Document Lperp784aZnOr0KDzjaQnE903

INTERIM REPORT #19 - Analysis of Ground Water Samples STUDY TITLE Analysis of Perfluorobutanesulfonate (PFBS), Perfluorohexanesulfonate (PFHS), and Perfluorooctanesulfonate (PFOS) in Water, Soil, Sediment, Fish, Clams, Vegetation, Small Mammal Liver and Small Mammal Serum Using LC/MS/MS for the 3M Decatur Monitoring Program DATA REQUIREMENTS EPA TSCA Good Laboratory Practice Standards 40 CFR 792 STUDY DIRECTOR Jaisimha Kesari P.E., DEE Weston Solutions, Inc. 1400 Weston Way West Chester, PA 19380 Phone: 610-701-3761 INTERIM REPORT COMPLETION DATE March 19,2007 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: 122 Interim Report #19 - Analysis o f Ground Water Samples Exygen Study No.: P0001131 GOOD LABORATORY PRACTICE COMPLIANCE STATEMENT Exygen Study Number P0001131, entitled "Analysis ofPerfluorobutanesulfonate (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. Exygen Research Jaisimha Kes Study Director Weston Solutions, Inc. Michael A. Santo: Sponsor Representative 3M Company Exygen Research Date Page 2 o f 122 Interim Report #19 - Analysis o f Ground Water Samples Exygen Study No.: P0001131 QUALITY ASSURANCE STATEMENT Exygen Research's Quality Assurance Unit reviewed Exygen Study Number P0001131, entitled, "Analysis of Perfluorobutanesulfonate (PFBS), Perfluorohexanesulfonate (PFHS), and Perfluorooctanesulfonate (PFOS) in Water, Soil, Sediment, Fish, Clams, Vegetation, Small Mammal Liver and Small Mammal Serum Using LC/MS/MS for the 3M Decatur Monitoring Program". All reviewed phases1 were inspected for conduct according to Exygen Research's Standard Operating Procedures, the Study Protocol, and all applicable Good Laboratory Practice Standards. All findings were reported to the Exygen Principal Investigator and Management and to the Study Director. Phase Date Inspected Date Reported to Date Reported to Principal Exygen Date Reported to Investigator Management Study Director 22. Raw Data Review and Interim Analytical Report Review 01/25-27/06 03/20/06 03/21/06 06/29/06 50. Raw Data Review and Interim Analytical Report Review 03/14,16/07 03/19/07 03/19/07 03/19/07 '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 o f 122 Interim Report #19 - Analysis o f Ground Water Samples Exygen Study No.: P0001131 CERTIFICATION OF AUTHENTICITY This interim report, for Exygen Study Number P0001131, is a true and complete representation of the raw data. Submitted by: Exygen Research 3058 Research Drive State College, PA 16801 (814) 272-1039 Principal Investigator, Exygen: Charles Simons Director of Analytical aboratory Operations Exygen Research Exygen Research Facility Management: R W I'M Richard A. Grazzini Executive Director of Analytical Sciences Exygen Research Study Director, Weston Solutions, Inc. Jaisimha Kesari P.E., DEE Weston Solutions, Inc. Sponsor Representative, 3M Company: lA iw jU 4 A r tW tP Michael A. Santoro Director of Regulatory Affairs Date 3\ m Date ^ i/id o r Date Exygen Research Page 4 of 122 Interim Report #19 - Analysis o f Ground Water Samples Exygen Study No.: P0001131 STUDY IDENTIFICATION Analysis of Perfluorobutanesulfonate (PFBS), Perfluorohexanesulfonate (PFHS), and Perfluorooctanesulfonate (PFOS) in Water, Soil, Sediment, Fish, Clams, Vegetation, Small Mammal Liver and Small Mammal Serum Using LC/MS/MS for the 3M Decatur Monitoring Program PROTOCOL NUMBER: P0001131 EXYGEN STUDY NUMBER: P0001131 TYPE OF STUDY: Residue SAMPLE MATRIX: Ground Water ' TEST SUBSTANCE: Perfluorobutanesulfonate (PFBS), Perfluorohexanesulfonate (PFHS), and Perfluorooctanesulfonate (PFOS) SPONSOR: 3M Company 3M Building 0236-01-B-10 St. Paul, MN 55144 STUDY DIRECTOR: Jaisimha Kesari P.E., DEE Weston Solutions, Inc. 1400 Weston Way West Chester, PA 19380 STUDY MONITOR: Michael A. Santoro 3M Company 3M Building 0236-01-B-10 St. Paul, MN 55144 PERFORMING LABORATORY: Exygen Research 3058 Research Drive State College, PA 16801 ANALYTICAL PHASE TIMETABLE: Study Initiation Date: 11/05/04 Interim Analytical Start Date: 12/16/05 Interim Analytical Termination Date: 06/14/06 Interim Report Completion Date: 03/19/07 Exygen Research Page 5 o f 122 Interim Report #19 - Analysis o f Ground Water Samples Exygen Study No.: P0001131 PROJECT PERSONNEL The Study Director for this project is Jaisimha Kesari at Weston Solutions, Inc. The following personnel from Exygen Research were associated with various phases of this interim portion of the study: Name Title Charles Simons Director, Analytical Laboratory Operations John Flaherty Vice President Karen Risha Laboratory Supervisor Christine Edwards Technician Mark Ammerman Sample Custodian Brian McAllister Sample Custodian Mindy Cressley Technician Brittany Kravets Technician Frances Crespi Technician Kimberly Hall Technician Krista Gallant Technician Exygen Research Page 6 o f 122 Interim Report #19 - Analysis o f Ground Water Samples Exygen Study No.: P0001131 TABLE OF CONTENTS Page TITLE PAGE....................................................................................................................... 1 GOOD LABORATORY PRACTICE COMPLIANCE STATEMENT............................. 2 QUALITY ASSURANCE STATEMENT..........................................................................3 CERTIFICATION OF AUTHENTICITY...........................................................................4 STUDY IDENTIFICATION................................................................................................5 PROJECT PERSONNEL.................................................................................................... 6 TABLE OF CONTENTS.....................................................................................................7 LIST OF 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..............................................................................12 5.0 REFERENCE MATERIAL........................................................................................ 12 6.0 DESCRIPTION OF ANALYTICAL METHOD........................................................ 13 6.1. Extraction Procedure............................................................................................... 13 6.2 Preparation of Standards and Fortification Solutions............................................... 13 6.4 Chromatography....................................................................................................... 14 6.5 Instrument Sensitivity............................................................................................... 14 6.6 Description of LC/MS/MS Instrument and Operating Conditions...........................15 6.7 Quantitation and Example Calculation..................................................................... 15 7.0 EXPERIMENTAL DESIGN...................................................................................... 17 8.0 RESULTS................................................................................................................... 17 9.0 CONCLUSIONS.........................................................................................................17 10.0 RETENTION OF DATA AND SAMPLES............................................................. 18 Exygen Research Page 7 o f 122 Interim Report #19 - Analysis o f Ground Water Samples Exygen Study No. : P0001131 Table I. LIST OF TABLES Page Summary of PFBS, PFHS and PFOS in Ground Water Samples..................20 Table n. Summary of PFBS and PFHS in Re-extracted Ground Water Samples........21 Table HI. Matrix Spike Recovery of PFBS, PFHS and PFOS in Ground Water Samples......................................................................................................... 22 Table IV. Matrix Spike Recovery of PFBS and PFHS in Re-extracted Ground Water Samples.............................................................................................. 24 Exygen Research Page 8 o f 122 Interim Report #19 - Analysis o f Ground Water Samples Exygen Study No.: P0001131 Figure 1. LIST OF FIGURES Page Typical Calibration Curve for PFBS in Reagent Water................................. 26 Figure 2. Extracted Standards of PFBS in Reagent Water, 0 ng/L and 25 ng/L, Respectively................................................................................................... 27 Figure 3. PFBS in Reagent Water, 50 ng/L Fortified Reagent Spk A, and 500 ng/L Fortified Reagent Spk B, Respectively.......................................................... 28 Figure 4. Chromatogram Representing a Ground Water Sample Analyzed for PFBS (Exygen ID: C0120251, Data Set: 121905D).................................................29 Figure 5. Typical Calibration Curve for PFHS in Reagent W ater................................ 30 Figure 6. Extracted Standards of PFHS in Reagent Water, 0 ng/L and 25 ng/L, Respectively....................................................................................................31 Figure 7. PFHS in Reagent Water, 50 ng/L Fortified Reagent Spk A, and 500 ng/L Fortified Reagent Spk B, Respectively...........................................................32 Figure 8. Chromatogram Representing a Ground Water Sample Analyzed for PFHS (Exygen ID: C0120251, Data Set: 121905D).................................................33 Figure 9. Typical Calibration Curve for PFOS in Reagent W ater................................ 34 Figure 10. Extracted Standards of PFOS in Reagent Water, 0 ng/L and 25 ng/L, Respectively....................................................................................................35 Figure 11. PFOS in Reagent Water, 50 ng/L Fortified Reagent Spk A, and 500 ng/L Fortified Reagent Spk B, Respectively...........................................................36 Figure 12. Chromatogram Representing a Ground Water Sample Analyzed for PFOS (Exygen ID: C0120251, Data Set: 121905D).................................................37 Exygen Research Page 9 o f 122 Interim Report #19 - Analysis o f Ground Water Samples Exygen Study No.: P0001131 LIST OF APPENDICES Appendix A Study Protocol P0001131 (Exygen Study No. P0001131) with Analytical Method V0001780: "Method of Analysis for the Determination of Perfluorooctanoic Acid (PFOA) in Water by LC/MS/MS", and Protocol Amendments............................ Page 38 Exygen Research Page 10 of 122 Interim Report #19 - Analysis o f Ground Water Samples Exygen Study No.: P0001131 1.0 SUMMARY Exygen Research extracted and analyzed ground water samples for the determination of perfluorobutanesulfonate (PFBS), perfluorohexanesulfonate (PFHS), and perfluorooctanesulfonate (PFOS) according to Exygen Method V0001780 (Appendix A). The limit of quantitation for PFBS, PFHS and PFOS in ground water was 25 ng/L. Analytical results and assessed accuracies for the analysis of PFBS, PFHS and PFOS found in ground water samples are summarized in Table I. The average percent recoveries standard deviations for PFBS, PFHS, PFOS in ground water samples were 90 9%, 124 33%, and 105 + 31%, respectively. Results for one sample for PFBS two samples for PFHS were not reported (NR) due to quality control failures. The samples were re-extracted in an attempt to obtain quantitative results. Analytical results and assessed accuracies for the analysis of PFBS and PFHS found in re extracted ground water samples are summarized in Table II. The average percent recovery standard deviation for PFHS in the re-extracted ground water samples was 107 11%. Quantitative results were obtained for all samples and analytes except for PFBS in one sample site. 2.0 OBJECTIVE The objective of the analytical part of this study was to determine levels of perfluorobutanesulfonate (PFBS), perfluorohexanesulfonate (PFHS), and perfluorooctanesulfonate (PFOS) in ground water according to Protocol P0001131 (Appendix A). 3.0 INTRODUCTION This report details the results of the analysis for the determination of PFBS, PFHS, and PFOS in ground water using the analytical 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 16, 2005, and the analytical termination date for this interim report was June 14,2006. Exygen Research Page 11 o f 122 Interim Report #19 - Analysis o f Ground Water Samples Exygen Study No.: P0001131 4.0 ANALYTICAL TEST SAMPLES Thirty-six ground water samples (Exygen ID C0120227 - C0120262) representing eight ground water sampling sites (the eight LOI wells), associated field quality control samples and one rinseate blank were received at ambient temperature on November 18, 2005, from Tim Frinak at Weston Solutions, Inc. The samples were logged in by Exygen personnel and placed in refrigerated storage. Sample log-in and chain of custody information is located in the raw data package associated with this interim report. Storage records will be kept at Exygen Research. 5.0 REFERENCE MATERIAL The analytical standards, PFBS and PFHS, were supplied by 3M. PFBS was received from 3M at Exygen on May 13, 2005. PFHS was received from 3M at Exygen on January, 20, 2003. PFOS was purchased from Fluka Corporation and was received at Exygen on April, 23, 2003. The available information for the reference materials is listed below. PFBS and PFHS were stored frozen and PFOS was stored refrigerated. Compound PFBS PFHS PFOS Exygen Inventory No. SP0005726 SP0002401 SP0002694 Lot # 101 SE036 430180-1 Purity (%) 96.7 98.6 101.2 Expiration Date 12/04/06 10/18/06 10/31/07 The molecular structures of PFBS, PFHS and PFOS are given below: PFBS Chemical Name: Perfluorobutanesulfonate Molecular Weight: 338 supplied as the potassium salt (C4FgS03`K+) Transitions Monitored: 299 - 99 Structure: F F F F F SO3 FFFF Exygen Research Page 12 o f 122 Interim Report #19 - Analysis o f Ground Water Samples Exygen Study No.: P0001131 PFHS Chemical Name: Perfluorohexanesulfonate Molecular Weight: 438 supplied as the potassium salt (C6Fi3S 0 3'K+) Transitions Monitored: 399 - 80 Structure: F F F F F F F SO3 FF FFF PFOS Chemical Name: Perfluorooctanesulfonate Molecular Weight: 538 supplied as the potassium salt (C8Fi7S 03"K+) Transitions Monitored: 499 -> 80 Structure: F F F F F F F F F SO3 FF F FFF 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 ground water 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 Exygen method V0001780. The stock standard solution was prepared at a concentration Exygen Research Page 13 of 122 Interim Report #19 - Analysis o f Ground Water Samples Exygen Study No.: P0001131 of 1000 pg/mL by dissolving 100 mg of each of the standards (corrected for purity and salt content, if necessary) in methanol. From this solution, a 100 pg/mL fortification standard solution was prepared by taking 10 mL of the stock and bringing the volume up to 100 mL with methanol. By taking 10 mL of the 100 pg/mL fortification standard and bringing the volume up to 100 mL with methanol, 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 methanol, a 1.0 pg/mL fortification standard was prepared. By taking 10 mL of the 1.0 pg/mL fortification standard and bringing the volume up to 100 mL with methanol, a 0.1 pg/mL fortification standard was prepared. By taking 10 mL of the 0.1 pg/mL fortification standard and bringing the volume up to 100 mL with methanol, a 0.01 pg/mL fortification standard was prepared. A set of standards containing PFBS, PFHS and PFOS were prepared in water and processed through the extraction procedure, identical to samples. The following concentrations were prepared: Cone, of Fort Fort Solution Volume (ng/mL)1 (PL) 00 10 100 10 200 10 400 100 100 100 200 100 400 1of PFBS, PFHS and PFOS Volume of Fortified Sample (mL) 40 40 40 40 40 40 40 Final Cone, of Calibration Std. (ng/L) 0 25 50 100 250 500 1000 The stock standard solution and all fortification and calibration standard solutions were stored in a refrigerator (4 2C) when not in use. Documentation of standard preparation is located in the raw data package associated with this interim report. 6.4 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, ~8.0 mins, and ~10.8 mins, respectively. Peaks above the LOQ were not detected in any of the reagent blank samples corresponding to the analyte retention time. 6.5 Instrument Sensitivity The smallest standard amount injected during the chromatographic run had a concentration of 25 ng/L of PFBS, PFHS and PFOS. Exygen Research Page 14 of 122 Interim Report #19 - Analysis o f Ground Water Samples Exygen Study No.: P0001131 6.6 Description of LC/MS/MS Instrument and Operating Conditions Instrument: API 4000 Biomolecular Mass Analyzer Interface: Turbo Ion Spray Liquid Introduction Interface Computer: DELL OptiPlex GX400 Software: Windows NT, Analyst 1.4.1 HPLC: Hewlett Packard (HP) Series 1100 HP Quat Pump HP Vacuum Degasser HP Autosampler HP Column Oven HPLC Column: Thermo Fluophase RP, 50 mm x 2.1 mm Column Temp.: -30 C Injection Voi.: 15 pL Mobile Phase (A): 2 mM Ammonium Acetate in water Mobile Phase (B): Methanol Time ('min') 0.0 1.0 8.0 10.0 11.0 18.0 Total run time: ~18 min %A 65 65 25 25 65 65 %B 35 35 75 75 35 35 Flow Rate: 0.3 mL/min Ions monitored: Analvte PFBS PFHS PFOS Mode negative negative negative Transition Monitored 299 - 99 399 80 499 80 Approximate Retention Time ('min') ~0.5 min. -8.0 min. -10.8 min. 6.7 Quantitation and Example Calculation Fifteen microliters of sample or calibration standard were injected into the LC/MS/MS. The peak area was measured and the standard curve was generated (using 1/x fit weighted linear regression) by Analyst software using six concentrations of standards. The concentration was determined from the following equations. Exygen Research Page 15 o f 122 Interim Report #19 - Analysis o f Ground Water Samples Exygen Study No.: P0001131 Equation 1 calculated the amount of analyte found (in ng/L, based on peak area) using the standard curve (linear regression parameters) generated by the Analyst software program. Equation 1: Analyte found (ng/L) = /Peak area - intercept! x DF slope Where: DF = Dilution Factor, factor by which the final volume was diluted, if necessary. For samples fortified with known amounts of PFBS, PFHS and PFOS prior to extraction, Equation 2 was used to calculate the percent recovery. Equation 2: Recovery (%) = /analyte found /ng/D - analyte in control (ng/Lri xl00% amount added (ng/L) An example of a calculation using an actual sample follows (calculation is for PFBS only): Ground water sample Exygen ID: CO120231 Spk F (Set: 121605A), fortified at 100000 ng/L with where: peak area = 14353 intercept = 0.000776 slope - 149 dilution factor = 1000 ng/L PFBS added (fort level) = 100000 amt in corresponding sample = 8240 (Set: 121605AR) From equation 1: Analyte found (ng/L) f14353 - 0.0007761 x 1000 149 From equation 2: % Recovery 96300 ng/L /96300 ne/L-8 2 4 0 ng/L) x 100% 100000 ng/L = 88% NOTE: Numbers may differ slightly from raw data due to rounding. Exygen Research Page 16 o f 122 Interim Report #19 - Analysis o f Ground Water Samples Exygen Study No.: P0001131 7.0 EXPERIMENTAL DESIGN For samples designated as field matrix spikes, PFBS, PFHS, and PFOS were also added at a known concentration to the bottles in the laboratory before being shipped to the field. The samples were filled to a 200 mL volumetric fill line in the field. The samples were extracted in three sets and one re-extraction set. Each set included one reagent blank, two reagent blanks fortified at known concentrations. Two sets contained three sample sites and one set contained two sample sites along with the field blank, the field blank spikes, and one rinse blank collected for the ground water samples. The re extraction set contained five samples sites along with the field blank and field blank spikes. For each site, a sample, a field duplicate and two-matrix field spikes were collected. For each site, a laboratory duplicate was extracted 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 ground water samples are summarized in Table I. The average percent recoveries standard deviations for PFBS, PFHS, PFOS in ground water samples were 90 9%, 124 33%, and 105 31%, respectively. Results for one sample for PFBS two samples for PFHS were not reported (NR) due to quality control failures. The samples were re-extracted in an attempt to obtain quantitative results. Analytical results and assessed accuracies for the analysis of PFBS and PFHS found in re extracted ground water samples are summarized in Table II. The average percent recovery standard deviation for PFHS in the re-extracted ground water samples was 107 + 11%. Quantitative results were obtained for all samples and analytes except for PFBS in one sample site. 