total number o f standards injected. 13.5 The correlation coefficient (R) for calibration curves generated must be 0.992 (RJ 0.985). I f calibration results Adi outside these limits, then appropriate steps must be taken to adjust instrument operation, and the standards or the relevant set o f samples should be reanalyzed. 13.6 Retention times between standards and samples must not drift more than 4 % within an analytical run. I f retention time drift exceeds this limit within an analytical run then the set must be reanalyzed. 14.0 Calculations 14.1 Use the following equation to calculate the amount o f PFOA found (in ng/mL, based on peak area) using the standard curve (linear regression parameters) generated by the Mass Lynx software program: PFOA found (ng/mL) (Peak area - intercept) slope 14.2 Use the following equation to convert the amount o f PFOA found in ng/mL to ng/g (ppb). PFOA found (ppb) - fPFQA found fn e/m D x final volume fm D x DF1 sample weight (g) DF factor by which the final volume was diluted, if necessary. P*ge 7 of 8 Page 43 o f 65 Exygen Research Page 78 o f 119 Interim Report #27 - Analysis o f Groundwater Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 Exygen Research Method Number V0001783 ANALYTICAL m e t h o d Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Fish and Clams by LC/MS/MS 14.3 For samples fortified with known amounts o f PFOA prior to extraction, use the following equation to calculate the percent recovery. Recovery (%) = [ total analyte found (ng/g) analyte found in control (ng/g)j analyte added (ng/g) Exygen Research Page 8 o f 8 Page 44 o f 65 Page 79 o f 119 Interim Report #27 - Analysis o f Groundwater Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 ANALYTICAL METHOD Method Number: V0001784 Method of Analysis for the Determination of Perfluorooctanoic A dd (P F O A ) in Vegetation by LC/MS/MS Analytical Testing Facility: Exygen Research 3058 Research Drive S u te College, PA 16801 Approved By: TLA COL Paul Connolly ' Technical Leader, LC-MS, Exygen Research q / z n / J d / ________ .ohn Flaherty ^ Vice President, Operations, Exygen Research _ Date Exygen Research T oul Pages: 7 Page 45 o f 65 Page 80 o f 119 Interim Report #27 - Analysis o f Groundwater Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 Exygen R u earch Method Number VOOOI7M ANALYTICAL METHOD 1 J Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Vegetation by LC/MS/MS 1.0 Scope This method is to be employed for the isolation and quantitation o f perfluorooctanoic acid by High Performance Liquid Chromatography coupled to a tandem Mass Spectrometric Detector (LC/MS/MS) in vegetation. 2.0 Safety 2.1 Always observe safe laboratory practices. 2.2 Consult the appropriate MSDS before handling any chemical for proper safety precautions. 3.0 Sample Requirement 3.1 At least 20 g o ftest sample for extraction. 3.2 Samples should b e processed before extraction. Place the frozen sample in a food processor and homogenize with dry ice. Place the samples in containers and leave open in frozen storage overnight to allow for carbon dioxide sublimation. Seal and place the samples in frozen storage until time of analysis. 3.3 Sample collection procedures will be specified in the sampling plan for this project. Reagents and Standards 4.1 W ater-H P L C grade 4.2 Acetonitrile - HPLC grade 4.3 Carbon ( 120*400 mesh) - Reagent grade 4.4 Methanol - HPLC grade 4.5 Silica gel (60-200 mesh) -R eag en t grade 4.6 Florisil (60*100 m esh)-R eagent grade 4.7 Superclean LC-NH: - Reagent grade 4.8 1-Octanol - HPLC grade 4.9 L*Ascorbic acid - Reagent grade 4.10 Dimethyldichlorosilane - Reagent grade 4.11 Toluene - Reagent grade 4.12 Ammonium Acetate - A.C.S. Reagent Grade 4.13 Perfluorooctanoic Acid - Sigma-Aldrich 5.0 Instrument and Equipment 5.1 A high performance liquid chromatograph capable o f pumping up to 2 solvents equipped with a variable volume injector capable of injecting 5*200 pL connected to a tandem Mass Spectrometer (LC/MS/MS). 5.2 A device to collect raw data for peak integration and quantitation. 5.3 Analytical balance capable o f reading to 0.00001 g. Page 2 of 7 Pag e 46 o f 65 Exygen Research Page 81 o f 119 Interim Report #27 - Analysis o f Groundwater Samples Exygen Study No.: P0001131 Exygen Protocol Number: POOO1131 Exygen Research Method Number V0001784 ___________________________ ANALYTICAL M ETH O D Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Vegetation by LC/MS/MS 5.4 Rotary evaporator. 5.5 125 mL pear-shaped flasks. 5.6 SO mL disposable polypropylene centrifuge tubes. 5.7 15 mL disposable polypropylene centrifuge tubes. 5.8 Disposable micropipets (50-1 OQuL, 100-200uL). 5.9 125-mL LDPE narrow-mouth bottles. 5.10 2 mL clear HPLC vial kit. 5.11 Disposable pipettes. 5.12 Autopipettes (100-1000 pL and 10-100 pL), w ith disposable Ups. 5.13 SPE tubes (20mL) (Supelco cat. no. N057177). 5.14 Wrist action shaker. 5.15 Centrifiige capable o f spinning 50 m L polypropylene tubes at 2000 rpm Chromatographic System 6.1 Analytical Column: FluophaseRP (Keystone Scientific), 2.1 mm x 50 mm, 5p (P/N: 82505-052130) 6.2 Temperature: 30"C 6.3 Mobile Phase (A) : 2 mM Ammonium Acetate in Water 6.4 Mobile Phase (B) : Methanol 6.5 Gradient Program: Time (min) 0.0 1.0 8.0 20.0 22.5 %A 65 65 25 25 65 Flow Rate % B falL/min) 35 0.3 35 0.3 75 0.3 75 0.3 35 0.3 6.6 Injection Volume: 15 pL (can be increased to as much as 50 pL). 6.7 Quantitation: Peak Area - external standard calibration curve. 6.8 Run Time: - 23 minutes. The above conditions are intended as a guide and m ay be changed in order to optimize the HPLC system. 7.0 MS/MS System 7.1 Mode: Electrospray Negative MRM mode, monitoring 413 --369 m/z for PFOA. Page 3 of 7 Page 47 o f 65 Exygen Research Page 82 o f 119 Interim Report #27 - Analysis o f Groundwater Samples Exygen Study N o.: P0001131 Exygen Protocol Number. P000I B I Exygen Reieirch Method Number V0001784 ANALYTICAL m e t h o d Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Vegetation by LC/MS/MS The above conditions are intended as a guide and m ay be changed in order to optimize the MSMS system. 8.0 Preparation o f Solutions 8.1 Mobile Phase 8.1.1 2 mM ammonium acetate in water is prepared by adding 0.IS4 g o f ammonium acetate to 1000 mL o f water. 8.2 Extraction Solutions 8.2.1 8.2.2 2% ascorbic acid in methanol is prepared by dissolving 2 g o f ascorbic acid in 100 mL o f methanol. 30% Dimethyldichlorosilane in toluene is prepared by bringing 3 mL o f dimethyldichlorosilane to a final volume o f 10 mL with toluene. Alternate volumes may be prepared. 9.0 Standard Preparation 9 .1 Standard Stock/Fortification Solution 9.1.1 9.1.2 9.1.3 9.1.4 9.1.5 Prepare a stock solution o f --100 jxg/mL o f PFOA by weighing 10 mg o f analytical standard (corrected for purity) and dilute to 100 mL with methanol in a 125-mL LDPE bottle. A 1.0 pg/mL fortification solution o f PFOA is prepared b y bringing 1 mL o f the 100 pg/mL solution to a final volume o f 100 with methanol in a 125 m L LDPE bottle. A 0.1 ng/mL fortification solution o f PFOA is prepared by bringing 10 mL o f the 1.0 pg/mL solution to a final volume oflOO with methanol in a 125 mL LDPE bottle. A 0.01 pg/mL fortification solution o f PFOA is prepared by bringing 10 mL o f the 0.1 pg/inL solution to a final volume o f 100 with methanol in a 125 mL LDPE bottle. The stock and fortification solutions are to be stored in a refrigerator at approximately 4C and are stable for a maximum period o f 6 months from the date o f preparation. 