Document V3pmDyV4zQn28OjgRbpxjzXrZ

INTERIM REPORT # 9-Analvsis of Ground Water Samles STUDY TITLE Analysis of Perfluorobutanesulfonate (PFBS), Perfluorohexanesulfonate (PFHS), and Perfluorooctanesulfonate (PFOS) in Water, Soil, Sediment, Fish, Clams, Vegetation, Small Mammal Liver and Small Mammal Serum Using LC/MS/MS for the 3M Decatur Monitoring Program DATA REQUIREMENTS EPA TSCA Good Laboratory Practice Standards 40 CFR 792 STUDY DIRECTOR Jaisimha Kesari P.E., DEE Weston Solutions, Inc. 1400 Weston Way West Chester, PA 19380 Phone: 610-701-3761 INTERIM REPORT COMPLETION DATE January 9,2007 PERFORMING LABORATORY Exygen Research 3058 Research Drive State College, PA 16801 Phone: 814-272-1039 STUDY SPONSOR 3M Company 3M Building 0236-01-B-10 St. Paul, MN 55144 Phone: 651-733-6374 PROJECT - Protocol Number: P0001131 Exygen Study Number: P0001131 T otal Pages: 121 Interim Report #9-Analysis of Ground Water Samples Exygen Study No.: P0001131 GOOD LABORATORY PRACTICE COMPLIANCE STATEMENT Exygen Study Number P0001131, entitled "Analysis of Perfluorobutanesulfonate (PFBS), Perfluorohexanesulfonate (PFHS), and Perfluorooctanesulfonate (PFOS) in Water, Soil, Sediment, Fish, Clams, Vegetation, Small Mammal Liver and Small Mammal Serum Using LC/MS/MS for the 3M Decatur Monitoring Program," conducted for 3M Company, is being performed in compliance with EPA TSCA Good Laboratory Practice Standards 40 CFR 792 by Exygen Research. Principal Investigator Exygen Research Jaisimha Kesari P.E., DEE Study Director Weston Solutions, Inc. Michael A. Santoro Sponsor Reprsentt 3M Company Exygen Research Date Page 2 o f 121 Interim Report #9-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 Studv Director 16. Raw Data Review and Draft Interim Analytical Report Review 07/18/05 07/26/05 07/26/05 07/26/05 17. Raw Data and Final Interim Analytical Report Review 07/21/05 07/26/05 07/26/05 07/26/05 46. Final Interim Analytical Report Review 01/05/07 01/09/07 01/09/07 01/09/07 Lydia Shaffer Technical Lead, Quality Assurance Unit Date '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 121 Interim Report #9-Analysis of Ground Water Samples Exygen Study No.: P0001131 CERTIFICATION OF AUTHENTICITY This interim report, for Exygen Study Number P0001131, is a true and complete representation of the raw data. Submitted by: Exygen Research 3058 Research Drive State College, PA 16801 (814)272-1039 Principal Investigator, Exygen: Exygen Research * K (p-y Date Exygen Research Facility Management: Exygen Research Study Director, Weston Solutions, Inc. Jaisimha Kesari P.E., DEE Weston Solutions, Inc. Sponsor Representative, 3M Company: Michael A. Santoro Director of Regulatory Affairs Exygen Research Page 4 o f 121 Interim Report #9-Analysis of 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: TYPE OF STUDY: SAMPLE MATRIX: TEST SUBSTANCE: SPONSOR: STUDY DIRECTOR: P0001131 Residue Ground Water Perfluorobutanesulfonate (PFBS), Perfluorohexanesulfonate (PFHS), and Perfluorooctanesulfonate (PFOS) 3M Company 3M Building 0236-01-B-10 St. Paul, MN 55144 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: 07/07/05 Interim Analytical Termination Date: 06/08/06 Interim Report Completion Date: 01109101 Exygen Research Page 5 o f 121 Interim Report #9-Analysis of 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 Chas Simons John Flaherty Karen Risha Christine Edwards Mark Ammerman Amy Sheehan Brian McAllister Brittany Kravets Krista Gallant Title Operations Manager Vice President Laboratory Supervisor Technician Sample Custodian Associate Scientist Sample Custodian Technician Technician Exygen Research Page 6 o f 121 Interim Report #9-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..........................14 6.7 Quantitation and Example Calculation....................................................................15 7.0 EXPERIMENTAL DESIGN......................................................................................16 8.0 