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. 9.0 CONCLUSIONS Except as noted above, the ground water samples were successfully extracted and analyzed for PFBS, PFHS and PFOS according to analytical method V0001780. Exygen Research Page 17 of 122 Interim Report #19 - Analysis o f Ground Water Samples Exygen Study No. : P0001131 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 18 of 122 Interim Report #19 - Analysis o f Ground Water Samples Exygen Study No.: P0001131 TABLES Exygen Research Page 19 o f 122 Interim Report #19 - Analysis o f Ground Water Samples Exygen Study No.: P0001131 Table I. Summary of PFBS, PFHS and PFOS in Ground Water Samples Exygen id C0120227 C0120227 Rep C0120228 C0120231 C0120231 Rep C0120232 C0120235 C0120235 Rep C0120236 C0120239 C0120239 Rep C0120240 C0120243 C0120243 Rep C0120244 C0120247 C0120247 Rep C0120248 C0120251 C0120251 Rep C0120252 C0120255 C0120255 Rep C0120256 C0120259 CO120262 Client Sample ID GW-220R-Q4-Y05-LF-0 GW-220R-Q4-Y05-LF-0* GW-220R-Q4-Y05-LF-DB GW-220L-Q4-Y05-LF-0 GW-220L-Q4-Y05-LF-0* GW-220L-Q4-Y05-LF-DB GW-226R-Q4-Y05-LF-0 GW-226R-Q4-Y05-LF-0* GW-226R-Q4-Y05-LF-DB GW-226L-Q4-Y05-LF-0 GW-226L-Q4-Y05-LF-0* GW-226L-Q4-Y05-LF-DB GW-327R-Q4-Y05-CP-0 GW-327R-Q4-Y05-CP-0* GW-327R-Q4-Y05-CP-DB GW-310R-Q4-Y05-CP-0 GW-310R-Q4-Y05-CP-0* GW-310R-Q4-Y05-CP-DB GW-317L-Q4-Y05-CP-0 GW-317L-Q4-Y05-CP-0* GW-317L-Q4-Y05-CP-DB GW-320L-Q4-Y05-CP-0 GW-320L-Q4-Y05-CP-0* GW-320L-Q4-Y05-CP-DB GW-TRIP-Q4-Y05-CP-0 GW-220L-Q4-Y05-RB-0 C4 Sulfonate PFBS C6 Sulfonate PFHS C8 Sulfonate PFOS P wfluofobutan-- ottonato_______Perfluorohexanesutfonata________ Parfluorooctanesulfonate Analyte Found (ppt, nfl/L) Assessed Accuracy (+/-%) Analyte Found (ppt, ng/L) Assessed Accuracy </-%) Analyte Found (ppt, ng/L) Assessed Accuracy (/-%> 5830 5610 5270 30 60400 30 57300 50 30 57200 30 57100 50 30 55400 30 60800 50 8240 7660 7510 30 92900 30 69300 30 30 88500 30 68400 30 30 88900 30 76300 30 NR NR 6240 30 15800 30 NR NR 5940 30 14900 30 NR NR 5680 30 16600 30 ND 30 ND 30 ND 30 ND 30 ND 30 ND 30 ND 30 ND 30 ND 30 97100 89000 92000 30 368000 30 348000 30 30 346000 30 331000 30 30 369000 30 310000 30 337000 30 696000 40 894000 50 323000 30 716000 40 787000 50 330000 30 688000 40 733000 50 ND 30 NR NR 196 30 ND 30 NR NR 183 30 ND 30 NR NR 188 30 ND 30 NR NR 64.8 40 ND 30 NR NR 63.3 40 ND 30 NR NR 61.3 40 ND 30 ND 30 ND 30 ND 30 ND 30 ND 30 "Laboratory Duplicate NR * Not reported due to quality control result failures, see Table II for re-extract data. ND = Not detected at or above the Limit of Quantitations (LOQ) of 25 ng/L. Exygen Research Page 20 of 122 Interim Report #19 - Analysis o f Ground Water Samples Exygen Study No.: P0001131 Table II. Summary of PFBS and PFHS in Re-extracted Ground Water Samples Exygen ID C0120235 C0120236 C0120252 C0120256 Client Sample ID G W -226R -Q 4-Y 05-LF-0 G W -2 26R -Q 4 -Y 05 -L F -D B G W -317L -Q 4-Y 05-C P -D B G W -320L -Q 4-Y 05-C P -D B C4 Sulfonate PFBS Perfluorobutanesulfonate Analyte Found (ppt, ng/L) Assessed Accuracy (+/-% ) NR NR - -- -- C6 Sulfonate PFHS PerfluorohexanesuHonate Analyte Assessed Found Accuracy (ppt, ng/L) (+/- %) 126 7.69 30 30 ` Laboratory Duplicate ND = Not detected at or above the Limit of Quantitations (LOQ) of 25 ng/L. NR = Not reported due to quality control failures. Exygen Research Page 21 o f 122 Interim Report #19 - Analysis o f Ground Water Samples Exygen Study No.: P0001131 Table III. Matrix Spike Recovery of PFBS, PFHS and PFOS in Ground Water Samples Sample Description GW-220R-Q4-Y05-LF-0 (C0120227 S pk C. 10000 ng /L Lab S pika) GW-220R-Q4-Y05-LF-0 (C0120227 S p k D. 100000 n g /L Lab S pika) GW -220R -Q 4 -Y05-LF-LS (C0120229,10000 n g /L F iald Spika) GW-220R-Q4-Y05-LF-HS <00120230,100000 ng/L Flald S pika) GW-220L-Q4-Y05-LF-0 <00120231 Spk E, 10000 ng/L Lab S pika) GW-220L-Q4-Y05-LF-0 (C0120231 Spk F, 100000 n g /L Lab Spika) GW-220L.-Q4-Y05-t.F-LS <00120233,10000 n g /L F iald Spika) GW-220L-Q4-Y05-LF-HS <00120234, 100000 ng/L Fiald S pika) GW-226R-Q4-Y05-LF-0 (C012023S S pk 0 .1 0 0 0 n g /L Lab Spika) G W -226R -Q 4-Y05-LF-0 (C012023S S pk H, 10000 ng /L Lab S pika) G W -226R -Q 4-Y05-LF-LS <00120237,1000 ng /L Fiald S pika) GW-226R-Q4-Y05-LF-HS <00120234,10000 n g /L F iald S pika) GW-226L-Q4-Y05-LF-0 <00120230 S p k C, 100 n g /L Lab S pika) GW-226L-Q4-Y05-LF-0 (C0120239 S pk D, 1000 n g /L Lab Spika) GW-226L-Q4-Y05-LF-LS (C0120241,100 n g /L F iald Spika) GW-226L-Q4-Y05-LF-HS (C0120242,1000 n g /L F iald Spika) GW-327R-Q4-Y05-CP-0 (C0120243 Spk E. 100000 n g /L Lab S pika) GW-327R-Q4-Y05-CP-0 (C0120243 Spk F, 1000000 n ^ L Lab Spika) GW-327R-Q4-Y05-CP-LS <00120249,100000 ng/L Fiald Spika) GW-327R-Q4-Y05-CP-HS <00120246,1000000 ng/L Fiald S pika) Spiked (ng/L) C4 Sulfonate PFBS________________ C6 Sulfonate PFHS________________ C8 Sulfonate PFOS Amount Found Amount Found Amount Found in Sample Recovered Recovery in Sample Recovered Recovery in Sample Recovery l[ (nfl/L) (non.) < *i (non.) (non.) (*> (nan.) (H) 10000 100000 10000 100000 5830 5830 5830 5830 14100 88100 13700 96600 83 82 79 91 60400 60400 60400 60400 45700 152000 66700 173000 92 113 57300 57300 57300 57300 70200 149000 66000 206000 92 * 149 10000 100000 10000 100000 8240 8240 8240 8240 1000 10000 1000 10000 NR NR NR NR 16800 96400 15800 102000 86 88 76 94 NR NR NR NR NR NR NR NR 92900 92900 92900 92900 6240 6240 6240 6240 72300 181000 78200 208000 88 * 115 4570 12700 5840 15500 . 65 . 93 69300 69300 69300 69300 15800 15800 15800 15800 92000 187000 97200 252000 118 * 183 17600 21500 15800 26800 . 57 110 100 1000 100 1000 ND ND ND ND 92.8 991 96.3 937 93 99 96 94 100000 97100 1000000 97100 100000 97100 1000000 97100 210000 1110000 203000 950000 113 101 106 85 ND 118 118 ND 1200 120 ND 134 134 ND 1240 124 368000 368000 368000 368000 405000 1550000 543000 1690000 . 118 * 132 ND 94.4 94 ND 926 93 ND 116 116 ND 1140 114 348000 348000 348000 348000 409000 1340000 621000 1110000 * 99 76 ' Sample residue exceeds the spiking level significantly; therefore, an accurate recovery value cannot be calculated ND * Not detected at or above the Limit of Quantitations (tOQ) of 25 ng/L. NR = Not reported due to quality control resuit failures, see Table IV for re-extract data. Note: Since this summary table shows rounded results, recovery values may vary slightly from the values in the raw data. Exygen Research Page 22 of 122 Interim Report #19 - Analysis o f Ground Water Samples Exygen Study No.: P0001131 Table III. Matrix Spike Recovery of PFBS, PFHS and PFOS in Ground Water Samples Continued Sample Description C4 Sulfonate PFBS________________ C6 Sulfonate PFHS________________ C8 Sulfonate PFOS Amount Amount Found Amount Amount Found Amount Amount Found Amount Spiked in Sample Recovered Recovery in Sample Recovered Recovery in Sample Recovered Recovery (ng/L) <*> (ng/L) (%) (ng/L) fng/U (%) GW-310R-Q4-Y05-CP-0 (C0120247 S pk 0 , 100000 n g /L L sb Spik) GW-310R-Q4-Y05-CP-0 (C0120247 S pk H, 1000000 n g /L Lab Spik) GW-310R-Q4-Y05-CP-LS (C012024 9,10 00 00 n g /L F l ld S p ik) GW-310R-Q4-Y05-CP-HS (C0120230,1000000 n g /L F ild Spik) 100000 1000000 100000 1000000 337000 337000 337000 337000 582000 1400000 461000 106 * 1130000 79 696000 822000 696000 696000 2610000 908000 191 * 696000 2100000 140 894000 854000 , 894000 894000 2320000 143 706000 894000 1380000 49 GW-317L-Q4-Y05-CP-0 (C0120251 Spk C, 500 n g /L L sb Spik) GW-317L-Q4-Y05-CP-0 (C0120251 Spk D, 5000 n g /L Lab Spik) GW-317L-Q4-Y05-CP-LS (C0120253,500 ng /L F M d Spik) GW-317L-Q4-Y05-CP-HS (C0120254,5000 n g /L F ikl S pik) 500 5000 500 5000 ND ND ND ND 417 4100 395 4150 63 82 79 83 NR NR NR NR NR NR NR- NR NR NR NR NR 196 560 73 196 4710 90 196 609 83 196 5550 107 GW-320L-Q4-Y05-CP-0 (C012Q255 S p k E , 100 n g /L L a b S p ik) GW-320L-Q4-Y05-CP-0 (C0120255 Spk F, 1000 ng/L Lab S p ik*) GW-320L-Q4-YG5-CP-LS (C0120257,100 ng/L F ild Spik) GW-320L-Q4-Y05-CP-HS (C0120255,1000 n g /L F M d Spik) 100 1000 100 1000 ND ND ND ND 87.7 874 91.5 1020 88 87 92 102 NR NR NR NR NR NR NR NR NR NR NR NR 64.8 64.8 64.8 64.8 141 1040 201 1540 76 98 136 148 G W -T R IP-Q 4-Y 05-C P-LS (C0120200,100 n g /L F M d Spik) G W -T R IP-Q 4-Y 05-C P-H S (C0120261,1000 n g /L F lald Spik) 100 1000 ND ND 89.5 881 90 88 ND 180 180 ND ND 1630 163 ND 111 1120 111 112 Average: Standard Deviation: 90 9 Average: Standard Deviation: 124 33 ` Sample residue exceeds the spiking level significantly; therefore, an accurate recovery value cannot be calculated NO = Not detected at or above the Limit of Quantitations (LOQ) of 25 ng/L. NR = Not reported due to quality control result failures, see Table IV for re-extract data. Note: Since this summary table shows rounded results, recovery values may vary slightly from the values In the raw data. Average: Standard Deviation: 105 31 Exygen Research Page 23 of 122 Interim Report #19 - Analysis o f Ground Water Samples Exygen Study No.: P0001131 Table IV. Matrix Spike Recovery of PFBS and PFHS in Re-extracted Ground Water Samples Sample Description GW-226R-Q4-Y05-LF-LS (C 0120237,1000 ng/L Field Spike) GW-317L-Q4-Y05-CP-LS (C0120252 Spk E, 500 ng/L Field Spike) G W -3 1 7 L-Q4-Y05-CP - HS (C0120252 Spk F, 5000 ng/L Field Spike) GW-317L-Q4-Y05-CP-LS (C0120253, 500 ng/L Field Spike) GW-317L-Q4-Y05-CP-HS (C0120254, 5000 ng/L Field Spike) GW-320L-Q4-Y05-CP-0 (C0120256 Spk G, 100 ng/L Lab Spike) GW-320L-Q4-Y05-CP-0 (C0120256 Spk H, 1000 ng/L Lab Spike) GW-320L-Q4-Y05-CP-LS (C 0120257,100 ng/L Field Spike) GW-320L-Q4-Y05-CP-HS (C 0120258,1000 ng/L Field Spike) Spiked (ng/L) 04 Sulfonate PFBS______________ C6 Sulfonate PFHS Amount Found Amount Found in Sample Recovered Recovery in Sample Recovered Recovery (ng/L) (ng/L) (%) (ng/L) (ng/L) (%) 1000 NR NR NR - -- 500 5000 500 5000 - . _ 126 621 99 126 5120 100 - - 126 639 103 - - 126 5090 99 100 1000 100 1000 . - _ 7.69 115 107 . _ 7.69 985 98 . _ 7.69 133 125 - - 7.69 1220 121 Average: Standard Deviation: NA NA Average: 107 Standard Deviation: 11 ` Sample residue exceeds the spiking level significantly; therefore, an accurate recovery value cannot be calculated ND = Not detected at or above the Limit of Quantitations (L0Q) 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. Exygen Research Page 24 of 122 Interim Report #19 - Analysis o f Ground Water Samples Exygen Study No.: P0001131 FIGURES Exygen Research Page 25 o f 122 Interim Report #19 - Analysis o f Ground Water Samples Exygen Study No.: P0001131 Figure 1. Typical Calibration Curve for PFBS in Reagent Water 1 2 1 6 0 5 A YOS.rdb (PFBS): "Linear" Regression C'1 / * * weighting): y - 146 x + 0 .0 0 0 7 7 6 ( r * 0 .9 9 8 8 ) Area, counts Exygen Research Page 26 o f 122 Interim Report #19 - Analysis o f Ground Water Samples Exygen Study No.: P0001131 Figure 2. Extracted Standards of PFBS in Reagent Water, 0 ng/L and 25 ng/L, Respectively XCOf06O6-O - PFBS (Standard) 29910/9910 anta aampia 1 o f 36 from f21905D.wiff $>aa/t not found) Tim e, min X C 0106 06-1 - PFBS (Standard) 2 9 9 .0 /9 0 .0 amu sample 2 of 36 from 121905D.w iff Area: 5 0 0 3 counts Height: 4 .3 0 e + 0 0 2 ops RT: 0 .5 1 4 min 0.51 CO * eCO c0> Exygen Research Page 27 of 122 Interim Report #19 - Analysis o f Ground Water 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 ftoagont Control - PFBS (Unknown) 299.W99.Qama -sample 9 o f 36 from 121905D.wiff (peak not found) 12.40 Tim e, min Reagent Spk A - PFBS (Q 0 )2 9 9 .0 /9 9 .0 a mu - sample 10 of 36 from 121905D.w iff Area: 8 9 5 9 counts Height: 7 .9 5 e + 0 0 2 ops RT: 0 .5 0 7 min 0.51 Tim e, min I Reagent Spk B - PFBS (QC) 2 9 9 .0 /9 9 .0 a m u -s a m p le 11 of 36 from 121903D.w iff Area: 8 5 9 8 3 counts Height: 8 .0 3 6 + 0 0 3 cps RT: 0 .5 0 8 min 0.51 Exygen Research Page 28 o f 122 Interim Report #19 - Analysis o f Ground Water Samples Exygen Study No.: P0001131 Figure 4. Chromatogram Representing a Ground Water Sample Analyzed for PFBS (Exygen ID: C0120251, Data Set: 121905D) I C0120251 - PFBS (Uataowa) 299.0/99.0 ama -tam p/ 16 o f 36 from 121905D.wiff Area: 3011 c o a a tt Heig/it: 1.11e+002ept RT: 1.21 mia Intensity, cps Exygen Research Page 29 of 122 Interim Report #19 - Analysis o f Ground Water Samples Exygen Study No.: P0001131 Figure 5. Typical Calibration Curve for PFHS in Reagent Water 1 2 1 6 0 5 A Y05.rdb (PFHS): "Lineai" Regression ("1 / x" weighting): y * 2 6 8 x + 0 .0 0 3 0 3 ( r 0 .8 8 5 0 ) Area, counts Exygen Research Page 30 o f 122 Interim Report #19 - Analysis o f Ground Water Samples Exygen Study No.: P0001131 Figure 6. Extracted Standards of PFHS in Reagent Water, 0 ng/L and 25 ng/L, Respectively I XC010606-0 - PFHS (Standard) 399.0/80.0 amu -sample 1 o f 36 from 121905D.wiff Steak not found) Intensity, cps T im e, min XC 010S 06-1 PFHS (Standard) 3 9 9 .0 /8 0 .0 amu sample 2 of 3 8 from 121005D .w iff Area: 5 4 8 0 counts Height: 3 .4 0 e + 0 0 2 cps RT: 8 .0 0 min Intensity, cps Exygen Research Page 31 o f 122 Interim Report #19 - Analysis o f Ground Water 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 Reagent Control -PFHS (Unknown)399l0/90.0am a sam ple 9 o f 36 from 121905D.wiff peak not found) 0.53 &c<ca>8 5 , 1 ?ft 2 78 3 e7',4.0A0 '425 -11 1068'~ 18 48 8 8 8 11-561185 I 13.40 14.84 J l / V W ^ A n aA t 7 H a ^ ^ a a /sa/ ^ 1 2 3 4 3 8 7 8 9 10 11 12 13 14 15 T im e, min R eagent Spk A - PFHS (Q C ) 3 9 9 .0 /8 0 .0 a m u -s a m p le 10 of 3 6 from 121905D.vuiff Area: 103 26 counts Height: 6 .2 4 e + 0 0 2 cps RT: 8 .0 9 min . 16 8.09 17.54 , A-* * `5co>5 c Area: 8 4 3 6 4 counts Height: 5 .8 3 e + 0 0 3 cps R T :8 .0 9 min 8.09 Exygen Research Page 32 of 122 Interim Report #19 - Analysis o f Ground Water Samples Exygen Study No.: P0001131 Figure 8. Chromatogram Representing a Ground Water Sample Analyzed for PFHS (Exygen ID: C0120251, Data Set: 121905D) C01202S1 - PFHS (Uahaowa) 399.0/80.0 ama -tamp!e 16 o f 36 from 121905D.wiff Area: 35766 count* Height: 2.76c+003cp* RT: a 00 mia 8.00 Intensity, cps Exygen Research Page 33 o f 122 Interim Report #19 - Analysis o f Ground Water Samples Exygen Study No.: P0001131 Figure 9. Typical Calibration Curve for PFOS in Reagent Water 1 2 1 6 0 5 A Y05.rdb (PFOS): "Lineai" Regression ("1 i x" weighting): y - 2 8 5 x + 0 .0 0 1 4 7 ( r * 0 .9 0 8 6 ) Area, counts Exygen Research Page 34 o f 122 Interim Report #19 - Analysis o f Ground Water Samples Exygen Study No.: P0001131 Figure 10. Extracted Standards of PFOS in Reagent Water, 0 ng/L and 25 ng/L, Respectively I XC121605-0 PFOS (Standard) 499L0/&O.0 amu -satnpte 2 o f 39 from 1216Q5A.wiff (peak not found) Intensity, cps T im e , min XC 12160S-1 - PFOS (Standard) 40 9 .0 /8 0 .0 amu - sam ple 3 of 3 9 from 121805A.w iff Area: 8 0 0 3 counts Height: 4 .6 7 e + 0 0 2 cps RT: 11.2 min 11.22 Intensity, cps Exygen Research Page 35 o f 122 Interim Report #19 - Analysis o f Ground Water 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 R eagent Centro/ PFOS (Unknown) 499.0/80.Offw tf - j am pfe 9 o f 38 from 121905A.wfff (peak not found) 11.33 V*) c0> Tim e, min | R eagent Spk A - PFOS (Q C ) 4 6 9 .0 /8 0 .0 a mu - sample 10 of 3 8 from 121905A.wiff Area: 140 28 counts Height: 7 .8 3 e + 0 0 2 cps RT: 11.2 min 11.21 Tim e, min Reagent Spk B PFOS (Q C )4 0 0 .0 /8 0 .0 amg sample 11 of 3 8 from 121005A.wiff Area: 137 1 8 6 counts Height: 6 .7 4 e + 0 0 3 cps RT: 11.2 min 11.23 & to Exygen Research Page 36 o f 122 Interim Report #19 - Analysis o f Ground Water Samples Exygen Study No. : P0001131 Figure 12. Chromatogram Representing a Ground Water Sample Analyzed for PFOS (Exygen ID: C0120251, Data Set: 121905D) I C0120251 - PFOS (Unknown) 499.0/80.0 antn -tam p l* 16 o f 36 from 121905D.wiff m Aron: 56521 count Height: 3.17**003 cps RT: 10.7m in 3000 10.74 2800 2600 2400 - 2200 2000 - 1800 1600 1400 I10.25 1200 - 1000 800- eoo- 400 200 0 -*-- ^ -- ..................................................... r ---nr.......................... 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 Tima, min Intensity, cps Exygen Research Page 37 of 122 Interim Report #19 - Analysis o f Ground Water Samples Exygen Study No.: P0001131 APPENDIX A Study Protocol P0001131 (Exygen Study No. P000I131) with Analytical Methods and Protocol Amendments Exygen Research Page 38 of 122 Interim Report #19 - Analysis o f Ground Water Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 STUDY PROTOCOL Study Title: Analysis of Perfluorobutanesulfonate (PFBS), Perfluorohexanesulfonate (PFHS), and Perfluorooctanesulfonate (PFOS) in 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 P erform ing Laboratory: Exygen Research 3058 Research D riv e State C ollege, P A 16801 Phone: (8 1 4 ) 272-1039 Sponsor Representative: M ich ae l A . Santoro D ire cto r o f R egu lato ry A ffa irs 3 M B u ild in g 0 2 3 6 -0 1 -B -1 0 St. P aul, M N 55144 Phone: (6 5 1 ) 7 3 3-6 374 Page I o f 65 Exygen Research Page 39 o f 122 Interim Report #19 - Analysis o f Ground Water Samples Exygen Study No.: P0001131 Exygen Pro to co l N um ber: P 0 0 0 1131 DISTRIBUTION: 1) Jaisimha Kesari, Study Director, Weston Solutions 2) John M . Flaherty, Principal Investigator, Exygen Research 3) M ichael A . Santoro, Sponsor Representative, 3M Company 4 ) Exygen Research Q uality Assurance Unit Exygen Research Page 2 o f 65 Page 40 of 122 Interim Report #19 - Analysis o f Ground Water Samples Exygen Study No.: P0001131 E xy g en Pro to co l N um ber: POOOl 131 PROTOCOL APPROVAL Study T itle : Analysis o f Perfluorobutanesulfonate (PFBS), Perfluorohexanesulfonate (PFH S), and Perfluorooctanesulfonate (PFOS) in W ater, Soil, Sediment, Fish, Clams, Vegetation, Small Mam mal Livers and Small M am m al Serum Using L C /M S /M S for the 3M Decatur M onitoring Program Exygen Protocol Number: POOOl 131 APPROVALS JaisimhauCesan, Study Director Weston Solutions m M ichael A . Safitoro, Sponsor Representative 3M Compaify A A ^ u /y ^ / Richard A . G: Exygen Resi sident, Facility Management U .______________________ iad, Q uality Assurance U nit Date D a te Paga 3 oJ 65 Exygen Research Page 41 of 122 Interim Report #19 - Analysis o f Ground Water Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 TABLE OF CONTENTS T IT L E P A G E ................................................................................................................................................................................1 D IS T R IB U T IO N ..........................................................................................................................................................................2 PR O T O C O L A P P R O V A L ........................................................................................................................................................3 T A B L E O F C O N T E N T S .......................................................................................................................................................... 