9.2 Standard Calibration Solutions 9.2.1 LC/MS/MS calibration standards are prepared in methanol via dilution o f the 1.0 pg/mL fortification solution. Page 4 ul Page 48 o f 65 Exygen Research Page 83 of 119 Interim Report #27 - Analysis o f Groundwater Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 Exygen ReMarch Method Number V0001784 A.NA LYHCAL M ETH O D Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Vegetation by LC/MS/MS 9.2.2 The following is a typical example: additional concentrations may be prepared as needed. Concentration o f Fortification Solution (us/mL) 10 1.0 1.0 Volume (mL) 5.0 2.5 1.0 Diluted to (mL) 100 100 100 Final Concentration W mL) 0.05 0.025 0.01 0.05 0.025 0.1 10 10 10 100 100 100 0.005 0.0025 0.001 0.005 10 100 0.0005 9.2.3 Store all calibration standards in 125-mL LDPE narrow-mouth bottles at 2*C to 6C, up to six months. 9.2.4 Alternate volumes and concentrations o f standards may be prepared as needed. 10.0 Batch Set Up 10.1 Each batch o f samples extracted (typically 20 or less) must include at least one untreated control and two untreated controls fortified at known concentrations (lab control spike) to verify procedural recovery for the batch 10.2 Requirements for field and laboratory duplicates and spikes will be specified in the quality assurance plan for this project. 11.0 Sample Extraction 11.1 Weigh 5 g o f frozen sample into SO mL polypropylene centrifuge tubes (fortify as needed, replace lid and mix well). 11.2 Add 30 m L o f acetonitrile and shake on a wrist action shaker for ~15 minutes. 11.3 Centrifuge the SO mL polypropylene tubes containing sample at -2000 rpm fo r-1 0 minutes. 11.4 Pack and condition the SPE tubes and silanize the pear-shaped flasks. 11.5 Pack the 20 m L SPE tubes in sequence with 2 g florisil, 2 g silica gel. 2 g carbon, and 1 g LC-NHj. Condition the columns with 20 mL o f methanol, then 20 mL o f acetonitrile. Discard all washes. Do not allow the column to dry. 11.6 Silanize the 125 mL pear-shaped flasks by nnsing with the 30% dimethyldichlorosilane in toluene solution. Rinse the flask with toluene once, followed by methanol (three times). Dry the flasks completely before use. either by air-drying or with stream o f nitrogen. 11.7 Decant the extract on to a conditioned SPE column fitted inside the mouth of the pear-shaped flask. Collect the eluate in the 125 m L silanized pear-shape flask. Page 5 o f Page 49 o f 65 Exygen Research Page 84 of 119 Interim Report #27 - Analysis o f Groundwater Samples Exygen Study No.: P0001131 Exygen Protocol Number: POOOl 131 Exygen Research Method Number V000I784 AiNALYTICAL M ETH O D | Method o f Analysis for the Determination o f Perfluoiooctanoic Acid (PFO A) in Vegetation by LC/MS/MS 11.8 Add 20 mL o f acetonitrile to the sample in the 50 raL centrifuge tube. 11.9 Shake the sample again fox -1 0 minutes on s wrist-action shaker. 11.10 Centrifuge the 50 mL polypropylene tubes containing sample at -2000 rpm fo r- 5 minutes. 11.11 Decant the extract onto the same SPE column. Collect the eluate into the same pear-shaped flask and combine with the eluent from the initial extraction. 11.12 Repeat steps 11.8 through 11.11 again. 11.13 Add 3-4 drops o f 1-octanol to the extract in the pear-shaped flask and evaporate at reduced pressure using a rotary evaporator (at < 40C). 11.14 Make the final volume, by adding 2 m L o f 2% ascorbic acid in methanol to the pear-shaped flask and swirl to mix/dissolvc. 11.15 Transfer the extracts to HPLC vials using disposable pipets. 11.16 Analyze samples using eleclrospray LC/MS/MS. 12.0 Chromatography 12.1 Inject the same amount o f each standard, sample and fortified sample into the LC/MS/MS system. A calibration standard must precede and follow all analyzed samples. 12.2 Standards o f PFOA corresponding to at least five or more concentration levels must be included in an analytical set. 12.3 An entire set o f extracted calibration standards must be included at (he beginning and at the end o f a sample set. Extracted standards must be interspersed between every 5-10 samples. As an alternative, an entire set o f extracted calibration standards may be injected at the beginning o f a set followed by extracted calibration standards interspersed every 5-10 samples (to account for a second set o f extracted standards). In either case, extracted calibration standards must be the 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 MassLynx 3.3 (or equivalent) software system. 12.5 Sample response should not exceed standard responses. Any samples that exceed standard responses should be further diluted and reanalyzed. 13.0 Acceptance Criteria 13.1 Chromatogram must show a peak o f a daughter ion at 369 amu from a parent o f 413 amu. The 413 amu parent corresponds to the PFOA anion, while the daughter ion (369 amu) represents the loss o f carbon dioxide. Page 6 of? Page 50 o f 65 Exygen Research Page 85 of 119 Interim Report #27 - Analysis o f Groundwater Samples Exygen Study No.: P0001131 E x y g e n P r o to c o l N u m b e r: P 0 0 0 1 131 Exygen Reteuch Method Number VOOOI784 ANALYTICAL m e t h o d ___________________________ Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Vegetation by LOMS/MS 13.2 Method blanks must not contain PFOA at levels greater than the LOQ. If a blank contain PFOA at levels greater than O.S ppb, then a new blank sample must be obtained and the entire set must be re-extracted. 13.3 Recoveries o f control spikes and matrix spikes must be between 70-130% of their known values. If a control spike fails outside the acceptable limits, the entire set o f samples should be re-extracted. 13.4 Any calibration standard found to be a statistical outlier by using the Huge Error Test, may be excluded from the calculation o f the calibration curve. However, the total number o f calibration standards that could be excluded must not exceed 20% o f the total number o f standards injected. 13.5 The correlation coefficient (R) for calibration curves generated must be 0.992 (R2 0.985). I f calibration results fall outside these limits, then appropriate steps must be taken to adjust instrument operation, and the standards or the relevant set o f samples should be reanalyzed. 13.6 Retention times between standards and samples must not drift more than 4 % within an analytical nut. If retention tim e drift exceeds this limit within an analytical run then the set must be reanalyzed. 14.0 Calculations 14.1 Use the following equation to calculate the amount o f PFOA found (in ng/mL. based on peak area) using the standard curve (linear regression parameters) generated by the Mass Lynx software program: PFOA found (ng/mL) - (Peak area - intercept! slope 14.2 Use the following equation to convert the amount o f PFOA found in ng/mL to ng/g (ppb). PFOA found (ppb) - [PFOA found (nn/mL^ x final volume fmL) x DF1 sample weight (g) DF - factor by which the final volume was diluted, if necessary. 14.3 For samples fortified with known amounts o f PFOA prior to extraction, use the following equation to calculate the percent recovery. Recovery (%) ( total analyte found (ng/g) - analyte found in control (ng/g)j qq analyte added (ng/g) Page 7 o l-' Page 5/ o f 65 Exygen Research Page 86 o f 119 Interim Report #27 - Analysis o f Groundwater Samples Exygen Study No.: P0001131 Exygen Protocol Number: P001131 ANALYTICAL METHOD Method Num ber V0001785 Method of Analysis for the Determination of Perfluorooctanoic Acid (PFOA) in Small Mammal Liver by LC/MS/MS Analytical Testing Facility: Exygen Research 3058 Research Drive State College, PA 16801 Approved By: V_c o L ____ Paul Connolly I Technical Leader, LC-MS, Exygen Research Date John Flaherty / Viiice President, Operations, Exygen Research Date Exygen Research Total Pages: 7 Page 52 o f 65 Page 87 o f 119 Interim Report #27 - Analysis o f Groundwater Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 Exygea Rewarch Method Number V001785 ANALYTICAL METHOD Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Small Mamma) Liver by LC/MS/MS ] 1.0 Scope This method is to be employed for the isolation and quantitation o f perfluorooctanoic acid by High Performance Liquid Chromatography coupled to a tandem Mass Spectrometric Detector (LC/MS/MS) in small mammal liver. 