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INTERIM REPORT #28 - 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 January 23, 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: 118 Interim Report #28 - Analysis of 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. ( Ch Principal Investigator Exygen Research Jaisimh Study Director Weston Solutions, Inc. Michael A. San Sponsor Representative SM: Company Date Exygen Research Page 2 of 118 Interim Report #28 - Analysis of 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 Date Inspected Date Reported to Date Reported to Principal Exygen Date Reported to Investigator Management Study Director 47. Raw Data and Final Interim Report Review 01/11,12/07 01/23/07 01/23/07 01/23/07 '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 118 Interim Report #28 - Analysis of 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, Exygen: Exygen Research ___ I Date Exygen Research Facility Management: Exygen Research Study Director, Weston Solutions, Inc. m A us JaisimhafCesari P.E., DEE Weston Solutions, Inc. Sponsor Representative, 3M Company: Michael A. Santoro Director of Regulatory Affairs Exygen Research Dane ^ Date Page 4 of 118 Interim Report #28 - Analysis of 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: 12/07/06 Interim Analytical Termination Date: 12/18/06 Interim Report Completion Date: 01/23/07 Exygen Research Page 5 of 118 Interim Report #28 - Analysis of 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 John Flaherty Chas Simons Karen Risha Amy Sheehan Mark Ammerman Brian McAllister Chrissy Edwards Krista Gallant Ling Ling Liu Title Vice President Operations Manager Laboratory Supervisor Scientist Sample Custodian Sample Custodian Technician Technician Technician Exygen Research Page 6 of 118 Interim Report #28 - Analysis of Groundwater Samples Exygen Study No.: P0001131 TABLE OF CONTENTS Page TITLE PAGE.......................................................................................................................1 GOOD LABORATORY PRACTICE COMPLIANCE STATEMENT............................. 2 QUALITY ASSURANCE STATEMENT......................................................................... 3 CERTIFICATION OF AUTHENTICITY.......................................................................... 4 STUDY IDENTIFICATION............................................................................................... 5 PROJECT PERSONNEL.................................................................................................... 6 TABLE OF CONTENTS.................................................................................................... 7 LIST OF 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 118 Interim Report #28 - Analysis of Groundwater Samples Exygen Study No.: 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 118 Interim Report #28 - Analysis of Groundwater Samples Exygen Study No. : P0001131 Figure 1. LIST OF FIGURES Page Typical Extracted Calibration Curve for PFBS in Reagent Water................22 Figure 2. Extracted Standards of PFBS in Reagent Water, 0 ng/L and 25 ng/L, Respectively................................................................................................... 23 Figure 3. PFBS in Reagent Water, 50 ng/L Fortified Reagent Spk A, and 500 ng/L Fortified Reagent Spk B, Respectively.......................................... 24 Figure 4. Chromatogram Representing a Groundwater Sample Analyzed for PFBS (Exygen ID: C0223366, Data Set: 120706A)................................................ 25 Figure 5. Typical Extracted Calibration Curve for PFHS in Reagent Water................ 26 Figure 6. Extracted Standards of PFHS in Reagent Water, 0 ng/L and 25 ng/L, Respectively................................................................................................... 27 Figure 7. PFHS in Reagent Water, 50 ng/L Fortified Reagent Spk A, and 500 ng/L Fortified Reagent Spk B, Respectively.......................................... 28 Figure 8. Chromatogram Representing a Groundwater Sample Analyzed for PFHS (Exygen ID: C0223370, Data Set: 120706A).................................................29 Figure 9. Typical Extracted Calibration Curve for PFOS in Reagent Water................ 30 Figure 10. Extracted Standards of PFOS in Reagent Water, 0 ng/L and 25 ng/L, Respectively................................................................................................... 31 Figure 11. PFOS in Reagent Water, 50 ng/L Fortified Reagent Spk A, and 500 ng/L Fortified Reagent Spk B, Respectively...........................................32 Figure 12. Chromatogram Representing a Groundwater Sample Analyzed for PFOS (Exygen ID: C0223369, Data Set: 120706A)..................................................... 33 Exygen Research Page 9 of 118 Interim Report #28 - Analysis of 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................................................................................................. 34 Exygen Research Page 10 of 118 Interim Report #28 - Analysis of 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 for the analysis of PFBS, PFHS and PFOS found in groundwater samples are summarized in Table I. Quantitative results were obtained for all samples and analytes except for PFBS in two groundwater sample sites that are 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 PFHS and PFOS in the groundwater samples were 102 4% and 108 11%, respectively. The average percent recovery standard deviation for PFBS in the trip blank matrix spike samples was 99 5%. 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 7, 2006, and the analytical termination date for this interim report was December 19, 2006. 4.0 ANALYTICAL TEST SAMPLES Eleven groundwater samples (Exygen ID C0223366 - C0223376) were received at ambient temperature on December 5, 2006 from Tim Frinak at Weston Solutions, Inc. Eight groundwater samples represented two groundwater sampling sites and associated field quality control samples. Three water samples represented one trip blank and two Exygen Research Page 11 of 118 Interim Report #28 - Analysis of Groundwater Samples Exygen Study No.: P0001131 trip blank spikes. All samples were logged in by Exygen personnel and placed in refrigerated storage. Sample log-in and chain of custody information is located in the raw data package associated with this interim report. Storage records will be kept at Exygen Research. 5.0 REFERENCE MATERIAL The analytical 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 (C4F9S03'K+) Transitions Monitored: 299 -->99 Structure: FF FF F SO3 FF FF PFHS Chemical Name: Perfluorohexanesulfonate Molecular Weight: 438 supplied as the potassium salt (C6Fi3S03'K+) Transitions Monitored: 399 - 80 Structure: FFF FF F F SO3 FFF FFF Exygen Research Page 12 of 118 Interim Report #28 - Analysis of Groundwater Samples Exygen Study No.: P0001131 PFOS Chemical Name: Perfluorooctanesulfonate Molecular Weight: 538 supplied as the potassium salt (CsFnSCVK^) Transitions Monitored: 499 -->80 Structure: FFFF FFFF F SO 3 FFFF FFFF 6.0 DESCRIPTION OF ANALYTICAL METHOD The analytical method "V0001780: Method of Analysis for the Determination of Perfluorooctanoic Acid (PFOA) in Water by LC/MS/MS" was used for 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 Ci8 SPE cartridge conditioned with 10 mL of methanol and 5 mL of water. The eluate was discarded. Approximately five milliliters of methanol was added to the cartridge. Five milliliters of eluate was collected into a graduated 15 mL polypropylene centrifuge tube. Each sample was analyzed by LC/MS/MS electrospray. 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 acetonitrile, a 0.1 pg/mL fortification standard was prepared. By taking 10 mL of the 0.1 Exygen Research Page 13 of 118 Interim Report #28 - Analysis of Groundwater Samples Exygen Study No.: P0001131 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 (ML) 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, -8.5 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: API 4000 Biomolecular Mass Analyzer Turbo Ion Spray Liquid Introduction Interface DELL OptiPlex GX400 Windows NT, Analyst 1.4.1 Exygen Research Page 14 of 118 Interim Report #28 - Analysis of Groundwater Samples Exygen Study No.: P0001131 HPLC: Hewlett Packard (HP) Series 1100 HP Quat Pump HP Vacuum Degasser HP Autosampler HP Column Oven HPLC Column: Thermo Fluophase RP, 50 mm x 2.1 mm Column Temp.: -30 C Injection Voi.: 15 pL Mobile Phase (A): 2 mM Ammonium Acetate in water Mobile Phase (B): Methanol Time (min') 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. -8.5 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 of 118 Interim Report #28 - Analysis of 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 PFHS only): Ground water sample Exygen ID: C0223366 Spk C (Set: 120706A), fortified at 100 ng/L with PFHS where: peak area intercept slope dilution factor ng/L PFHS added (fort level) amt in corresponding sample = = = = = = 101016 0.000222 398 1 100 153 (Set: 120706A) From equation 1: Analyte found (ng/L) = fl01016 --0.0002221 x 1 398 = 254 ng/L From equation 2: % Recovery = (254 ng/L - 153 ng/L) x 100% 100 ng/L - 101 % 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. Exygen Research Page 16 of 118 Interim Report #28 - Analysis of Groundwater Samples Exygen Study No.: P0001131 The samples were extracted in one set. The set included one reagent blank and two reagent blanks fortified at known concentrations. The set also contained two sample sites and the field blank and the field blank spikes collected for the groundwater samples. For each site, a sample, a field duplicate and two-matrix field spikes were collected. For each site, a laboratory duplicate and two laboratory matrix spikes were also extracted. 8.0 RESULTS Analytical results 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 in two groundwater sample sites that are not reported (NR) due to quality control failures. Fortification recoveries for PFBS, PFHS, and PFOS in groundwater samples are detailed in Table II. The average percent recoveries standard deviations for PFHS and PFOS in the groundwater samples were 102 4% and 108 11%, respectively. The average percent recovery standard deviation for PFBS in the trip blank matrix spike samples was 99 5%. 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 118 Interim Report #28 - Analysis of Groundwater Samples Exygen Study No.: P0001131 TABLES Exygen Research Page 18 of 118 Interim Report #28 - Analysis of Groundwater Samples Exygen Study No.: P0001131 Table I. Summary of PFBS, PFHS and PFOS in Groundwater Samples Exygen ID Client Sam ple ID C4 Sulfonate PFBS Perfluorobutanesulfonate Analyte Found (ppt, ng/L) Assessed Accuracy (+/- %) C6 Sulfonate PFHS C8 Sulfonate PFOS Perfluorohexanesulfonate______ Perfluorooctanesulfonate Analyte Found (ppt, ng/L) Assessed Accuracy (+/- %) Analyte Found (ppt, ng/L) Assessed Accuracy (+ /-% ) C0223366 C0223366 Rep C0223367 DAL-G W S -6 0 4 S -0 -0 6 1201 D A L -G W S -604S -0-061201 D A L -G W S -604S -D B -061201 C0223370 C0223370 Rep C0223371 D A L -G W S -6 0 4L -0-0 6 1201 DAL-G W S -604L -0-061201 * D AL-G W S -604L -D B -061201 NR NR NR NR NR NR 153 30 51.4 30 - 170 30 54.5 30 - 154 30 58.6 30 _ 214 30 725 30 - 211 30 709 30 208 30 746 30 C0223374 D A L -G W S -T R IP -0 -0 6 1 201 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 118 Interim Report #28 - Analysis of Groundwater Samples Exygen Study No.: P0001131 Table II. Matrix Spike Recovery of PFBS, PFHS and PFOS in Groundwater Samples Sample Description DAL-GWS-604S-0-061201 (C0223366 S p kC , 100 ng/L Lab Splka) DAL-GWS-604S-0-061201 (C02233S6 Spk D, 1000 ng/L Lab Splka) DAL.-GWS-604S-LS-061201 (C02233M, 100 ng/L Fiald Splka) DAL-GWS-604S-HS-061201 (C0223309,1000 ng/L Field Splka) DAL-GWS-604L-0-061201 (C0223370 Spk E, 100 ng/L Lab Spike) DAL-GWS-604L-0-061201 (C0223370 Spk F, 1000 ng/L Lab Spike) DAL-GWS-604L- LS-061201 (C0223372,100 ng/L Field Spike) DAL-GWS-604L-HS-061201 (C0223373, 1000 ng/L Field Spike) DAL-GWS-TRIP-LS-061201 (C022337S, 100 ng/L Field Spike) DAL-GWS-TRIP-HS-061201 (C0223370,1000 ng/L Field Spike) C4 Sulfonate PFBS_____________ C6 Sulfonate PFHS_____________ C8 Sulfonate PFOS Amount Amt Found Amount Amt Found Amount Amt Found Amount Spiked in Sample Recovered Recovery in Sample Recovered Recovery in Sample Recovered Recovery H 'L ) (ng/L) (ng/L) (%) (ng/L) (ng/L) (%) (ng/L) (ng/L) (%) 100 1000 100 1000 100 1000 100 1000 NR NR NR NR NR NR NR NR NR NR 153 254 101 51.4 162 111 NR NR 153 1110 96 51.4 1030 98 NR NR 153 254 101 51.4 158 107 NR NR 153 1180 103 51.4 1170 112 NR NR 214 323 109 725 850 . NR NR 214 1200 99 725 1950 123 NR NR 214 323 109 725 928 NR NR 214 1230 102 725 1950 123 100 ND 103 103 ND 98.9 99 ND 94.9 95 1000 ND 956 96 ND 1010 101 ND 982 98 Average: Standard Deviation: 99 5 Average: Standard Deviation: 102 4 'Sample residue exceeds the spiking level significantly (3x spiking level); therefore, an accurate recovery value cannot be calculated. ND = Not detected at or above the Limit of Quantitation (LOQ) of 25 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. Average: Standard Deviation: 106 11 Exygen Research Page 20 of 118 Interim Report #28 - Analysis of Groundwater Samples Exygen Study No.: P0001131 FIGURES Exygen Research Page 21 of 118 Interim Report #28 - Analysis of Groundwater Samples Exygen Study No. : P0001131 Figure 1. Typical Extracted Calibration Curve for PFBS in Reagent Water 120706A itv l.rd b (PFBS): "U n ta r ' R tg rts io n w tigM ing): y 119 x + 375 (f * 0.9949) Area, counts Exygen Research Page 22 of 118 Interim Report #28 - Analysis of Groundwater Samples Exygen Study No.: P0001131 Figure 2. Extracted Standards of PFBS in Reagent Water, 0 ng/L and 25 ng/L, Respectively I X C m 7 0 t' C -P FB S (Standard) 299.