4 IN T R O D U C T IO N ....................................................................................................................................................................... 5 T E S T M A T E R IA L S .................................................................................................................................................................. 5 O B JE C T IV E .................................................................................................................................................................................6 T E S T IN G F A C IL IT Y ................................................................................................................................................................ 6 ST U D Y D IR E C T O R .................................................................................................................................................................. 7 SP O N S O R R E PR E SE N T A T IV E ............................................................................................................................................ 7 PR IN C IPA L IN V E S T IG A T O R .............................................................................................................................................. 7 PR O PO SE D EX P E R IM E N T A L ST A R T A N D TE R M IN A T IO N D A T E S ............................................................... 7 ID E N T IFIC A T IO N A N D JU S TIF IC A TIO N O F T H E T E S T S Y S T E M .................................................................... 8 SA M PLE P R O C U R E M E N T . R E C E IPT A N D R E T E N T IO N ....................................................................................... 8 SA M PLE ID E N T IF IC A T IO N .................................................................................................................................................9 A N A L Y T IC A L PR O C E D U R E S U M M A R Y ......................................................................................................................9 V E R IF IC A T IO N O F A N A L Y T IC A L P R O C E D U R E ...................................................................................................... 9 M E T H O D F O R C O N T R O L O F B U S ..................................................................................................................................11 ST A T IS T IC A L M E T H O D S .....................................................................................................................................................11 G L P S T A T E M E N T .................................................................................................................................................................... 11 R E P O R T ........................................................................................................................................................................................ 11 SA FE TY A N D H E A L T H ......................................................................................................................................................... 12 A M E N D M E N T S T O P R O T O C O L ........................................................................................................................................13 D A TA R EC O RD K E E P IN G ....................................................................................................................................................13 Q U A LIT Y A S S U R A N C E ........................................................................................................................................................ 14 R E T E N T IO N O F D A T A A N D A R C H IV IN G ....................................................................................................................14 A P P E N D IX I. A N A L Y T IC A L M E T H O D S .........................................................................................................................15 Page 4 o f 65 Exygen Research Page 42 o f 122 Interim Report #19 - Analysis o f Ground Water Samples Exygen Study No.: P0001131 E x y g en Pro to co l N um ber: P 0 0 0 1131 INTRODUCTION 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 using L C /M S /M S for the 3M Decatur M onitoring Program. The study w ill be audited for compliance w ith EPA TS C A Good Laboratory Practice Standards 40 C FR 792 by the Q uality Assurance U n it 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 M olecular W eight: 338 supplied as the potassium salt (CiF^SO s'K*) Lot Number: 101 Purity: 96.7% Transitions Monitored: 299 - * 99 Structure: FF FF FF F PFHS Chemical Name: Perfluorohexanesulfonate M olecular W eight: 438 supplied as the potassium salt (CFnSO a'K*) Lot Number: SE036 Purity: 98.6% Transitions Monitored: 399 -> 80 Structure: Page 5 o f 65 Exygen Research Page 43 o f 122 Interim Report #19 - Analysis o f Ground Water Samples Exygen Study No.: P0001131 Exygen Pro to co l N um ber: P 0 0 0 1131 PFOS Chemical Name: Perfluorooctanesulfonate M olecular W eight: 538 supplied as the potassium salt (CgF^SC^'K*) Lot Number: 217 Purity: 86.9% Transitions Monitored: 499 - 99 Structure: OBJECTIVE The purpose o f this study is to perform analysis for perfluorobutanesulfonate (PFBS), perfluorohexanesulfonate (PFHS) and perfluorooctanesulfonate (PFO S) in water, soil, sediment, fish, clams, vegetation, sm all mammal livers and small mammal serum for the 3M Decatur M onitoring Program using the current versions o f the follow ing Exygen analytical methods: V0001780: V0001781: V0001782: V0001783: V0001784: V0001785: V0001786: "Method o f Analysis for the Determ ination o f Perfluorooctanoic Acid (PFO A ) in W ater by L C /M S /M S " "Method o f Analysis for the Determ ination o f Perfluorooctanoic Acid (PFO A ) in Soil by L C /M S /M S " "Method o f Analysis for the Determ ination o f Perfluorooctanoic Acid (PFO A ) in Sediment by L C /M S /M S " "Method o f Analysis for the Determ ination o f Perfluorooctanoic Acid (PFO A ) in Fish and Clams by L C /M S /M S " "Method o f Analysis for the Determ ination o f Perfluorooctanoic Acid (PFO A ) in Vegetation by L C /M S /M S " "Method o f Analysis for the Determ ination o f Perfluorooctanoic Acid (PFO A ) in Small Mam m al Liver by L C /M S /M S " "Method o f Analysis for the Determ ination o f Perfluorooctanoic Acid (PFO A ) in Small Mam m al Serum by L C /M S /M S " TESTING FACILITY Exygen Research 3058 Research D rive State College, PA 16801 Phone: (814) 272-1039 Page 6 o f 65 Exygen Research Page 44 o f 122 Interim Report #19 - Analysis o f Ground Water Samples Exygen Study No.: P0001131 E xy g en P ro to co l N um ber: P 0 0 0 1 131 STUDY DIRECTOR Jaisimba Kesari P.E., DEE Weston Solutions, Inc. 1400 Weston W ay West Chester, PA 19380 Phone: (610) 701-3761 Fax: (610) 701-7401 j .kesari@westonsolutions.com SPONSOR REPRESENTATIVE M ichael A . Santoro 3M Company Director o f Regulatory Affairs 3 M Building 0236-01-B -10 St. Paul, M N 55144 Phone: (651) 733-6374 PRINCIPAL INVESTIGATOR John M . Flaherty Exygen Research 3058 Research Drive State College, PA 16801 Phone: (814)272-1039 j ohn.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 45 o f 122 Interim Report #19 - Analysis o f Ground Water Samples Exygen Study No.: P0001131 E x y g en P ro to co l N um ber: P 0 0 0 1 131 IDENTIFICATION AND JUSTIFICATION OF THE TEST SYSTEM The following are the test systems for this study: W ater (groundwater and surface water) Soil Sediment Fish Clams Vegetation Small Mam m al Liver Small Mam mal Serum The samples w ill be collected by Weston Solutions. The control samples w ill be purchased and prepared by the testing facility. Purchase and processing details for the control samples w ill be included in the final report associated w ith this study. The test systems were chosen to access the environmental impact o f PFBS, PFHS and PFOS in the Decatur, Alabama area. SAMPLE PROCUREMENT, RECEIPT AND RETENTION W ater, soil, sediment, fish, clam, vegetation, small mammal liver and small mammal serum samples w ill 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 W ork Plan for Sampling Environmental M edia." The number and types o f samples collected w ill vary depending availability in the field. The total number o f samples received and analyzed for each m atrix w ill be documented in the final report associated w ith this study. W ater, soil, and sediment samples w ill be used as received without further processing at Exygen. These samples w ill be stored refrigerated at 2C -8C . Fish, clam, vegetation and small mammal liver samples w ill be processed according to the appropriate analytical method (see Appendix I). These samples w ill be stored frozen at < -10"C. Sm all mammal whole blood samples w ill be centrifuged in the field at the tim e o f collection and the serum fraction w ill be used for the study. Small mammal serum w ill be stored frozen at S - IC C . The receipt and processing o f the samples w ill be documented in the final report and raw data associated w ith the study. Page 8 o f 65 Exygen Research Page 46 o f 122 Interim Report #19 - Analysis o f Ground Water Samples Exygen Study No.: P0001131 E xy g en P ro to co l N um ber: P 0 0 0 1131 SAMPLE IDENTIFICATION Prior to analysis, each sample w ill be assigned a laboratory sample reference number. The reference number w ill be unique and w ill distinguish each laboratory sample that is processed throughout the analytical procedure. Chromatographic data w ill be identified by the laboratory sample reference number. Sample storage conditions and locations w ill be documented throughout the study. ANALYTICAL PROCEDURE SUMMARY References: V0001780: "Method o f Analysis for the Determ ination o f Perfluorooctanoic Acid (PFO A ) in W ater by L C /M S /M S " V0001781: "Method o f Analysis for the Determ ination o f Perfluorooctanoic Acid (PFO A ) in Soil by L C /M S /M S " V0001782: "Method o f Analysis for the Determ ination o f Perfluorooctanoic Acid (PFO A ) in Sediment by L C /M S /M S " V0001783: "Method o f Analysis for the Determ ination o f Perfluorooctanoic Acid (PFO A ) in Fish and Clams by L C /M S /M S " V0001784: "Method o f Analysis for the Determ ination o f Perfluorooctanoic Acid (PFO A ) in Vegetation by L C /M S /M S " V0001785: "Method o f Analysis for the Determ ination o f Perfluorooctanoic Acid (PFO A ) in Small Mam m al Liver by L C /M S /M S " V0001786: "Method o f Analysis for the Determ ination o f Perfluorooctanoic Acid (PFO A ) in Small Mam m al Serum by L C /M S /M S " The above methods use analytical conditions capable o f separating the isomers o f PFBS, PFHS and PFOS. The final report w ill include the isomers summed into total PFBS, total PFHS, and total PFOS found. VERIFICATION OF ANALYTICAL PROCEDURE A laboratory control sample w ill be used for the preparation o f fortified control samples. The test substance w ill be made into solutions as per the method, and added to the matrices via a micropipette. For water sampling, Exygen w ill supply one bottle per sample collected. The bottles w ill be 500 m L precleaned Sci/Spec Prem ier w ide mouth H D PE bottles. These bottles have been routinely used for fluorochemical sample Page 9 o f 65 Exygen Research Page 47 o f 122 Interim Report #19 - Analysis o f Ground Water Samples Exygen Study No. : P0001131 E xygen Pro to co l N um ber. P 0 0 0 1 131 collection at the testing facility and have been shown to be free o f PFBS, PFHS and PFOS. Samples w ill be added to each container to a volum etric fill line at 200 m L. A field duplicate, a low field spike and a high field spike o f each sample w ill be collected. The low and high field spike bottles w ill contain PFBS, PFHS and PFOS as w ell as perfluorooctanoic acid (P FO A ) and 1.2-13C perfluorooctanoic acid ( l3C PFO A ). PFO A and l3C PFOA are included in the solutions used to spike the samples. The results for PFOA and l3C PFOA w ill not be reported in this study. Exygen w ill supply one field blank (control water) and two field blank spikes (control w ater fortified with PFBS, PFHS and PFOS at a low and high level) for every twenty samples collected. A t the testing facility, each water sample (excluding field duplicates and field spikes) w ill be extracted in duplicate and w ill also be fortified at a low and high concentration w ith 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 w ill supply one 500 mL precleaned Sci/Spec Premier wide mouth HDPE bottle per sample collected or a zip-seal bag. A ll containers/bags used for sample collection w ill be shipped to the sample location. Samples w ill be added to each container or bag in the field. A t the testing facility, each sample w ill be extracted in duplicate and w ill also be fortified at a known concentration w ith 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 w ill supply a 50 m L polypropylene centrifuge tube. For small mammal serum, Exygen w ill supply a collection kit for each sample containing serum separator tubes (red top), vacutainers, needle holders and needles, transfer pipettes, and polypropylene tubes. A t the testing facility, each liver and serum sample w ill be extracted in duplicate and w ill also be fortified at a known concentration w ith 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 a trix Low Spiking Level H ig h Spiking Level W ater 5 0 0 n g /L 5000 ng/L Soil 4 ng/g 40 ng/g Sediment 4n g /g 40 ng/g Fish 10 ng/g 100 ng/g Clams 10 ng/g 100 ng/g Vegetation 10 ng/g 100 ng/g Small Mam m al Liver 10 ng/g 100 ng/g Small Mam m al Serum 10 ng/mL 100 ng/m L Page 10 o f 65 Exygen Research Page 48 of 122 Interim Report #19 - Analysis o f Ground Water Samples Exygen Study No.: P0001131 E xy g en P ro to co l N um ber: P 0 0 0 1 131 Recoveries are anticipated to be between 70% and 130% o f the fortified levels; however, the exact precision and accuracy w ill be determined by the analysis o f the quality control samples described above. A statement o f accuracy w ill be included in the final report. METHOD FOR CONTROL OF BIAS Control o f bias w ill be addressed by taking representative sub-samples from a homogeneous m ixture o f each m atrix from untreated control samples, and by analyzing at least two levels o f fortifications. STATISTICAL METHODS Statistics w ill be lim ited to those specified in the subject methods and to the calculation o f average recoveries, as applicable. GLP STATEMENT A ll aspects o f this study shall be performed and reported in compliance with EPA TS C A Good Laboratory Practice Standards 40 C FR 792. The final report or data package (supplied to the Sponsor) shall contain a statement that the study was conducted in compliance w ith current and applicable GLP standards and w ill outline any deviations in the study from those standards. This statement w ill be signed by the Study Director and Sponsor Representative. REPORT A final report w ill be prepared by the principal investigator or their designee at the conclusion o f the study. The report w ill include, but w ill not be lim ited to, the following: The name and address o f the Study Director, Sponsor Representative, and o f 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 6i Exygen Research Page 49 of 122 Interim Report #19 - Analysis o f Ground Water Samples Exygen Study No.: P0001131 E xygen P ro to co l N um ber: POOO1131 The signed and dated statement by the Exygen Research Q uality Assurance U n it 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. I f the subject method was followed exactly, it is necessary to include only a copy o f the analytical method. A ny modifications to this method w ill be incorporated into the report. I f the method is photo-reduced, the project number and page number must be included on each page. Description o f the instrumentation used and operating conditions. A ll results from all sets analyzed. Control and fortified samples w ill be identified and the data table w ill include sample number and fortification level. Representative chromatograms for each analyte in each m atrix, including chromatograms o f a standard and a control sample, and a chromatogram at a fortification level. The location o f the analyte peaks w ill be clearly identified in all chromatograms. A ll circumstances that may have affected the quality or integrity o f the data w ill 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 w ill be signed and dated by the Study Director and the Sponsor Representative. A ll applicable requirements for reporting o f study results as per 40 CFR 792.185. SAFETY AND HEALTH Laboratory personnel w ill practice good sanitation and health habits. Every reasonable precaution shall be taken to prevent inadvertent exposure o f personnel and the environment to the test or reference substance(s). Page 12 o f 65 Exygen Research Page 50 of 122 Interim Report #19 - Analysis o f Ground Water Samples Exygen Study No.: P0001131 E xy g en P ro to co l N um ber: POOO1131 AMENDMENTS TO PROTOCOL A ll significant changes to the analytical protocol outlined here w ill be expressed in w riting, signed and dated by the Study Director and Sponsor Representative. Amendments usually w ill be issued prior to initiation o f study plan change. However, when a change is required without sufficient tim e for the issue o f a w ritten amendment, that change may be effected verbally with supporting documentation signed and dated by the Study Director and followed w ith a w ritten amendment as soon as possible. In this case, the effective date o f the w ritten amendment w ill be the date o f the documented change. Copies o f the signed amendments w ill be appended to all distributed study plan copies. The original amendment w ill be maintained w ith the original study plan. Any deviations from the study plan or from the analytical method as provided w ill be documented and reported prom ptly 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 A ll 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 A ny other documentation necessary for the reconstruction o f the study Chromatograms- A ll chromatograms w ill 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 w ill include the amount o f analyte added and the sample number o f the sample that was fortified. Analytical standard chromatograms w ill additionally include the concentration (e.g., pg/m L). Page 13 o f 65 Exygen Research Page 51 o f 122 Interim Report #19 - Analysis o f Ground Water Samples Exygen Study No.: P0001131 E xy g en Pro to co l N um ber: P 0 0 0 1 131 As part o f the documentation the follow ing sheets w ill be included in each analytical set: a run sheet listing the samples to be run in the set, and an instrument conditions sheet dseribing the instrument type and operating conditions. QUALITY ASSURANCE The Q A U nit o f Exygen Research w ill inspect the study at intervals adequate to assure compliance w ith G LP's, and w ill report the findings o f audits to the Study Director, Exygen Management, and the Sponsor Representative. RETENTION OF DATA AND ARCHIVING A ll hard copy raw data, including, but not lim ited to, the original chromatograms, worksheets, correspondence, and results shall be included w ith the data package submitted to the Study Director. These w ill be archived w ith the original study plan, amendments, final report, and all pertinent information &om the Sponsor. The testing facility shall keep a ll electronic raw data and any instrument, equipment, and storage logs for the period o f tim e specified in 40 CFR 792.195. An exact copy o f the materials submitted to the study director w ill also be kept at Exygen Research. Exygen w ill obtain permission from the study director before discarding or returning samples. Exygen Research Page 4 o f 65 Page 52 of 122 Interim Report #19 - Analysis o f Ground Water Samples Exygen Study No. : P0001131 E xy g en P rotocol N um ber: POOOl 131 APPENDIX I ANALYTICAL METHODS V0001780: "Method o f Analysis for the Determ ination o f Perfluorooctanoic Acid (PFO A ) in W ater by L C /M S /M S " V 0001781: "Method o f Analysis for the Determ ination o f Perfluorooctanoic Acid (PFO A ) in Soil by LC /M S /M S " V0001782: "Method o f Analysis for the Determ ination o f Perfluorooctanoic Acid (PFO A ) in Sediment by L C /M S /M S " V0001783: "Method o f Analysis for the Determ ination o f Perfluorooctanoic Acid (PFO A ) in Fish and Clams by L C /M S /M S " V 0001784: "Method o f Analysis fo r the Determ ination o f Perfluorooctanoic Acid (PFO A ) in Vegetation by L C /M S /M S " V0001785: "Method o f Analysis for the Determ ination o f Perfluorooctanoic Acid (PFO A ) in Small Mam m al L iver by L C /M S /M S " V0001786: "Method o f Analysis for the Determ ination o f Perfluorooctanoic Acid (PFO A ) in Small Mam m al Serum by L C /M S /M S " Exygen Research Page IS o f 65 Page 53 o f 122 Interim Report #19 - Analysis o f Ground Water Samples Exygen Study No.: P0001131 E x y g en P ro to co l N um ber: P000 U 31 ANALYTICAL METHOD Method Number: VQ001780 Method o f Analysis for the Determination o f Perfluorooctanolc Acid (PFOA) in Water by LC/MS/MS Analytical Testing Facility: Exygen Research 3038 Research Drive State College, PA 16801 Approved By: _______ Paul Connolly ' Technical Leader, LC-MS, Exygen Research Date / lohn Flaherty / ' Vice President, Operations, Exygen Research Exygen Research T out Pages: 7 Page !6 o f 65 Page 54 o f 122 Interim Report #19 - Analysis o f Ground Water Samples Exygen Study No.: P0001131 E xy g en P ro to co l N um ber: P 0 0 0 1 131 Exygea R u en c Method Number VOOOI' SU ANALYTICAL METHOD Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in W ater by LC/MS/MS 1.0 Scope This method is to be employed for the isolation and quantitation o f perfluorooctanoic a d d by High Performance Liquid Chromatography coupled to a iandem Mass Spectromctric Detector (LC/MS/MS) in water. 