2.0 Safety 2.1 Always observe safe laboratory practices. 2.2 Consult the appropriate MSDS before handling any chemical for proper safety precautions. 3.0 Sample Requirement 3.1 At least 5 g o f test sample for extraction. 3.2 Samples should be processed before extraction. Place the frozen sample in u food processor and homogenize with dry ice. Place the samples in containers and leave open in frozen storage overnight to allow for carbon dioxide sublimation. Sea! and place the samples in frozen storage until time of analysis. Alternately, if there is an insufficient amount o f sample H e s s than S g). then no processing is necessary and the sample can be used as supplied 3.3 Sample collection procedures will be specified in the sampling plan for this project. 4.0 Reagents and Standards 4.1 W ater-H P L C grade 4.2 Methanol - HPLC grade 4.3 Acetonitrile - HPLC grade 4.4 Ammonium Acetate - A.C.S. Reagent Grade 4. S Perfluorooctanoic Acid - Sigma-Aldrich 5.0 Instrument and Equipment 5.1 A high performance liquid chromatograph capable o f pumping up to 2 solvents equipped with a variable volume injector capable o f injecting 5*200 fiL connected to a tandem Mass Spectrometer (LC/MS/MS). 5.2 A device to collect raw data for peak integration and quantitation. 5.3 Analytical balance capable o f reading to 0.00001 g. 5.4 50 mL disposable polypropylene centrifuge tubes. 5.5 15 mL disposable polypropylene centrifuge tubes. 5.6 Disposable micropipets (50-lOOuL, 100-200uL). 5.7 125-mL LDPE narrow-mouth bottles. 5.8 2 raL clear HPLC vial kit. Page 2 o f Page 53 o f 65 Exygen Research Page 88 o f 119 Interim Report #27 - Analysis of Groundwater Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 Exygen Reiesrch Method Number VOOO1785 ANALYTICAL M ETH O D _________________________ Method o f Analysis for the Determination o f Periluorooctanoic Acid (PFOA) in Small Mammal Liver by LC/MS/MS 5.9 Disposable pipettes. 5.10 Autopipettes (100-1000 pL and 10-100 pL), with disposable tips. 5.11 Waters Sep Pak Vac 6 cc (lg ) tC18 SPE cartridges. 5.12 SPE vacuum manifold. 5.13 Tissuemizer. 5.14 Wrist-action shaker. 5.15 Centrifuge capable o f spinning 15 mL polypropylene tubes at 3000 rpm. 6.0 Chromatographic System 6.1 Analytical Column: Fluophase RP (Keystone Scientific), 2.1 mm x 50 mm. 5p (P/N: 82505-052130) 6.2 Temperature: 30C 6.3 Mobile Phase (A) : 2 mM Ammonium Acetate in Water 6.4 Mobile Phase (B) : Methanol 6.5 Gradient Program: Time (min) 0.0 1.0 8.0 20.0 22.5 %A 65 65 25 25 65 Flow Rate % B (mL/min) 35 0.3 35 0.3 75 0.3 75 0.3 35 0.3 6.6 Injection Volume: 15 pL (can be increased to as much as 50 pL). 6.7 Quantitation: Peak Area - external standard calibration curve. 6.8 RunTim e: - 2 3 minutes. The above conditions are intended as a guide and may be changed in order to optimize the HPLC system. 7.0 MS/MS System 7.1 Mode: Electrospray Negative MRM mode, monitoring 413 --369 m/z for PFOA. The above conditions are intended as a guide and may be changed in order to optimize the MSMS system. Page 3 of 7 Pag e 54 o f 65 Exygen Research Page 89 o f 119 Interim Report #27 - Analysis of Groundwater Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 Exygen Reaaarch 1Method Number VOOO 785 I AN.vLVTICAl-TlETHOD Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Small Mammal Liver by LC/MS/MS 5.0 Preparation o f Solutions 8.1 Mobile Phase 8.1.1 2 mM ammonium acetate in water is prepared by adding 0 .154 g of ammonium acetate to 1000 m L o f water. Alternate volumes may be prepared. 9.0 Standard Preparation 9.1 Standard Stock/Fortification Solution 9.1.1 Prepare &stock solution o f -1 0 0 pg/raL o f PFOA by weighing 10 ing o f analytical standard (corrected for purity) and dilute to 100 mL with methanol in a 125-mL LDPE bottle. 9.1.2 A 1.0 pg/raL fortification solution o f PFOA is prepared by bringing I mL o f the 100 p ^ m L solution to a final volume o f 100 with methanol in a 12S mL LDPE bottle. 9.1.3 A 0.1 pg/mL fortification solution o f PFOA is prepared by bringing 10 mL o f the 1.0 pg/mL solution to a final volume o f 100 with methanol in a 125 m L LDPE boule. 9.1.4 The stock and fortification solutions are to be stored in a refrigerator at approximately 4C and are stable for a maximum period o f 6 months from the date o f preparation. 9.2 Standard Calibration Solutions 9.2.1 9.2.2 LC/MS/MS calibration standards are prepared in methanol via dilution o f the 0.1 pg/mL fortification solution. The following is a typical example: additional concentrations may be prepared as needed. Concentration Final of Fortification Volume Diluted to Concentration Solution fna/raL) 100 100 (mL) 5.0 2.0 (mL) 100 100 (ntt/mU 5.0 2.0 100 1.0 too 5.0 10 (00 1.0 0.5 2.0 10 100 0.2 1.0 10 100 0.1 9.2.3 Store all calibration standards in 125-mL LDPE narrow-mouth bottles at 2*C to 6*C, up to six months. 9.2.4 Alternate volumes and concentrations o f standards may be prepared as needed. Page 4 of 7 Page 55 o f 65 Exygen Research Page 90 of 119 Interim Report #27 - Analysis o f Groundwater Samples Exygen Study N o.: P0001131 Exygen Protocol Number: P0001131 Exygen Reteuch Method Number V 0001785 I ANALYTICAL METHOD Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Small Mammal Liver by LC/MS/MS 10.0 Batch Set Up 10.1 Each batch o f samples extracted (typically 20 o r less) must include at least one untreated control and two untreated controls fortified at known concentrations (lab control spike) to verify procedural recovery for the batch. 10.2 Requirements for field and laboratory duplicates and spikes will be specified in the quality assurance plan for this project. 11.0 Sample Extraction 11.1 Weigh 1 g o f sample into a 50 mL polypropylene centrifuge tubes (fortify as needed, replace lid and mix well). Note that alternate weights o f liver may be measured depending on die sample size available for use. 11.2 Add water to the sample for a final volume o f 10 mL. 11.3 Homogenize sample using etissuem izcr for ~1 minute. 11.4 Transfer 1 mL o f die sample using a disposable pipette into a 15 mL disposable centrifuge tube. 11.5 Add 5 mL o f acetonitrile and shake for - 2 0 m inutes on a wrist-action shaker. 11.6 Centrifuge the tubes at -3 0 0 0 rpm for - 5 minutes. 11.7 Decant the supernatant into a 50 m L disposable centrifuge tube and add 3$ mL o f water. 11.8 Condition the Cis SPE cartridges (1 g, 6 mL) by passing 10 mL methanol followed by 5 mL o f HPLC water (~ 2 drop/sec). Do not let column run dry 11.9 Load the sample on conditioned C u SPE cartridge. Discard eluate. 11.10 Elute with - 2 mL o f methanol. Collect 2 mL o f eluate into a graduated 15 mL polypropylene centriftige tube (final volume * 2 mL). 1l .l 1 Analyze samples using electrospray LC/MS/MS. 12.0 Chromatography 12.1 Inject the same amount o f each standard, sample and fortified sample into the LC/MS/MS system. A calibration standard must precede and follow all analyzed samples. 12.2 Standards o f PFOA couesponding to at least five or more concentration levels must be included in an analytical set. 12.3 An entire set o f calibration standards m ust be included at the beginning and <u the end o f a sample set. Standards must be interspersed between every 5-IU samples. As an alternative, an entire set o f calibration standards may be injected at the beginning o f a set followed by calibration standards interspersed every 5-10 samples (to account for a second set o f standards). In either case, calibration standards must 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 Page 5 o f 7 Page 56 o f 65 Exygen Research Page 91 o f 119 Interim Report #27 - Analysis o f Groundwater Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 Exygen Research Method Number VOOOI785 I ANALYTICAL METHOD Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Small Mammal Liver by LC/MS/MS versus calibration standard concentration using MassLynx 3.3 (or equivalent) software system. 