*99.0 mir -$amph 1 o f 34from 1207&6A.wiff Arta: 375 counts Height: 163e+0OTtp* RT: 0.535 min 13.78 ,,14.13 Intensity, cps XC 120706-1 PFBS (S tandrd)209.0/99.0 mu sample 2 of 3 4 from 12070GA.wiff A rt : 5831 count* Height: 4.58e+002 cps RT: 0.544 min 0.54 Intensity, cps Exygen Research Page 23 of 118 Interim Report #28 - Analysis of 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 I Rnagint Control-P FBS (Unknown) 299.0/99.0a m n -w m p to 9 o f 4 from 12Q706A.wiff not found) 12.75 Intensity, cps Intensity, cps Area: 6720 counts Height: 4.80e+002 cps RT: 0.540 min 0.55 Reagent Spk B PFBS (QC) 299.0/00.0 amu sample 11 of 3 4 from 120706A.wiff Area: 50324 counts Height: 4.20e+003 cps RT: 0.551 min 0 .5 5 Intensity, cps Exygen Research Page 24 of 118 Interim Report #28 - Analysis of Groundwater Samples Exygen Study No.: P0001131 Figure 4. Chromatogram Representing a Groundwater Sample Analyzed for PFBS (Exygen ID: C0223366, Data Set: 120706A) I C02233C6 PFBS (UH*kowh) 299L199L0 jm it . ta m p / H o f 34from 120706A.wiff Aroa: 25205 counts Height: 1.74o+0Q3cp* RT: A S w /jf 0.54 Intensity, cps Exygen Research Page 25 of 118 Interim Report #28 - Analysis of Groundwater Samples Exygen Study No.: P0001131 Figure 5. Typical Extracted Calibration Curve for PFHS in Reagent Water 120706A rtv l.rd b (PFHS): HL in ji" R tgrtssion ("1 / x " weighting): y 398 x + 0.000222 (( 0 .9 9 9 9 ) Area, counts Exygen Research Page 26 of 118 Interim Report #28 - 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 XC1207M-4 PFHS (Stundard) 399.0/90.6amu tam p f* 1 o f 54from 120706A,wiff H O t fOM M d) Intensity, cps XC 120706-1 PFHS (Stand ard) 399.0/80.0 amu - sample 2 of 3 4 from 120706A.wiff Area: 10744 counts Height: 4 .74e*002 cps RT: 8.30 m in 8.30 Intensity, cps Exygen Research Page 27 of 118 Intensity, cps Interim Report #28 - Analysis of 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 R t jg m t Control PFHS (IMtmowm) 399.6/90.0 amtit -sam p/* 9 o f 34from 120796A.wiff 4>m Amot foutu/) 13.49 R * j g t n t Spk A PFHS (QC) 39*9.0/80.0 im u sam pl* 10 of 3 4 from 120700A.wiff Aroa: 19138 counts Hoight: 9 .69*+ 002 cps RT: 8.41 min Tim , min 8.41 A ro i: 191950 counts HoigM: 9.92*+ 003 ops RT: 8.40 min Intensity, cps Intensity, cps Exygen Research Page 28 of 118 Interim Report #28 - Analysis of Groundwater Samples Exygen Study No.: P0001131 Figure 8. Chromatogram Representing a Groundwater Sample Analyzed for PFHS (Exygen ID: C0223370, Data Set: 120706A) I C*22337Q' P f:HS(UH*ttown)399.Ort*.0JMU - tJ m p fo 2 4 o f 34 from 120796A.wtff Araa: 85005 couato H tig kt: 4 8 fr+ 0 0 3 cp RT: 9.41 m in 8.41 Intensity, cps Exygen Research Page 29 of 118 Interim Report #28 - Analysis of Groundwater Samples Exygen Study No.: P0001131 Figure 9. Typical Extracted Calibration Curve for PFOS in Reagent Water 120706A fv1 .rdb (PFOS): " U n i i i " Regression C'1 / ** weightin']): y * 33G x + -0.0008 (r * 0.0905) Area, counts Exygen Research Page 30 of 118 Interim Report #28 - Analysis of Groundwater Samples Exygen Study No.: P0001131 Figure 10. Extracted Standards of PFOS in Reagent Water, 0 ng/L and 25 ng/L, Respectively X C n n 6 4 -P F O S (Standard) 499.<V80,0amn pvaA not found) 1o f 34 from 12070M.wiff XC 120706-1 PFOS (Standard) 409.0/80.0 a m u -s a m p l 2 of 3 4 from 120706A.wiff Area: 8556 counts Height: 5.G0e+002 cps RT: 11.0 min T im *, min 11.02 Intensity, cps Intensity, cps Exygen Research Page 31 of 118 Intensity, cps Interim Report #28 - Analysis of 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 I Roagont Control PFOS (Unknown) 499.W90.9imM -ta m p h 9 o f ^4 from 120709A.wiff A r t* : 343counts Height: 2.0U+001 cps RT: 11.0 m i* Area: 15423 counts Height: 1.0Qe+003 cps RT: 11.1 min I Reagent Spk B PFS (QC) 499.0/80.0 amu sample 11 of 3 4 from 120706A.wiff Area: 155515 counts Height: 9.91e+003 cps RT: 11.1 min Intensity, cps Intensity, cps Exygen Research Page 32 of 118 Interim Report #28 - Analysis of Groundwater Samples Exygen Study No.: P0001131 Figure 12. Chromatogram Representing a Groundwater Sample Analyzed for PFOS (Exygen ID: C0223369, Data Set: 120706A) Intensity, cps C0223369* PFOS (Q Q 4 9 9 .0 M .6 a m u -ta m p / 20o f 34 from 12070SA.wiff Ara: 39220 c o u n ts HtfgOt: 2.47*+IM3cpt RT: 11.1 milt 11.08 2400 2300 2200 2100 2000 1900 1800 1700 1600 1500 1400 1300 1200 1100 1000 900 800 10.58 700 600 500 400 300 200 100 0 J----------- 1-----------1----- ------ 1............ . 12 3 4 ........................... .............. 5 6 7 8 9 10 11 12 13 14 15 16 17 T im t, min Exygen Research Page 33 of 118 Interim Report #28 - Analysis of Groundwater Samples Exygen Study No.: P0001131 APPENDIX A Study Protocol P0001131 (Exygen Study No. P0001131) with Analytical Methods, Protocol Amendments, and Deviations Exygen Research Page 34 of 118 Interim Report #28 --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 Performing Laboratory: Exygen Research 3058 Research Drive State College, PA 16801 Phone: (814) 272-1039 Sponsor Representative: M ichael A. Santoro Director o f Regulatory Affairs 3M Building 0236-01-B-10 St. Paul, MN 55144 Phone: (651) 733-6374 Page / oj 6S Exygen Research Page 35 of 118 Interim Report #28 - Analysis of Groundwater Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 DISTRIBUTION: 1) Jaisimha Kesari, Study Director, Weston Solutions 2) John M. Flaherty, Principal Investigator, Exygen Research 3) Michael A. Santoro, Sponsor Representative, 3M Company 4) Exygen Research Quality Assurance Unit Exygen Research Page 2 o f 65 Page 36 of 118 Interim Report #28 - Analysis of Groundwater Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 PROTOCOL APPROVAL Study Title: Analysis o f Perfluorobutanesulfonate (PFBS), Perfluorohexanesulfonate (PFHS), and Perfluorooctanesulfonate (PFOS) in Water, Soil, Sediment, Fish, Clams, Vegetation, Small Mammal Livers and Small Mammal Serum Using LC/MS/MS for the 3M Decatur Monitoring Program Exygen Protocol Number: P0001131 APPROVALS Jaisimha Weston Solutions Michael A. Safitoro, Sponsor Representative 3M Company ^ / Richard A. Grj Exygen Rese: President, Facility Management ead, Quality Assurance Unit Date 2$ -C & ' Date Page 3 oj 65 Exygen Research Page 37 of 118 Interim Report #28 - Analysis of Groundwater Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 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 BJECTIVE..................................................................................................................................................................... 6 TESTING FACILITY......................................................................................................................................................b STUDY DIRECTOR........................................................................................................................................................7 SPONSOR REPRESENTATIVE................................................................................................................................... 7 PRINCIPAL INVESTIGATOR..................................................................................................................................... 7 PROPOSED EXPERIMENTAL START AND TERMINATION D A TES........................................................... 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 ETHODS........................................................................................................................................... 11 GLP STA TEM ENT..........................................................................................................................................................11 REPO RT............................................................................................................................................................................ 11 SAFETY AND HEA LTH ............................................................................................................................................... 12 AMENDMENTS TO PROTOCOL............................................................................................................................... 13 DATA RECORD K EE PIN G ..........................................................................................................................................13 QUALITY A SSURANCE.............................................................................................................................................. 14 RETENTION OF DATA AND ARCHIVING............................................................................................................ 14 APPENDIX I, ANALYTICAL M ETHODS.................................................................................................................15 Page 4 o f 65 Exygen Research Page 38 of 118 Interim Report #28 - Analysis of Groundwater Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 INTRODUCTION The purpose of this study is to perform analysis for perfluorobutanesulfonate (PFBS), perfluorohexanesulfonate (PFHS) and perfluorooctanesulfonate (PFOS) in water, soil, sediment, fish, clams, vegetation, small mammal livers and small mammal serum using LC/MS/MS for the 3M Decatur Monitoring Program. The study will be audited for compliance with EPA TSCA Good Laboratory Practice Standards 40 CFR 792 by the Quality Assurance Unit o f Exygen Research. TEST MATERIALS The test materials are perfluorobutanesulfonate (PFBS), perfluorohexanesulfonate (PFHS) and perfluorooctanesulfonate (PFOS) and are all supplied by 3M. PFBS Chemical Name: Perfluorobutanesulfonate Molecular Weight: 338 supplied as the potassium salt (C4 F9 SO3I O Lot Number: 101 Purity: 96.7% Transitions Monitored: 299 -> 99 Structure: PFHS Chemical Name: Perfluorohexanesulfonate Molecular Weight: 438 supplied as the potassium salt (CFnSCL'lO Lot Number: SE036 Purity: 98.6% Transitions Monitored: 399 -> 80 Structure: Page 5 o f 65 Exygen Research Page 39 of 118 Interim Report #28 - Analysis of Groundwater Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 PFOS Chemical Name: Perfluorooctanesulfonate Molecular Weight: 538 supplied as the potassium salt (CgFnSCVlO Lot Number: 217 Purity: 86.9% Transitions Monitored: 499 -* 99 Structure: FFFF FFFF FFFF FFFF OBJECTIVE The purpose o f this study is to perform analysis for perfluorobutanesulfonate (PFBS), perfluorohexanesulfonate (PFHS) and perfluorooctanesulfonate (PFOS) in water, soil, sediment, fish, clams, vegetation, small mammal livers and small mammal serum for the 3M Decatur Monitoring Program using the current versions of the following Exygen analytical methods: V0001780: "Method of Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Water by LC/MS/MS" V0001781: "Method of Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Soil by LC/MS/MS" V0001782: "Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Sediment by LC/MS/MS" V0001783: "Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Fish and Clams by LC/MS/MS" V0001784: "Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Vegetation by LC/MS/MS" V0001785: "Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Small Mammal Liver by LC/MS/MS" V0001786: "Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Small Mammal Serum by LC/MS/MS" TESTING FACILITY Exygen Research 3058 Research Drive State College, PA 16801 Phone: (814) 272-1039 Page 6 o f 65 Exygen Research Page 40 of 118 Interim Report #28 - Analysis of Groundwater Samples Exygen Study No.: P0001131 Exygen Protocol Number: POOOl 131 STUDY DIRECTOR Jaisimha Kesari P.E., DEE Weston Solutions, Inc. 1400 Weston Way West Chester, PA 19380 Phone: (610) 701-3761 Fax: (610) 701-7401 j .kesari@westonsolutions.com SPONSOR REPRESENTATIVE Michael A. Santoro 3M Company Director o f Regulatory Affairs 3M Building 0236-01-B-10 St. Paul, MN 55144 Phone: (651) 733-6374 PRINCIPAL INVESTIGATOR John M. Flaherty Exygen Research 3058 Research Drive State College, PA 16801 Phone: (814)272-1039 john.flaherty@exygen.com PROPOSED EXPERIMENTAL START AND TERMINATION DATES It is proposed that the analytical portion o f this study be conducted from October 01, 2004 to December 31, 2005. The actual experimental start and termination dates w ill be included in the final report. Page 7 o f 65 Exygen Research Page 41 of 118 Interim Report #28 - Analysis of 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: Water (groundwater and surface water) Soil Sediment Fish Clams Vegetation Small Mammal Liver Small Mammal Serum The samples will be collected by Weston Solutions. The control samples will be purchased and prepared by the testing facility. Purchase and processing details for the control samples will be included in the final report associated with this study. The test systems were chosen to access the environmental impact of PFBS, PFHS and PFOS in the Decatur, Alabama area. SAMPLE PROCUREMENT, RECEIPT AND RETENTION Water, soil, sediment, fish, clam, vegetation, small mammal liver and small mammal serum samples will be received at Exygen directly from Weston Solutions. The details o f sample procurement for this study are outlined in the 3M work plan entitled "Phase 2 Work Plan for Sampling Environmental Media." The number and types o f samples collected will vary depending availability in the field. The total number o f samples received and analyzed for each matrix will be documented in the final report associated with this study. Water, soil, and sediment samples will be used as received without further processing at Exygen. These samples will be stored refrigerated at 2C-8C. Fish, clam, vegetation and small mammal liver samples will be processed according to the appropriate analytical method (see Appendix I). These samples will be stored frozen at < -10"C. Small mammal whole blood samples will be centrifuged in the field at the time o f collection and the serum fraction will be used for the study. Small mammal serum will be stored frozen at S -10C. The receipt and processing of the samples will be documented in the final report and raw data associated with the study. Page 8 o f 65 Exygen Research Page 42 of 118 Interim Report #28 - Analysis of Groundwater Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 SAMPLE IDENTIFICATION Prior to analysis, each sample will be assigned a laboratory sample reference number. The reference number will be unique and will distinguish each laboratory sample that is processed throughout the analytical procedure. Chromatographic data will be identified by the laboratory sample reference number. Sample storage conditions and locations will be documented throughout the study. ANALYTICAL PROCEDURE SUMMARY References: V0001780: "Method of Analysis for the Determination of Perfluorooctanoic Acid (PFOA) in Water by LC/MS/MS" V0001781: "Method of Analysis for the Determination of Perfluorooctanoic Acid (PFOA) in Soil by LC/MS/MS" V0001782: "Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Sediment by LC/MS/MS" V0001783: "Method of Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Fish and Clams by LC/MS/MS" V0001784: "Method of Analysis for the Determination of Perfluorooctanoic Acid (PFOA) in Vegetation by LC/MS/MS" V0001785: "Method o f Analysis for the Determination of Perfluorooctanoic Acid (PFOA) in Small Mammal Liver by LC/MS/MS" V0001786: "Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Small Mammal Serum by LC/MS/MS" The above methods use analytical conditions capable o f separating the isomers o f PFBS, PFHS and PFOS. The final report will include the isomers summed into total PFBS, total PFHS, and total PFOS found. VERIFICATION OF ANALYTICAL PROCEDURE A laboratory control sample will be used for the preparation o f fortified control samples. The test substance will be made into solutions as per the method, and added to the matrices via a micropipette. For water sampling, Exygen will supply one bottle per sample collected. The bottles will be 500 mL precleaned Sci/Spec Premier wide mouth HDPE bottles. These bottles have been routinely used for fluorochemical sample Page 9 o f 65 Exygen Research Page 43 of 118 Interim Report #28 - Analysis of Groundwater Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 collection at the testing facility and have been shown to be free o f PFBS, PFHS and PFOS. Samples will be added to each container to a volumetric fill line at 200 mL. A field duplicate, a low field spike and a high field spike of each sample will be collected. The low and high field spike bottles will contain PFBS, PFHS and PFOS as well as perfluorooctanoic acid (PFOA) and 1.2-13C perfluorooctanoic acid (13C PFOA). PFOA and 13C PFOA are included in the solutions used to spike the samples. The results for PFOA and 13C PFOA will not be reported in this study. Exygen will supply one field blank (control water) and two field blank spikes (control water fortified with PFBS, PFHS and PFOS at a low and high level) for every twenty samples collected. At the testing facility, each water sample (excluding field duplicates and field spikes) will be extracted in duplicate and will also be fortified at a low and high concentration with PFBS, PFHS and PFOS and processed through the described procedure to determine method accuracy and to check for bias. For soil, sediment, clams, and vegetation, Exygen will supply one 500 mL precleaned Sci/Spec Premier wide mouth HDPE bottle per sample collected or a zip-seal bag. All containers/bags used for sample collection will be shipped to the sample location. Samples will be added to each container or bag in the field. At the testing facility, each sample will be extracted in duplicate and will also be fortified at a known concentration with PFBS, PFHS and PFOS at both a low and high level and processed through the described procedure to determine method accuracy and to check for bias. For small mammal liver, Exygen will supply a 50 mL polypropylene centrifuge tube. For small mammal serum, Exygen will supply a collection kit for each sample containing serum separator tubes (red top), vacutainers, needle holders and needles, transfer pipettes, and polypropylene tubes. At the testing facility, each liver and serum sample will be extracted in duplicate and will also be fortified at a known concentration with PFBS, PFHS and PFOS at both a low and high level and processed through the described procedure to determine method accuracy and to check for bias. Low and high spiking levels for each matrix are defined below: M atrix Low Spiking Level High Spiking Level Water 500 ng/L 5000 ng/L Soil Sediment Fish Clams Vegetation Small Mammal Liver 4ng/g 4 ng/g lOng/g lOng/g 10ng/g 10ng/g 40ng/g 4 0 ng/g 100ng/g 100ng/g 100ng/g 100ng/g Small Mammal Serum 10 ng/mL 100 ng/mL Page 10 o f 65 Exygen Research Page 44 of 118 Interim Report #28 - Analysis of Groundwater Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 Recoveries are anticipated to be between 70% and 130% o f the fortified levels; however, the exact precision and accuracy will be determined by the analysis o f the quality control samples described above. A statement of accuracy will be included in the final report. METHOD FOR CONTROL OF BIAS Control o f bias will be addressed by taking representative sub-samples from a homogeneous mixture o f each matrix from untreated control samples, and by analyzing at least two levels o f fortifications. STATISTICAL METHODS Statistics will be limited to those specified in the subject methods and to the calculation o f average recoveries, as applicable. GLF STATEMENT All aspects o f this study shall be performed and reported in compliance with EPA TSCA Good Laboratory Practice Standards 40 CFR 792. The final report or data package (supplied to the Sponsor) shall contain a statement that the study was conducted in compliance with current and applicable GLP standards and will outline any deviations in the study from those standards. This statement will be signed by the Study Director and Sponsor Representative. REPORT A final report will be prepared by the principal investigator or their designee at the conclusion o f the study. The report will include, but will not be limited to, the following: The name and address o f the Study Director, Sponsor Representative, and of the testing facility. A statement o f GLP compliance (any related documentation, such as chain-of-custody records, must be in the study records). Page I! o f 65 Exygen Research Page 45 of 118 Interim Report #28 - Analysis of Groundwater Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 The signed and dated statement by the Exygen Research Quality Assurance Unit regarding dates o f study inspections and dates findings were reported to the Study Director and Management. A description o f the exact analytical conditions employed in the study. If the subject method was followed exactly, it is necessary to include only a copy o f the analytical method. Any modifications to this method will be incorporated into the report. If the method is photo-reduced, the project number and page number must be included on each page. Description of the instrumentation used and operating conditions. All results from all sets analyzed. Control and fortified samples will be identified and the data table will include sample number and fortification level. Representative chromatograms for each analyte in each matrix, including chromatograms o f a standard and a control sample, and a chromatogram at a fortification level. The location o f the analyte peaks will be clearly identified in all chromatograms. All circumstances that may have affected the quality or integrity o f the data will be documented in the report. Locations where raw data and the final report are to be archived. Additions or corrections to the final report shall be in the form o f an amendment signed by the Study Director. The amendment shall clearly identify that part o f the report that is being altered and the reasons for the alterations. The amendment will be signed and dated by the Study Director and the Sponsor Representative. All applicable requirements for reporting o f study results as per 40 CFR 792.185. SAFETY AND HEALTH Laboratory personnel will practice good sanitation and health habits. Every reasonable precaution shall be taken to prevent inadvertent exposure o f personnel and the environment to the test or reference substance(s). Page 12 o f 65 Exygen Research Page 46 of 118 Interim Report #28 - Analysis of Groundwater Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 AMENDMENTS TO PROTOCOL All significant changes to the analytical protocol outlined here will be expressed in writing, signed and dated by the Study Director and Sponsor Representative. Amendments usually will be issued prior to initiation o f study plan change. However, when a change is required without sufficient time for the issue o f a written amendment, that change may be effected verbally with supporting documentation signed and dated by the Study Director and followed with a written amendment as soon as possible. In this case, the effective date o f the written amendment will be the date o f the documented change. Copies o f the signed amendments will be appended to all distributed study plan copies. The original amendment will be maintained with the original study plan. Any deviations from the study plan or from the analytical method as provided will be documented and reported promptly to the Sponsor Representative. DATA RECORD KEEPING Records to be maintained include the following (as appropriate): Sample tracking sheet(s) Sample receipt records, storage history, and chains o f custody History and preparation o f standards (stock, fortification, calibration) Description o f any modifications to the method Instrument run sheets, bench-sheets or logs Analytical data tables All chromatographic and instrumental conditions Sample extraction and analysis dates A complete listing o f study personnel, signatures and initials Chronological presentation o f all study correspondence Any other documentation necessary for the reconstruction o f the study Chromatograms- All chromatograms will contain the following: Sample identification, injection date, arrow or other indication o f the area o f interest, and injection number corresponding to the run. Additionally, fortifications will include the amount o f analyte added and the sample number o f the sample that was fortified. Analytical standard chromatograms will additionally include the concentration (e.g., pg/mL). Page 13 o f 65 Exygen Research Page 47 of 118 Interim Report #28 - Analysis of Groundwater Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 As part o f the documentation the following sheets will be included in each analytical set: a run sheet listing the samples to be run in the set, and an instrument conditions sheet describing the instrument type and operating conditions. QUALITY ASSURANCE The QA Unit o f Exygen Research will inspect the study at intervals adequate to assure compliance with GLP's, and will report the findings o f audits to the Study Director, Exygen Management, and the Sponsor Representative. RETENTION OF DATA AND ARCHIVING All hard copy raw data, including, but not limited to, the original chromatograms, worksheets, correspondence, and results shall be included with the data package submitted to the Study Director. These will be archived with the original study plan, amendments, final report, and all pertinent information from the Sponsor. The testing facility shall keep all electronic raw data and any instrument, equipment, and storage logs for the period o f time specified in 40 CFR 792.195. An exact copy o f the materials submitted to the study director will also be kept at Exygen Research. Exygen will obtain permission from the study director before discarding or returning samples. Exygen Research Page 14 o f 65 Page 48 of 118 Interim Report #28 - Analysis of Groundwater Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 APPENDIX I ANALYTICAL METHODS V0001780: "Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Water by LC/MS/MS" V0001781: "Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Soil by LC/MS/MS" V0001782: "Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Sediment by LC/MS/MS" V0001783: "Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Fish and Clams by LC/MS/MS" V0001784: `Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Vegetation by LC/MS/MS" V0001785: "Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Small Mammal Liver by LC/MS/MS" V0001786: "Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Small Mammal Serum by LC/MS/MS" Exygen Research Page IS o f 6S Page 49 of 118 Interim Report #28 - Analysis of Groundwater Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 ANALYTICAL METHOD Method Number: V00I780 Method o f Analysis for the Determ ination o f Perfloorooctaaolc A d d (PFOA) io W ater by LC/MS/MS Analytical Tearing Facility: Exygen Research 3058 Research Drive State College, PA 16801 Approved By: " v U c A ______ Paul Connolly ' Technical Leader, LC-MS, Exygen Research '/ John Flaherty / Vice President, Operations, Exygen Research to|z.w/oW Date &L Exygen Research Total Pages: 7 Page 16 o f 65 Page 50 of 118 Interim Report #28 - Analysis of Groundwater Samples Exygen Study No.: P0001131 Exygen Protocol Number: POOO1131 Exygen Research Method Number V000P8O t a n a ly tic a l m e th o d Method o f Analysis for the Determination ofPerfluorooctanoic Acid (PFOA) in W ater 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 Spcctrometric 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 safely precautions. 3.0 Sample Requirement 3.1 At least 40 mL o f test sample for attraction. 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 Water - HPLC 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 reiuimg to 0.00001 g. 5.4 50 mL disposable polypropylene centrifuge tubes. 5.5 15 mL disposable polypropylene centrifuge lubes. 5.6 Disposable micropipets (50-100uL, 100-200uL). 5.7 125-mL LDPE narrow-mouth bottles. 5.8 2 m L clear HPLC vial kit. 5.9 Disposable pipettes. 5.10 Autopipettes (100-1000 pL and 10-100 pL), with disposable tips. 5.11 Waters Sep Pak Vac 6 cc (Ig ) tC l8 SPE cartridges. Page 2 o f 1 Page 7 o f 65 Exygen Research Page 51 of 118 Interim Report #28 - Analysis of Groundwater Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 Exygen JUtevch Method Number VOOO17SQ ANALYTICAL METHOD Method o f Analysis for the Determination ofPerfluorooctanoic Acid (PFOA) in Water by L C /M S/M S 5 12 SPE vacuum manifold. 5.13 Centrifuge capable o f spinning 50 m L polypropylene tubes at 3000 rpm. 6.0 Chromatographic System 6.1 Analytical Column: Fluophaae RP (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 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 Rale % B (m ^m jnl 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. The above conditions are intended as a guide and may be changed m order to optimize foe MSMS system. 