2.0 Safety 2.1 Always observe safe laboratory practices. 2.2 Consult the appropriate MSDS before handling any chemical for proper safety precautions. 3.0 Sample Requirement 3.1 At least 40 m L o f test sample for extraction. 3.2 No sample processing is needed for water samples. 3.3 Samples stored refrigerated should be allowed to equilibrate to room temperature. 3.4 All samples must be thoroughly mixed before being sampled for extraction 3.5 Any samples containing particles should be centrifuged at -3 0 0 0 rpm for -5 minutes and the supernatant used for the extraction. 3.6 Sample collection procedures will be specified in the sampling plan for this project. 4.0 Reagents and Standards 4.1 W ater - 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 tandem Mass Spectrometer (LC/MS/MS). 5.2 A device to collect raw dam for peak integration and quantitation. 5.3 Analytical balance capable o f trading to 0.00001 g. 5.4 50 mL disposable polypropylene centrifoge tubes. 5.5 1S mL disposable polypropylene centrifoge tubes. 5.6 Disposable micropipets (50-1 OOuL, 100-200uL). 5.7 125-mL LDPE narrow-mouth bottles. 5.8 2 m L clear HPLC vial kit. 5.9 Disposable pipettes. 5.10 Autopipettes (100-1000 pL and 10-100 pL), with disposable tips. 5.11 Waters Sep Pak Vac 6 cc (Ig ) tC18 SPE cartridges. Page 2 of ? Page 17 o f 65 Exygen Research Page 55 of 122 Interim Report #19 - Analysis o f Ground Water Samples Exygen Study No. : P0001131 E xy g en P ro to co l N um ber: P 0 0 0 1 131 Exygen Reaearch Method Number V000178Q ANALYTICAL m e t h o d Method o f Analysis foe the Determination o f Perfluorooctanoic Acid (PFOA) in W ater by L C /M S /M S 5 1 2 SPB vacuum manifold. 5.13 Centrifoge capable o f spinning 50 m L polypropylene tubes at 3000 rpm. 6.0 Chromatographic System 6.1 Analytical Column: Floophase RP (Keystone Scientific), 2.1 mm x 50 mm. 5h (P/N: 82505-052130) 6.2 Temperature: 30C 6.3 Mobile Phase (A ) : 2 m M 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 %A 65 65 25 25 65 Flow Rate m (mUminl 35 0.3 35 0.3 75 0.3 75 0.3 35 0.3 6.6 Injection Volume: 15 pL (can b e 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, monitoring 413 -> 369 m/z. The above conditions are intended u 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 aaimooium acetate in water is prepared by adding 0.154 g o f ammonium acetate to 1000 mL o f water. Alternate volumes may be prepared. Page 3 ui Page /8 o f 65 Exygen Research Page 56 of 122 Interim Report #19 - Analysis o f Ground Water Samples Exygen Study N o.: P0001131 E xy g en P ro to co l N um ber: P 0 0 0 1 131 Exygea Research Method Number V0001780 ANALYTICAL METHOD Method o f Analysis for the Determination o f Perfluorooctanoic A d d (PFOA) in Water by L C /M S/M S 9.0 Standard Preparation 9.1 Standard Stock/Fortificatian Solution 9.1.1 Prepare a stock solution o f ~ 100 pg/mL o f PFOA by weighing 10 mg o f analytical standard (corrected for purity) and dilute to 100 mL with methanol in a 12S-mL LDPE bottle. 9.1.2 A 10 pg/mL fortification solution o f PFOA is prepared by bringing 10 m L o f the 100 pg/mL solution to a final volume o f 100 with methanol in a 123 mL LDPE bottle. 9.1.3 A 1.0 pg/mL fortification solution o f PFOA is prepared by bringing 10 m L o f the 10 jig/mL solution to a final volume o f 100 with methanol in a 123 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 w ith methanol in a 123 m L LDPE bottle. 9.1.3 A 0.01 pgftnL fortification solution o f PFOA is prepared by bringing 10 mL o f the 0.1 pg/raL solution to a final volume o f 100 with methanol In a 125 mL LDPE bottle. 9.1.6 The stock and fortification solutions are to be stored in a refrigerator at approximately 4*C and are stable for a maximum period o f 6 months from the date o f preparation. 9.2 Standard Calibration Solutions 9.2.1 9.2.2 LC/MS/MS calibration standards are prepared in HPLC water. The calibration standards are processed through die extraction procedure, identical to samples. The following is a typical example: additional concentrations may be prepared as needed. Final Concentratimi Fortification Volume of Concentration of Calibration o f Fortification Volume Fortified Control Calibration Standard ID Solution (nob) (uL> Sample (mL) Standard (not)* (example) 0 0 40 0 XCmmddyy-0 10 100 40 10 200 40 10 400 40 100 too 40 23 XCmmddyy-1 50 XCmmddyy-2 too XCmraddyy-3 250 XCmmddyy*4 100 200 40 300 XCmroddyy-5 100 400 40 1000 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. Pige 4 oJ'n Page 79 o f 65 Exygen Research Page 57 of 122 Interim Report #19 - Analysis o f Ground Water Samples Exygen Study No.: P0001131 Exygen P ro to co l N um ber: P 0 0 0 1131 Exygen Rewwch Method Number V00017SO ANALYTICAL METHOD Method o f Analysis for the Determination o f PerfhiorODCtanoic Acid (PFOA) in Water by L C /M S/M S 9.2.3 9.2.4 9.2.5 A zero ttandard aolution (reagent blank) m ust be prepared with each set o f standards extracted. Store all extracted calibration standards in 15-mL polypropylene tubes at 2*C to 6C, up to two weeks. Alternate volumes and concentrations o f standards m ay be prepared as needed. 10.0 Batch Set Up 10.1 Bach 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 o r a portion o f sample diluted to 40 mL with water into 50 m L polypropylene centrifoge tubes (fortify as needed, replace lid and mix well). 11.2 Condition the C u SPE cartridge! (1 g, 6 mL) by passing 10 mL methanol followed b y 5 m L o f HPLC water (~ 2 drop/sec). Do not let column run dry 11.3 Load sample on conditioned C u SPE cartridge. Discard eluate. 11.4 Elute with - 5 m L 100% methanoL Collect 5 mL o f eluate into graduated 15 m L polypropylene centrifoge tubes (final volume - 5 mL). 11.5 Analyze samples using electrosprmy LC/MS/MS. 12.0 Chromatography 12.1 Inject the same amount o f each standard, sample and fortified sample into the LC/MS/MS system. A calibration standard m ust precede and follow all analyzed samples. 12.2 Standards o f PFOA corresponding to at least five o r m ore concentration levels must be included in an analytical set. 12.3 A n entire set o f extracted calibration standards m ust b e included at the beginning and i t the end o f a sample set. Extracted standards must be interspersed between every 5*10 samples. A s an alternative, an entire set of extracted calibration standards m ay be injected at the beginning o f a set follow) by extracted calibration standards interspersed every 5*10 samples (to account for a second set o f extracted standards). In either case, extracted calibration standards m ust be the first and last injection in a sample set. 12.4 Use linear standard curves for quantitation. Linear standard curves are generated for the analyte by linear regression using 1/x weighting o f peak area Page 5eP Page 20 o f 65 Exygen Research Page 58 o f 122 Interim Report #19 - Analysis o f Ground Water Samples Exygen Study No.: P0001131 E x y g en P ro to co l N um ber: P 0 0 0 1131 Exygen Reiawch Method Number V0001780 ANALYTICAL METHOD Method o f Analysis for the Determination o f Ferfluorooctanoic Acid (PFOA) in Water by LC/MS/MS venue calibration etandard concentration using MaasLynx 3.3 (or equivalent) software system. 12.5 Sample response should not exceed standard responses. Any samples that exceed standard responses should be flirther diluted and reanalyzed. 13.0 Acceptance Criteria . 13.1 Chromatogram must show a peak o f a daughter ion at 369 amu from a parent o f 413 amu. The 413 amu parent corresponds to the PFOA anion, while the daughter ion (369 amu) represents the loss o f carbon dioxide. 13.2 Method blanks must not contain PFOA at levels greater than the LOQ. If a blank contains PFOA at levels greater than 50 ng/L, then a new blank sample must be obtained and the entire set must be re-extracted. 13.3 Recoveries o f control spikes and matrix spikes must be between 70-130% o f their known values. I f a control spike (alls outside the acceptable limits, the entire set o f sample* should be re-extracted. Any matrix spike outside 70 130% should be evalusted 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 ber o f extracted standards injected. 13.5 The correlation coefficient (R) for calibration curves generated must be 0.992 (R3 0.985). If calibration results fall outside these limits, then appropriate steps must be taken to adjust instrument operation, and the standards or the relevant set o f samples should b e reanalyzed. 13.6 Retention times between standards and samples must not drift more than 4 % within an analytical run. I f retention time drift exceeds this limit within an analytical run then the set must be reanalyzed. 14.0 Calculations 14.1 Use the following equation to calculate foe amount o f PFOA found (in ngi'L, based on p e ik area) using foe standard curve (linear regression parameters) generated by foe Masa Lynx software program: PFOA found (ng/L) - (Peak area - intercept) x DF slope DF " factor by which foe final volume was diluted, if necessary. Pge 6 of 7 Page 2J o f 65 Exygen Research Page 59 of 122 Interim Report #19 - Analysis o f Ground Water Samples Exygen Study No. : P0001131 E xy g en P ro to co l N um ber: P 0 0 0 1 131 Exygen Research Method Number VQOQ178Q | ANALYTICAL M ETHOD ~| Method o f Analyst! for the Detemiinatlon ofPerfluorooctanoic Acid (PFOA) in Water by L C /M S/M S 14.2 For sample! fortified with known amounts o f PFOA prior to extraction, use the following equation to calculate the percent recovery. Recovery (%) [to tal analyte found (ng/L) analyte found in control (ng/L)] analyte added (ng/L) Exygen Research Page 7 of? Page 22 o f 65 Page 60 o f 122 Interim Report #19 - Analysis o f Ground Water Samples Exygen Study No. : P0001131 Exygen P ro to co l N um ber: P 0 0 0 1 131 ANALYTICAL M ETHOD Method N um ber V0001781 Method o f Analysis for the D eterm lu lk w o f Perfluorooctu oic Acid (PFOA) Id Soil by LC /M S/M S Analytical Testing Facility: Exygen Research 3058 Research Drive State College. PA 16801 Approved By: H A C-Ji, Paul Connolly ' Technical Leader, LC-MS, Exygen Research Date ohn Flaherty f 'Vice Prsidait, Operations, Exygen Research Date Exygen Research Total Pages: 7 Page 23 o f 65 Page 61 o f 122 Interim Report #19 - Analysis o f Ground Water Samples Exygen Study No. : P0001131 E x y g en Pro to co l N u m b e r P 0 0 0 1131 E s y y Romrch Method Number V0001781 ANALYTICAL METHOD Method o f Analysis for the Determination ofPerfluorooctanoic Acid (PFOA) in Soil by L C /M S/M S 1.0 Scope Thie method is to be employed for the isolation and quantitation o f perfluorooctanoic a d d by High Performance Liquid Chromatography coupled to a tandem Mass Spcctrometric Detector (LC/MS/MS) in soil. 2.0 Safety 2.1 Always observe safe laboratory practices. 2.2 Consult the appropriate MSDS before handling any chemical for proper safety precautions. 3.0 Sample Requirement 3.1 At least 1S g o f test sample for extraction. 3.2 No sample processing is needed for soil samples. 3.3 Samples stored refrigerated should be allowed to equilibrate to room temperature. 3.4 All samples must be thoroughly mixed before being sampled for extraction. 3.5 Sample collection procedures will be specified in the sampling plan for this project. 4.0 Reagents and Standards 4.1 Water - HPLC grade 42 M ethanol- 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 iqjector 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 centrifiige tubes. 5.6 Disposable micropipet! (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 jiL and 10*100 pL), with disposable tips. 5.11 Waters Sep Pak Vac 6 cc (lg ) tC18 SPE cartridges. 5.12 SPE vacuum manifold. 5.13 Ultrasonic bath. Page 2 of ? Page 24 o f 65 Exygen Research Page 62 of 122 Interim Report #19 - Analysis o f Ground Water Samples Exygen Study No.: P0001131 E x y g en Pro to co l N um ber: POOQl 131 Exygen Ratexeb Method Number V0Q01781 I ANALYTICAL METHOD Method o f Analysis for the Determination ofPerfluorooctanoic Acid (PFOA) in Soil by L C /M S/M S 5.14 Wrist-action shaker. 5.15 Centrifuge capable o f spinning SO m L polypropylene tubes al 5000 rpm. 6.0 Chromatographic System 6.1 Analytical Column: Fhiophase 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 %A 65 65 25 25 65 Flow Rate 2LB fmL/min) 35 0.3 35 0.3 75 0.3 75 0.3 35 0.3 6.6 Injection Volume: 15 pL (can be increased to as m uch as 50 jiL). 6.7 Quantitation: Peak Area - external standard calibration curve. 6.8 RunTim e: - 2 3 minutes. The above condition! are intended aa a guide and may be changed in order to optimize the HPLC system. 7.0 MS/MS System 7.1 Mode: BlectrosprayN egativeM RM m ode,m onitoring4 1 3 - 3 6 9 m /zfor PFOA. The above conditions are intended as a guide and may be changed in order to optimize tiie MSMS system. 8.0 Preparation o f Solutions 8.1 Mobile Phase 8.1.1 2 mM ammonium acetate in water is prepared by adding 0.154 g of ammonium acetate to 1000 m L o f water. Alternate volumes may be prepared. 3Page o f 7 Page 25 o f 65 Exygen Research Page 63 o f 122 Interim Report #19 - Analysis o f Ground Water Samples Exygen Study No.: P0001131 E xy g en P ro to co l N um ber: P 0 0 0 1 131 Exygen lUaaarcb 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/Fortification Solution 9.1.1 Prepare a stock solution o f 100 pg/mL o f PFOA by weighing 10 mg o f analytical standard (corrected for purity) and dilute to 100 mL with methanol in a 125-mL LDPE bottle. 9.1.2 A 10 pg/mL fortification solution o f PFOA is prepared by bringing 1 o mL o f the 100 pg/mL solution to a final volume o f 100 with methanol in a 125 mL LDPE bottle. 9.1.3 A 1.0 pg/mL fortification solution o f PFOA is prepared by bringing 10 mL o f the 10 p ^ m L solution to a final volume oflOO with methanol m a 125 m L LDPE bottle. 9.1.4 A 0.1 p ^ m L 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 m L LDPE bottle. 9.1.5 A 0.01 pg/mL fortification solution o f PFOA is prepared by bringing 10 m L o f foe 0.1 pg/mL solution to a fins] volume o f 100 with methanol in a 125 mL LDPE bottle. 9.1.6 The stock and fortification solutions am to be stored in s refrigerator at approximately 4C and am stable for a maximum period o f 6 months from foe date o f preparation. 9.2 Standard Calibration Solutions 9.2.1 9.2.2 LC/MS/MS calibration standards are prepared in HPLC water. The calibration standards are processed through the extraction procedure, identical to samples. The following is a typical example: additional concentrations may be prepared as needed. Concentration of Fortification Solution (rob) Fortification Volume of Volume Fortified Control (PL) Semole (mL) Final Concentration of Calibration Standard (rot)* Calibration Standard ID (example) 00 10 100 10 200 10 400 100 100 100 200 100 400 40 40 40 40 40 40 40 0 XCmmddyy-0 25 XCmmddyy-1 50 XCmmddyy-2 100 XCmmddyy-3 250 XCmmddyy-4 500 XCmmddyy>5 1000 XCmmddW'6 * The extracted concentration o f foe calibration standard is equal to 8x its initial concentration, due to the concentration o f the standard during the extraction (SPE). XC extracted calibration standard. h 4 of7 Page 26 o f 65 Exygen Research Page 64 of 122 Interim Report #19 - Analysis o f Ground Water Samples Exygen Study No. : P0001131 Z "* E x y g en P ro to co l N um ber. POOQ1131 Exygen R n n re k Method Number V 000178I ANALYTICAL METHOD Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Soil by LC/MS/MS 9.2.3 9.2.4 9.2.5 A zero standard solution (reagent blank) must be prepared with each set o f standards extracted. Store all extracted calibration standards in 15-mL polypropylene tubes at 2*C to 6*C, up ti> 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 mL o f methanol) and two reagent controls fortified at known concentrations (lab control spike) to verify procedural reoovery for the batch. 10.2 Requirements for field and laboratory duplicates and spikes will be specified in the quality assurance plan for this project. 11.0 Sample Extraction 11.1 Weigh 5 g o f sample into 50 m L polypropylene centrifuge tubes (fortify as needed, replace lid and mix well). 1U112 Add 5 mL o f methanol and shake on a wrist action shaker fo r-1 5 minutes. Transfer the tubes to an ultrasonic bath and sonicate for ~15 minutes. 11.4 Bring the volume up to 40 m L with water in the 50 m L polypropylene centrifuge tube. 11.5 Centriftige fo r- 1 0 minutes at -3 0 0 0 rpm. 11.6 Condition the C m SPE cartridge (1 g, 6 m L) by passing 10 mL methanol followed by 5 mL o f HPLC water ( - 2 drop/sec). Do not let column run dry 11.7 Load (decant) the sample on the conditioned C u SPE cartridge. Discard eluate. 11.8 Elute with - 5 m L 100% methanol. Collect 5 m L o f eluate into graduated 15 mL polypropylene centrifUge tubes (final volume * 5 mL). 11.9 Analyze samples using electrospray LC/MS/MS. 12.0 Chromatography 12.1 Inject the same amount o f each standard, sample and fortified sample into the LC/MS/MS system. A calibration standard m ust precede and follow all analyzed samples. 