12.5 Sample response should not exceed standard responses. Any samples that exceed standard responses should be further diluted and reanalyzed. 13.0 Acceptance Criteria 13.1 Chromatogram must show a peak o f a daughter ion at 369 &mu from a parent o f 413 amu. The 413 amu parent corresponds to the PFOA anion, while the daughter ion (369 amu) represents the loss o f carbon dioxide. 13.2 Method blanks must not contain PFOA at levels greater than the LOQ. If a blank contains PFOA at levels greater than 10 ng/g, then a new blank sample must be obtained and the entire set must be re-extracted. 13.3 Recoveries o f control spikes and matrix spikes must be between 70-130/ of their known values. If a control spike falls outside the acceptable limits, (he entire set o f samples should be re-extracted. Any matrix spike outside 70 130% should be evaluated by the analyst to determine if re-extraction is warranted. 13.4 Any calibration standard found to be a statistical outlier by using the Huge Error Test, may be excluded from the calculation o f the calibration curve However, the total number o f calibration standards that could be excluded must not exceed 20% o f the total number o f standards injected. 13.5 The correlation coefficient (R) for calibration curves generated must be 0.992 (RJ 20.985). I f calibration results fall outside these limits, then appropriate steps must be taken to adjust instrument operation, and the standards or the relevant set o f samples should be reanalyzed. 13.6 Retention times between standards and samples must not drift more than 4 % within sn analytical run. If retention time drift exceeds this limit within an analytical run then the set must be reanalyzed. 14.0 Calculations 14.1 Use the following equation to calculate the amount o f PFOA found (in ng/mL, based on peak area) using the standard curve (linear regression parameters) generated by the Mass Lynx software program: PFOA found (ng/m L) (Peak area - intercept') x D F x aliquot factor slope DF factor by which the final volume was diluted, if necessary. Aliquot factor " 10 Page 6 o f 7 Page 57 o f 65 Exygen Research Page 92 of 119 Interim Report #27 - Analysis o f Groundwater Samples Exygen Study No.: P0001131 Exygen Protocol Number: POOOl 131 Exygen Research Method Number VOOO1785 I a n a ly t ic a l m e t h o d Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Small Mammal Liver by LC/MS/MS 14.2 For samples fortified with known amounts o f PFOA prior to extraction, use the following equation to calculate the percent recovery. Recovery (%) [ total analyte found (ng/mL) - analyte found in control (ng/mL)] ^ ^ analyte added (ng/mL) 14.3 Use the following equation to convert the amount o f PFOA found in ng/mL tu ng/g (ppb). PFOA found (ppb) - fPFOA found fn e/m U x final volume fmL)l sample weight (g) Exygen Research Page 7 o f 7 Page 58 o f 65 Page 93 of 119 Interim Report #27 - Analysis o f Groundwater Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 ANALYTICAL METHOD Method N um ber V00G1786 M ethod o f Analysis for the Determ ination of Perfiuorooctanoic Acid (PFOA ) in Small Mammal Serum by LC/MS/MS Analytical Testing Facility: Exygen Research 3058 Research Drive State College, PA 16801 Approved By: ^ ^ C-AAvi Paul Connolly I Technical Leader, LC-MS, Exygen Research \//v / & / ihn Flaherty * Vice President, Operations, Exygen Research u l i-fc/o'l Date _&M r Date Exygen Research Total Pages: 7 Page 59 o f 65 Page 94 o f 119 Interim Report #27 - Analysis o f Groundwater Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 Exygen R etevch Method Number V 0001786 I ANALYTICAL M ETHOD Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFO A) in Small Mammal Serum by LC/MS/MS 1.0 Scope This method is to be employed for the isolation and quantitation o f perfluorooctanoic acid by High Performance Liquid Chromatography coupled to a tandem Mass Spectramctric Detector (LC/MS/MS) in small mammal serum. 2.0 Safety 2.1 Always observe safe laboratory practices. 2.2 Consult the appropriate MSDS before handling any chemical for proper safety precautions. 3.0 Sample Requirement 3.1 At least 1 mL o f test sample for extraction. 3.2 No sample processing is needed for serum samples. However, frozen serum samples must to allowed to completely thaw to room temperature before use. 3.3 Sample collection procedures will be specified in the sampling plan for this project. 4.0 Reagents and Standards 4.1 W ater-H P L C grade 4.2 Methanol - HPLC grade 4.3 Acetonitrile - HPLC grade 4.4 Ammonium Acetate - A.C S. Reagent Grade 4.5 Perfluorooctanoic Acid - Sigma-Aldrich 5.0 Instrument and Equipment 5.1 A high performance liquid chromatograph capable o f pumping up to 2 solvents equipped with a variable volume injector capable o f injecting 5-200 fiL connected to a tandem Mass Spectrometer (LC/MS/MS). 5.2 A device to collect raw data for peak integration and quantitation. 5.3 Analytical balance capable o f reading to 0.00001 g. 5.4 50 mL disposable polypropylene centrifuge tubes. 5.5 15 mL disposable polypropylene centrifUge tubes. 5.6 Disposable micropipets (5(M00uL, 100-200uL). 5.7 125-mLLDPE narrow-mouth bottles. 5.8 2 mL clear HPLC vial kit. 5.9 Disposable pipettes. 5.10 Autopipettes (100-1000 pL and 10-100 pL), with disposable dps. 5.11 Waters Sep Pak V ac 6 cc (Ig ) tC!8 SPE cartridges. 5.12 SPE vacuum manifold. 5.13 Vortexer. Page 2 iii'7 Page 60 o f 65 Exygen Research Page 95 o f 119 Interim Report #27 - Analysis o f Groundwater Samples Exygen Study No.: P0001131 Exygen Protocol Number: POOO1131 E x y g so lL e te sr e h Method Number V00017<> | A lW V n C A L METHOD Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Small Mammal Serum by LC^MS/MS 5.14 Wrist-action shaker. 5.15 Centrifuge capable o f spinning 15 mL polypropylene tubes at 3000 rpm 6.0 Chromatographic System 6.1 Analytical Column: Fluophase RP (Keystone Scientific), 2.1 mm x 50 mm. 5p (P/N: 82505-052130) 6.2 Temperature: 3Q#C 6.3 Mobile Phase ( A ) : 2 mM Ammonium Acetate in Water Mobile Phase (B) : Methanol Gradient Program: T m O nitl 0.0 1.0 8.0 20.0 22.5 %A 65 65 25 25 65 Flow Rate % B (mL/min) 35 0.3 35 0.3 75 0.3 75 0.3 35 0.3 6.6 Injection Volume: 15 pL (can be increased to as much as 50 pL). 6.7 Quantitation: Peak Area - external standard calibration curve. 6.8 RunTim e: - 2 3 minutes. The above conditions are intended as a guide and may be changed in order 10 optimize the HPLC system. 7.0 MS/MS System 7.1 Mode: Electrospray Negative MRM mode, monitoring 413 -+ 369 m/z for PFOA. The above conditions are intended as a guide and m ay be changed in order to optimize the MSMS system. 8.0 Preparation o f Solutions 8.1 Mobile Phase 8.1.1 2 mM ammonium acetate in water is prepared by adding 0.1S4 g o f ammonium acetate to 1000 mL o f water. Alternate volumes may be prepared. Page 3 u f 7 Page 6J o f 65 Exygen Research Page 96 o f 119 Interim Report #27 - Analysis o f Groundwater Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 Exygen Research Method Number V0001786 a n a ly tica l m e t h o d Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Small Mammal Serum by LC/MS/MS 9.0 Standard Preparation 9.1 Standard Stock/Fortification Solution 9.1.1 Prepare a stock solution o f -1 0 0 pg/mL o f PFOA by weighing 10 mg o f analytical standard (corrected for purity) and dilute to 100 mL with methanol in a 125-mL LDPE bottle. 9.1.2 A 1.0 pg/mL fortification solution o f PFOA is prepared by bringing I mL o f the 100 pg/raL solution to a final volume o f 100 with methanol in a 125 mL LDPE bottle. 