RESULTS...................................................................................................................17 9.0 CONCLUSIONS.........................................................................................................17 10.0 RETENTION OF DATA AND SAMPLES.............................................................17 Exygen Research Page 7 of 121 Interim Report #9-Analysis of Ground Water Samples Exygen Study No.: P0001131 Table I. LIST OF TABLES Page Summary of PFBS, PFHS and PFOS in Ground Water Samples...................19 Table II. Summary of PFBS, PFHS and PFOS in Re-extracted Ground Water Samples.......................................................................................................... 20 Table HI. Matrix Spike Recovery of PFBS, PFHS and PFOS in Ground Water Samples.......................................................................................................... 21 Table IV. Matrix Spike Recovery of PFBS, PFHS and PFOS in Re-extracted Ground Water Samples.................................................................................. 23 Exygen Research Page 8 o f 121 Interim Report #9-Analysis o f Ground Water Samples Exygen Study No. : P0001131 Figure 1. LIST OF FIGURES Page Typical Calibration Curve for PFBS in Reagent Water................................. 25 Figure 2. Extracted Standards of PFBS in Reagent Water, 0 ng/L and 25 ng/L, Respectively................................................................................................... 26 Figure 3. PFBS in Reagent Water, 50 ng/L Fortified Reagent Spk A, and 500 ng/L Fortified Reagent Spk B, Respectively.......................................................... 27 Figure 4. Chromatogram Representing a Ground Water Sample Analyzed for PFBS (Exygen ID: C0074420 Rep, Data Set:070705E)................................ 28 Figure 5. Typical Calibration Curve for PFHS in Reagent W ater................................ 29 Figure 6. Extracted Standards of PFHS in Reagent Water, 0 ng/L and 25 ng/L, Respectively................................................................................................... 30 Figure 7. PFHS in Reagent Water, 50 ng/L Fortified Reagent Spk A, and 500 ng/L Fortified Reagent Spk B, Respectively...........................................................31 Figure 8. Chromatogram Representing a Ground Water Sample Analyzed for PFHS (Exygen ID: C0074404, Data Set: 070705E).......................................32 Figure 9. Typical Calibration Curve for PFOS in Reagent W ater................................ 33 Figure 10. Extracted Standards of PFOS in Reagent Water, 0 ng/L and 25 ng/L, Respectively....................................................................................................34 Figure 11. PFOS in Reagent Water, 50 ng/L Fortified Reagent Spk A, and 500 ng/L Fortified Reagent Spk B, Respectively...........................................................35 Figure 12. Chromatogram Representing a Ground Water Sample Analyzed for PFOS (Exygen ID: C0074404, Data Set:070705 E ).......................................... 36 Exygen Research Page 9 o f 121 Interim Report #9-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 37 Exygen Research Page 10 o f 121 Interim Report #9-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. Analytical results and assessed accuracies for the analysis of PFBS, PFHS and PFOS found in re-extracted ground water samples are summarized in Table II. Fortification recoveries for PFBS, PFHS and PFOS in the ground water samples are detailed in Table III. The average percent recoveries standard deviations for PFBS, PFHS, PFOS in ground water samples were 110 27%, 106 20%, and 123 35%, respectively. Fortification recoveries for PFBS, PFHS and PFOS in the re-extracted ground water samples are detailed in Table IV. The average percent recoveries standard deviations for PFBS, PFHS, PFOS in re-extracted ground water samples were 99 33%, 81 14%, and 104 43% respectively. 