12.2 Standards o f PFOA corresponding to at least five or m ore concentration levels must be included in an analytical set. 12.3 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 Page 5 o f? Page 27 o f 65 Exygen Research Page 65 of 122 Interim Report #19 - Analysis o f Ground Water Samples Exygen Study No.: P0001131 E xy g en P ro to co l N um ber: POOOl 131 Exygea Research Method Number VOOO1781 I ANALYTICAL METHOD Method o f Analysis for the Determination ofPerfluorooctanoic Acid (PFOA) in Soil by LC /M S/M S ] extracted calibration standards may be injected at the beginning o f a set followed by extracted calibration standards interspersed every 5-10 samples (to acoount for a second set o f extracted standards). In cither case, extracted calibration standards must be the first and last injection in a sample set. 12.4 Use linear standard curves for quantitation. Linear standard curves are generated for the analyte by linear regression using 1/x weighting o f peak area versus calibration standard concentration using MassLynx 3.3 {or equivalent) software system. 12.5 Sample response should not exceed standard responses. Any samples that exceed standard responses should be further diluted and reanalyzed. 13.0 Acceptance Criteria 13.1 Chromatogram must show a peak o f a daughter ion at 369 amu from a parent o f 413 amu. The 413 amu parent corresponds to the PFOA anion, while the daughter ion (369 amu) represents the loss o f carbon dioxide. 13.2 Method blanks m ust 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 extracted. 13.3 Recoveries o f control spikes and matrix spikes must b e between 70-130% of their known value. If a control spike falls outside the acceptable limits, the entire set o f samples should be re-extracted. Any matrix spike outside 70 130% should be evaluated by the analyst to determine if re-extraction is warranted. 13.4 Any calibration standard found to be a statistical outlier by using the Huge Error Teat, may be excluded from the calculation o f the calibration curve. However, the total number o f extracted calibration standards that could be excluded must not exceed 20% o f the total num ber o f extracted standards injected. 13.5 The correlation coefficient (R) for calibration curves generated must be 20.992 (R3 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 m ust not drift more than 4 % within an analytical run. I f retention tim e drift exceeds this limit within an analytical run then the set must be reanalyzed. Page 6 o f 7 Page 28 o f 65 Exygen Research Page 66 of 122 Interim Report #19 - Analysis o f Ground Water Samples Exygen Study No.: P0001131 E x y g en P ro to c o l N um ber: P 0 0 0 1 131 Exygen R n a ic h Method Number V0001781 | .................. ANALYTICAL METHOD ........... Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Soil by LC/MS/MS 14.0 Calculations 14.1 Use the following equation to calculate the amount o f PFOA found (in ng/L, based on peak area) using foe standard curve (linear regression parameters) generated by foe Mass Lynx software program: PFOA found (ng/L) - (Peak irea - imercem) x DF slope DF factor by which the final volume was diluted, i f necessary. 14.2 For samples fortified with known amounts o f PFOA prior to extraction, use the following equation to calculate 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 .0 4 m sample weight (5 g) 14.4 Use the following equation to calculate the amount o f PFOA found in pph based on dry weight. PFOA found (ppb) dry weight * PFOA found (ppb) x [100% / total solids(%)) P ag7of7 Page 29 oj 65 Exygen Research Page 67 o f 122 Interim Report #19 - Analysis o f Ground Water Samples Exygen Study No.: P0001131 E x y g en P ro to co l N um ber: P 0 0 0 1 131 ANALYTICAL METHOD Method N um ber V0001782 M ethod o f Aaalysls fo r th e D eterm ination o f PerfluorooctanoJc Acid (PFO A ) in Sedlmeot by LC/MS/MS Analytical Testing Facility: Exygen Research 3058 Research Drive State College, PA 16801 Approved By: CJL Paul Connolly *1 Technical L ead, LC-MS, Exygen Research __ Date / iolohhrn Flaherty / Vice President, Operations, Exygen Research Date Exygen Research Total Pages: 7 Page 30 o f 65 Page 68 o f 122 Interim Report #19 - Analysis o f Ground Water Samples Exygen Study No.: P0001131 Exygen Protocol Number; P0001131 Bxygao fUaearch Method Number VOO1782 I ANALYTICAL METHOD Method of Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Sediment by L C /M S/M S 1.0 Scope This method i t to be employed for the isolation and quantitation o f perfluorooctanoic acid by High Performance Liquid Chromatography coupled to a tandem Mass Spectremetric Detector (LC/MS/MS) in sedim ent 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 processing is needed for sediment samples. 3.3 Samples stored refrigerated should b e allowed to equilibrate to room temperature. 3.4 All samples must be thoroughly m ixed before being sampled for extraction. 3.5 Sample collection procedures will be specified in the sampling plan for this project 4.0 Reagents and Standards 4.1 W ater-H P L C grade 4.2 M ethanol-H PLC grade 4.3 Acetic A d d - Reagent grade 4.4 Ammonium Acetate - A.C.S. Reagent Grade 4.5 Perfluorooctanoic Acid - Sigma-Aldrich 5.0 Instrument and Equipment 5.1 A high performance liquid chromatograph capable o f pumping up to 2 solvents equipped with a variable volume injector capable o f injecting 5*200 pL connected to a tandem Maas Spectrometer (LC/MS/MS). 5.2 A device to collect raw data forpeak integration and quantitation. 5.3 Analytical balance capable o f reading to 0.00001 g. 5.4 50 mL disposable polypropylene centrifoge tubes. 5.5 15 m L disposable polypropylene centrifuge tubes. 5.6 Disposable micropipets (50*100uL, 100*200uL). 5.7 125*mL LDPB narrow-mouth bottles. 5.8 2 m L clear HPLC vial kit. 5.9 Disposable pipettes. 5.10 Autopipettes (100*1000 p L and 10-100 pL), with disposable Ups. 5.11 Waters Sep Pak Vac 6 cc (lg ) tC18 SPE cartridges. 5.12 SPE vacuum manifold. Pag 2 of 7 Page 31 o f 65 Exygen Research Page 69 of 122 Interim Report #19 - Analysis o f Ground Water Samples Exygen Study No. : P0001131 E xy g en P ro to co l N um ber: P 0 0 0 1 131 Exygea Research Method Number V0001782 ANALYTICAL METHOD Method o f Analysis for the Deterednation o f Perfluorooctanoic Acid (PFOA) in Sediment bv L C /M S/M S 5.13 Voitexer. 5.14 Wrist-action shaker. 5.15 Centrifiige capable o f spinning 50 m L polypropylene tubes at 3000 rpm. 6.0 Chromatographic System 6.1 Analytical Column: Fluophase RP (Keystone Scientific), 2.1 mm x 50 mm. $p (P/N: 82505*052130) 6.2 Temperature: 30*C 6.3 Mobile Phase (A ) : 2 m M 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 2LA 65 65 25 25 65 Flow Rate 2Lfi fmL/minl 35 0.3 35 0.3 75 0.3 75 0.3 35 0.3 6.6 Injection Volume; 15 p L (can be increased to as m uch as 50 pL). 6.7 Quantitation: Peak Area - external standard calibration curve. 6.8 RunTim e: - 2 3 minutes. The above conditions are intended as 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 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 it prepared by adding 0.154 g of ammonium acetate to 1000 m L o f water. Page 3 of 7 Page 32 o f 63 Exygen Research Page 70 of 122 Interim Report #19 - Analysis o f Ground Water 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 B x y g c a R M M ic fe M ethod N um ber VOOO1782 j ANALYTICAL METHOD | Method o f Analysis for the Determination ofPexfluorooctanoic Acid (PFOA) in Sediment by L C /M S/M S 8.2 Extraction Solutions 8.2.1 1% acetic acid in water is prepared by adding 10 m L o f acetic acid to 1000 mL o f water. Alternate volumes may be prepared. 9.0 Standard Preparation 9.1 Standard Stock/Foitification Solution 9.1.1 Prepare a stock solution o f -100 pg/m L o f PFOA by weighing 10 mg o f analytical standard (corrected for purity) and dilute to 100 mL with methanol in a 125-mL LDPB 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 final volume o f 100 with methanol in a 125 mL LDPB bottle. 9.1.3 A 1.0 pg/mL fortification solution o f PFOA is prepared by bringing 10 mL o f tile 10 pg/mL solution to a final volume o f 100 with methanol in a 125 m L LDPB bottle. 9.1.4 A 0.1 pg/mL fortification solution o f PFOA is prepared by bringing 10 m L o f the 1.0 p g fa L solution to a final volume o f 100 with methanol in a 125 m L LDPB bottle. 9.1.5 A 0.01 p ^ m L fortification solution o f PFOA is prepared by bringing 10 o L o f the 0.1 pg/mL solution to a final volume o f 100 with methanol in a 125 m L LDPB bottle. 9.1.6 The stock and fortification solutions are to be stored in a refrigerator at approximately 4*C and are stable for a m aximum 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 a m p le : additional concentrations may be Concentration o f Fortification Solution (ns/mL) 100 100 too 10 5 2 Volume (mL) 10 5 2 10 10 10 Diluted to (mL) 100 100 100 100 100 100 Final Concentration (ng/mL) 10.0 5.0 2.0 t.O o.s 0.2 Page 4 of 7 Page 33 o f 65 Exygen Research Page 71 o f 122 Interim Report #19 - Analysis o f Ground Water Samples Exygen Study No.: P0001131 E x y g en P ro to co l N um ber: P 0 0 0 1131 Exygm ftw strch Method Number V0001782 I ANALYTICAL M ETHOD | Method o f Analysis to r the Determination ofPerfluorooctanoic Acid (PFOA) in Sediment by L C /M S/M S 9.2.3 9.2.4 Store all calibration standarda in 125-raL LDPE narrow-mouth bottles at 2*C to 6C, up to six months. Alternate volumes and concentrations o f standards m ay be prepared as needed. 10.0 Batch Set Up 10.1 Each batch o f samples extracted (typically 20 o r le u ) 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 duplicate and spikes wilt be specified in the quality plan for this project. 11.0 Sample Extraction 11.1 W eigh 5 g o f sample into 50 m L polypropylene centrifuge tubes (fortify as needed, replace lid and mix well). 11.2 Add 35 mL o f \% acetic acid, cap, vortex and shake on a wrist action shaker for h 50 minutes A 11.3 Centrifirge the tubes at -3 0 0 0 xpm for - 2 0 minutes. 11.4 Condition the C n SPE cartridges ( l g, 6 mL) by passing 10 mL methanol followed b y 2 0 m L o f HPLC water ( - 2 drop/sec). Do not let column run dry 11.5 Load (decant) the sample on die conditioned C n SPE cartridge. Discard eluate. 11.6 Add 20 m L o f methanol to the sediment left in the bottom o f the 50 mL centrifuge tube. Cap, vortex and shake o n wrist action shaker for -3 0 minutes. 11.7 Centrifiige the tubes at -3 0 0 0 rpm for - 2 0 minutes. 11.8 Decant foe methanol onto the same SPE cartridge. Collect the eluate. 11.9 Wash the column w ith 4 mL o f methanol. Collect the eluate and add it to the eluate collected in step 11.8. 11.10 Condition a second C u SPE cartridge (1 g. 6 m L) by passing lO m L 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 m L o f water and load on the second conditioned SPE cartridge. 11.12 Elute with - 5 mL 100% methanol. Collect 5 m L o f eluate into graduated 15 mL polypropylene centrifiige tubes (final volume 5 mL). 11.13 Analyze samples using electrospray LC/MS/MS. Page S o f 7 Page 34 o f 65 Exygen Research Page 72 o f 122 Interim Report #19 - Analysis o f Ground Water Samples Exygen Study N o.: P0001131 E xygen P ro to co l N um ber: P 0 0 0 1 131 Exygen Research Method Number VOOO1782 | ANALYTICAL METHOD \ Method o f Analysis for the Determination ofPerfluorooctanoic Acid (PFOA) in Sediment by L C /M S/M S 12.0 Chrom atoFsphy 12.1 12.2 12.3 12.4 12.3 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 levels must be included in an analytical set. An entire set o f extracted calibration standards must be included at the beginning and at the end o f a sample set. Standards m ust be interspersed between every 3-10 samples. As an alternative, an entire set o f calibration standarda may be injected at die beginning o f a set followed by calibration standards interspersed every 5-10 samples (to account for a second set o f standards). In either case, calibration standards must be the first and Iasi injection in a sample set. Use linear standard curves for quantitation. Linear standard curves arc generated for die analyte by linear regression using 1/x weighting o f pede area versus calibration standard concentration using MassLynx 3 3 (or equivalent) software system, Sample response should not exceed standard responses. Any samples that exceed standard responses should b e Author diluted and reanalyzed. 13.0 Acceptance Criteria 13.1 Chromatogram must show a peak o f a daughter ion at 369 amu from a parent o f 413 amu. The 413 amu parent corresponds to the PFOA anion, while the daughter ion (369 amu) represents the loss o f carbon dioxide. 13.2 Method blanks must not contain PFOA at levels greater than the LOQ. If a blank contains PFOA at levels greater than 0.2 ng/mL, then a new blank sample must be obtained and the entire set must be re-extracted. 13.3 Recoveries o f control spikes and matrix spikes m ust be between 70-130% o f their known values. I f a control spike falls outside the acceptable limits, the entire set o f samples should be re-extracted. Any matrix spike outside 70 130% should be evaluated by the analyst to determine if re-extraction is warranted. 13.4 Any calibration standard found to be a statistical outlier by using the Huge Error Test, may be excluded from the calculation o f the calibration curve However, the total number o f extracted calibration standards that could be excluded must not exceed 20% o f the total num ber o f extracted standards injected. 13.3 The correlation coefficient (R) for calibration curves generated must be 20.992 (RJ 20.983). If calibration resulta fall outside these limits, then appropriate steps must b e taken to adjust instrument operation, and the standards o r the relevant set o f samples should be reanalyzed. P ig6on Page 35 o f 65 Exygen Research Page 73 of 122 Interim Report #19 - Analysis o f Ground Water 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 Exygea Re-- rch Method Number V0001782 I ANALYTICAL METHOD | Method o f A nalyse for the Determination o f Perfluoroocianoic Acid (PFOA) in Sediment by L C /M S /M S 13.6 Retention times between etandarda 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 n g /m l. based on peak area) using the standard curve (linear regression parameters) generated by foe Maes Lynx software program: PFOA found (ng/mL) * (Peak area - intercept) x DF slope DF * factor by which the final volume was diluted, if necessary. 14.2 For samples fortified with known amounts o f PFOA prior to extraction, use the following equation to calculate the percent recovery. Recovery (%) - [ total analyte found (ng/mL) - analyte found in co ntrol(n^m L )] . }QQ analyte added (ngftnL) 14.3 Use the following equation to convert the amount o f PFOA found in ng/mL to ng/g (ppb). PFOA found (ppb) - (PFOA found (nc/mL) x final volume fS mL)I sample weight (S g) 14.4 U se 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 [1 0 0 % / total soIids(%)) Page 7 of 7 Page 36 o f 65 Exygen Research Page 74 of 122 Interim Report #19 - Analysis o f Ground Water Samples Exygen Study No.: P0001131 Exygen P rotocol N um ber: P 0 0 0 1131 ANALYTICAL METHOD Method Number: V0001783 Method o fA iify a ii for the Dctennlaatioa o f Perfluorooctanolc Acht (PFOA) in Fish ed Clami by LC/MS/MS Analytical Testing Facility: Exygen Research 3058 Research Drive State College PA 16801 Approved By: C--ftJL____ Paul Connolly 1 Technical Leader LC-MS, Exygen Research '/w /Uy' Flaherty Vice President, Operations, Exygen Research Date Date Exygen Research Total Pages: 8 Page 37 o f 65 Page 75 o f 122 Interim Report #19 - Analysis o f Ground Water Samples Exygen Study No.: P0001131 Exygen Pro to co l N um ber: P 0 0 0 1 131 Exygen Rasetrch Method Number V0Q017 ANALYTICAL m e t h o d Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Fish and Claras by LC/MS/MS 1.0 Scope This method is to be employed for the isolation and quantitation o f perfluorooctanoic acid by High Performance Liquid Chromatography coupled to a tandem Mass Spectrometric Detector (LC/MS/MS) in fish and clams. 2.0 Safety 2.1 Always observe safe laboratory practices. 2.2 Consult the appropriate MSDS before handling any chemical for proper safety precautions. 3.0 Sample Requirement 3.1 At least 20 g o 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 subLimadon. Seal and place foe samples in frozen storage until rime o f analysis. 3.3 Sample collection procedures will be specified in foe sampling plan for this project Reagents and Standards 4.1 W ater-H P L C grade 4.2 Acetonitrile - HPLC grade 4.3 Carbon (120-400 mesh) - Reagent grade 4.4 Methanol - HPLC grade 4.3 Silica gel (60*200 mesh) - Reagent grade 4.6 Floriiil (60-100 mesh) - Reagent grade 4.7 Superclean LC-NHj - Reagent grade 4.8 l-Octanol - HPLC grade 4.9 L-Ascotbic acid - Reagent grade 4.10 DimefoytdichlorosUane - Reagent grade 4.11 Toluene - Reagent grade 4.12 Ammonium Acetate - A.C.S. Reagent Grade 4.13 Perfluorooctanoic Acid - Sigma-Aldricb 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 iqjoctor capable o f injecting 3-200 pL connected to a tandem Mass Spectrometer (LC/MS/MS). 5.2 A devioe to collect raw data for peak integration and quantitatioa 5.3 Analytical balance capable o f reading to 0.00001 g. Page 2 of 8 Page 36 o f 65 Exygen Research Page 76 o f 122 Interim Report #19 - Analysis o f Ground Water Samples Exygen Study No.: P0001131 E xy g en Pro to co l N um ber: P 0 0 0 1 131 Exygea Research [ ANALYTICAL METHOD Method Number V001783 Method o f Analysis for the Detenninttion o f Perfluorooctanoic A d d (PFOA) in Fish and C tarns by LC/MS/MS 5.4 Rotary evaporator. 5.5 Tisaumizer. 5.6 122 m L pear-shaped flasks. 5.7 50 mL disposable polypropylene centrifuge tubes. 5.8 15 m L disposable polypropylene centrifuge tubes. 5.9 Disposable micropipets (50-100uL, 100-200uL). 5.10 125-mL LDPE narrow*mouth bottles. 5.11 2 mL clear HPLC vial kit. 5.12 Disposable pipettes. 5.13 Autopipettes (100*1000 pL and 10-100 pL), with disposable tips. 5.14 SPE tubes (20mL) (Supelco cat. no. N057177). 5.15 Wrist action shaker. 5.16 Centrifuge capable o f spinning 50 m L polypropylene tubes at 2000 rpm. 6.0 Chromatographic System 6.1 Analytical Column: 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 raM Ammonium Acetate in W ater Mobile Phase (B) : Methanol Gradient Program: Thna /mirri 0.0 1.0 8.0 20.0 22.5 %Jk 65 65 25 25 65 Flow Rate fmL/minl 35 0.3 35 0.3 75 0.3 75 0.3 35 0.3 6.6 Injection Volume: 15 pL (can be increased to as much as 50 pL). 6.7 Quantitation: Peak Area - external standard calibration curve. 6.8 Run Time: ~ 23 minutes. The above conditions are intended as a guide and may be changed in o rd er to optimize the HPLC system. Page 3 of8 Page 39 o f 65 Exygen Research Page 77 o f 122 Interim Report #19 - Analysis o f Ground Water Samples Exygen Study No.: P0001131 S0**' E xy g en P ro to co l N um ber: P 0 0 0 1 131 Exygen R am rch M ethod Num ber V 0001783 I ANALYTICAL METHOD | Method o f Analysis (r the Detenninatioa ofPerfluorooctm oic Acid (PFOA) in Fish and Clams by LC/MS/MS 7.0 MS/MS System 7. ] Mode: Electrocpray Negative MRM m ode, monitoring 4 1 3 - 369 m/z for PFOA. The above conditions are intended as a guide and may be changed in order to optimize the MSMS system. 8.0 Preparation o f Solutions 8.1 Mobile Phase 8.1.1 2 mM ammonium acetate la water U prepared by adding 0.154 g o f ammonium acetate to 1000 m L o f water. 82 Extraction Solutions 8.2.1 8.2.2 2% ascorbic acid in methanol is prepared by dissolving 2 g o f ascorbic acid in 100 mL o f methanol. 