9.1.3 A 0.1 pg/mL fortification solution o fPFO A is prepared by bringing 10 m L o ftb e 1.0 pg/mL solution to a final volume o f 100 with methanol in a 125 mL 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 maximum period o f 6 months from the date o f preparation. 9.2 Standard Calibration Solutions 9.2.1 9.2.2 LC/MS/MS calibration standards are prepared in methanol via dilution o f the 0.1 pg/mL fortification solution. The following is a typical example: additional concentrations may be prepared os needed. Concentration Final o f Fortification Volume D iluted to Concentration Solution (ng/mL) (mL) (mL) (ng/m L ) 100 5.0 100 5.0 100 2.0 100 2.0 100 1.0 100 1.0 5.0 10 too 0.5 2.0 10 100 0.2 1.0 10 100 0.1 9.2.3 Store all calibration standards in 125-mL LDPE nam>w-mouth bottles at 2C to 6C, up to six months. 9.2.4 Alternate volumes and concentrations o f standards may be prepared as needed. 10.0 Batch Set Up 10.1 Each batch o f samples extracted (typically 20 o r less) must include at least one untreated control and two untreated controls fortified at known concentrations (lab control spike) to verify procedural recovery for the batch 10.2 Requirements for field and laboratory duplicates and spikes will be specified in the quality assurance plan for this project. Page 4 of? Page 62 o f 65 Exygen Research Page 97 o f 119 Interim Report #27 - Analysis o f Groundwater Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 Exygen Research Method Number V000J786 f ANALYTICAL M ETHOD Method o f Analysts for the Determination o f Perfluorooctanoic Acid (PFOA) in Small Mammal Serum by LC/MS/MS 11.0 Sample Extraction 11.1 Measure 1 mL o f sample into a 50 mL polypropylene centrifuge tubes (fortify as needed, replace lid and mix well). Note that alternate volumes o f serum may be measured depending on die sample size available for use. 11.2 Add water to the sample for a final volume o f 20 mL. Cap tightly 11.3 Vortex for -1 minute. 11.4 Transfer 1 m L o f the sample using a disposable pipette into a 15 mL disposable centrifuge tube. 11.5 Add S mL o f acetonitrile and shake for -2 0 minutes on a wrist-action shaker 11.6 Centrifuge the tubes at -3 0 0 0 rpm for ~5 minutes. 11.7 Decant the supernatant into a SO m L disposable centrifuge tube and add 35 m L o f water. 11.8 Condition the C u SPE cartridges (1 g, 6 mL) by passing 10 mL methanol followed b y 5 m L o f HPLC water (~ 2 drop/sec). Do not let column run dry 11.9 Load the sample on conditioned C u SPE cartridge. Discard eluate. 11.10 Elute with ~2 mL o f methanol. Collect 2 m L o f eluate into a graduated 15 m L polypropylene centrifuge tube (final volume - 2 mL). 11.11 Analyze samples using electrospray LC/MS/MS. 12.0 Chromatography 12.1 Inject the same amount o f each standard, sample and fortified sample into the LC/MS/MS system. A calibration standard must precede and follow all analyzed samples. 12.2 Standards o f PFOA corresponding to at least five or more concentration levels must be included in an analytical set. 12.3 An entire set o f calibration standards must be included at the beginning and at the end o f a sample set. Standards must be interspersed between every 5-U> samples. As an alternative, an entire set o f calibration standards may be injected at the beginning o f a set followed by calibration standards interspersed every 5-10 samples (to account for a second set o f standards), in either case, calibration standards must 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 MassLynx 3.3 (or equivalent) software system. 12.5 Sample response should not exceed standard responses. Any samples that exceed standard responses should be further diluted and reanalyzed. Page 5 of ' Page 63 o f 65 Exygen Research Page 98 o f 119 Interim Report #27 - Analysis o f Groundwater Samples Exygen Study N o.: P0001131 Exygen Protocol Number: P0001131 Exygen Rcaevcb Method Number VOOOI786 ANALYTICAL METHOD Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Small Mammal Serum by LC/MS/MS 13.0 Acceptance Criteria 134 Chromatogram must show a peak o f a daughter ion at 369 amu from a parent o f 413 amu. The 413 amu parent corresponds to the PFOA anion, while the daughter ion (369 amu) represents the loss o f carbon dioxide. 13.2 Method blanks must not contain PFOA at levels greater than the LOQ. if a blank contains PFOA at levels greater than 10 ng/mL, then a new blank sample must be obtained and the entire set must be re-extracted. 13.3 Recoveries o f control spikes and matrix spikes must be between 70>130% of their known values. I f a control spike falls outside the acceptable limits, the entire set o f samples should be re-extracted. Any matrix spike outside 70 130% should be evaluated by the analyst to determine if re-extraction is warranted. 13.4 Any calibration standard found to be a statistical outlier by using the Huge Error Test, may be excluded from the calculation o f the calibration curve However, the total number o f calibration standards that could be excluded must not exceed 20% o f the total number o f standards injected. 13.5 The correlation coefficient (R) for calibration curves generated must be 0.992 (R2 20.985). I f calibration results fall outside these limits, then appropriate steps must be taken to adjust instrument operation, and the standards o r the relevant set o f samples should be reanalyzed. 13.6 Retention times between standards and samples must not drib more that] 4 % within an analytical run. If retention time drift exceeds this limit within an analytical run then the set must be reanalyzed. 14.0 Calculations 144 Use the following equation to calculate the amount o f PFOA found (in ng/mL. based on peak area) using the standard curve (linear regression parameters) generated by the Mass Lynx software program: PFOA found (ng/mL) (Peak area - intercept) x DF x aliquot factor slope DF * factor by which the final volume waa diluted, if necessary. Aliquot factor " 20 14.2 For samples fortified with known amounts o f PFOA prior to extraction, use the following equation to calculate the percent recovery. Recovery (% ) [ total analyte found (ng/mL) - analyte found in control (ng/mL)] __________________ analyte added (ng/mL)______________________________ Page 6 o f 7 Page 64 o f 65 Exygen Research Page 99 o f 119 Interim Report #27 - Analysis o f Groundwater Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 Exygea Research Method Number VOOO1786 I AINaI vT IC A L m e t h o d Method o f Analysis for the Determination ofPerfluorooctanoic A d d (PFOA) in Small Mammal Serum by LC/MS/MS I 14.3 Use the following equation to convert the amount o f PFOA found in ng/mL to ppb. PFOA found (ppb) - fPFOA found (ng/mL) x final volume ftnLl] sample volume (mL) Exygen Research fPage 7 o f Page 65 o f 65 Page 100 of 119 Interim Report #27 - Analysis o f Groundwater Samples Exygen Study No.: P0001131 3058 Research Drive Phone: 814-272-1039 State College, PA 16801 Fax: 814-231-1580 PROTOCOL AMENDMENT Amendment Number:_1__ Effective Date: 01/19/05 Exygen Study Number: P0001131 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 ID: W 0 0 0 1 226-6 ADM INISTRATIVE FORM Exygen Research Page 101 o f 119 Interim Report #27 - Analysis o f Groundwater Samples Exygen Study No.: P0001131 RESEARCH 3058 Research Drive Phone: 814-272-1039 State College, PA 16801 Fax: 814-231-1580 Amendment Number: Effective Date: Exygen Study Number PROTOCOL AMENDMENT 2 03/07/05 ' P0001131 Client Study Number: Page 1 of 1 None DESCRIPTION OF AMENDED SECTION 1) Report, page 11 of 65 2) Test Materials, page 6 o f 65: PFOS transition monitored 499 -> 99. AMENDED TO 1) Instead of one final report, interim reports will be issued. 