8.0 Preparation o f Solutions 8.1 Mobile Phase 8.1.1 2 mM ammonium acetate in water is prepared b y adding 0.154 g o f ammonium acetate to 1000 mL o f water. Alternate volumes rosy be prepared. Exygen Research Page 18 of 65 Page 52 of 118 Interim Report #28 - Analysis of Groundwater Samples Exygen Study No.: P0001131 Exygen Protocol Number: POOO1131 Exygen Research Method Number VQOOI78Q | ANALYTICAL M ETHOD j 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 P repares 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-rnL 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 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 the 1.0 pg/raL solution to a final volume o f 100 with methanol in a 125 m L LDPE bottle. 9.1.5 A 0.01 pg/mL fortification solution o f PFOA is prepared by bringing 10 mL o f the 0.1 p ^ m L 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. Concentration ofFortificttion Solution (nob) 0 10 10 10 100 100 100 Fortification Volume of Volume Fortified Control (PL) Semole (ml/) 0 40 100 40 200 40 400 40 too 40 200 40 400 40 Final Concentration of Calibration Standard (ppt>* 0 25 so 100 250 500 1000 Calibration Standard ID (example) XCmroddyy-0 XCnunddyy-] XCmmddyy-2 XCmmddyy-3 XCmmddyy-4 XCmmddyy-5 XCmmddw-6 * The extracted concentration o f the calibration standard is equal to 8* its initial concentration, due to the concentration o f the standard during the extraction (SPE). XC " extracted calibration standard. Page 4 oi ' Page 9 o f 65 Exygen Research Page 53 of 118 Interim Report #28 - Analysis of Groundwater Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 Exygntemrch Method Number V000I780 I ............ ANALYTICAL METHOD ~ Method o f Analysis for the Determination o f PerHuorooctanoic 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 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 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 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 Measure 40 mL o f sample or a portion o f sample diluted to 40 mL with water into 50 mL polypropylene centrifuge tubes (fortify as needed, replace lid and mix well). 11.2 Condition the C u SPE cartridges (1 g, 6 mL) by passing 10 mL methanol followed by 5 m L o f HPLC water (~ 2 drop/sec). Do not let column run dry 11.3 Load sample on conditioned Ci i SPE cartridge. Discard eluate. 11.4 Elute with -5 m L 100% methanol. Collect 5 mL o f eluate into graduated 15 m L polypropylene centriftige tubes (final volume - 5 mL). I t . 5 Analyze samples using elcctrospray LC/MS/MS. 12.0 Chromatography 12.1 12.2 12.3 12.4 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. Standards o f PFOA corresponding to at least five o r m ore concentration level s must be included in an analytical set. An entire set o f extracted calibration standards m ust be included at the beginning and at the end o f a sample set. Extracted standards must be interspersed between every 5-10 samples. As an alternative, an entire set o f extracted calibration standards may be injected at the beginning o f a set followed by extracted calibration standards interspersed every 5-10 samples (to account for a seoond set o f extracted standards). In either case, extracted calibration standards must be the first and last injection in s sample set. 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 PSe 5 of ? Page 20 o f 65 Exygen Research Page 54 of 118 Interim Report #28 - Analysis of Groundwater Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 Exygen Research Method Number V0001780 ANALYTICAL METHOD___________________________ Method o f Analysis for the Determination ofPerfluorooctanoic Acid (PPOA) in Water 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 amu from a parent o f 413 arau. 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. I f 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 extracted calibration standards that could be excluded must not exceed 20% o f the total num b: 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 inatrument operation, and the standards o r the relevant set o f samples should be reanalyzed. 13.6 Retention times between standsrds 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 o f 7 Page 2/ o f 65 Exygen Research Page 55 of 118 Interim Report #28 - Analysis of Groundwater Samples Exygen Study No.: P0001131 Exygen Protocol Number: POOOl 131 Exygea Research Method Number V000I780 | ANALYTICAL M ETHOD ......... 1 Method o f Analysis for the Determination o f Perfluorooctanoic 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 (Va) [ total analyte found (ng/L) - analyte found in control (ng/L)] | ^ analyte added (ng/L) Exygen Research Pig 7 of " Page 22 o f 65 Page 56 of 118 Interim Report #28 - Analysis of Groundwater Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 ANALYTICAL METHOD Method Num ber V0001781 M ethod o f Analysis fo r the D e te rm in a te s o f Perfluorooctanoic Acid (PFOA ) Id Soil by LC /M S/M S Analytical Testing Facility: Exygen Research 30S8 Research Drive State College, PA 16801 Approved By: C -JL P*aaul ConnnooUllyv Technical Leader, LC-MS, Exygen Research ia ln ,/c W Date Date Exygen Research Total Pages: 7 Page 23 o f 65 Page 57 of 118 Interim Report #28 - Analysis of Groundwater Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 Eygen Research_________________________________________________Method Number V000178J f ANALYTICAL METHOD Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Soil by LC/MS/MS 1.0 Scope Thie method is to be employed for the isolation and quantitation o f perfluorooctanoic acid by High Performance liq u id Chromatography coupled to a tandem Mass Spectrometric Detector (LC/MS/MS) in s o il 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 A tleast IS g o ftestsam p lefo rex traclio n . 3.2 No sample processing is needed for soil samples. 3.3 Samples stored refrigerated should be allowed to equilibrate to room temperature. 3.4 Alt 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. 4.0 Reagents and Standards 4.1 Water - HPLC 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-mL LDPE narrow-mouth bottles. 5.8 2 mL cletr HPLC vial kit. 5.9 Disposable pipettes. 5.10 Autopipettes (100-1000 pL and 10-100 pL), with disposable tips. 5.11 Waters Sep P ik Vac 6 cc (Ig ) tC18 SPE cartridges. 5.12 SPE vacuum manifold. 5.13 Ultrasonic bath. Page 2 of 7 Page 24 o f 65 Exygen Research Page 58 of 118 Interim Report #28 - Analysis of Groundwater Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 Exygen Research MethodNumber VOO1781 ANALYTICAL METHOD Method o f Analytic for the Determination o f Perfluorooctanoic Acid (PFOA) in Soil by L C /M S/M S 5.14 Wrist-action shaker. 5.15 Centrifuge capable o f spinning 50 m L polypropylene tubes &l 5000 rpm. 6.0 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: T im e /'m in i 0.0 1.0 8.0 2 0 .0 2 2 .5 %A 65 65 25 25 65 F low R ate K B fm L/m inl 35 0.3 35 0.3 75 0.3 75 0.3 35 0.3 6.6 Injection Volume: 1S pL (can be increased to as m uch 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: ElectrosprayN egativeM RM m ode,m onitoring413 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 o f ammonium acetate to lOOOmLofwater. Alternate volumes m ay be prepared. 3Page of? Page 25 o f 65 Exygen Research Page 59 of 118 Interim Report #28 - Analysis of Groundwater Samples Exygen Study No.: P0001131 Exygcn Protocol Number: P0001131 Exygca Research Method Number V0001781 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/Foitification 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 p ^ m L fortification solution o f PFOA is prepared by bringing 10 mL o f the 10 p ^ m L solution to a final volume o f 100 with methanol in a 125 m L LDPE bottle. 9.1.4 A 0.1 pg/mL fortification solution o fPFO A is prepared by bringing 10 mL o f foe 1.0 pg/mL solution to a final volume o f 100 with methanol in a 125 mL LDPE bottle. 9.1.5 A 0.01 pg/mL fortification aolution o fP F O A is prepared by bringing 10 mL o f the 0.1 pg/tnL 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. Concentration ofFortification Solution (rob) 0 10 10 10 100 100 100 Fortification Volume of Volume Fortified Control (uL) Sample (mL) 0 40 100 40 200 40 400 40 100 40 200 40 400 40 Final Concentration of Calibration Standard (dpO* 0 25 so 100 250 500 1000 Calibration Standard ID (example) XCmmddyy-0 XCmmddyy-l XCmmddyy-2 XCmmddyy-3 XCmmddyy-4 XCmmddyy-5 XCmmddW'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 f 7 Page 26 o f 65 Exygen Research Page 60 of 118 Interim Report #28 - Analysis of Groundwater Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 ExygaoEUwarcb MethodNuntor VOOOI78I I ANALYTICAL METHOD Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Soil by LC/MS/MS 9.2.3 9.2.4 9.2.5 A zero standard solution (reagent blank) must be prepared with each set o f standards extracted. Store all extracted calibration standards in l5mL polypropylene tubes 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 S m L o f methanol) and two reagent controls fortified at known concentrations (lab control spike) to verify procedural recovery for the batch. 10.2 Requirements for field and laboratory duplicates and spikes will be specified in the quality assurance plan for this project. 11.0 Sample Extraction 1 U Weigh 5 g o f sample into 50 m L polypropylene centrifuge tubes (fortify as needed, replace lid and mix well). 112 Add 5 m L o f methanol and shake on a wrist action shaker for -1 5 minutes U J Transfer the tubes to an ultrasonic bath and sonicate fo 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 u SPE cartridges (1 g, 6 mL) by passing 10 mL methanol followed by 5 m L o f HPLC water ( - 2 drop/sec). Do not let column run dry 11.7 Load (decant) the aample on the conditioned C u SPE cartridge. Discard eluate. 11.8 Elute with - 5 mL 100% methanol. Collect 5 mL o f eluate into graduated 15 mL polypropylene centrifuge tubes (final volume S 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 o f PageS o f 7 Page 27 o f 65 Exygen Research Page 61 of 118 Interim Report #28 - Analysis of 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 Research Method Number VOOQ1781 I ANALYTICAL METHOD Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Soi I by LC/MS/MS extracted calibration standards m ay be injected at the beginning o f a set followed by extracted calibration standards interspersed every 5-10 samples (to account for a second set o f extracted standards). In either case, extracted calibration standards must be the first and last injection in a sample set. 12.4 Use linear standard curves for quantitation. Linear standard curves are generated for the analyte by linear regression using 1/x weighting o f peak area versus calibration standard concentration using M assLynx 3 3 (or equivalent) software system. 12.5 Sample response should not exceed standard responses. Any samples that exceed standard responses should be further diluted and reanalyzed. 13.0 Acceptance Criteria 13.1 13.2 13.3 13.4 13.5 13.6 Chromatogram must show a peak o f a daughter ion at 369 amu from a parent o f 413 amu. The 413 amu parent corresponds to the PFOA anion, while the daughter ion (369 amu) represents the loss o f carbon dioxide. Method blanks must not contain PFOA at levels greater than the LOQ. If a blank contains PFOA at levels greater than 50 ng/L, then a new blank sample must be obtained and the entire set must be re-extracted. Recoveries o f control spikes and matrix spikes must be between 70-130% of their known values. 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 detennine if re-extraction is warranted. Any calibration standard found to be a statistical outlier by using the Huge Error Test, may be excluded from the calculation o f 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 The correlation coefficient (R) for calibration curves generated must be 20.992 (RJ 20.985). If calibration results fall outside these limits, then appropriate steps must be taken to adjust instrument operation, and ihc standards or the relevant set o f samples should be reanalyzed. Retention times between standards and samples must not drift more than 4 % within an analytical run. I f retention tim e drift exceeds this limit within an analytical run then the set must be reanalyzed. Pag* 6 of 7 Page 28 o f 65 Exygen Research Page 62 of 118 Interim Report #28 - Analysis of Groundwater Samples Exygen Study No.: P0001131 Exygen Protocol Number: POOO1131 Exygeo R iM iie h Method Number V0001781 ANALYTICAL METHOD__________________________ Method o f Analysis for the Determination o f Perfiuorooctanoic 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. 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 foe 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) - fPFOA found fna/L) x volume extracted (0.04D1 sample weight (5 g) 14.4 Use the following equation to calculate foe 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 o f 7 Page 29 oj 65 Exygen Research Page 63 of 118 Interim Report #28 - Analysis of Groundwater Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 ANALYTICAL METHOD Method N um ber V0001782 M ethod o f Analysis for the D eterm ination o f Perflnorooctanoic Acid (PFOA ) io Sediment by LC/MS/MS Analytical Testing Facility: Exygen Research 3058 Research Drive State College, PA 16801 Approved By: C Jl. Paul Connolly I Technical Leader, LC-MS, Exygen Research /( * /________ /ohn Flaherty / Vice Prsidait, Operations, Exygen Research --------lS,lzbM Date Date Exygen Research Total Pages: 7 Page 30 o f 65 Page 64 of 118 Interim Report #28 - Analysis of Groundwater Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 Exygen Reeearch Method Number V0001782 | ANALYTICAL METHOD | Method o f Antlyii for the Determination o f Perfluorooctanoic Acid (PFOA) in Sediment by L C /M S/M S 1.0 S cope 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 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 teat sample for extraction. 3.2 No sample proceasing is needed for sediment simples. 3.3 Samples stored refrigerated should be allowed to equilibrate to room temperature. 3.4 All sample* must be thoroughly mixed 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 Water -H P L C grade 4.2 Methanol - HPLC 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 w ith a variable volume injector capable o f injecting 5-200 jiL 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'tnL L D P E n arrow -m outh 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. S.l 1 Waters Sep Pak Vac 6 cc (Ig) tC18 SPE cartridges. S.12 SPE vacuum manifold. Page 2 o f 7 Page 31 o f 65 Exygen Research Page 65 of 118 Interim Report #28 - Analysis of Groundwater Samples Exygen Study No.: P0001131 E x y g e n P ro to c o l N u m b e r: P 0 0 0 1 131 Exygca Rmtrch Method Number V0QO1782 ANALYTICAL M ETHOD Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Sediment bv LC/MS/MS 5.13 Vortexer. 