30% Dimethytdichiorosilane in toluene is prepared by bringing 3 mL ofdimethyldichlorosilane 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/m L o f PFOA by weighing 10 mg o f analytical standard (corrected for purity) and dilute to 100 mL with methanol in a 125-mL LDPE bottle. A 1.0 pg/mL fortification solution o f PFOA is prepared by bringing 1 mL o f die 100 pg/mL solution to a final volume o f 100 with methanol in a 12S m L LDPE bottle. A 0.1 ggfaiL fortification solution o f PFOA is prepared by bringing Io m L o f the 1.0 p g fa L solution to a final volume o f 100 with methanol in a 125 raL LDPE bottle. A 0.01 pg/mL fortification solution o f PFOA is prepared by bringing 10 mL o f the 0.1 pg/mL solution to a final volume o f 100 with methanol in a 125 mL LDPE bottle. The stock and fortification solutions are to be stored in a refrigerator at approximately 4*C and are stable for a maximum period o f 6 months from the date o f preparation. Page 4 o f 8 Page 40 o f 65 Exygen Research Page 78 o f 122 Interim Report #19 - Analysis o f Ground Water 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 Exygen Research [ ANALYTICAL METHOD Method Number VQOO1783 Method o f Analysis for the Detenniaation ofPerfluorooctonoic Acid (PFOA) in Fish and G a o s 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 pg/mL fortification solution. The following is a typical example: additional concentrations may be prepared as needed. Concentration o f Fortification Vohim Solution (us/mL) (mL) Diluted to (mL) Final Concentration G*fi/mL) 1.0 5.0 100 1.0 2.5 100 0.05 0.025 1.0 1.0 100 0.01 0.0$ 0.025 10 10 100 100 0.005 0.002S 0.1 0.005 10 10 100 100 0.001 0.0005 9.2.3 Store all calibration standards In 125-mL LDPE narrow-mouth bottles at 2C to 6*C, up to six months. 9.2.4 Alternate volumes and concentrations o f standards may be prepared as needed. 10.0 Batch Set tip 10.1 Each batch o f samples extracted (typically 20 o r leas) must include at least one untreated control and two untreated controls fortified at known concentrations (lab control spike) to verify procedural recovery for the batch. 10.2 Requirements for field and laboratory duplicates and spikes will be specified in the quality assurance plan for this project. 11.0 Sample Extraction 11.1 W eigh 5 g o f frozen sample into SO m L polypropylene centrifuge tubes (fortify as needed, replace ltd and mix well). 11.2 Add 30 m L o f acetonitrile end shake on a wrist action shaker for - 1 S minutes 11.3 Place the tubes in a freezer for - 1 hour. 11.4 Pack and condition the SPE tubes and silanize the pear-shaped flasks. 11.5 Pack the 20 m L SPE tubes in sequence w ith 2 g florisil, 2 g silica gel, 2 g carbon, and 1 g LC-NH;. Condition the columns with 20 mL o f methanol, then 20 m L o f acetonitrile. Discard all washes. Do not allow the column to dry. U .6 Silanize the 123 mL pear-shaped flasks b y rinsing with the 30% dimethyldichloroalane in toluene solution. Rinse the flask with toluene once, followed by methanol (three times). Dry the flasks completely before use. either by air-drying o r with a stream o f nitrogen. Pge5 ofx Page 4! o f 65 Exygen Research Page 79 of 122 Interim Report #19 - Analysis o f Ground Water Samples Exygen Study No.: P0001131 E xy g en Pro to co l N um ber: P 0 0 0 1 131 Exygen Research Method Number V000I783 ANALYTICAL M ETHOD Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Fish and Clams by LC/MS/MS 11.7 Centrifuge the SO m i. polypropylene lubes 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 eluate 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 frit phase using a tiesumizer for - 3 0 seconds and rinse the tiasuroizer with -lO m L o f acetonitrile into the tube. 11.10 Shake the sample again fo r- 1 0 minutes on a wrist-action shaker. 11.11 Place the tubes in a freezer for - 1 hour more. 11.12 Centrifrige the 50 m L polypropylene tubes containing sample at -2 0 0 0 rpm fo r- 1 0 minutes. 11.13 Decant the attract 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 m L o f acetonitrile through the SPE column and combine the eluate in the same pear-stuped flask. 11.15 Add 3-4 drops o f l-octano! to the extract in the pear-shaped flask and evaporate at reduced pressure using a rotary evaporator (at < 40C). 11.16 M ake the final 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 pipeU. 11.18 Analyze samples using electrospray LC/MS/MS. 12.0 Chromatography 12.1 Ipject die same amount o f each standard, sample and fortified sample into the LC/MS/MS system. A calibration standard m ust precede and follow all analyzed samples. 12.2 Standards o f PFOA corresponding to at least five o r more concentration levels must be included in an analytical set. 12.3 An entire set o f calibration standards must be included at the beginning and at the end o f a sample set. Standards must be interspersed between every 5-10 samples. As an alternative, an entire set o f calibration standards may be injected at the beginning o f t set followed by calibration standards interspersed every 5-10 samples (to account fbr 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 fbr quantitation. Linear standard curves are generated for the analyte by linear regression using 1/x weighting o f peak area versus calibration standard concentration using M assLynx 3.3 (or equivalent) software system. Page 6 ofS Page 42 o f 65 Exygen Research Page 80 o f 122 Interim Report #19 - Analysis o f Ground Water Samples Exygen Study No.: P0001131 E xy g en Pro to co l N um ber: P 0 0 0 1 131 Exygen Research Method Number V0001783 _________ ANALYTICAL M ETH O D Method o f Analysis for the Determination ofPerfluorooctanoic Acid (PFOA) in Fish and Clams by LOMS/MS 12.3 Sample response should not exceed standard responses- Any samples that exceed standard responses should be forther diluted and reanalyzed. 13.0 Acceptance Criteria 13.1 Chromatogram must show s peak o f a (laughter 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 p e ste r than 0.5 ppb, then a new blank sample must be obtained and the entire set m ust 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 foils outside the acceptable limits, the entire set o f samples should b e re-extracted. 13.4 Any calibration standard found to be a statistical outlier by using the Huge Error Test, may be excluded from the calculation o f the calibration curve However, the total number o f calibration standards that could be excluded must not exceed 20% o f the total number o f standards injected. 13.3 The correlation coefficient (R) for calibration curves generated must be 20.992 (R3 20.985). I f calibration results fall outside these limits, then appropriate steps must be taken to adjust instrument operation, and the standards o r the relevant set o f samples should be reanalyzed. 13.6 Retention times between standards and samples must not drift more than 1 4 %within an analytical nm. I f m ention 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, 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) 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 (ng/mL) x final volume (mL) x DF1 ample weight (g) OF factor by which the final volume was diluted, if necessary. Page 7 o f8 Page 43 o f 63 Exygen Research Page 81 o f 122 Interim Report #19 - Analysis o f Ground Water Samples Exygen Study No.: P0001131 E xy g en Pro to co l N um ber: P 0 0 0 1 131 Exygen RMcarch M ethod Number VOOOt 78.3 | A X A lA Tie-U . METHOD | Method o f Analysis for (be Determination o f Perfluorooctanoic Acid (PFOA) in Fish and Clams by LC/MS/MS 14.3 For samples fortified with known amounts o f PFOA prior to extraction, use foe following equation to calculate the percent recovery. Recovery (%) - [ total analyte found (ng/g) analyte found in control (ng/g)l analyte added (ng/g) Exygen Research Page 8 o f 8 Page 44 o f 65 Page 82 o f 122 Interim Report #19 - Analysis o f Ground Water Samples Exygen Study No.: P0001131 E xy g en Pro to co l N um ber: P 0 0 0 1 131 ANALYTICAL METHOD Method Num ber V0001784 M ethod o f Analysis fo r the D eterm ination o f Perflnorooetanolc A d d (PFOA ) in Vegetation by LC/MS/MS Analytical Testing Facility: Exygen Research 3058 Research Drive State College, PA 16801 Approved By: "vt-A. C J iL Puil Connolly Technical Leader, LC-MS, Exygen Research o Z /n f/U /________ .John Flaherty ' ' v i c e President, Operations, Exygen Research ___ l O f c t . M Date Date Exygen Research Total Pages: 7 Page 45 o f 65 Page 83 of 122 Interim Report #19 - Analysis o f Ground Water Samples Exygen Study No.: P0001131 E x y g en P ro to co l N um ber: P 0 0 0 1131 Exygen Rem rch M ethod Number V000174 ANALYTICAL m e t h o d Method o f Analysis for the Determination ofPerfluorooctanoic Acid (PFOA) in Vegetation by LC/MS/MS 1.0 Scope Thia method is to be employed for foe isolation and quantitation o f perfluoroocianoic 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 foe appropriate MSDS before handling any chemical for proper safety precautions. 3.0 Simple 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 sample* 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 foe 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 Florisil (60-100 mesh) - Reagent grade 4.7 Superclean LC-NH* - Reagent grade 4.9 1-O ctanol-H PLC grade 4.9 L-Ascorbic acid - Reagent grade 4.10 Dimefoyldichtorosilane - Reagent grade 4.11 Toluene - Reagent grade 4.12 Ammonium Acetate - A.C.S. Reagent Grade 4.13 Pcrfluorooctanote 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 pL connected to a tandem Maas Spectrometer (LC/MS/MS). 5.2 A device to collect raw data for peak integration and quantitation. 5J Analytical balance capable o f reading to 0.00001 g. Page 2 o f 7 Page 46 o f 65 Exygen Research Page 84 of 122 Interim Report #19 - Analysis o f Ground Water Samples Exygen Study No.: P0001131 E x y g en Pro to co l N um ber: P 0 0 0 1 131 Exygen lUsMfcb Method Number V0001784 ANALYTICAL METHOD | Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Vegetation by LC/MS/MS 5.4 Rotary evaporator. 5.5 125 m L pear-shaped flask. 5.6 50 mL disposable polypropylene centrifuge tubes. 5.7 IS mL disposable polypropylene centrifuge tubes. 5.8 Disposable micropipets (50-IQOuL, 100-200uL). 5.9 125-mL LDPE narrow-mouth bottles. 5.10 2 m L clear HPLC vial kit. 5.11 Disposable pipettes. 5.12 Autopipettes (100-1000 p L and 10-100 pL), w ith disposable tips. 5.13 SPE tubes (20mL) (Supeico c a t no. N057177). 5.14 Wrist action ahaker. 5.15 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: 30*C 6.3 Mobile Phase (A ): 2 mM Ammonium Acetate in W ater 6.4 Mobile Phase (B) : Methanol 6.5 Gradient Program: Time fminl 0.0 1.0 8.0 20.0 22.5 'Ah 65 65 25 25 65 Flow Rale % B (mL/minl 35 0.3 35 0.3 75 0.3 75 0.3 35 0.3 6.6 Injection Volume: IS pL (can be increased to aa m uch aa 50 pL). 6.7 Quantitation: Peak Area - external standard calibration curve. 6.8 RunTim e: ~ 2 3 minute*. The above conditions are intended as a guide and m ay be changed in order to optimize the HPLC system. 7.0 MS/MS System 7.1 Mode: Electrospray Negative MRM mode, m onitoring 413 - 369 m/z for PFOA. Pige 3 of 7 Page 47 o f 65 Exygen Research Page 85 o f 122 Interim Report #19 - Analysis o f Ground Water Samples Exygen Study No. : P0001131 E xy g en P ro to co l N um ber: P 0 0 0 1131 Exygen lU scvcb M ethod Number V 0001784 ANALYTICAL METHOD Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Vegetation by LC/MS/MS The above conditions are intended as a guide and m ay 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 8.2.1 122 2%ascoibic acid in methanol is prepared by dissolving 2 g o f ascorbic a d d in 100 m L o f methanol. 30% Dimethyldichlorosilane in toluene ia prepared by bringing 3 mL o f dunetiiytdichloroBtiane to a final volum e o f 10 mL with toluene. Alternate volumes m ay be prepared. 9.0 Standard Preparation 9.1 Standard Stock/Fortification Solution 9.1.1 Prepare a stock solution o f ~ 100 pg/mL o f PFOA by weighing 10 mg o f analytical standard (corrected for purity) and dilute to 100 mL with methanol in a 125-mL LDPE bottle. 9.1.2 A 1.0 p ^ m L fortification solution o f PFOA is prepared b y bringing I mL o f foe 100 pg/mL solution to a final volume o f 100 with methanol in a 125 m L LDPE bottle. 9.1.3 A 0.1 pg/mL fortification solution o f PFOA is prepared by bringing 10 m L o f die 1.0 pg/mL solution to a final volume o f 100 with methanol in a 125 m L LDPE bottle. 9.1.4 A 0.01 p g ta L fortification solution o f PFOA is prepared by bringing 10 mL o f the 0.1 pg/mL solution to final volume o f 100 with methanol in a 125 m L LDPE bottle. . 9.1.5 The stock and fortification solutions are to b e stored in a refrigerator a: approximately 4C and are stable for a maximum period o f 6 months from die date o f preparation. 9.2 Standard Calibration Solution 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 u< * Page 48 o f 65 Exygen Research Page 86 of 122 Interim Report #19 - Analysis o f Ground Water Samples Exygen Study No.: P0001131 Exygen P ro to co l N um ber: P 0 0 0 1131 ExygcaR tievcb M ethod Number V 0001784 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 9.2.2 The following ie a typical example: additiMial concentrations may be prepared as needed. Concentration Final o f Fortification Solution (us/mL) 1.0 1.0 1.0 Volume (mL) 3.0 2.5 1.0 Diluted to (mL) 100 too 100 Concentration (m/mL) 0.05 0.023 0.01 0.03 0.023 0.1 10 10 10 100 100 100 0.005 0.0025 0.001 0.005 10 100 0.0005 9.2.3 Store all calibration standards in 125-mL LDPE narrow-mouth bottles at 2*C to 6C, up to six months. 9.2.4 Alternate volumes and concentrations o f standards m ay be prepared as needed. 10.0 Batch Set Up 10.1 Each batch o f samples extracted (typically 20 or less) must include si 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 30 m L polypropylene centrifuge tubes (fortify as needed, replace lid and mix well). 11.2 Add 30 tnL o f acetonitrile and shake on a wrist action shaker for -1 3 minutes. 11.3 Centrifuge the 30 mL polypropylene tubes containing sample at -2 0 0 0 rpm fo r- 1 0 minutes. 11.4 Pack and condition the SPE tubes and ailanize the pear-shaped flasks. 11.3 Pack the 20 mL SPE tubes in sequence with 2 g florisil, 2 g silica gel. 2 g carbon, and 1 g LC-NHj. Condition the columns with 20 m L o f methanol, then 20 mL o f acetonitrile. Discard all washes. D o not allow the column to dry. 11.6 Silanize the 123 mL pear-shaped flasks by rinsing with the 30% diraethyldichloroeiline in toluene solution. Rinse the flask with toluene once, followed by methanol (three times). D ry the flasks completely before use. either by air-drying or with stream o f nitrogen. 11.7 Decant the extract mi to a conditioned SPE column fitted inside the mouth of the pear-shaped flask. Collect the eluate in the 123 m L silanizod pear-shape flask. Page 5 o f Page 49 o f 65 Exygen Research Page 87 o f 122 Interim Report #19 - Analysis o f Ground Water Samples Exygen Study No.: P0001131 E xygen P ro to co l N um ber: P 0 0 0 1 131 Exygen Reieuch Method Number V0001784 ANALYTICAL m e t h o d Method o f Analysis for the Determination o f Perfluorooctanoic A d d (PFOA) in Vegetation by LC/MS/MS 11.8 Add 20 m L o f acetonitrile to the sample in the 50 m L centrifUge tube. 11.9 Shake the (ample again for *10 minutes on a wrist-action shaker. 11.10 CentrifUge the 50 mL polypropylene tubes containing sample at -2 0 0 0 rpm fo r- 5 minutes. 11.11 Decant the extract onto the same SPE column. Collect the eluatc into the same pear-shaped flask snd 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 M ai the final volume, by adding 2 m L o(2% ascorbic acid m methanol to the pear-shaped flask and swirl to mix/dissolve. 11.15 Transfer the extracts to HPLC vials using disposable pipets. 11.16 Analyze samples using electrospray LC/MS/MS 12.0 Chromatography 12.1 Inject the same amount o f each standard, sample and fortified sample into the LC/MS/MS system. A calibration standard must precede and follow all analyzed samples. 12.2 Standards o f PFOA corresponding to at least five o r m ore concentration levels must be included in an analytical set. 12.3 An entire set o f extracted, calibration standards must be included at the beginning and at the end o f a a m p le set. Extracted standards must be interspersed between every 5-10 samples. As an alternative, an entire set o f 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 l/x weighting o f peak area versus calibration standard concentration using M sssLynx 3.3 (or equivalent) software system. 12.5 Sample response should not exceed standard responses. Any samples that exceed standard responses should be further diluted and reanalyzed. 13.0 Acceptance Criteria 13.1 Chromatogram must show a peak o f a daughter ion at 369 amu from a parent o f 413 amu. The 413 amu parent corresponds to the PFOA anion, while the daughter ion (369 amu) represents the loss o f carbon dioxide. Page 6 o f 7 Page SOo f 65 Exygen Research Page 88 of 122 Interim Report #19 - Analysis o f Ground Water Samples Exygen Study No.: P0001131 E xy g en Pro to co l N um ber: P 0 0 0 1 131 E xygttR etw ch Method Number V0001784 ANALITICA!* METHOD Method o f Analysis for die Determination o f Perfluorooctanoic Acid (PFOA) in Vegetation byL C /M S/M S 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 Te-extracted. 13.3 Recoveries o f control spikes and matrix ip ik ei 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 b e re-extracted. 13.4 Any calibration standard found to be statistical outlier by using the Huge Error Test, may b e excluded firom 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 foe 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 foe relevant set o f samples should be reanalyzed. 13.6 Retention times between standards and samples must not drift more than * 4 % within an analytical run. I f retention tim e drift exceeda 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) - fPFOA found fna/m L* final vnlume (m D x DF1 sample weight (g) DF factor by which the Anal 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 (%) [to tal analyte found (ng/g) - analyte found in control (ng/g)] ^ ^ analyte added (ng/g) Page 7 oi 7 Page SI o f 65 Exygen Research Page 89 of 122 Interim Report #19 - Analysis o f Ground Water Samples Exygen Study No. : P0001131 Exygen P rotocol N um ber: P 0 0 0 1 131 ANALYTICAL METHOD M dhod Num ber V0001785 M ethod o f Analysis fo r the Determ lnatloo o f P erflaorooctaaoic Acid (PFO A ) in Smell M amm al Liver by LC/MS/MS Analytical Testing Facility: Exygen Research 3058 Research Drive State College, PA 16801 Approved By: _c --aLj____ Paul Connolly I Technical Leader, LC-MS, Exygen Research 4L John Flaherty / Vice President, Operations, Exygen Research Date /M y Date Exygen Research Total Pages: 7 Page 52 o f 65 Page 90 o f 122 Interim Report #19 - Analysis o f Ground Water Samples Exygen Study No.: P0001131 E x y g en Pro to co l N um ber: P 0 0 0 1 131 Exygta Research M ethod N umber VOOO1785 | ANALYTICAL M ETHOD Method o f A nalytii for the Determination ofPerfluorooctaooic Acid (PFOA) in Small Mamma} Liver b y LC/MS/MS 1.0 Scope Thia method i* to be employed for the ieolation and quantitation o f perfluorooctanoic acid by High Performance Liquid Chromatography coupled to a tandem M a ss Spectrometric Detector (LC/MS/MS) in small 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 S g o f test aarople 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. 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 PLC grade 4.3 Acetonitrile - HPLC grade 4.4 Ammonium Acetate - A.C.S. Reagent Grade 4.5 Perfluorooctanoic Acid - Sigma-Aldrich 5.0 Instrument and Equipment 5.1 A high performance liquid chromatograph capable o f pumping up to 2 solvents equipped with a variable volume injector capable o f injecting 5-200 pL connected to a tandem M an Spectrometer (LC/MS/MS). 5.2 A device to collect raw data fix peak integration and quantitation. 5.3 Analytical balance capable o f reading to 0.00001 g. 5.4 50 mL disposable polypropylene centriftige tubes. 5.5 15 m L disposable polypropylene centrifuge tubes. 5.6 Disposable micropipets (S0100uL, 100-200uL). 