2) PFOS transition monitored m ay also be 499 -> 80. RATIONALE 1) Due to the excessive sizes of the data sets, interim reports will be issued to allow the client to receive data in a tim elierm an * 'm *" / * / - -2W 2) The API 4000 LC/MS/MS systems detect the 499 -> 80 PFOS transition with greater sensitivity than the 499 -> 99 transition. IMPACT ON STUDY 1) The client will be able to receive and review the data more quickly. 2) The 499 -> 80 transition can be detected with greater sensitivity; therefore, giving better chromatography. M ai Study Director Signature y d />? d /a d f rincipal Investigator Signature sJ * /* D a te Study Director M anagem ent Signature a te LIB R A R Y ID: V 0 0 0 1 2 2 6 -8 Exygen Research A D M IN IS T R A T IV E F O R M Page 102 o f 119 Interim Report #27 - Analysis o f Groundwater Samples Exygen Study No.: P0001131 JU L .2 3 .2 0 0 5 0:56fiM EXYGEN RESEARCH N0.r?4 P.3 Ewgserr<2L 3058 Research Drive Phone: 814-272-1039 State College, PA 16801 Fax: 814-23M580 RE SEARCH Amendment Number Effective Date: Exygen Study Number PROTOCOL AMENDMENT 3 07/18/05 P1131 Client Study Number: Pas*1 f i NA DESCRIPTION OF AMENDED SECTION Verification of Analytical Procedure, page 10 of protocol. AMENDED TO . The field duplicate can be used for the laboratory spikes and replicate when the primary sample volume is limited. RATIONALE 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 spikes 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 needed. IMPACT ON STUDY No negative impact on the study. Using the duplicate sample allows for the full QC of the sample site to be completed. QJAhh- Study OjrtdKr Signature . q L /v fta c f /Principal Investigator Signatura MllDirector Mi Exygen M a r^ c i j Sponsor S ig n atu re^ required) 7/iA ^ Oste Date U - jtn -*S Date 7/z s /o Data Exygen QAU Review /'?>' 1v LIBRARY ID: V000122S4 ADMINISTRATIVE FORM Exygen Research Page 103 of 119 Interim Report #27 --Analysis o f Groundwater Samples Exygen Study No.: P0001131 'T Ilrlj-2005 04:22pm From-WESTON SOLUTIONS NOVi22.S005 4:58PM EXTGEN RESEARCH T-67T P .003/003 F-713 N u.ai4 r .a 3058 Research Drive Phone; 814-172-1039 State College, PA 1680T Fax; 814-231-1580 Amendment Number. Effective Date: Exygen Study Number PROTOCOL AMENDMENT 4 ^11/22/05 ' P1131 Client Study Number. Pa?9 1 of 1 NA d e s c r ip t io n o f a m e n d e d S E c n o tT Analytical Procedure Summary V000l7$0:"Methcd ofAnalysis for the Determination of PerfluoroooctarmicArid (PFOA5 in Water by LCJMS/MS,* Section 11.0 Ofthe method. AMENDED TO Section 11.0. Samples may be diluted before going through the extraction procedure. RATIONALE If a 40 mL portion of sample win not load onto the Ci> SPE cartridge, a predilution can be prepared and extracted. 1 IMPACT-ON STUpy " No negative impact on the study. Mors usable data may be obtained. Sponsor Signature fifrequired) Date Exvaen QAU Rsvfew (fr - I. i i UBRARYID: V00012ZM R E C E IV E D T IM E N O V .2 3 . 5 :4 0 P M ADMINISTRATIVE FORM P R IN T T IM E N O V .2 3 . 5 :4 1 P M 1 Exygen Research Page 104 of 119 Interim Report #27 - Analysis o f Groundwater Samples Exygen Study No.: P0001131 ffcHEM EHSR 236 1B 651 733 1958 J U L |S -8 0 0 3 0 5 :1 2 FR0M :3f1 EM J. LPB 6 51 7 70 6 1 7 6 OV.ZZ.2005 4:58PP1 EXYSEN RESERROI 11/23 '05 14:12 NO.979 03/03 . H J:96517331950 P:Kt MO.9 1 4 P .3 3058 Research Drive Phone: 814-272-1039 State College, PA 16801 Fax: BM-23M5KJ Amendment Number. Effective Date: Exygen StudyNumber PROTOCOL AMENDMENT 4 11/22I0S P1131 Client StudyNumber: Page 1of1 NA ` OaCRIgQQNOFANEMDESP_sJfedLQN Analytical Procedure Summary; V003l780:'Methocf ofAnalysis for the Determination of PerfluoroooctanoicAdd (PFOA) in Water by IC/M S/M Sr Section 11.0 ofthe method. ~ ~~ AM EN PEPT5 section 11.0. Samples may be diluted before going through 0 extraction procedure. If a 40 mL portion of sample will not toad onto the C ii SPE cartridge, a pre-dilution can be prepared and extracted. IMPACT ON STUDY t\ No negative Impact on the study. More usable data rrwy be obtained. Study O lre a o rsg n a tu r mie b st ~~ -- _____ O ats U ^ *n r- c J oats A///, IS . WS Oste Exygen QAU R e vie w i I l LIBRARY IO : V0001S36-6 ADMINISTRATIVE FORM R E C E IV E D T IM E N O V .2 3 . 3 :3 2 P M s- -s PR IN T TIM E N O V .2 3 . 3 :3 3P M Exygen Research Page 105 of 119 Interim Report #27 - Analysis of Groundwater Samples -20BB 04:08pm Frora-WESTON SOLUTIONS Exygen Study No.: P0001131 H 52 P00|/0l F-ITB Ilf ,11 RE5 RCH 3058 ResearchOrive Staw C b l t a j , P A 1 * 8 0 1 Ptiorw B14-Z7Z-1037 P 14-231*1580 : Amendment Nu iben Effective O ste Exygen Study Number VOCOU AMENDMENT a _______ 12/01/05 P0001131 Client Study Numtjer Pagel of.1 N A ! DESCRIPTION OF A M K E P S E C j^ " Teat Materials, page 5 of Protocol. AMENDED TO An alternate lof of PFOSnvy sl used forthis study. ............. M P m e The PFOS used previously in this study (let numoer 217 from 8M ) non out and i necessary ta use a nsw tot No negative impact on study. IM E A C T O ^S T U gT Study DlieiMi ffljWaiutn U m - i r r y MndpglWvHtgab.' ionaiu v M ih b O tt ' Date on jT > /M 4 A 'j(Q 6 T5S----------------- Bsygan QAU InrtTOte G &~-- Js k < IsS s. UBRARVID: V D O a" 'K M RECEIV ED TIM E 1AR. i l -H 8P M ADMINISTRATIVEFORM PR IN T TIM E I M FIR .21. 5 :2 9 P M Exygen Research Page 106 o f 119 Interim Report #27 - Analysis o f Groundwater Samples Exygen Study No.: P0001131 A M A RESEARCH 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 05/17/06 P0001131 ' Client Study Number Page 1 of 1 _____ NA DESCRIPTION OF AMENDED SECTION Appendix I, Analytical Method V0001782 AMENDED TO *4 ' As per client request, samples in login L4026 that have been tagged for re-extraction by the sponsor will be re-extracted using the following method: Direct Injection Method: Before the samples are weighed for the extraction, they are mixed thoroughly by vigorously shaking the container. A one-gram portion of sediment is weighed Into a 15-milliliter centrifuge tube for the extraction. Ten milliliters o f 1% acetic acid in methanol is added to each sample. The samples are then shaken by hand, vortexed, and sonicated for thirty minuts. The samples are then centrifuged for -10 minutes at -3000 rpm. Each sample is analyzed by LC/MS/MS electrospray. RATIONALE More usable data will be obtained by using an alternate method. IMPACT ON STUDY No negativexmpact on study. Study Dire&orSIgnature Q l -- ^Pnnclpal Investigator Signature ' Ak Sponsor Signature (iUequired) Data Date D a te /A Dal . fai Exygen QAU Init/Date f t " LIBRARY ID: V0001226-9 ' AD M INISTRATIVE FORM Exygen Research Page 107 o f 119 Interim Report #27 - Analysis of Groundwater Samples Exygen Study N o.: P0001131 EARC H 3058 Research Drive Phone: 814-272-1039 State College, PA 16801 Fax: 814-231 -1580 Am endm ent Number: Effective Date: Exygen Study Num ber PROTOCOL AMENDMENT 8 07/06/06 P0001131 C lient Study N um ber: Page 1 of 1 ______ N A DESCRIPTION OF AMENDED SECTION Appendix I, Analytical Method V0001782. AMENDED TO As per client request, sediment samples re-tagged for re-extraction by the sponsor will be re-extracted using the following method: D ire ct Injection M ethod: Before the samples are weighed for the extraction, they are mixed thoroughly by vigorously shaking the container. A one-gram portion o f sediment is weighed into a 15-milliliter centrifuge tube for the extraction. Ten milliliters o f 1% acetic acid in methanol is added to each sample. The samples are then shaken by hand, vortexed, and sonicated for thirty minutes. The samples are then centrifuged for - 1 0 minutes at -3 0 0 0 rpm. Each sample is analyzed by LC/MS/MS electrospray. RATIONALE More usable data will be obtained by using an alternate method. IMPACT ON STUDY No negative impact on study. Study Director Signature /7 .