5.14 Wrist-action shaker. 5. 13 Centriflige capable o f pinning 50 m L polypropylene tubes al 3000 rpm. 6.0 Chromatographic System 6.1 Analytical Column: Fluophase RP (Keystone Scientific), 2.1 mm x SO mm. 5m (P/N: 82505-052130) 6.2 Temperature: 30*C 6.3 Mobile Phase (A) : 2 mM Ammonium Acetate in Water 6.4 Mobile Phase (B) : Methanol 6.5 Gradient Program: Time (min) 0.0 1.0 8.0 20.0 22.5 'A& 65 65 25 25 65 Flow Rate 2U1 (mUmin) 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 mh 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 mL o f water. Page 3 o f7 Page 32 o f 63 Exygen Research Page 66 of 118 Interim Report #28 - Analysis of Groundwater Samples Exygen Study No.: P0001131 Exygen Protocol Number: PQ001131 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 8.2 Extraction Solutions 8.2.1 1% acetic acid in water is prepared by adding 10 mL o f acetic acid lo 1000 oiL o f water. Alternate volumes m ay b e prepared. 9.0 Standard Preparation 9.1 Standard Stock/Fortification Solution 9.1.1 Prepare a stock solution o f MOO jig/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 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 1 0 ng/mL fortification solution o f PFOA is prepared by bringing 10 mL o f the 10 pgfaiL solution to a final volume o f 100 with methanol in a 125 mL LDPE bottle. 9.1.4 A Q .l pgrinL fortification solution o f PFOA 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 0.0! jig/mL fortification solution o f PFOA is prepared by bringing 10 mL o f the 0.1 p ^ m L 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 s 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 Concentration of Fortification Soluti) irm/mL) too 100 100 10 5 2 Volume (mL) 10 5 2 10 10 10 Diluted to (mL) 100 100 100 100 100 100 Final Concentration (nK/mL) to o 50 2.0 1.0 0.5 0.2 Page 4 of 7 Page 33 o f 63 Exygen Research Page 67 of 118 Interim Report #28 - Analysis of Groundwater Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 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 all 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 o r less) must include at least one untreated control and two untreated controls fortified at known concentrations (lab control spike) to verify procedural recovery for the batch. 10.2 Requirements for field and laboratory duplicates and spikes will be specified in the quality assurance plan for this project. 11.0 Sample Extraction 11.1 Weigh 5 g o f sample into 50 mL polypropylene centrifuge tubes (fortify as needed, replace lid and mix well). 11.2 Add 35 mL o f 1% acetic acid, cap, vortex and shake on a wrist action shaker for - 6 0 minutes. 11.3 Centrifuge the tubes at -3000 ipm 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) the sample on foe conditioned C u SPE cartridge. Discard eluate. 11.6 Add 20 m L o f methanol to the sediment left in foe bottom o f foe 50 mL centrifuge tube. Cap, vortex and shake on a wrist action shaker for -3 0 minutes. 11.7 Centrifuge the tubes si -3 0 0 0 rpm for - 2 0 minutes. 11.8 Decant the methanol onto foe same SPE cartridge. Collect foe eluate. 11.9 Wash the column with 4 m L o f methanol. Collect foe eluate and add it to the eluate collected in step 11.8. 11.10 Condition a seco n d C is 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 - 5 mL 10054 methanol. Collect 5 m L 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 o f 7 Page 34 o f 65 Exygen Research Page 68 of 118 Interim Report #28 - Analysis of Groundwater Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 Exygen Rmxrcfa Method Number VOOOI7B2 1 ANALYTICAL M ETHOD Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFO A) in Sediment bv 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 o r 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. Standards must be interspersed between every 5-10 sample*. 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 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 0.2 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 20V* o f the total number o f extracted standards injected. 13.5 The correlation coefficient (R) for calibration curves generated must be 0.992 (R3 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 7 Page 35 o f 65 Exygen Research Page 69 of 118 Interim Report #28 - Analysis of Groundwater Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 Exygea Rctearch Method Number VOOO1732 1 ANALYTICAL METHOD | Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Sediment by LC/MS/MS 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 limn 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, baaed 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. 142 For samples foitified 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 to ng/g (ppb). PFOA found (ppb) ~ fPFQA found (ng/mL) x final volume fS mLll sample weight (5 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 sohds(%(] Pafl 7 f 7 Page 36 o f 65 Exygen Research Page 70 of 118 Interim Report #28 - Analysis of Groundwater Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 ANALYTICAL METHOD Method Number: V001783 M ethod o f Analysis fo r the Determ ination o f Perflnorooctanolc A d d (PFOA ) io Fish ad Clame by LC/MS/MS Analytical Testing Facility: Exygen Research 3058 Research Drive SUte College, PA 16801 Approved By: Paul Connolly Technical Leader, LC-MS, Exygen Research '/r> d S 'ohn Flaherty Vice President, Operations, Exygen Research Dale Date Exygen Research Total Pages: 8 Page 37 o f 65 Page 71 of 118 Interim Report #28 - Analysis of Groundwater Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 Exygen Resetrch Method Number V0001783 analytical method Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Fish and C lanu by LC/MS/MS 1.0 Scope This method it 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 safely precautions. 3.0 Sample Requirement 3.1 At least 20 g o f test sample for extraction. 3.2 Samples should be processed before extraction. Place the frozen sample in a food processor and homogenize 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 4.0 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*200 pL connected to a tandem Masa 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 8 Page 38 of 65 Exygen Research Page 72 of 118 Interim Report #28 - Analysis of Groundwater Samples Exygen Study No. : P0001131 Exygen Protocol Number: P0001131 Exygen Reaearch Method Number V0001783 ANALYTICAL METHOD 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 m L pear-shaped flasks. 5.7 SOm L disposable polypropylene centrifuge tubes. 5.8 15 mL disposable polypropylene centrifuge tubes. 5.9 Disposable micropipets (50-100uL, 100-200uL). 5.10 125-mLLDPE narrow-mouth bottles. 5.11 2 mL clear HPLC vial kit. 5.12 Disposable pipettes. 5.13 Autopipettes (100-1000 pL and 10-100 |iL), with disposable tips. 5.14 SPE tubes (20mL) (Supelco cat. no.N 057l77). 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: 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 raM Ammonium Acetate in Water 6.4 Mobile Phase ( B ) : Methanol 6.5 Gradient Program: Time (mini 0.0 1.0 8.0 20.0 22.5 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 A rea-'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. Page 3 of 8 Page 39 o f 65 Exygen Research Page 73 of 118 Interim Report #28 - Analysis of Groundwater Samples Exygen Study No.: P0001131 s'***1 Exygen Protocol Number: P00Q1131 Exygen Research Method Number V0Q01783 ANALYTICAL M ETHOD Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Fish and Clams by LC/MS/MS 7.0 MS/MS System 7.1 Mode: Electrospny 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 o f ammonium acetate to 1000 m L o f water. 8.2 Extraction Solutions 8.2.1 8.2.2 2% ascorbic acid in methanol is prepared by dissolving 2 g o f ascorbic acid in 100 mL o f methanol. 30% Dimethyldichlorosilane in toluene is prepared by bringing 3 m l o f dimethyldichlorosilane to a final volume o flO mL with toluene. Alternate volumes may be prepared. 9.0 Standard Preparation 9.1 Standard StodtfFortification 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 (corrected for purity) and dilute to 100 mL with methanol in a 125-mL LDPE bottle. A 1.0 pg/mL fortification solution o f PFOA is prepared by bringing t mL o f the 100 pg/raL 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/raL solution to a final volume o f 100 with methanol in a 125 mL LDPE bottle. A 0.01 pgfaiL 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 firom the date o f preparation. Page 4 of 8 Page 40 o f 65 Exygen Research Page 74 of 118 Interim Report #28 - Analysis of Groundwater Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 Exygen Research Method Number V0001783 [ ANALYTICAL M ETHOD Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Fish and G am a 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 1.0 p.g/mL fortification solution. The following is a typical example: additional concentrations may be prepared aa needed. Concentration o f Fortification Solution (ue/rnL) 1.0 1.0 1.0 0.05 0.025 0.1 0.005 Volume (mL) S.O 2.5 1.0 10 10 10 10 Diluted to (mL) 100 100 100 100 100 100 100 Final Concentration (ua/mL) 0.05 0.025 0.01 0.005 0.0025 0.001 0.0005 9.2.3 Store all calibration standards in 125-mL LDPE narrow-mouth bottles at 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 o r less) must include at least one untreated control and two untreated controls fortified at known concentrations (lab control spike) to verify procedural recovery for the batch. 10.2 Requirements for field and laboratory duplicates and spikes will be specified in the quality assurance plan for this project. 11.0 Sample Extraction 11.1 Weigh 5 g o f frozen sample into 50 mL polypropylene centrifuge tubes (fortify aa needed, replace lid and mix well). 11.2 Add 30 mL o f acetonitrile and shake on a wrist action shaker f o r -15 minutes 11.3 Place the tubes in a freezer fo r-1 hour. 11.4 Pack and condition the SPE tubes and silanize the pear-shaped flasks. 11.5 Pack the 20 m L SPE tubes in sequence with 2 g florisil, 2 g silica gel, 2 g carbon, and 1 g LC-NH*. Condition tbc 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% 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 a stream o f nitrogen. Page < ofK Page 41 o f 65 Exygen Research Page 75 of 118 Interim Report #28 - Analysis of Groundwater Samples Exygen Study No.: P0001131 Exygea Protocol Number: P0001131 Exygtn Research Method Number V000178J ANALYTICAL M ETHOD Method o f Analysis for the Determination o f Perfhiorooctanoic Acid (PFOA) in Fish and Clams by LC/MS/MS 11.7 CeotrifUge the 50 mL polypropylene tubes containing sample at -2000 rpm for - 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 eluste in the 125 m L silanized pear-shape flask. 11.9 Add 10 mL o f acetonitrile to the sample in the 50 m L centrifuge tube. Homogenize the frozen fat phase using a tissumizer for - 3 0 seconds and rinse the tissumizer with - 1 0 mL o f acetonitrile into the tube. 11.10 Shake the sim ple again f o 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 m L 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. 11.14 Pass 20 mL o f acetonitrile through the SPE column and combine the eluate in the same pear-shaped flaik. 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 Iqject the same amount o f each standard, sample and fortified sample into the LC/MS/MS system. A calibration standard must precede and follow all analyzed samples. 12.2 Standards o f PFOA corresponding to at least five 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 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 M assLynx 3.3 (or equivalent) software system. Pag 6 of* Page 42 o f 65 Exygen Research Page 76 of 118 Interim Report #28 - Analysis of Groundwater Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 Exygen Research Method Number VOOO1763 ANALYTICAL m e t h o d Method o f Analysis for the Determination o f Periluorooctanoic Acid (PFOA) in Fish and Clams by LC/MS/MS 12.5 Sample response should not exceed standard responses. Any samples that exceed standard responses should be farther 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% 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. 13.4 Any calibration standard found to be a statistical outlier by using the Huge Error Test, may be excluded from the calculation o f the calibration curve. However, the total number o f calibration standards that could be excluded must not exceed 20% o f the total number o f standards injected. 13.5 The correlation coefficient (R) for calibration curves generated must be 20.992 (RJ 20.983). I f calibration results fall outside these limits, then appropriate steps must be taken to adjust instrument operation, and the standards o r the relevant set o f samples should be reanalyzed. 13.6 Retention times between standards and samples must not drift more than 4 % within an analytical run. I f retention time drift exceeds (his 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 M aw Lynx software program: PFOA found (ng/mL) fPeak 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) - IPFQA found (ng/mL) x final volume fmLI x DF1 sample weight (g) DF factor by which the final volume was diluted, if necessary. Page 7 of S Page 43 o f 63 Exygen Research Page 77 of 118 Interim Report #28 - Analysis of Groundwater Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 Exygen Research Method Number V0001783 I ANALYTICAL m e t h o d Method o f Analysis for the Determination o f Perfiuorooctanoic 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)] , [QQ analyte added (ng/g) Exygen Research Page 8 ot'S Page 44 o f 65 Page 78 of 118 Interim Report #28 Analysis of Groundwater Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 ANALYTICAL METHOD Method Number: V0001784 M ethod o f Analysts for the D eterm ination o f Perfluorooctauoic Acid (PFOA ) in Vegetation by LC/MS/MS Analytical Testing Facility: Exygen Research 3058 Research Drive S u te College. PA 16801 Approved By: H - X C O U ___ Paul Connolly ' Technical Leader, LC-MS, Exygen Research n//r> f U / ________ John Flaherty ^ Vice President, Operations, Exygen Research ___ l a t M Date Date Exygen Research ToUi Pages: 7 Page 45 o f 65 Page 79 of 118 Interim Report #28 - Analysis of Groundwater Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 Exygen Remrch Method Number VOOOI' M ANALYTICAL METHOD Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Vegetation by LC/MS/MS 1.0 Scope This method c to be employed for the isolation and quantitation o f perfluorooctanoic acid by High Performance Liquid Chromatography coupled to a tandem Mass Spectromtrie Detector (LC/MS/MS) in vegetation. 