5.7 125-raL LDPE narrow-mouth bottles. 5.8 2 m L clear HPLC vial kit. Page 2 o f ? Page 53 o f 65 Exygen Research Page 91 of 122 Interim Report #19 - Analysis o f Ground Water Samples Exygen Study No.: P0001131 E x y g en Pro to co l N um ber: P 0 0 0 1 131 Exygea R am rch M ethod N um ber VQOQI7B5 | ANALYTICAL METHOD Method o f Analysis for the Determination ofPerfluorooctanoic Acid (PFOA) in Small Mammal l iv e r by LC/MS/MS 5.9 Disposable pipettes. 5.10 Autopipettes (100-1000 p L and 10-100 p i ) , w ith disposable tips. 5.11 Waters Sep Pak Vac 6 c c (lg )tC 1 8 S P B cartridges. 5.12 SPE vacuum manifold. 5.13 Tissuemizsr. 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 nun 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 ZA 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: IS pL (can b e 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 ire intended as a guide and m ay b e changed in order to optimize foe HPLC system. 7.0 MS/MS System 7.1 Mode: Electrospray Negative M RM m ode, monitoring 413 369 m/z for PFOA. The above conditions are intended as a guide and m ay b e changed in order to optimize the MSMS system. Page 3 o f? Page 54 o f 65 Exygen Research Page 92 o f 122 Interim Report #19 - Analysis o f Ground Water Samples Exygen Study No. : P0001131 E xy g en Pro to co l N um ber: POOO1131 Exygo R uttT ch M ethod Number V 0001785 I a N/UATICALMETHOD Method o f Analysis fbr the Determination o f Perfluorooetanoic Acid (PFOA) in Smali Mammal Liver by LC/MS/MS 8.0 Preparation o f Solutions 8.1 Mobile Phase 8.1.1 2 mM ammonium acetate in water ia prepared by adding 0 154 g of ammonium acetate to 1000 mL o f water. Alternate volumes may be prepared. 9.0 Standard Preparation 9.1 Standard Stock/Fortification Solution 9.1.1 Prepara a stock solution o f M 0 0 p g /m L o f PFOA by weighing 10 ing o f analytical standard (corrected for purity) and dilute to 100 mL with methanol in a 125-mL LDPE bonlc. 9.1.2 A 1.0 pg/mL fortification solution o f PFOA is prepared by bringing I m L o f the 100 pg/mL solution to a final volume o f 100 with methanol in a 125 m L LDPE bottle. 9.1.3 A 0.1 pg/mL fortification solution o f PFOA ia 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 123 m L LDPE bottle. 9.1.4 Tbe stock and fortification solutions are to b e stored in a refrigerator at approximately 4*C and are stable fbr a maximum period o f 6 months from die 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 Diluted to Concentration Solution ins/mL) (mL) (mL) (na/mL) 100 5.0 100 5.0 100 2.0 100 2.0 too 1.0 100 1.0 5.0 to 100 2.0 10 100 0.5 0.2 1.0 10 100 0.1 9.2.3 Store all calibration standards in 125-mL LDPE narrow-mouth bottles at 2*C to 6*C, up to six months. 9.2.4 Alternate volumes and concentrations o f standards m ay be prepared as needed. Page 4 o f 7 Page 55 o f 65 Exygen Research Page 93 o f 122 Interim Report #19 - Analysis o f Ground Water Samples Exygen Study No.: P0001131 E xy g en P ro to co l N um ber: P 0 0 0 1131 Exyfcn Reecareh M ethod N umber VOOO178$ I ANALYTICAL METHOD Method o f Analyris for the Determination o f Perfhiorooctanoic Acid (PFOA) in Small Mammal Liver b y LC/MS/MS 10.0 Batch Set Up 10.1 Each batch o f samples extracted (typically 20 o r less) must include at least one untreated control and two untreated controls fortified at known concentrations (lab control spike) to verify procedural recovery for the batch. 10.2 Requirements for field and laboratory duplicates and spikes will be specified in the quality assurance plan for this project. 11.0 Sim ple Extraction 11.1 Weigh 1 g o f sample into a 50 raL polypropylene centrifuge tu b es (fortify as needed, replace lid and mix well). Note that alternate weights o f liver m ay be measured depending on the sample size available for use. 11.2 Add water to the sample for a final volume o f 10 mL. 11.3 Homogenize sample using a tissuemizer for minute. 11.4 Transfer 1 mL o f the sample using a disposable pipette into a 15 mL disposable centriAige tube. 11.5 Add 5 m L o f acetonitrile and shake for - 2 0 m inutes on a wrist-action shaker. 11.6 Centrifuge the tubes at 3000 rpm for - 5 minutes. 11.7 Decant the supernatant into a 50 m L disposable centrifuge tube and add 35 mL o f water. 11.8 Condition the C u SPE cartridges ( l g, 6 mL) by pairing 10 mL methanol followed b y 5 m L ofH PL C water ( 2 drop/sec). Do not let column run dry 11.9 Load the sample on conditioned C u SPE cartridge. Discard eluate. U .10 Elute with 2 mL of methanol Collect 2 mL o f eluate into a graduated 15 m L polypropylene centrifoge tube (final volume 2 mL). 1l.l 1 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 m ust precede and follow all analyzed samples. 12.2 Standards o f PFOA corresponding to at least five or m ore concentration levels must b e included in an analytical a c t 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 m ust be interspersed between every 5-tu 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 fu st 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 regrearion using l/x weighting o f peak area Page 5 o f 7 Page 56 o f 65 Exygen Research Page 94 o f 122 Interim Report #19 - Analysis o f Ground Water Samples Exygen Study No.: P0001131 E xy g en Pro to co l N um ber: P 0 0 0 1 131 Exy*o Rem icfc I ...... ........... a n a l y t i c a l m e t h o d Method Number V0001785 Method o f Analysis for the Determination ofPerfhiorooctanoic Acid (PFOA) in Small Mammal U ver by LGMS/MS venue 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 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 10 ng/g, then a new blank sample must be obtained and the entire set must be re-extracted. 13.3 Recoveries o f control spikes and matrix spikes must be between 70-130% of their known values. I f a control spike falls outside the acceptable limits, the entire set o f samples should be re-extracted. Any matrix spike outside 70 130% should be evaluated by the analyst to determine i f re-extraction is warranted. 13.4 Any calibration standard found to be a statistical outlier by using the Huge Error Test, may be occluded from die 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 (R1 20.985). I f calibration results fall outside these limits, then appropriate step 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 m ust not drift more than 4 % w ithin an analytical run. I f retention time drift exceeds this limit within an analytical run then the set must be reanalyzed. 14.0 Calculations 14.1 Use the following equation to calculate the amount o f PFOA found (in ng/mL, based on peak area) using the standard curve (linear regression parameters) generated b y the M ass Lynx software program: PFOA found (ng/mL) - /Peak area - intercept') x D F x aliquot factor slope DF * factor by which the final volume was diluted, if necessary. Aliquot factor 10 Page 6of 7 Page 57 o f 65 Exygen Research Page 95 of 122 Interim Report #19 - Analysis o f Ground Water Samples Exygen Study No.: P0001131 E xy g en P ro to co l N um ber: P 0 0 0 1 131 ExytimiUtMich________________________________Math) Number V000I78S I A N A IA ilC 'A L M ETHOD Method o f A nalyii. for the Detonnination o f Perfluorooctenoic Acid (PFOA) in Small Mammal Liver b y LC/MS/MS 14.2 For samples fortified with known amounts o f PFOA prior to extraction, use the following equation to calculate the percent recovery. Recovery (%) - [ total analyte found (ngfaL ) 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 n g /j (ppb). PFOA found (ppb) - fPFOA found fn a /m t.t final volume fmLtl sample weight (g) Exygen Research Page 7 o f 7 Page SS o f 65 Page 96 of 122 Interim Report #19 - Analysis o f Ground Water Samples Exygen Study No.: P0001131 E x y g en Pro to co l N um ber: P 0 0 0 1 131 ANALYTICAL METHOD Method N um ber VOOOI786 M ethod o f A nalysts fo r th e Determ ination o f Perfluorooctanoic Acid (PFO A ) in Small M amm al Serum by LC/MS/MS Analytical Testing Facility: Exygen Research 3058 Research Drive State College, PA 16801 Approved By: c--tL/) Paul Connolly | Technical Leader, LC-MS, Exygen Research 4L\/ / 7 ) / / J6 h n Flaherty / Vice President, Operations, Exygen Research Date /'Jr Date Exygen Research Total Pages: 7 Page 59 o f 65 Page 97 of 122 Interim Report #19 - Analysis o f Ground Water Samples Exygen Study No. : P0001131 z0"*' E x y g en Pro to co l N um ber: P 0 0 0 1 131 ExygmRtmrch Method Number VDOO[786 I "Tw AJLVTICAL M ETH O D ~ Method o f Analysis for the Determination o f Perflucrooctanoic Acid (PFOA) in Small Mammal Serum by LC/MS/MS 1.0 Scope This method la to b e employed for foe isolation and quantitation o f perfluorooctanoic acid by High Performance Liquid Chromatography coupled to a tandem Mass Spectrometric Detector (LC/MS/MS) in small mammal serum. 2.0 Safety 2.1 Always observe safe laboratory practices. 2.2 Consult the appropriate MSDS before handling any chemical for proper safety precautions. 3.0 Sample Requirement 3.1 At least 1 raL o f test sample for extraction. 3.2 No sample processing is needed for serum samples. However, frozen serum samples must to allowed to completely thaw to room temperature before use. 3.3 Sample collection procedures will be specified in the sampling plan for this project 4.0 Reagents and Standards 4.1 W ater - HPLC grade 4.2 M ethanol-H PLC grade 4.3 Acetonitrile - HPLC grade 4.4 Ammonium Acetate - A.C.S. Reagent Grade 4.5 Perfluorooctanoic Acid - Sigma-Aldrich 5.0 Instrument and Equipment 5.1 A high performance liquid chromatograph capable o f pumping up to 2 solvents equipped with a variable volume injector capable o f injecting 5*200 pL connected to a tandem Maas Spectrometer (LC/MS/MS). 5.2 A device to collect raw data for peak integration and quantitation. 5.3 Analytical balance capable o f reading to 0.00001 g. 5.4 50 mL disposable polypropylene centrifoge tubes. 5.5 15 mL disposable polypropylene centriftige tubes. 5.6 Disposable micropipets (50-lOOuL, 100*200uL). 5.7 125-raL LDPE narrow-mouth bottles. 5.8 2 mL clear HPLC vial kit. 5.9 Disposable pipettes. 5.10 Autopipettes (100*1000 p L and 10-100 pL), w ith disposable tips. 5.11 Waters Sep Pak Vac 6 c c (lg )tC 1 8 S P E cartridges. 5.12 SPE vacuum manifold. 5.13 Vortexer. Page 2 o f 7 Page 60 o f 65 Exygen Research Page 98 o f 122 Interim Report #19 - Analysis o f Ground Water Samples Exygen Study No.:P0001131 E xy g en Pro to co l N um ber: P 0 0 0 1 131 Exygen Research M ethod N umber V000178& [ A 1 W VOCAL METHOD Method o f Analysis for Um Determination o f Perfluorooctanoic Acid (PFOA) in Small Mammal Serum by LCftfS/MS 5.14 Wrist-action shaker. $.15 Centrifuge capable o f spinning 15 mL polypropylene tubes at 3000 rpm. 6.0 Chromatographic System 6.1 Analytical Column: Fluopbase RP (Keystone Scientific), 2.1 mm x 50 mm. Sp (P/N: 82505-052130) 6.2 Temperature: 30*C 6.3 M obik Phaae (A) : 2 mM Ammonium Acetate in Water 6.4 Mobile Phaae (B) : Methanol 6.5 Gradient Program: Time (min) 0.0 1.0 8.0 20.0 22.5 %A 65 65 25 25 65 Flow Rate %B (mL/min) 35 0.3 35 0.3 75 0.3 75 0.3 35 0.3 6.6 Injection Volume: 15 pL (can be increased to as much aa 50 pL). 6.7 Quantitation: Peak Area - external standard calibration curve. 6.8 RunTim e: ~ 2 3 minutes. The above conditions are intended as a guide and m ay be changed in order to optimize the HPLC system. 7.0 MS/MS System 7.1 Mode: Electrospray Negative MRM mode, m onitoring 4 1 3 - 369 m/z for PFOA. The above conditions are intended as a guide and m ay be changed in order to optimize the MSMS system. 8.0 Preparation o f Solutions 8.1 Mobile Phase 8.1.1 2 mM ammonium acetate in water is prepared by adding 0.154 g o f ammonium acetate to 1000 m L o f water. Alternate volumes may be prepared. Page 3 o f 7 Page 61 o f 65 Exygen Research Page 99 of 122 Interim Report #19 - Analysis o f Ground Water Samples Exygen Study No. : P0001131 Exygen P ro to co l N um ber: P 0 0 0 1 131 ExygeaRncifcb Method Number VOOOI78S I ANALYTICAL MUTHOD Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Small Mammal Serum by LC/MS/MS 9.0 Standard Preparation 9.1 Standard Stock/Fortification Solution 9.1.1 Prepare a stock solution o f -1 0 0 pg/mL o f PFOA by weighing 10 mg o f analytical standard (corrected for purity) and dilute to 100 mL with methanol in a 125-mL LDPE bottle. 9.1.2 A 1.0 ji^ m L fortification solution o f PFOA is prepared by bringing l mL o f the 100 pg/raL solution to a final volume o f 100 with methanol in a 125 m i LDPE bottle. 9.1.3 A 0.1 pgfrnL fortification solution o fP F O A is prepared by bringing 10 m L o f foe 1.0 jig/mL solution to a final volume o f 100 with methanol in a 125 mL LDPE bottle. 9.1.4 The stock and fortification solutions are to be stored in a refrigerator at approximately 4*C and are stable for a maximum period o f 6 months from the date o f preparation. 9.2 Standard Calibration Solutions 9.2.1 9.2.2 LC/MS/MS calibration standard are prepared in methanol vie dilution o f foe 0.1 pg/mL fortification solution. The following ia a typical example: additional concentrations may be prepared a t needed. Concentration Final o f Fortification Volume Diluted to Concentration Solution fat/mL) (mL) (mL) (n*/mL) 100 5.0 too 2.0 100 100 5.0 2.0 100 1.0 100 1.0 5.0 10 100 2.0 10 100 0.5 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 6BC, 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. Pag 4 of? Page 62 o f 65 Exygen Research Page 100 of 122 Interim Report #19 - Analysis o f Ground Water Samples Exygen Study No. : P0001131 E x y g en P rotocol N um ber: P 0 0 0 1 131 Exygen JU teuch Method Number V0001786 I a n a l y t ic a l m e t h o d Method o f A iu ly iu for the Detomunititm o f Porfluoroocttnoic Acid (PFOA) in Small Mammal Seram by LC/MS/MS 1\ .0 Sample Extraction 11.1 Measure l rnL o f sample into a 50 m L polypropylene centrifuge tubes (fortify as 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 for - 1 minute. 11.4 Transfer i 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 20 m inutes on a wrist-action shaker. 11.6 Centrifuge the tubes at 3000 rpm for 5 minutes. 11.7 Decant the supernatant into a SO m L disposable centrifuge tube and add 3S m L o f water. 11.8 Condition the C u SPE cartridges (1 g, 6 mL) by passing 10 mL methanol followed b y 5 m L o f HPLC water ( 2 drop/sec). Do not let column run dry 11.9 Load the sample on conditioned C n SPE cartridge. Discard eluate. 11.10 Elute with 2 mL o f methanol. Collect 2 m L o f eluate into a graduated IS m L polypropylene centrifugo 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 m ore concentration levels must be included in n analytical set. 12.3 An entire set o f calibration standards must be included at the beginning and at the end o f a sim ple set. Standards m ust 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 $10 samples (to account for a second set o f standards), in either case, calibration standards must be the first and last injection in a sample set. 12.4 Use linear standard curves for quantitation. Linear standard curves are generated for the analyte by linear regression using 1/x weighting o f peak area versus calibration standard concentration using MassLynx 3.3 (or equivalent) software system. 12.5 Sample response should not exceed standard responses. Any samples that exceed standard responses should be further diluted and reanalyzed. Page 5 o f ` Page 63 o f 65 Exygen Research Page 101 of 122 Interim Report #19 - Analysis o f Ground Water Samples Exygen Study No.: P0001131 E x y g en P ro to co l N um ber: P 0 0 0 1131 Exygen R am reh Method Number V0001786 I AWALYT1CALMICTHOD Method o f Analysis for the D etenniiution o f Perftuorooctanoic Acid (PFOA) in Small Mammal Serum by LC/MS/MS ] 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 tlw daughter ion (369 amu) represents the loss o f carbon dioxide. 13.2 Method blanks must not contain PFOA at levels greater than the LOQ. if a blank contains PFOA at levels greater than 10 ng/mL, then a new blank sample must be obtained and the entire set must be re-extracted. 13.3 Recoveries o f control spikes and matrix spikes must b e between 70-130% o f 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 apike 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, die 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 o r the relevant set o f samples should be reanalyzed. 13.6 Retention times between standards and samples must not drift more thun 4 % within an analytical run. I f retention time drift exceeds this limit within an analytical ran then the set must be reanalyzed. 14.0 Calculations 14.1 Use the following equation to calculate the amount o f PFOA found (in ng/mL. baaed on peak area) using the standard curve (linear regression parameters) generated by the Mass Lynx software program: PFOA found (ng/mL) * (Peak area - intercept) x OF x aliquot factor slope DF factor by which the final volume waa diluted, if necessary. Aliquot factor - 20 14.2 For samples fortified with known amounts o f PFOA prior to extraction, use the following equation to calculate the percent recovery. Recovery (%) [ total analyte found (ng/mL) - analyte found to control (ng/mL)] ^ ^^ analyte added (ng/mL) Page 6 o f7 Page 64 o f65 Exygen Research Page 102 o f 122 Interim Report #19 - Analysis of Ground Water Samples Exygen Study No.: P0001131 E xy g en P ro to co l N um ber: P 0 0 0 1131 Exygen Research Method Number V0001 I AHa LVTICAI. METHOD ~1 M ethod o f Analysis for the Determination o f Perfluorooctanoic 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) - fPPO A found fng/m L l * final volum e fm U j sample volume (mL) Exygen Research page ? o f' Page 65 o f 65 Page 103 o f 122 Interim Report #19 - Analysis o f Ground Water Samples Exygen Study No.: P0001131 3058 Research Drive Phone: 814-272-1039 State College, PA 16801 Fax: 814-231-1580 PROTOCOL AMENDMENT Amendment Number: 1 Effective Date: 01/19/05 Exygen Study Number: P0001131 Client Study Number: Page 1 of 1 DESCRIPTION OF AMENDED SECTION 1) Analytical Procedure Summary V0001780:Sectlon 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-determlned 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. siglos DafeR' oj joj s' Date Date j LIBRARY ID: W 0 0 0 1 226-6 .ADMINISTRATIVE FORM Exygen Research Page 104 o f 122 Interim Report #19 - Analysis o f Ground Water 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 DESCRIPTIO N OF AMENDED SECTIO N 1) Report, page 11 of 65 2) Test M aterials, page 6 of 65: PFOS transition monitored 499 -> 99. AMENDED TO 1) Instead of one final report, Interim reports will be issued. 2) PFOS transition monitored may also be 499 -> 80. R A T IO N A LE 1) Due to the excessive sizes of the data sets, interim reports will be issued to allow the client to receive data in a tim elier manor-, *uuutt>~ e s jn /w 2) The API 4000 LC/M S/M S systems detect the 499 -> 80 PFO S transition with greater sensitivity than the 499 -> 99 transition. IMPACT ON STUDY 1) The client will be able to receive and review the data more quickly. 