//V /A c C ________________ ^Principal Investigator Signature D a te ___ 7 jj D a te Study Director M anagem ent Signature Exygen M anagem ent Signature D a te D a te Sponsor Signature (if required) D a te Exygen QAU Init./Date /Qfc- / ^ L ld e LIB R A R Y ID: V 0 0 0 1 2 2 6 -9 A D M IN IS T R A TIV E FO RM Exygen Research Page 108 of 119 Interim Report #27 - Analysis o f Groundwater Samples Exygen Study No.: P0001131 CHEM EHSR 236 1% 651 733 1958 07-11-2006 03:5tpn FrmrKESTON SOLUTIONS 07/11 '06 14:09 NO.142 02/02 T-520 P .001/002 F-545 3058ftesesrthOrWe ^ StateCollege, PA16801 Fan: fll4-231;l5 JrT sT a R C H . .. Arfienditienl Nurtiber EffiactiveDate: Exygen Study Nurtiber protocol amendment `- t . .... 07/08/06-- __ __ P0001131~ Client,Stufe Number. ' . .:!f a j i i ` f i : |a OB 8 Appendix I, Analytical Method V0001782. . .. ----------- ---------------- i--------- ' AMENDEb TP ^ 7717 As percHent request. tedimant eamples ra-tagged for re-extradon by the sponsor win be re-extracted using the follqwlng method: . ' Direct injection Method: Before' the samples are weighed,for foe extrectloa fe jy jjf* mixed thoroughly by vigorously shaking the container.. A one-gram p or^n of W im oni Isvmtghed Into a 15-mlltiWer centrifuge tube,for the extraction- Ten mUliTitom or .77 soatlc add In methanol is added to.e a c h sample. The samples are then sM kdnby hand, vortexed, and sonicated for thirty minutes. The samples are then centnfugsc tor >10 minutes at - 300O rpm. Each'sample is analyzed by LC/MS/MS olwtrospray. .... -------- RATIONALE . Mora usable date will be obtained ,by using,an altoroatemethod. No negative impact on study. TWPAgrfiNSTUBS" .. . * ." ' " StudyDNSMorSignatur ' ' ------------------- r : . bet ' . t : . /i// A h o ld mwMfoatognatln m . .i; SlUdyOMdtorMtnMytnent,Signature x 'Exyfltn SponsorSignatur (IT required) ; Mi ; ~ . . - V i ' - . iV - . : / V - r . ' .. ' . . a - j a w * w S b ': p m . ; :i - . . . . . :. 7 1 Exygen o A :i ero s fe fe :,7 i'M e.. -- ------- ;-- u : .r ; : - f , 7 UaRAAViP:V000iaZM R E C EIV E D T IM E J U L . l l . 4 :2 7P M ' AOMfowraATKiepruaM ' P R IN T T IM E J U L .l l . 4 :2 8 P M Exygen Research Page 109 of 119 Interim Report #27 - Analysis o f Groundwater Samples Exygen Study No.: P0001131 01-06-2007 03:59pm Froa-KSTON SOLUTIONS T-021 P.002/005 F-058 IT RESEARCH 3058 Research Drive Phone: 814-272-1039 State College, PA 16801 Fax: 814-23M 580 . Amendment Number: Effective Date: Exygen Study Number PROTOCOL AMENDMENT 11 12/21/06 - P0001131 Client Study Number: Pag' 1 P0001131 LIBRARY ID: VD0O1S2S4 R E C E IV E D T IM E JA N . 8 . 4 :0 2 P M ADMINISTRATIVE FORM P R IN T T IM E JA N . 8 . 4 :0 4 P M Exygen Research Page 110 o f 119 Interim Report #27 - Analysis o f Groundwater Samples Exygen Study No.: P0001131 S E P .2 0 .2 0 0 5 l:5 2 P M EXYGEN RESEARCH N O .3 7 7 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 o f Occurrence: 03/16/05 Exygen Project # : P760/P1131 Deviation # : 1/1 Client P ro je ct# : NA R e fe re n ce # : 05-122 Reoulatorv Driver: X _____ GMP GLP Other None Sample Description: Deviation Type: (Include Vft fo r methods and SOPs) Protocol Method X SOP V#: 0001658-3 Notebook reference: NA L o g in # : NA Container#: NA L o t# : NA Summary o f Deviation; This deviation pertains to all soil and sediment samples analyzed for percent solids before 07/07/05. a. No blanks or duplicates were run as required by section 9.3. b. Some sample weights exceed the allowable range (> 10g). Cause: ____ Preparation_____ A n a ly s is _____ in stru m e n t____ Client Request X Other Im pact: There has been no negative impact on the study. All o f the percent solid values that were determined during the time period in question are considered valid, although the SOP was not followed. In the newly revised version o f the SOP blanks and duplicates are no longer required. Also, in the new SOP, the allowable amount of sample to be used is < 20 g. All of the samples in question in this deviation weighed less than 20 g. The technician analyzing the samples for percent solids was following the new procedure before it was formally approved. Corractlva Actions: A new version of the SOP has been issued and approved (V000Q427-3). Q ulaa littyy ,Assurance LIBRARY ID: V000164Q-6 3/l/og Date Mi Sponsor Representative __________ Sponsor Management ' Date Date Date ADMINISTRATIVE FORM Exygen Research Page 111 of 119 Interim Report #27 - Analysis o f Groundwater Samples Exygen Study No.: P0001131 S E P .2 0 .2 B 0 5 1 :52PM EXYGEN RESEflRCH BQenR E S E A R C H 3058 Research Drive State College, PA 16801 N O .3 7 7 P .3 Phone; 814-272-1039 Fax: 814-231-1580 DEVIATION FORM General: X Project Specific Deviation Exygen Project # : P760/P1131 Facility Deviation Pane 1 of 1 Date of Occurrence: 06/29/05 Deviation # : 2/2 Client Project # : NA Reference# : Regulatory Driver: Deviation Type: (Include VUfor methods and SOPs) CMP CLP O ther None Sample Description: X Protocol _____ Method V#: NA Notebook reference: NA ____ SOP Login#: L4254/L4256 Container#: C0056480-85 L o t# : NA Summary of Deviation: The protocol states that control and fortified control samples of each matrix will be analyzed; however, control dam was not obtained. Fish was used as the control for the analysis of these six clam samples. Cause: Preparation Analysis Impact: No negative impact on this study. Instrument____Client Request X Other Corrective Actions: Deviation issued. al Investigator Qhjuhl, idy Director Quality Assurance LIB R A R Y ID; V 0 0 0 1 6 4 0 -6 Date Sponsor Management Date A D M IN IS T R A TIV E FORM Exygen Research Page 112 of 119 Interim Report #27 - Analysis o f Groundwater Samples Exygen Study No.: P0001131 S E P .2 0 .2 0 0 5 1 :5 2P M EXYGEN RESEARCH en RESEARCH 3058 Research Drive State College, PA 16601 N O .3 7 7 P .4 Phone: 814-27Z-1039 Fax: 814-231-1580 General: X Project Specific Deviation DEVIATION FORM Facility Deviation Paga 1 of 1 Date of Occurrence: 12/27/04 Exygen Project if : P760/P1131 Deviation # : 3/3 Client Project # : NA Reference # : O S '-f3 9" Regulatory Driver: Deviation Type: (Include Vft for methods end SOPs) GMP GLP Other None Samala Description: Protocol V#: 202-20 Section 5.2.3 Notebook reference: IMA Method X_SOP Login#: _______NA Container#: NA L o t# : NA Summary o f Deviation: SL2114 (30% Dimethyldichlorosilane 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 tor the fish extraction from 12/27/04 through 01/07/05. Cause: Preparation Analysis Instrument Client Request X Other Imoact: No negative Impact on the study. Toluene was used only as a solvent for the glassware preparation. Dimsthyldlchlorosilane, which is the coating agent, was not expired. Corrective Actions: Deviation issued. Signatures: /> PrinoipaJJnvesfigat6r Study Director Quality Assurance /) f ' -------- Date "' Exygen kj^nagdment mm tate/ UN Sponsor Representative Date UN Sponsor Management Asd&~rs Date Date Date LIBR AR Y ID: V 0 0 0 1 640 -6 A D M IN IS T R A TIV E FO RM Exygen Research Page 113 o f 119 Interim Report #27 - Analysis o f Groundwater Samples Exygen Study No.: P0001131 I I RC H 3058 Research Drive Phone: 814-272-1039 State College, PA 16801 Fax:814-231-1580 DEVIATION FORM ___________________________________________________________________ Pag 1of 2 General: X P roject S pecific D eviation _____Facility D eviation Date of Occurrence: 04/26/06 Exygen Project # : P 7 6 0 /P 1 1 3 1 D e via tion # : ______5 C lient P roject # : ________ N A ________ Reference # : 06-076 Reoulatorv Driver: D e v ia tio n T y p e : (Include V # for methods and SOPs) ______ X ______ ___ __ GMP GLP Other None X Protocol ______ M eth od V#: NA Notebook reference: NA Sam ple Description: ~ ~ ______ S O P L og in#: L0008191 C ontainer#: N A L o t # : ___________ N A Summary of Deviation: T h e