2.0 Safety 2 .1 Always observe safe laboratory practices. 2.2 Consult the appropriate MSDS before handling any chemical for proper safety precautions. 3.0 Sample Requirement 3.1 At least 20 g o f test sample for extraction. 3.2 Samples should be processed before extraction. Place the frozen sample in a food processor and homogenize with dry ice. Place 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 Water - HPLC grade 4.2 Acetonitrile - HPLC grade 4.3 Carbon (120400 mesh) - Reagent grade 4.4 Methanol - HPLC grade 4.5 Silica gel (60-200 mesh) - Reagent grade 4.6 Florisil (60-100 mesh) -R eag en t grade 4.7 Superclean LC-NH2 - Reagent grade 4.8 1-O ctanol-H PLC grade 4.9 L-Ascorbic a d d - 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 S.O Instrument and Equipment 5.1 A high performance liquid chromatograph capable o f pumping up to 2 solvents equipped with variable volume injector capable o f injecting 5-200 pL connected to a tandem Mass Spectrometer (LC/MS/MS). 5.2 A device to collect raw data for peak integration and quantitation. 5.3 A nalyticalbalancecapableofreadingtoO .00001 g. Page 2 O Page 46 o f 65 Exygen Research Page 80 of 118 Interim Report #28 - Analysis of Groundwater Samples Exygen Study No.: P0001131 Exygen Protocol Number: POOOl 131 Exygm Research Method Number V0Q017M ___________________________ ANALYTICAL M ETH O D Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Vegetation by LC7MS/MS 5.4 Rotary evaporator. 5.5 125 mL pear-shaped flasks. 5.6 50 mL disposable polypropylene centrifuge tubes. 5.7 15 mL disposable polypropylene centrifuge tubes. 5.8 Disposable micropipets (50-100uL, 100-200uL). 5.9 125-mL LDPE narrow-mouth bottles. 5.10 2 mL clear HPLC visl kit. 5.11 Disposable pipettes. 5.12 Autopipettes (100-1000 p L and 10-100 pL), with disposable tips. 5.13 SPE tubes (20mL) (Supelco cat. no. N057177). 5.14 Wrist action shaker. 5.15 Centrifiige capable o f spinning 50 mL polypropylene tubes at 2000 rpm 6.0 Chromatographic System 6.1 Analytical Column: Fluophase RP (Keystone Scientific), 2.1 mm x 50 mm, 5p (P/N: 82505-052130) 6.2 Temperature: 30C 6.3 Mobile Phase (A ): 2 mM Ammonium Acetate in Water 6.4 Mobile Phase ( B ) : Methanol 6.5 Gradient Program: Time (min) 0.0 1.0 8.0 20.0 22.5 SLA 65 65 25 25 6S 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 Run Time: - 23 minutes. The above conditions are intended as a guide and may be changed in order to optimize the HPLC system. 7.0 MS/MS System 7.1 Mode: Electrospray Negative MRM mode, m onitoring 413 -* 369 m/z for PFOA. Page 3 of 7 Page 47 o f 65 Exygen Research Page 81 of 118 Interim Report #28 - Analysis of Groundwater Samples Exygen Study No.: P0001131 Exygen Protocol Number: POOO1131 Exygen Research 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 may b e 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 o f ammonium acetate to 1000 mL o f water. 8.2 Extraction Solutions 8.2.1 %22 2% ascorbic acid in methanol is prepared by dissolving 2 g o f ascorbic a d d in 100 mL o f methanol. 30% Dimcthytdichlorosilane 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 -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. A 1.0 pg/mL 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 mL LDPE bottle. A 0.1 pg/mL fortification solution o f PFOA is prepared by bringing 10 m L o fth e 1.0 pg/mL solution to 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 pgftnL 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 di Page 48 o f 65 Exygen Research Page 82 of 118 Interim Report #28 - Analysis of Groundwater Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 Exygea iUievcb Method Number V0001784 ANALYTICAL m e t h o d Method o f Analysis for the Determination o f Perfluorooctsnoic 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 Final ofFortification Solution (ua/mL) 1.0 Volume (mL) 5.0 Diluted to (mL) 100 Concentration (nt/mLi 0.05 1.0 2.5 100 0.025 1.0 1.0 100 0.01 0.05 10 100 0.005 0.025 10 100 0.0025 0.1 10 100 0.001 0.005 10 100 0.0005 9.2.3 Store all calibration standards in 125-tnL LDPE narrow-mouth bottles at 2*C to 6C, 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 (Isb 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. 1 1 .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 50 mL polypropylene tubes containing sample at -2000 rpm for -1 0 minutes. 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 1 g LC-NH2. Condition the columns with 20 mL o f methanol, then 20 mL o f acetonitrile. Discard all washes. Do not allow the column to diy. 11.6 Silanize the 125 raL 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 a 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 49 o f 65 Exygen Research Page 83 of 118 Interim Report #28 - Analysis of Groundwater Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 Exygen Research Method Number V0001784 ANALYTICAL m e t h o d I Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Vegetation by LC/MS/MS 11.8 Add 20 mL o f acetonitrile to the sample in the 50 raL centrifuge tube. 11.9 Shake the sample again for - 1 0 minutes on a wrist-action shaker. 11.10 Centrifuge the SO mL polypropylene tubes containing sample at -2000 rpm for 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/disaolve. 11.15 Transfer the extracts to HPLC vials using disposable pipets. 11.16 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 se t Extracted standards must be interspersed between every 5-10 samples. As an alternative, an entire set of extracted calibration standards m ay be injected at the beginning o f a set followed by extracted calibration standards interspersed every 5-10 samples (to account for a second set o f extracted standards). In either case, extracted calibration standards 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 peak o f a daughter ion at 369 amu from a parent o f 413 amu. The 413 amu parent corresponds to the PFOA anion, while the daughter ion (369 amu) represents the loss o f carbon dioxide. Pag* 6 of 7 Page SO o f 65 Exygen Research Page 84 of 118 Interim Report #28 - Analysis of Groundwater Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 Exygen R*uarch Method Number V000I7S4 analytical method___________________ Method o f Analysis for the Detennination o f Perfluorooctanoic Acid (PFOA) in Vegetation by LC/MS/MS 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 falla outside the acceptable limits, the entire set o f samples should be re-extracted. 13.4 Any calibrati) standard found to be a statistical outlier by using the Huge Error Test, may be excluded from the calculation o f the calibration curve. However, the total number o f calibration standards that could be excluded must not exceed 20% o f the total number o f standards injected. 13.5 The correlation coefficient (R) for calibration curves generated must be 20.992 (R* 20.985). I f calibration results fall outside these limits, then appropriate steps must be taken to adjust instrument operation, and the standards or the relevant set o f samples should be reanalyzed. 13.6 Retention times between standards and samples must not drift more than 4 % within an analytical run. 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 (na/mL) x final volume (mL) 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)] ^ ^ analyte added (ng/g) Page 7 o f > Page 51 o f 65 Exygen Research Page 85 of 118 Interim Report #28 - Analysis of Groundwater Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 ANALYTICAL METHOD Method Num ber V0001785 M ethod o f AnalysU fo r the D eterm ination o f Perfloorooetanoic Acid (PFOA ) in Small M amm al Liver by LC/MS/MS Analytical Testing Facility: Exygen Research 3058 Research Drive State College, PA 16801 Approved By: C-- ____ Paul Connolly I Technical Leader, LC-MS, Exygen Research </ A 4 L John Flaherty / Vice President, Operations, Exygen Research Date Date Exygen Research Total Pages: 7 Page 52 o f 65 Page 86 of 118 Interim Report #28 - Analysis of Groundwater Samples Exygen Study No.: P0001131 Exygen Protocol Number: P 0 0 0 131 Exygen Reacaich Method Number VOOO1785 | ...... ANALYTICAL M ETH OD Method o f Analyst* for the Determination o f Perfluorooctanoic Acid (PFOA) in Small Mammal Liver by LC/MS/MS 1.0 Scope This method is to be employed for the isolation and quantitation o f perfluorooctanoic acid by High Performance Liquid Chromatography coupled to a tandem Mass Spectrometnc Detector (LC/MS/MS) in total] mammal liver. 2.0 Safoty 2.1 Always observe safe laboratory practices. 2.2 Consult the appropriate MSDS before handling any chemical for proper safety precautions. 3.0 Sample Requirement 3.1 At least 5 g o f test sample for extraction. 3.2 Samples should be processed before extraction. Place the frozen sample in a food 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 o f analysis. Alternately, i f there is an insufficient amount o f sample (-less than 5 g), then no processing is necessary and the sample can be used as supplied 3.3 Sample collection procedures will be specified in the sampling plan for this project. 4.0 Reagents and Standards 4.1 W ater-H P L C grade 4.2 M ethanol-H PL C grade 4.3 A cetonitrile-H PLC grade 4.4 Ammonium Acetate - A.C.S. Reagent Grade 4. $ Perfluorooctanoic Acid - Sigma-Aldrich 3.0 Instrument and Equipment 5.1 A high performance liquid chromatograph capable o f pumping up to 2 solvents equipped with a variable volume injector capable o f injecting 3-200 pL connected to a tandem Mass Spectrometer (LC/MS/MS). 5.2 A device to collect raw data for peak integration and quantitation. 3.3 Analytical balance capable o f reading to 0.00001 g. 5.4 30 m L disposable polypropylene centrifiige tubes. 3.3 15 mL disposable polypropylene centrifuge tubes. 5.6 Disposable micropipets (50-100uL, !00-200uL). 5.7 125-mL LDPE narrow-mouth bottles. 5.8 2 mL clear HPLC vial k it Page 2 o p Page 53 o f 65 Exygen Research Page 87 of 118 Interim Report #28 - Analysis of Groundwater Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 Exygen R o w c h Method N umber VOOOI785 ANALYTICAL M ETHOD Metbod o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Small Mammal Liver by LC/MS/MS $.9 Disposable pipettes. 5.10 Autopipettes (100-1000 pL an d 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: 30"C 6.3 Mobile Phase (A) : 2 mM Ammonium Acetate in Water 6.4 Mobile Phase (B) : Methanol 6.5 Gradient Program: Time (mini 0.0 1.0 8.0 20.0 22.5 65 65 25 25 65 Flow Rate % B fmL/min'i 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 u 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, m onitoring 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. yPage o f ' Page 54 o f 65 Exygen Research Page 88 of 118 Interim Report #28 - Analysis of Groundwater Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 Exygen R u n rch 1Method Number VOOO 785 a n a ly t ic a l m e t h o d Method o f Analysis for the Detennination o f Periluorooctanoic Acid (PFOA) in Small Mammal Liver by LC/MS/MS J 8.0 Preparation o f Solutions 8.1 Mobile Phase 8.1.1 2 tnM ammonium acetate in water is prepared by adding 0 154 g of ammonium acetate 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/raL o f PFOA by weighing 10 mg o f analytical standard (corrected for purity) and dilute to 100 mL with methanol in a l25mL LDPE bottle. 9.1.2 A 1.0 pg/mL 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 mL LDPE bottle. 9.1.3 A 0.1 pgAnL fortification solution o f PFOA is prepared by bringing 10 mL o f 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 4C and are stable fbr a maximum period o f 6 months from file 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 o f Fortification Volume D iluted to Concentration Solution (na/m L) (m L) (m L) (ne/m L) 100 5.0 100 5.0 100 2.0 100 2.0 100 1.0 100 1.0 5.0 10 100 0.5 2.0 10 100 0.2 1.0 10 100 0.1 9.2.3 Store all calibration standards in 125-mL 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 needed. Pge 4 o f 7 Page 55 o f 65 Exygen Research Page 89 of 118 Interim Report #28 - Analysis of Groundwater Samples Exygen Study No.: P0001131 Exygen Protocol Number: POOO1131 Exygea Reweb Method Number VOOO1785 | .............. ANALYTICAL METHOD Method o f Analysis few 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 sample* 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 aasurance plan for this project. 11.0 Sample Extraction 11.1 Weigh 1 g o f sample into a SO m L polypropylene centrifuge tubes (fortify as needed, replace lid and mix well). Note that alternate weights o fliv er may be measured depending on the sample size available for use. 11.2 Add water to the sample for a final volume o f 10 mL. 11.3 Homogenize sample using a tissuemizer for ~1 minute. 11.4 Transfer 1 mL o f the sample using a disposable pipette into a IS mL disposable centrifuge tube. 11.5 Add 5 mL o f acetonitrile and shake for - 2 0 minutes on a wrist-action shaker. 11.6 Centrifuge the tubes at -3 0 0 0 rpm for - 5 minutes. 11.7 Decant foe supernatant into a SO m L disposable centrifuge tube and add 3S mL o f wata1. 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 m L o f eluate into a graduated IS mL 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 m ust 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 -1o 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 S-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 l/x weighting o f peak area Pg 5 of 7 Page 56 o f 65 Exygen Research Page 90 of 118 Interim Report #28 - Analysis of Groundwater Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 Exygen Research Method Number V0001785 I ANALYTICAL M ETHOD 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.S Sample response should not exceed standard responses. Any samples that exceed standard responses should be ftuther diluted and reanalyzed. 