2) The 499 -> 80 transition can be detected with greater sensitivity; therefore, giving better chromatography. Study Director Signature y rincipal Investigator Signature Date Date S - aaW -- oS Date i/tt/e f Date Exygen QAU Review L k f 03lo S ltS LIBRARY ID: V 0001226-8 ADMINISTRATIVE FORM Exygen Research Page 105 of 122 Interim Report #19 - Analysis o f Ground Water Samples Exygen Study No. : P0001131 JU L .2S .200S 8 :5 6 m EXYGEN RESEARCH N O .7 7 4 P .3 n RESEARCH 3058 Research Drive Phone: 814-272-1039 State College, pa 1801 Fax: 814-231-1580 Amendment Number Effective Date: Exygen Study Number PROTOCOL AMENOMENT 3 07/13/05 P1131 Client Study Number: Page 1 et 1 NA rsspw etom of amenpep Vrification ofAnalytical Procedure, page 10 of protocol. AMENDED T The field duplicate can be used for the laboratory spikes and replicate when the primary sample volume is limited. BATIQMftlE The sample size for a water sample Is 200 m l. If a sample site requires re-extraction for any reason, there would not be enough of the primary sample to repeat two laboratory spikes and a replicate. The field duplicate Is technically the samesample as the primary sample and therefore, can bs used tor laboratory spikes end replicates as needed. M^ ,, IMPACT ON STUDY No negative Impact on the study. Using the duplicate sample ellows for the fell QC of the sample site to be completed. QJKL JyKSwMi Exygen Maqrfi X, Sponsor StgntturVOfrequired^ Oats Z / -M L - IS Date l / l s / p'OS Exygen QAU Review /T k .d v Vas-AS" LIBRARY ID: V000122S4 ADMINISTRATIVE FORM l 1 Exygen Research Page 106 of 122 Interim Report #19 --Analysis o f Ground Water Samples Exygen Study No.: P0001131 'T I1-23-2005 0 4 :2 2p* fron-HESTON SOLUTIONS NOVi,2 2 .2 0 0 5 4 :5 6 m EXYGEN RE9ERRCH T-S7T P.003/003 F-713 N U .3 1 * r.-o E xygen 3091 Research Drive Phone.1814-272-1039 State College, PA16801 Fex: 814-231-158) % / ^ J rTsearch Amendment Number. Effective Date: Exygen Study Number PROTOCOL AMENDMENT 4 11/22/05 P1131 Client Study Number. P a g a i of1 NA HESO tlEDOMg E ^ ^ & R B E P Iffitt Analytical Prcoedure Summary; VOCOi780:-Methcd ofAnalysis fe rita Determination of PerfluoroooctanoicAdd (PFOA) in Water by LCfMS/MS ' Section 11.0 ofthe method. AMENDED TO Section 11.0. Samples may be diluted before going through the extraction procedure. -- r a tio n a le -- ---- -- . If e 40 mL portion of sample will not load onto dm CwSPE cartridge, e pre-dilution can be prepared and extracted. ' " IS a S lS ~ No negative Impact on the study. More usable dote may be obtained. studyD*ttwanature BcygsnMani Spw aer Slanature (Ifrequired) Dale Date v-*f 3T Exygen QAU Review f f r - V W -*" UBRARYID: VQ00122M RECEIVED TIME NOV.23. S:40PM ADMINISTRATIVEFORM PRINT TIME NOV.23. 5:41PM Exygen Research Page 107 o f 122 Interim Report #19 - Analysis o f Ground Water Samples Exygen Study No.: P0001131 ffcHEM EHSR 236 1B 651 733 1958 JULsS-2083 0 5 :1 2 FR0M:3M ENJ. LflB 651 778 6176 IV.2E.28B 5 4S0PH EXY5CN RESEARCH 11/23 '05 14:12 NO.979 03/03 . 10:96517331350 N O .9 1 4 P .3 IT 3058 Research Drive Phene: 814-272-1039 State Cottage, PA 16801 Fax: 814*231-1580 RESEARCH Amendment Number Effective Date: Exygen Study Number PROTOCOL AMENDMENT 4. ii a o B 1*1131 Client Study Number: Page 1 e fi NA ' aacpignoN ofaMshpep^Icupn Analytical Procedure Summary: V000l780^Method ofAnalysis for the Pcterniinafloii of PerfluoreooctanoicAa (PFOA) inWater by IOMS/MS,' section 11.0 ofthe method. Section 11.0. Samples may be dllutad before goingthroughme extraction procedure. If a 40 mL portion of sample will not toad onto the C,,SPE cartridge, a pre-dilution can ba prepared and extracted. IMPACT ON STUDY i\ No negative Impacton the study. Mon usable data may be obtained. Study PlriaorsgnaBirR B w lw P W B P rM W |p ilP llg ESK RSie"" S tow n Mswy w rsiagt De 53T D ew U r-N V ri? ONi to //, l i . w s Mr E xv o a n Q A tl R eview U BR M V ID rV O O O lU S-S ADMINISTRATIVE FORM RECEIVED TIM E NO V.2 3 . 3:32P M PR IN T TIM E NOV.2 3 . 3 :3 3 P M S' \ Exygen Research Page 108 o f 122 Interim Report #19 - Analysis o f Ground Water Samples '-2008 04:08mi Frw-KSTON SOLUTIONS + Exygen Study No.: P0001131 T--152 HU 3 tsa ResearchOm Ptwne: m - m -I O Stau College, PA16801 F *14-231*1380 R ; :,'R C H .Amendment Nu iber Effective D ate: Exygen Study Number " T'-OCOU AMENDMENT 1 2 /o )t . poom i ClientStudyNumber pagel 1 NAl Teat Materials, page S of Protocol. '.T T T iT irill* An alternate lot o>PFOS in -y loused forthisstudy. MfmsThe PFOS used previously in this study Get nnuummiber 217 from SM> nm out and S was necessary to use a new lo t No negativeimpacton study. IM PACT M ? U8RARV ID: VDOO""S!M RECEIVED TIME KIR. I l -=8PM , M bM M ISTW nV EFOIW F'R IN T TIM E I M R R .21. 5:29P M Exygen Research Page 109 of 122 Interim Report #19 - Analysis o f Ground Water Samples Exygen Study No.:P0001131 3058 Research Drive Phone: 814-272-1039 State College, PA 16801 Fax: 814-231-1580 Amendment Number: Effective Date: Exygen Study Number PROTOCOL AMENDMENT 6 05/17/06 ' P0001131 Client Study Number. Page 1 of 1 NA DESCRIPTION OF AMENDED SECTION Appendix I, Analytical Method V0001782 AMENDED TO . * As per client request samples in login L4026 that have been tagged for re-extraction by the sponsor will be re-extracted using the following method: - Direct Injection Method: Before the samples are weighed for the extraction, they are mixed thoroughly by vigorously shaking the container. A one-gram portion of sediment is weighed into a 15-milliliter centrifuge tube for the extraction. Ten milliliters 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 -3000 rpm. Each sample is analyzed by LG/MS/MS electrospray. RATIONALE More usable data will be obtained by using an alternate method. IMPACT ON STUDY No negativeimpact on study. nQLKiJ/ Study DlroAOTSfenature Pnndpal Investigator Signature ' Date S7/V Data Sponsor Signature (iUbquIred) Date 3 t-n * A ^.Z O C , Date idinku Dais' ' E xyg en Q A U In it/D ate / V 'V o fc LIBRARY ID: V0001226-9 ' ADMINISTRATIVE FORM Exygen Research Page 110 o f 122 Interim Report #19 - Analysis o f Ground Water Samples Exygen Study No.: P0001131 RESEARCH 3058 Research Drive Phone: 814-272-1039 State College, PA 16801 Fax: 814-231-1580 Amendment Number Effective Date: Exygen Study Number PROTOCOL AMENDMENT 8 07/06/06 P0001131 Client Study Number: Page 1 of 1 NA DESCRIPTIO N O F AMENDED SECTIO N 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 Injection M ethod: 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-m illiliter centrifuge tube for the extraction. Ten milliliters 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/M S/M S electrospray. R A T IO N A LE More usable data will be obtained by using an alternate method. IMPACT ON STUDY No negative impact on study. Study Director Signature yj/v? /u cC incipal Investigator Signature Study Director Management Signature Exygen Management Signature Sponsor Signature (if required) Date 7 A /'t Date Date Date Date Exygen QAU Init./Date /Q g - / 7 L in fe LIBRARY ID: V 0001226-9 ADMINISTRATIVE FORM Exygen Research Page 111 of 122 Interim Report #19 - Analysis o f Ground Water Samples Exygen Study No.: P0001131 CHEM EHSR 236 lfe 651 733 1958 0 7 -1 I-0 0 5 03:57p FrorKSTO N SOLUTIONS 07/11 '06 14:09 NO.142 02/02 + T-55S P.001/007 F-5 3058 teireh WW* Phons^*?'!? Stats CoUeje, PATH01 Fa 1.4-231-1580 RESEARCH , ,/y. i y .. Amendment Number: EffacthraDate: Bitygin Study Nurtbsr ^ R tO iia AMNDMENT -t 5 . . P 0 0 0 il3 r Oient:Study Number. TNlaM -MA_ BE Appendix f, Analytical Method VD017S2. : : F ,-: " fig B ib E b tb ^ . ,:-f: , - y . ; .'y .' ' . * , , . At percflent request sedlmant uan^les re-tagged for're-extraetton bythe sponsorwin be raextracted uemg the follqwlnqmethod: . Dtrect.lhjaetlon Metnod: Beforethe sample are weighed fortbo extraction, jhiy. are mixed,thoroughly byvigorously shaking the container. Aone-gram portion.ofrudiment Isymighed Into a 15-fflltBlttercentdfiigs tuta.fbrthe extraction. tenmmiierpeffK acetic seid Inmethanolix added .each sample. Thetemples are thenhafkiinby ,. hand, vortaxad, and sonicated forthirty minutes. The samples art than centrifuged fpr -10 mlnutsa at -3000 rpm. Eachsample is analyzed byIC/MS/MS eleetroeprey.' .. B& Mori ueebte data will be obtainedby using,an altamtemethod. P^JPNBTUQT LiaWAflVID:V000122M RECEIVED TIME JL.11. 4:27PM AONtdPWTWTTVPPrwU ' PRINT TIME J U L .ll. 4:28PM Exygen Research Page 112 of 122 Interim Report #19 - Analysis o f Ground Water Samples Exygen Study No.: P0001131 01-0B-2007 03:SBpn Froo-WESTON SOLUTIONS T-821 t. 002/005 f-058 Exvoen y k f ^ J researci 3098 Research Drfve Phene: 814-272-1039 State College, PA 16801 Fax: 814-231-1580 . Amendment Number Effective Date: Exygen Study Number PROTOCOL AMENDMENT 11 12^1/ . POPP1131 Client Study Number Pas 1 1 P0001131 P P 9 C R IP T 1 0 N A M E N G E P ^P T IQ S ~ Protocol Distribution Section; 2) John M. Flaherty, Principal Investigator, Exygen Research 2} Charles Simons, Principal Investigator, Exygen Research ~; ~ RATIONALE John Flaherty retired and Chas Simons lus taken over John's role as Principal Invastigatorfbr the study. No negative impact im p a c t Q N s r y j j T JiA. W B M M n ftij Sponsorsnat** Oats M t Je^8.7ml Exygen QAU In itia te L J S / O lfttln LIBRARY ID:V00012M RECEIVED TIME JON. 8 . 4:02PM ADMINISTRATIVE FORM PRINT TIME JON. 8 . 4:04PM Exygen Research Page 113 of 122 Interim Report #19 - Analysis o f Ground Water Samples Exygen Study No.: P0001131 SEP.20 .20 0 5 1:52PM EXYGEN RESEARCH ;en RESEARCH 3058 Research Drive State College, PA 16801 NO.377 P .2 Phone: 814-272-1039 Fax: 814-231-1580 S an erai: X Project Specific Deviation DEVIATION FORM Facility Deviation Pape 1 of 1 Date of Occurrence: 03/16/05 Exygen Project # ; P760/P1131 Deviation # : 1/1 Client Project # : NA Reference # : 05-122 Regulatory Driver: aviation Tvna: (In clu d e V# fo r m eth o d s a n d S O P s) GMP GLP Other None Sample Description: _____ Protocol ______ Method V # 0001658-3 N otebook refe re n ce: NA X SOP Login#: _______ NA C o n ta in e r# : NA L o t# : NA Summary of 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 Impact: Them 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 Ore new SOP, the allowable amount of sample to be used Is < 20 g. All of the samples In question in this deviation weighed less than 20 g. The technician analyzing the samples for percent solids was following the new procedure before it was formally approved. Coiractlw Actions: A new version of the SOP has been issued and approved (V0000427-3). Signatures: . a/ Principal Indfistigator / Assurance LIBRARY ID: V0001640-6 w s- .Date -a/iAff Date Sponsor Representative M. Sponsor Management Date Data ADMINISTRATIVE FORM Exygen Research Page 114 o f 122 Interim Report #19 - Analysis o f Ground Water Samples Exygen Study No.: P0001131 SEP.20.2EB5 52PM EXYGEN RESEflRCH NO.377 P .3 3058 Research Drive Phone: 814-272-1039 State College, PA 16801 Fax: 814-231-1580 DEVIATION FORM Sanerai: X Project Specific Deviation Exygen Project# : P760/PH31 Client Project#: NA Facility Deviation Page 1 of 1 Date of Occurrence: 06/29/05 Deviation # : 2/2 Reference#: Q & - / 3 Regulatory Driver: Deviation Tvoe: (Include W fo r m ethods and SOPs) GMP GLP Other None Sample Description: X Protocol _____ Method V#:NA Notebook reference: NA ___ SOP Login#: L4254/L4256 Container#: C0056480-65 L o t#: NA Summary of Deviation; The protocol states that control and fortified control samples of each matrix will be analyzed; however, control dam was not obtained. Fish was used as the control fo r the analysis of these six clam samples. Cause: ____Preparation_____Analysis_____Instrument_____Client Request X Other Impact: No negative Impact on this study. Siantium ; , jfg Ak r t>al Investigator ody Director A aI A ' Quality Assurance f/lo /iS " Date Sponsor Representative Jk. Sponsor Management Date LIBRARY ID: VD001640-S ADMINISTRATIVE FORM Exygen Research Page 115 of 122 Interim Report #19 - Analysis o f Ground Water Samples Exygen Study No.: P0001131 SEP.20.2005 1:52PM EXYGEN RESEARCH ExvgOn 3058 Research Drive State College, PA 16801 RESEARCH NO.377 P .4 Phone: 814-272-1039 Fax: 814-231-1580 DEVIATION FORM General; X Project Specific Deviation ____Facility Deviation Exygen Protect # : P760/P1131 Client Project # : NA Paoe 1 of 1 ate of Occurrence: 12/27/04 Deviation # : 3/3 Reference # : o S ~ ~ f / T . Regulatory Driver: Deviation Type: (Inude W fo r m ethods and SOPs) GMP X GLP ________ Other None Sample Description: Protocol V#: 202-20 Section 5.2.3 Notebook reference: NA Login # : NA Container# : Method NA lo t # : X SOP NA SL2114 (30% Oimethyldichlorosllane 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 lha fish extraction from 12/27/04 through 01/07/05. Cause: ____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. Dimethytdlchlorosilane. which is the coating agent, was not expired. Corrective Actions: Deviation Issued. Signatures: / /Prfncipajflnvestigator Study Director Quality Assurance Date Sponsor Representative J L ... Sponsor Management Date Date Date LIBRARY ID: V 0 001640-6 ADMINISTRATIVE FORM Exygen Research Page 116 of 122 Interim Report #19 - Analysis o f Ground Water Samples Exygen Study No.: P0001131 3058 Research Drive Phone: 814-272-1039 State College, PA 16801 Fax: 814-231-1580 DEVIATION FORM _____________________ ___________________________________________________ Pao lo f 2 SSSSIfiL X Project Specific Deviation ___ Facility Deviation Date of Occurrence: 04/26/06 Exygen Project # : P760/P1131 Deviation#: S Oient Project#: ______NA_____ Reference#: 06-076 Reaulatorv Driver: Deviation Type: (In d u rfo V# lo r m ethods e nd S O P sI X ____ GMP GLP Other None Sample Description: X Protocol V#:NA Notebook reference: NA Method SOP Login#: L0008191 Container#: NA Lot#: ________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 WC 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, vortexed, and sonicated fo r thirty minutes. The samples were then centrifuged for -10 minutes at -3000 rpm. Each sample was analyzed by LC/MS/MS electrospray. Using this method acceptable data was obtainedfor PFOS, but the recoveries for PFOA and UC PFOA were still poor. Another alternative method was then used for PFOA and l3C PFOA, as follows: Alternative SPE Method: The samples that were prepared in 1% acetic acid for the direct injection method were used for this extraction. Five mitiliters of each sample was aliquoted into a SO-mL polypropylene centrifuge tube and the volumewas taken to 40 mL with water. The samples were then centrifuged for -10 minutes at -3000 rpm. The supernatant was then loaded onto a Cl( SPE cartridge conditioned with 10 mL of methanol and SmLofwater. The eluale was discarded. Approximately five milliliters of methanol was added to the cartridge. Five milliliters of eluale was collected into a graduated 16 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 to-. V0001640-6 ADMINISTRATIVE FORM Exygen Research Page 117 o f 122 Interim Report #19 - Analysis o f Ground Water Samples Exygen Study No.: P0001131 CHEN EHSR 236 1B 651 733 1958 O M M M 01:11pa FrcrWJTOI HUTOB 05/01 '06 13:46 NO.106 03/03 + M M P.OCI/WJ M IO ' CH fO H ta a v iM v i S M * Collap, M IO P I P hanr Fic*1fJJM I ` rw H H :,. , UMMTIRVSatlMM . .V" "" ' ' v ' '{:' v '* " .-I . : . ' . ; . '. V v "> > / i - ' *. : *' . ;V. *... *'* -v , .r . / . v yffyy. * ...;.y- -vh:i ::-v: :- . ' .-C.&lW-': m z . v .vv Exygen Research Page 118 of 122 Interim Report #19 - Analysis o f Ground Water Samples Exygen Study No.: P0001131 EHS OPNS ENVIRONMENTAL LAB 1651 778 4226 07/11/06 11:46 0 :04/07 N0:681 RESEARCH 3058 Research Drive Phone: 814-272-1039 State College, PA 16801 Fax: 814-231-1580 OBVIATION FORM i L X Project Specific Deviation Faculty Deviation Data' of Occurrence: 04/Z0AD6 Exygen PrOiect # : P780/P1131 Deviation# ; ' . j- " '; . Client Project di : NA~ - ' Rftanoe#: V .'M A ftC RtmUWvPrivar DevUHpn Vyoe: (Indudt W formathtda and SOPs) QMP GLP Other None _____ Protocol X Memo# VO: VQ06427-3 Notebook reference: NA a u m rttP rtP lP tiw Login#; _______NA Container#: C001S9446; Lot#: M. BtimnitfygfPavlatfgn; :.1 One aoN ample (C00158446) was weighed at -Sg for percent solid analyti, rather.thanet -20g which I* stated In the method. ' ' " : . .; Ceiiee: ____Prparation_____Anelysl____ Instrument __ __ Client Request Other Impact; ; . No negative Impact An accurate perdent solid number was obtained by slightly altering th calculation used. . .' ' ., CorrectlVp Actions; Deviation Issued. in e iiK ee: . ^SUppjAjQiveet-lgfUator f c ' f MAh/, - - Date , / Exygen ^neg em e MM Exygen Research Page 119 of 122 Interim Report #19 - Analysis o f Ground Water Samples Exygen Study No.: P0001131 EHS OPNS ENVIRONMENTAL LAB fjT 651 778 4226 07/11/06 11:46 g :06/07 NO:681 RESEARCH 3058 Research Drive Phone; 814-272-1039 State College, PA 16801 Fax; 814-231-1580. DEVIATION FORM fltunk X p)|aet Specific Deviation ___^ Facility Oevtotioii /' F t l ' f . t f l - O ita of Occurrence: 68/28/06 Exygen Project # ; P76Q/P1131 : Deviation#: ' ' f f l y ' Client Project 4 : NA . Referenoe * : Retmlalcrv Driver: OsvIsttOnTVoa: (Includa VPformethods and SOPa) ;___ X ____ __ . ' GMP GLP Other None Ornai,Pmcirtion; Login#: NA ,, ____Protocol ____ _ Method ' V#: 1800 Notebook reference: - X -' SOP Container#: NA . L o t#: m. 3 q ffim irv o f Deviation: . Peer review of raw data was not documented per SOP VlfiOO prior to 8/28/06. Ceuee: Preparation___ Analysis____ Instrument ' :-------- :---------;---------------- :__ ' Client Request Raw data will be more thoroughly evaiuoled and reviewed before QA Inspection. : Other . >* Corrective Action: ; .. .' A note to He will be Issued to ail subsequent reports stating dial overall summaries hBVe Oden peer reviewed. ' ' ' . . . ' T p M /L L f Bnndoto Investigator/ ; x lL M ' 'Oefe / Exygen I k|afcft ' Date Sp'pntr Rpfesentstlve M ________ Sponsor Management Date Exygen Research Page 120 o f 122 Interim Report #19 - Analysis o f Ground Water Samples Exygen Study No.: P0001131 EHS OPNS ENVIRONMENTAL LAB '651 778 4226 07/11/06 11:46 0 :07/07 N0:681 RESEARCH 3058 Research Olive Phone: Si4-$2-1039 State College, PA 16801 Fax: 814-231-1580 QEVIATION FORM Qsnerel: . X Project Specific Deviation Facility Deviation P e s e te n D ite of Occrrano* 06CBI8 Exygen Project # : P76/PH31 .'D eviation#: 8 8 - - Client Project # : NA : Reference it : . f l i k - i l l a ' . fteuHtery Priver: Devtetioh Tvae: (Includa VM to r m ethods and SO PsI ___ ' OMP X OLP ^ Other __Norie OTPlt PilBflrtlMi X Protocol _____ Meihod V A N A ".. Notebook reference: ___SOP Login#: L000S121 LOO8O01 Container # : .. aummervof Pevfatlon; Samples were filled to 250 mL Ineteadof 200 mL. C06B347 C016B354 00171086 : L ot#: NA Cauee: X Preparation __ ; AhiWyai , Ihetrumeht ' ' Client Request Imoact: .: . Samples could not be extracted accordiitg to the protocol. Other SonaglftfiArttenir Splklng.'lavels were adjusted to accommodate the alternative vbfmf. SlflQlt! m A is r pal Investigator ~ / MUthl/ 8tudy.DlrsH9r ^Lei'i^J ualfty Assurance litZl(Xr Date Sponsor Management i&L. Date Exygen Research Page 121 o f 122 Interim Report #19 - Analysis o f Ground Water Samples Exygen Study No.: P0001131 01-08-2007 03:S0pi Fros-HESTON SOLUTIONS T-021 P.005/005 F-050 Bsyseir / ^ /R E S E A R C H 3058 ResearchDrive Phon 814-272-1039 Stete College, PA16801 Fax: 814*231-1580 X Project Spedito Deviation DEVIATION FORM Fadny Deviation FteeJ.cM Date ofOccurrence: Nov. 2006Jan. 2007 Exygen Project # : POPPI131 Deviation#: 10 Client Project#: POPPI131 Reference#: ffl-Q Q L Aenuliwoiv P rtv ST Povtrton Vvee: (in eiu tts V8 Itrm oO n ds an d SQ Ps) GMP GLP Other Sareele baacrlettanT _____ Protocol X Metro# Vtfc V0001780 Notebook reference: ___ Login#: L0010155 L0010165 UOPI0254 Container#: Lot: Stock siamfenlt and fortlloaton standards used In the extraction Ibrtiie M logine were ecatanMe Instead a lmdtianal. tn Came: X Preparation___ Analysis____ Instrument Client Request Other tonegative Impact. Compounds are eompletBlysoluble In acetonitrile as Preyare In methanoL gonecdva Actions: evtatton issued. No actiontaken. 4 UBRARV ID:W0001640-7 RECEIVED T IM E JAN. 8 . 4 :0 2 P M ADMINISTRATIVE FORM PR IN T TIM E JAN. 8 . - 4 :0 4 P M I Exygen Research Page 122 o f 122