three sed im ent sam ples in L8191 <C017 2 8 9 2 - C 0 1 7 2 8 9 4 ) w e re o riginally e x tra c te d using the sed im ent m ethod V 0 0 0 1 7 8 2 . Poor recoveries w ere obtained for P F O S , P F O A a n d 13c P F O A . Because o f this, the study sponsor requested the use of an alternative extraction for these compounds, as follows: D irect In jec tio n M eth o d : Before Ihe sam ples w ere weighed for the extraction, they w ere m ixed thoroughly by vigorously shaking the container. A o ne -gram portion o f sed im ent w a s w eig h ed Into a 15-m illiliter centrifuge tube for the extraction. T e n milliliters of 1 % acetic acid in m ethanol w a s add ed to each sam ple. T h e sam ples w ere then shaken by hand, vortexed, and sonicated for thirty minutes. The sam ples w ere then centrifuged for - 1 0 m inutes at - 3 0 0 0 rpm . Each sam ple w as analyzed by LC /M S /M S electrospray. Using this m ethod a cc eptable d a ta w a s obtained for P F O S , but the recoveries fo r P F O A and ,3C P F O A w e re still poor. A nother alternative m ethod w a s then used for P F O A a n d ,3C P F O A , as follows: A lte rn a tiv e S P E M e th o d : T h e sam ples that w e re prepared in 1 % a c e tic acid for th e direct injection m ethod w e re used for this extraction. Five milliliters of each sam ple w as aliquoted into a 50-m L polypropylene centrifuge tube and the volum e w as taken to 4 0 m L with w ater. T h e sam ples w ere then centrifuged for - 1 0 minutes at -3 0 0 0 rpm. The supernatant was then loaded onto a C ,, S P E cartridge conditioned with 10 m L o f m ethanol and 5 m L o f w ate r. T h e e lu a te w a s discarded . A p proxim a tely five milliliters o f m ethanol w a s a dd ed to the cartridge. Five milliliters o f e lu a te w a s colle cte d into a graduated 15 m L polypropylene centrifuge tube. Each sam ple was analyzed by L C /M S /M S electrospray. C a u s e : ____ P r e p a r a tio n _____ A n a ly s is _____ In s tru m e n t____ C lien t R e q u e s t X O ther Im p a c t: More usable data w as obtained. LIBRARY ID: V0001640-6 ADMINISTRATIVE FORM Exygen Research Page 114 of 119 Interim Report #27 - Analysis o f Groundwater Samples Exygen Study No.: P0001131 CHEM EHSR 236 1B 651 733 1958 05-01*2006 OMIm FrwiSTC* 50LUTI05S 05/01 '06 13:46 NO.106 03/03 M H MC3/00J F-31B U M R a m re h lM v i H M rir SW CoUfft, M 10P1 f m SlfiJM M O SEAUCtf O i^ T IG W F O W a . FatfUfrPtvMfcn flMHofOcou[i a rU F o fic t* : H EBSHSffinEBwHS 5 * " VeS, ym-iti StudyDtmelor Q/ fuadty/takcustnuc*U x ~ 7W *&*..CM/t . 'flfeorj!M..Mtafttft.ffl'trft '> . 7>J$V:f-Vv . N- > ' -.s. t . jf. . .. ` ' u '' , i 1 ' ' * . " **`,N* , . '. 't , 11 . "{ i ' /.* 1 . , y iS fX : U nM K V IftV B O tiaO M -S ' Exygen Research Page 115 of 119 Interim Report #27 - Analysis o f Groundwater Samples Exygen Study No.: P0001131 EHS OPNS ENVIRONMENTAL LAB f f 651 778 4226 07/11/06 11:46 S :04/07 NO:681 RESEARCH 3058 Research Drive Phne: 114-212*1039. State College, PA 16801 Fax: 814-231*1580 nerfli ' " X Project Specific Deviation DEVIATION FORM Facility Deviation ft---lai Data of Occurrence: 04/26/06 Exygen Project # : P760/P1131 Deviation # : ' tfe '; . Client Project : ____ n a . : Refi-- noe#: '0 6 -T f8 :. . R m u litw v P riv a r Deviation Vyoe: /Include V# for methods and SOPa) jT ' __ GMP OLP Other None Sample Description: _____ Protocol X Method V#: VC000427-3 Notebook reference: NA Login#: _______NA Container # : ' C00159446: Lot # : NA Bummer of Deviation: .' . ^ One sod sample (C00159446) was weighed at -Bg for percent solid analysis, rether.than at -20g which Is stated In the method. ' " Causa: ___ Preparation_____A nalysis____ Instrument __ __ Client Request ' X ' Other Im p act; ' .. . .. No negative Impact An accurate percent solid number was obtained by slightly altering the calculation used. ' ' y ... C oiractlVi Actione: Deviation issued. ainnitiwi; j . .K m k f PfincIpaUnvestlgator ' ... .... Data / Exygenanijji StudyCfiflractor Quality Assurance / - Date Spoonnsscor Management Exygen Research Page 116 o f 119 Interim Report #27 - Analysis o f Groundwater Samples Exygen Study N o.: P0001131 EHS OPNS ENVIRONMENTAL LAB 651 778 4226 0 7 /1 1 /0 6 1 1 :4 6 0 : 0 6 /0 7 NO:681 RESEARCH 3058 Research Drive Phone: 814-272-1039 State College, P 16801 Fax: 814-231-1580 D eviation form Panarmi: X Project Specific Deviation ___ Facility Deviation ia ti Osta of Occurrence: 68/2EWD6 Exygen Project # ; P7601P1131 Dviation # : 17 Client Project 4 : NA . Referanoe # : r i f c - U - t Reolatorv Driver: Deviation Tvpa: (Includa V# for methods and SOPs) ___ t X ____ ___ . OMP OLP Other None gamble Deaeri otlon: _____ Protocol ____ _ Method V#: 1800 Notebook reference: X. S0P L o gin#: ______ NA Container # : NA L o t# :' NA a ffiai rv f o tiitifg n : ': . Peer review of raw data was not documented per SOP V1800 prior to 6/28/06. Cauee: Im cict: Preparation___ Analysis_____ Instrument Client Request <'" Other Raw date will be more thoroughly evaluated end reviewed before QA Inspection. Corrective Actions: A note to fie will be Issued to all subsequent reports stating that overall eummartes have been pear reviewed. ' ' ' . .. " ' '< MM Exygen M^njgraen} l/l/juA/l.q. Data Sponsr Management .Dat Exygen Research Page 117 o f 119 Interim Report #27 - Analysis o f Groundwater Samples Exygen Study No.: P0001131 EHS OPNS ENVIRONMENTAL LAB 6 5 1 778 4226 07/11/06 11:46 0 :07/07 N0:681 RESEARCH 3058 Research Drive Phone: 814-272-1039 State College, PA 16801 Fax: 814-231-1580 Penerai: . X Project Specific Deviation DEVIATION FORM Pape t'c f 1 Facility Deviation Date of Occtinenoa: 06/28/06 Exygen Project # : P760/P1131 ' Deviation # : ; &/S - Client Project # : N. : Reference*: > V -Ilia temlitonr Privtn ___ ' GMP . X- GLP ... Other ___ None Bamnle Deacrhttlon; Deviation Tvoo: (Include V# lor methods and SOPsl , ; X Protocol Method ___SOP VA NA \ Notebook rference: Login#: L0008121 LO0Q8O81 Container # : Summery o f Dviation: Semple were filled to 280 mL Instead of 200 mL. CO160347 001.60354 00171088 : L o t# : NA Cause: X Preparation ___ Analysis ___ Instrument Impact: . : . .; Samples could not be extracted according to the protocol. Client Request___ Other 6 o m ;U v iiA til9 n P i SpIklngTavela were adjusted to accommodate the alternative volume. Signatures T h /tk r :lpsl Investigator ~ jUt Study Director uiM^Assurfince A lslPfp Data Exygen Sponsor Reprsentative JL Sponsor Management 6 ~ * tU S i - W M y l/n J o i> 1 04 Data Exygen Research Page 118 o f 119 Interim Report #27 - Analysis o f Groundwater Samples Exygen Study No.: P0001131 01-08-2007 03:59pit Fro-*EST0N SOLUTIONS T-021 P.005/005 F-OS0 IT research 3058 Research Drive Phone: 814-Z72-1039 State College, PA 16801 Fax: 814*231-1580 DEVIATION FORM . General: X Project Specific Deviation Facility Deviation ____ ____ Page 1 cf1 Date of Occurrence: N o v.2006Jan. 2007 Exygen Project#: P0001131 D e viatio n #: 10 Client Prqjeet#: P0001131 ftafaraneB#: Rgulawrv Drivw T" DavtarionTvpe: (Inckrte V# ftrmsVmds and SOPs) _____ X _____ _____ GMP GLP Other None Sample Description: _____ Protocol X Method Vif: V001780 Notebook reference: ____ SOP Login#: L0010155 Container#: L ot#: L0010165 ______________ LOOT0254 _____________ . Summary of Deviation: Stock standods and fertMIeation standards usad In the extraction for these logins ware prepared in acetonitrile Instead of m in eral. Causa: X Preparation____Analysis____ Instrument _____Client Request ____ Other Im pact ~ No negative Impact. Compounds are completely soluble In acetonitrile as they are In methanol. Cwradhre Action Deviation Issued. No action taken. LIBRARYID: V0001640-7 R E C EIV E D T IM E JA N . 8 . 4 :0 2 P M ADMINISTRATIVE FORM P R IN T T IM E JA N . 8 . 4 :0 4 P M Exygen Research Page 119 o f 119