13.0 Acceptance Criteria 13.1 Chromatogram must show 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 toss 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 tbe 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 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, tbe local 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 (R1 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 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/mL) (Peak area - intercept) x DF x aliquot factor slope DF " factor by which tbe final volume was diluted, i f necessary. Aliquot factor - 10 Page 6 of 7 Page 57 o f 65 Exygen Research Page 91 of 118 Interim Report #28 - Analysis of Groundwater Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 Exygea Rneareb Mtihod Number VOOOI785 | ANALYTICAL 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 (%) - f 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 lo ng/g (ppb). PFOA found (ppb) - iPFOA found (ng/mL) x final volume <mU] sample weight (g) Exygen Research Page 7 o f 7 Page 58 o f 65 Page 92 of 118 Interim Report #28 - Analysis of Groundwater Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 ANALYTICAL METHOD Method Number. V0001786 M ethod o f Analysis for the D eterm ination o f Perfluorooctanoic Acid (PFOA ) in Small Mammal Serum by LC/MS/MS Analytical Testing Facility: Exygen Research 3058 Research Drive State College, PA 16801 Approved By: ^v--?--V. C--lAvi Paul Connolly I Technical Leader, LC-MS, Exygen Research \//Z> / / ihn Flaherty Vice President, Operations, Exygen Research Date /'A J ? Date Exygen Research Total Pages: 7 Page 59 o f 65 Page 93 of 118 Interim Report #28 - Analysis of Groundwater Samples Exygen Study No.: P0001131 Exygen Protocol Number: P 000J131 Exygen Research Method Number V0001786 | A N A LiTlCA L M ETHOD Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Small Mammal Serum by LC/MS/MS 1.0 Scope This method is to be employed for the isolation and quantitation o f perfluorooctanoic acid by High Performance Liquid Chromatography coupled to a tandem Mass Spectrumetric Detector (LC/MS/MS) in small mammal serum- 2.0 Safety 2.1 Always observe safe laboratory practices. 2.2 Consult the appropriate MSDS before handling any chemical for proper safety precautions. 3.0 Sample Requirement 3.1 At least 1 m L o f teat sample for extraction. 3.2 No sim ple 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 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 iryector 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-lOOuL. 100-200uL). 5.7 125-mL LDPE narrow-mouth bottles. 5.8 2 m L d e a r HPLC vial kit. 5.9 Disposable pipettes. 5.10 Autopipettes (100-1000 p L and 10-100 pL), with disposable tips. 5.11 Waters Sep Pak Vac 6 cc (Ig ) tC18 SPE cartridges. 5.12 SPE vacuum manifold. 5.13 Vortexer. Page 2 o f ? Page 60 o f 65 Exygen Research Page 94 of 118 Interim Report #28 - Analysis of Groundwater Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 Exygen Rctearch Method Number V00UI78& I A ,V .',, ~ 1 C A L M ETH O D 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: PluophaseRP (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 faiL/min) 35 0.3 35 0.3 75 0.3 75 0.3 35 0.3 6.6 Injection Volume: 15 jiL (can be increased to as m uch as 50 tiL). 6.7 Quantitation: Peak Area - externa) 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, m onitoring 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 mL o f water. Alternate volumes may be prepared. Page 3 u f 7 Page 61 o f 65 Exygen Research Page 95 of 118 Interim Report #28 - Analysis of Groundwater Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 Exygen Research Method Number VOOOP86 I ANALYTICAL METHOD Method o f Analysis for the Determination o f Periluorooctanoic 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 bottle9.1.2 A 1.0 pg/mL fortification solution o f PFOA is prepared by bringing 1 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 f PFOA is prepared b y bringing IU 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 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 foe 0.1 pg/mL fortification solution. The following is a typical example: additional concentrations may be prepared as needed. Concentration o f Fortification Volum e D iluted to Final Concentration Solution (na/m L) (m L) (m L) (na/m L) 100 5.0 100 2.0 100 1.0 100 100 100 5.0 2.0 1.0 5.0 10 100 0.5 2.0 10 100 0.2 1.0 10 100 0.1 9.2.3 Store all calibration standards in 125-mL 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 needed. 10.0 Batch Set Up 10.1 Each batch o f camples 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 96 of 118 Interim Report #28 - Analysis of Groundwater Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 Exygta Research Method Number VOOO1786 | ANALYTICAL M ETHOD Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Small Mammal Serum by LC/MS/MS 11.0 Sample Extraction 11.1 Measure 1 mL o f sample into a 50 mL polypropylene centrifuge tubes (fortify u needed, replace lid and mix well). Note that alternate volumes o f serum may be measured depending on the 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 fo r-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 5 mL o f acetonitrile and shake for - 2 0 minutes on a wrist-action shaker. 11.6 Centrifuge the tubes at -3 0 0 0 rpm for - 5 minutes. 11.7 Decant the supernatant into 50 mL disposable centrifuge tube and add 33 m L o f water. 11.8 Condition the C n 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 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 o r more concentration levels must b e included in an analytical aet. 12.3 An entire set o f calibration standards must be included at the beginning and ai the end o f a sample set. Standards must be interspersed between every 5 -to 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 u 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 o Page 63 o f 65 Exygen Research Page 97 of 118 Interim Report #28 - Analysis of Groundwater Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 Exygen Research Method Number VOOOJ 7*6 [ 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 13.1 13.2 13.3 13.4 13.5 13.6 Chromatogram must show a peak o f a daughter ion at 369 amu from a parent o f 413 amu. The 413 amu parent corresponds to the PFOA anion, while the daughter ion (369 amu) represents the loss o f carbon dioxide. Method blanks must not contain PFOA a! 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. 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 aet 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. 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, die total number o f calibration standards that could be excluded must not exceed 20% o f the total number o f standards injected. The correlation coefficient (R) for calibration curves generated must be 0.992 (RJ 0.985). I f calibration results fall outside these limits, then appropriate steps must be taken to adjust instrument operation, and the standards o r the relevant set o f samples should be reanalyzed. Retention times between standards and samples must not drift more than 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 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 x aliquot factor slope DF factor by which foe final volume was 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 of? Page 64 o f 65 Exygen Research Page 98 of 118 Interim Report #28 - Analysis of Groundwater Samples Exygen Study No.: P0001131 Exygen Protocol Number: POOOI131 Exyjen Research Method Number VOQO1786 I AN/vL yTIC'AL M E TH O D ~! Method o f Analysis for the Determination ofPerfluorooctanoic Acid (PFOA) in Small Mammal Serum by LC/MS/MS 14.3 Use the following equation to convert the amount o f PFOA found in ng/mL to ppb. PFOA found (ppb) fPFQA found (ng/mL) x final volume fmLll sample volume (mL) Exygen Research Page 7 of 7 Page 65 o f 65 Page 99 of 118 Interim Report #28 - Analysis of Groundwater Samples Exygen Study No.: P0001131 E RESEARCH 3058 Research Drive Phone: 814-272-1039 State College, PA 16801 Fax: 814-231-1580 PROTOCOL AMENDMENT Amendment NumberM 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 IO: W 0001226-6 . ADMINISTRATIVE FORM Exygen Research Page 100 of 118 Interim Report #28 - Analysis of Groundwater Samples Exygen Study No.: P0001131 E 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 o f 65 2) Test Materials, page 6 o f 65: PFOS transition monitored 499 -> 99. AMENDED TO 1) Instead o f 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 o f the data sets, interim reports will be issued to allow the client to receive data in a timelier-maneF. Dianne' a/ci/cs2) 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 belter chromatography. tL. Study Director Signature J 2 /m /siu L f Tincipal Investigator Signature Date Study Director Management Signature ate LIBRARY IO: VQ001226-8 Exygen Research ADMINISTRATIVE FORM Page 101 of 118 Interim Report #28 - Analysis of Groundwater Samples Exygen Study No.: P0001131 JUL.ES.2005 0:56AM EXYGEN RESEARCH NO.774 P .3 3058 Research Drive Phone: 814-272*1039 State Collese, PA 16801 Fax:814-231-1580 Amendment Number Effective Date: Exygen Study Number PROTOCOL AMENDMENT 3 07/18/05 P1131 Client Study Number: DESCRIPTION OF AMENDED SECTION Verification of Analytical Procedure, page 10 of protocol. Page 1 on NA The field duplicate can be used for the laboratory spikes and replicate when the primary sample volume is limited- _, b at iq n ale The sample size for a water sample is 200 ml- If a sample site requires re-extraclion 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 tor laboratory spikes Bnd replicates as reeded. 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. QJAjL Study OjredMffSignatum i l / y f/o c r Principal Investigator Sigruityrd A _________ Sponsor Signatur1?rarpimd) 7 / > A ' Oala Oate 2 / ~ ji/L ->S Date ~ ) /lS 0'os Date Exygen QAU Review LIBRARY ID: V 0 0 0 1 2 * ADMINISTRATIVE FORM 5 Exygen Research Page 102 of 118 Interim Report #28 - Analysis of Groundwater Samples Exygen Study No.: P0001131 r 11-23-2005 04:22p Fron-WESTON SOLUTIONS N O V i2 2 .2 0 0 5 4!5 B P H EXYGEN RESEARCH T-67T P.003/003 F-713 r .a Amendment Number: Effective Date: Exygen Study Num ber 3058 Research Drive Phonei 814-272-1039 State College, PA 16801 Fax; 814-231-1580 H PROTOCOL AMENDMENT 4 ~ \im m P1131 Client Study Number. Paa1 1 NA __ DESCRIPTION OF AMENDED SECTION Analytical Procedure Summary: vaooi780:*Method or Analysis torthe Determination of PerftuoroooctahoicAcid (PFOA} in Water by LC/MS/MST Section 11,0 Ofthe method. AMENDED TO Section 11.0. Samples may be diluted before going through the extraction procedure. B A TIQ N A il " ~~ If a 40 mL portion of sample win not load onto the Cia SPE cartridge, a pre-dllubon can be prepared and extracted. V ' IMPACT ON STUDY No negative impact on the study. Mora usable data may be obtained. SponsorSignature (ifrequired) Date Exvoen QAU Review ( b - I. i t LIBRARY ID: V000122M RECEIVED TIM E NOV.2 3 . 5:40PM ____________ ADMINI6TRATIVE FORM PRINT TIME NOV.2 3 . 5:41PM 1 Exygen Research Page 103 of 118 Interim Report #28 - Analysis of Groundwater Samples Exygen Study No.: P0001131 ffcHEM EHSR 236 1B 651 733 1958 JUL1-4-8003 05:IS FROM=3 ENU. LPB 651 778 6176 P tCV.Z2.2005 4:58PM EXY6ENRESEflROt 11/23 '05 14:12 NO.979 03/03 . 10:56517331538 NO.314 P.3 3058 Research Drive Rhone: 814-272-1039 State College, PA 16801 Fax: 814*231-1580 Amendment Number. Effective Date: Exygen study Number PROTOCOL AMENDMENT 4 11/22/OS P1131 Client Study Number: Pee 1 of i NA DESCRIPTION OF AMENDED sfeCTJON Analytical Procedure Summary; V000i780^MeRiod ofAnalysis for tha Pateimlnatimi of Perfluoroooctanoic Acid (PFOA) in Water by LC/MS/MS,' Section 11.0 oTthe method. am ended t6 Section 11.0. Samples may be diluted before going throughtne traction procedure. stornai lfa 4 0 m L portion of sample will not load onto the C, SPE cartridge, a pre-dilution can ba prepared and extracted. IMPACT ON SOUPY i\ No negative Impact on the study. More usable data may be obtained. 'i StudyDirenarsnatur* ~ oste i I t ~~ ~ ~ Dal* Z ls-A iV r-cf o a ts fi///. IS .w S oSS Exvoan QAL) Review f a . LIBRARYID:VOOOIMW RECEIVED TIME NOV.2 3 . 3:32PM ADMINISTRATIVE FORM PRINT TIM E NOV.2 3 . 3:33PM Exygen Research Page 104 of 118 Interim Report #28 - Analysis of Groundwater Samples ' -2006 0 4 :09pm Fna-KESTON SOLUTIONS + Exygen Study No.: P0001131 T-152 P . 0 0 |/ 0 ^ F-II9 J k E; 1R C hi 3058 ResearchO rt Mwn: 8H-Z7Z-10# State Colleso, PA16801 F 814-Z3t-1580 Amendment Nu iben E__ff_e_c_ti_v_e_D_a_t_e_: Exygen Study Number T-'OCOL AMENDMENT J ________ 12/01/05 . P00011S1 Client Study Number pa9^ o fi NA ------ DgSCRlCTONJOF AMENDCTSfcgnqrt Teat MatefMs, page & of Protocol. \ \ a iy -a T li An alternate lot of PFOSiTi 'v st used for thisstudy. ............RATINALE The PF08 used previously in this study Cot numeer 217 from SM) f*n out and t necessaryto u n a new lot No negative impact on study. _ _ I '^SfaJdyotremM sMvrMuutuin M W TM -*aSB. gnifndjpLalWvjmzrfsuito S' gUraiu. ivi on g -n u H*UC 'o l 4 Exygsn qaU InlUOate J-3k> IsSa. UBRARTID: VDOD"TWW RECEIVED TIME 1PR. 1 '28PM . ADMINISTRATIVEform PRINT TIME I MAR.21. 5:29PM Exygen Research Page 105 of 118 Interim Report #28 - Analysis of Groundwater Samples Exygen Study No.: P0001131 3058 Research Drive Phone: 814-272-1039 State College, PA 16801 Fax: 814-231-1580 Amendment Number: Effective Date: Exygen Study Number PROTOCOL AMENDMENT 6 05/17/06 ' P0001131 Client Study Number Page 1 oM NA DESCRIPTION OF AMENDED SECTION Appendix I, Analytical Method V0001782 AMENDED TO . ' As per client request, samples in login L4026 that have been tagged for re-extraction by the sponsor win 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 -1 0 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 negativeimpact on study. LIBRARY ID: V0001226-9 Exygen QAU Init./Date #>- / S fa /e to ' ADMINISTRATIVE FORM Exygen Research Page 106 of 118 Interim Report #28 - Analysis of Groundwater Samples Exygen Study No.: P0001131 3058 Research Drive Phone: 814-272-1039 State College, PA 16801 Fax: 814-231-1580 Amendment Number: Effective Date: Exygen Study Number PROTOCOL AMENDMENT 8 07/06/06 P0001131 Client Study Number: Page 1 of 1 NA 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 irect Inje ctio n 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 m illiliters of 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 Date Study Director Management Signature Exygen Management Signature Sponsor Signature (if required) LIBRARY ID: V0001226-9 Date Date Date Exygen QAU lnit./Date tCt- / lib ia te ADMINISTRATIVE FORM Exygen Research Page 107 of 118 Interim Report #28 - Analysis of Groundwater Samples Exygen Study No.: P0001131 CHEM EHSR 236 ife 651 733 1958 O H 1 -2 0 0 6 0 3 :5 tp frn H U T O T W IU T IO IM 07/11 '06 14:09 N0.142 02/02 + T-50 P-OO/OOZ W p ^R E SE A R C H 305BResearchOftve '^ 8 2 1 3 State College, PA16801 Fa; B14-231-1580 . . protocol amendment ArtiMditierrt Number EliiiJva Dat: B wmfm icygn Study Nuttiber ' P Q 0 0 ii3 Z Client:Study Number Q r ::psi*i*i Appendix I, Analytical Method V0001782. A i per pRant request. sedinteot samptes re-tagged for' ip-extractlon by the sponsor pW . be re-extracted using the following method: . Otrtiet Ihjsedon M e m o * Baftre the samples ere weighed,for the extraction mixed thoroughly by Vigorously shaking the container. A one-gram P ^ W ,n t la fcatghed Into a 15-mllRWer centrifuge tube.fer the extraction. Ten mfflflitewof acetic add in methanol Is added '.each sample. The samples ere men stw en m r hand, vortexed, and sonicated for thirty minutes. The samples are then ceotnwgeo tor -1 0 minutes at -30 00 rpm. Bach sample is analyzed by LC/MS/M5 aleetrtispray. ..., ' ------ ' ; RESALE .. . . i! Moris usable date wm be obtained .by using,an alternate mettwd. IMPACTON S r tS T No negative impact on study. e tu ^ D n M rsiB n sn w S p o n s o r S f jj n t f u r (IT m o v l r id ) . * 7 'j`: . . : r '-! ' im *SS 4M. OpM - . !. ' P l l * . , It -M W r W S 6 ' : Oats .. W < - -- : .. . /'.* ' ExygsmQU,1.ftfrtJs*s LiaAAflViP:VD0012SM RECEIVED TIME J U L . l l . 4:27PM " : '"'T *r"M o m k tK t n i/^ PRINT TIME JU L. 11. 4:28PM . ; V.5 Exygen Research Page 108 of 118 Interim Report #28 - Analysis of Groundwater Samples Exygen Study No.: P0001131 01-08-2007 03:59pn Froa-ESTON SOLUTIONS T-021 P .002/005 F-058 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 11 12/21/06 . P0001131 Client Study Number: Pag1 o f1 P0001131 LIBRARY ID:V0001S2M R EC EIV ED TIM E JAN. 8 . 4:02PM ADMINISTRATIVE FORM PRIN T TIM E JAN. 8 . 4:04PM Exygen Research Page 109 of 118 Interim Report #28 - Analysis of Groundwater Samples Exygen Study No.: P0001131 S E P .2 0 .2 0 0 5 1!52PM EXYGEN RESEfiRCH BqenR E S E A R C H NO.377 P .2 3058 Research Drive Phone: 814-272-1039 State College, PA 16801 Fax: 814-231-1580 DEVIATION FORM P e n e ra i: X Project Specific Deviation Exygen Project # : P760/P1131 Facility Deviation Pane 1 of 1 Date of Occurrence: 03/16/05 Deviation # : 1/1 Client Project # : NA Reference # : 05-122 Reoulatorv Driver: Deviation Tvne: (Include V# fat methods and SOPs) GMP GLP Other None Sample Description: _____ Protocol _____ Method V#: 0001658-3 Notebook reference: NA X SOP Login#: _______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 Analysis Instrument Client Request X Other Im aas; There has been no negative impact on the study. All of 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 of 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 man 20 g. The technician analyzing the samples for percent solids was following the new procedure before it was formally approved. C onractlve A c tio n s: A new version of the SOP has been Issued and approved (V0000427-3). Sianafegsa O , / /T ; , 'Principal liW sstigator / O M a ). ' ' Study D irutiST ft Quality Assurance / x y . /S f. * 1 ///* $ * .Date >' Exygen Madagepafent dm yJk J Dite Sponsor Representative - -ih I& S ' Date NA Sponsor Management Date Date Date LIBRARY ID: V0001540-6 ADMINISTRATIVE FORM Exygen Research Page 110 of 118 Interim Report #28 - Analysis of Groundwater Samples Exygen Study No.: P0001131 S E P .2 0 .2 00 5 i:52Pt1 EXYGEN RESERRCH N O .3 7 7 P .3 3058 Research Drive Phone: 814-272-1039 State College, PA 16801 Fax: 814-231-1580 DEVIATION FORM General: X Project Specific Deviation Exygen Project # : P760/P1131 Facility Deviation Paoe 1 of 1 Date of Occurrence: 06/29/05 Deviation # : 2/2 Client Project # : NA Reference#: o z J J _ Regulatory Driver: Deviation Tvoe: (Include Vti tor methods and SOPs) GMP GLP Other None Sam olo Description: X Protocol _____ Method \m: NA Notebook reference: NA _____SOP Login#: L4254/L4256 Container#: C0056480-85 L o t# : NA Summary o f 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_____Instrument_____Client Request X Other Impact: No negative impact on this study. C orrective Actions: Deviation issued. 5fflpywee; / / y arfl$jpal Investigator SAfoAi/ idy Director Quality Assurance W Ci Exygen Mnagefhent Jl Sponsor Representative J3L Date Sponsor Management U ^-O S Date Date Date LIBRARY ID: V0001640-6 ADMINISTRATIVE FORM Exygen Research Page 111 of 118 Interim Report #28 - Analysis of Groundwater Samples Exygen Study No.: P0001131 SEP. 2 0 .2 0 0 5 H52PM EXYGEN RESEARCH en RESEARCH 358 Research Drive State College, PA 16801 N O .3 7 7 P .4 Phone: 814-272*1039 Fax: 814-231-1580 General: X Project Specific Deviation DEVIATION FORM Facility Deviation Paae 1 of 1 Date o f Occurrence: 12/27/04 Exygen Project # : P760/P1131 Deviation # : 3/3 Client Project#: NA Reference # : oS ~~f Y ^ Regulatory D river: Deviation Tvne: (Include V# for methods end SOPs) GMP G IP Other None Samle Description: Protocol V#: 202-20 Section 5.2.3 Notebook reference: NA Method X SOP Login#: NA Container#: NA L o t# : NA Summary o f Deviation: SL2114 (30% Dimethyldichlorosllane in Toluene) was given the expiration date of 02/13/05, but RE544 (Toluene) used to make SL2114 expired on 03/28/04. SL2114 was used to silanlzed glassware prepared for the fish extraction from 12/27/04 through 01/07/05. Causa: ____Preparation_____Analysis____ Instrument_____Client Request X Other Impact: No negative impact on the study. Toluene was used only as a solvent for the glassware preparation. Dimethyldlchlorosilane, which is the coating agent, was not expired. Corrective Actions: Deviation issued. Signatures: J /Principa*investigat6r K o k !l k / / Study Director Quality Assurance ^Date ate/ Date " ' Exygen k^nggdment UN Sponsor Representative UN Sponsor Management *~ts Date Date Date LIBRARY ID: V0C01640-6 ADMINISTRATIVE FORM Exygen Research Page 112 of 118 Interim Report #28 - Analysis of Groundwater Samples Exygen Study No.: P0001131 RESEARCH 3058 Research Drive Phone: 814-272-1039 State College, PA 16801 Fax: 814-231-1580 DEVIATION FORM General: X Project Specific Deviation ____Facility Deviation Pag 1 of 2 Date of Occurrence: 04126/06 Exygen Project # : P760/P1131 Client Project#: _______NA Deviation#: Reference#: 5 06-076 Regulatory Driver: _____ X _____ _____ GMP GLP Other None Sample Description: Deviation Type: (In c lu d e V# fo r m e th o d s e n d S O P s ) X Protocol _____ Method _____ SOP V#:NA Notebook reference: NA Login#: L0008191 Container#: _______NA______ L o t# : __________ NA Summary of Deviation: The three sediment samples in L8191 (C0172892 - C0172894) were originally extracted using the sediment method V0001782. Poor recoveries were obtained for PFOS, PFOA and ,3C PFOA. Because of this, the study sponsor requested the use of an alternative extraction for these compounds, as follows: Direct Injection Method: Before the samples were weighed for the extraction, they were mixed thoroughly by vigorously shaking the container. A one-gram portion of sediment was weighed into a 15-milliliter centrifuge tube for the extraction. Ten milliliters of 1% acetic acid in methanol was added to each sample. The samples were then shaken by hand, voriexed, and sonicated for thirty minutes. The samples were then centrifuged for -1 0 minutes at -3000 rpm. Each sample was analyzed by LC/MS/MS electrospray. Using this method acceptable data was obtained for PFOS, but the recoveries for PFOA and ,3C PFOA were stiU poor. Another alternative method was then used for PFOA and ,3C PFOA, as follows: Alternative SPE Method: The samples that were prepared in 1% acetic acid fo r the direct injection method were used for this extraction. Five milliliters of each sample was aliquoted into a 50-mL polypropylene centrifuge tube and the volume was taken to 40 mL with water. The samples were then centrifuged for -1 0 minutes at -3000 rpm. The supernatant was then loaded onto a C ,, SPE cartridge conditioned with 10 mL of methanol and 5 mL of water. The eluale was discarded. Approximately five milliliters of methanol was added to the cartridge. Five milliliters of eluale was collected into a graduated 15 mL polypropylene centrifuge tube. Each sample was analyzed by LC/MS/MS electrospray. Cause: ____ Preparation____ Analysis____ Instrument____ Client Request X Other Impact: More usable data was obtained. LIBRARY ID: V0001640-8 ADMINISTRATIVE FORM Exygen Research Page 113 of 118 Interim Report #28 - Analysis of Groundwater Samples Exygen Study No.: P0001131 CHEH EHS8 236 1B 651 733 1958 U M H I N O M Ipa FrM HTC* SOIUHOIIS 05/01 '06 13:46 N0.106 03/03 + m m r.m /im H U jONRnaarehiMva SM* Catton. M SEAfCK ' M V tftT m F O fW .. Phorir RW f 1f.lJ t.lM 0 O M M ft * * . , '' X U d llB adtodM M an . ParinDavMfen b w ip m M t# : . j a s a a a L - ' Obnimfiet: d-- I--U B BSSBnSwE ' `* . ' ,' . DttDatton; ? '',f` * PM W ; UdyDnrar QuriSytaaunane* M * I tl fa M k MWi< im b W " 'T6B > ///* ..Cm m M ^ek jfefcpnyB S *,.. . #peridiM.Mftnitm * S' . . .tto... .. . _ u n M triftv a sa ia ttM Exygen Research Page 114 of 118 Interim Report #28 - Analysis of Groundwater Samples Exygen Study No.: P0001131 EHS OPNS ENVIRONMENTAL LAB t t 651 778 4226 07/11/06 11:46 (3 :04/07 N0:681 RESEARCH 3058 Research Drive Phone: 814-2.72-1039 State College, PA 16801 Fax: 814-231-1580 DEVIATION FORM aneral: '" X Project Specific Deviation Faculty Deviation Data of Occurrence: 04/28/06 Exygen Project # : P760/P1131 Deviation Mi ' . t i " ' ; . Client Project M: ____ Na, ' Refrenoe # : M - m - . '' RllUllltW Y P rlY tr OMP QLP Other None SatMlt DrtPlirtHm; Devtsttpn Vyoe: /Include VMfor methods and SOPs) Protocol X Method V#: V000427-3 Notebook reference: NA Login 8 : _______NA Container # : ` C00159446 ' Lot # : NA Summary of Deviation: : ./. , One sod ample (C00159446) was weighed al -8g for percent solid analysis, rather than at -20g which I*stated In ths method. ' ' " . . . Cause; Preparation____Analysis____ Instrument __ __ Citent Requaet X Other Impact; . . ;' . No negative Impact An accurate percent solid number was obtained by slightly altering the calculation used. \' .. ' . ' Corrective 'Actions : Deviation issued. 8l^ / F/ 9 fh l& C rmncoi^aMtinpvveeasttlogaatoorr / fl/j/o L --r DDate / Exvydgent{tfainagei ment Studjffitredor . Htlilk Dan H fL U ft fc Sponsor Raprei Quality Assures ^ __ oh.trfob . _^(A Date Sponsor Management Date .Data.. Exygen Research Page 115 of 118 Interim Report #28 - Analysis of Groundwater Samples Exygen Study No.: P0001131 EHS OPNS ENVIRONMENTAL LAB ff 651 778 4226 07/11/Q6 11:46 0 :06/07 NO:681 irr RESEARCH 3058 Research Driv Phone: 814-272-T03 State College, P16801 Fax: 814-231-1580 ; VIATION FORM general: ' X . ' Project Spcifi Deviation ____ Facility Deviation FjWfgi Odta of Occurrence: fl8C8ff)6 Exygen Project # : P760/P1131 Dviation # : .v l i t Client Project # : _______ NA , ' Reference # : r if e - ll* -* Renleterv Driver: ___ _ X ____ ___ . ' QMP GLP Other None Devlstlon Tvpb; (Include Vtt for methods end SOPa) ______Protocol ______ Method X OP' V#: 1800 Notebook reference: L o g in #: m .NA Container # : L o t#:' Peer review of raw data was not documented per SOP V1800 prior to 6/28/D6. J/L c ue: ____ P reparation_____ A n a to le ____ . Instrum ent____ Client Request Vt" Other Impact; Raw data will be more thoroughly evaluated and reviewed before QA Inspection. ' ' Corrective Actione: A note to file will be Issued to sir subsequent reports stating that overall summaries have been peer reviewed. ' '' . Dele / Exygen Mrfn h h sk x e. D sta Sponsor RpifeaentaUve OjA Sponsor ManfiQarnant . Date D a t Exygen Research Page 116 of 118 Interim Report #28 - Analysis of Groundwater Samples Exygen Study No.: P0001131 EHS OPNS ENVIRONMENTAL LAB 651 778 4226 07/11/06 11:46 0 :07/07 NO:681 RESEARCH 3058 Research Drive Phone: B4-?7-1039 State College, PA 16801 Fax: 814-231-1580 DEVIATION FORM 0 farai: . X Project Specific Deviation ___ Facility Deviation F i 1PM Dele of Occurrence: 06/28/06. Exygen Project # : P76/P131 .' Deviation # : ; (Wft ' Client Project # : NA. : Rettrano#': r a l l i a . NenulatOrv Driver: Dviation'Twee: (Includa Vil tor methods end SOPal ___ GMP .X ... ' OLP Other Norie amate D aecrirtlon: X Protocol V#: NA Notebook reference: _____ Meihocf ____ P Login*: L0008121 L0008081 Contornar#: urnmarv of Pevfatlon: Semples were filled to 250 mL Instead of 200 mL. 00160347 C0169354 C017088 : L o t* : NA Cause: X Preparation ___ ;AniWyels j___ Instrument ' ' Client Regim ai Im pact: .: .; Samples could not be extracted according to the protocol. Qther g w T tflftg A stia m i Splklng.ievela vera adjusted to accommodate the alternative volume. fflaHMMi , j cillpel IInnvveesstiiggaator m&L Study.Dli lality Assurance . j, : < /m ' Date. mm liM & e. D ate jM i J k Sponsor Representative Sponsor Management D ate Exygen Research Page 117 of 118 Interim Report #28 - Analysis of Groundwater Samples Exygen Study No.: P0001131 01-08-2007 0 3 :S9pn Fros-KSTO N SOLUTIONS T-021 P .005/005 F-0SB IT research 3058 Research Drive Phone: 814-272-1039 State College, PA 16801 Fax: 814-231-1580 DEVIATION FORM Generil: X Project Specific Deviation Facility Deviation _________ Paaelofl Date of Occurrence: Nov. 2006Jan. 2007 Exygen Project#: P0001131 Deviation#: 10 Client ftqject#: P0001131 Reference#: (\l~ O O i Rsouistorv Driver? Dovtotton Type: (l/ldudv V#formethodsand SGPs) _____ X ____ _____ GMP GLP Other None Sample Description: _ ____Protocol X Method Vfc VM01780 Notebook reference: ____ SOP Login#: 10010155 Container#: Lot#: L0010165 _____________ LOOT0 2 5 4 ____________ ._________________________________ . . Summary o f O uvtrton: Stock standards and fodUIcaticn standards uasd Inthe extraction farthese logine were prepared in aoatonhrOe Instead of methanol. Cause; X Preparation___ Analysis____ Instrument____ Client Request ____ Other Impact: 1 No negative Impact. Compounds are completelysoluble Inacetonitrile as they are m methanol. LIBRARYID: V0081640-7 RECEIVED TIM E JAN. 8 . 4:02PM ADMINISTRATIV8 FORM PRINT TIM E JAN. 8 . 4:04PM Exygen Research Page 118 of 118
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