2.0 OBJECTIVE The objective of the analytical part of this study was to determine levels of perfluorobutanesulfonate (PFBS), perfluorohexanesulfonate (PFHS), and perfluorooctanesulfonate (PFOS) in ground water according to Protocol P0001131 (Appendix A). 3.0 INTRODUCTION This report details the results of the analysis for the determination of PFBS, PFHS, and PFOS in ground water using the analytical 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 July 7, 2005, and the analytical termination date for this interim report was July 20,2005. Exygen Research Page 11 o f 121 Interim Report #9-Analysis o f Ground Water Samples Exygen Study No.: P0001131 4.0 ANALYTICAL TEST SAMPLES Thirty-nine ground water samples (Exygen ID C0074404-C0074442) representing nine ground water sampling sites and associated field quality control samples, were received on wet ice on June 15, 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 (C4F9S03'K +) Transitions Monitored: 299 - 99 Structure: FF FF F SO 3 FF FF PFHS Chemical Name: Perfluorohexanesulfonate Molecular Weight: 438 supplied as the potassium salt (CFuSOs'K ^ Transitions Monitored: 399 - 80 Exygen Research Page 12 o f 121 Interim Report #9-Analysis o f Ground Water Samples Exygen Study No.: P0001131 Structure: FFF FFF F SO3 FFF FFF PFOS Chemical Name: Perfluorooctanesulfonate Molecular Weight: 538 supplied as the potassium salt (CgFnSOsTC*) Transitions Monitored: 499 - 80 Structure: FFFF FFFF F S03 FFFF FFFF 6.0 DESCRIPTION OF ANALYTICAL METHOD The analytical method "V0001780: Method of Analysis for the Determination of Perfluorooctanoic Acid (PFOA) in Water by LC/MS/MS" was used for the 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 o f methanol and 5 mL o f 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 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 Exygen Research Page 13 o f 121 Interim Report #9-Analysis o f Ground Water Samples Exygen Study No.: P0001131 volume up to 100 mL with methanol, a 1.0 pg/mL fortification standard were 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 were 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 o f PFBS, PFHS and PFOS were --5.1 mins, --10.8 mins, and --12.9 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. 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 Exygen Research Page 14 o f 121 Interim Report #9-Analysis o f Ground Water Samples Exygen Study No.: P0001131 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 ('mint 0.0 1.0 8.0 10.0 11.0 18.0 Total run time: ~18min %A 65 65 25 25 65 65 %B 35 35 75 75 35 35 Flow Rate: 0.3 mL/min Ions monitored: Analvte PFBS PFHS PFOS Mode negative negative negative Transition Monitored 299 -99 399 -80 499 - 80 Approximate Retention Time (min) -5.1 min. -10.8 min. -12.9 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. Equation 1 calculated the amount of analyte found (in ng/L, based on peak area) using the standard curve (linear regression parameters) generated by the Analyst software program. Equation 1: Analyte found (ng/L) = (Peak area - intercept) x DF slope Where: DF = Dilution Factor, factor by which the final volume was diluted, if necessary. Exygen Research Page 15 of 121 Interim Report #9-Analysis o f Ground Water Samples Exygen Study No.: P0001131 For samples fortified with known amounts of PFBS, PFHS and PFOS prior to extraction, Equation 2 was used to calculate the percent recovery. Equation 2: Recovery (%) = (analyte found (ng/L) - analyte in control (ng/L)) xl00% amount added (ng/L) An example of a calculation using an actual sample follows (calculation is for PFBS only): Ground water sample Exygen ID: C0074404 Spk C (Set: 070705E), fortified at 10000 ng/L with where: peak area = 19515 intercept = 0.00171 slope = 96.1 dilution factor = 100 ng/L PFBS added (fort level) = 10000 amt in corresponding sample = 8800 (Set: 070705ER) From equation 1: Analyte found (ng/L) From equation 2: % Recovery = IT9515 - 0.001711 x 100 96.1 20300 ng/L (20300ng/L - 8800ng/L) x 100% 10000 ng/L = 115% 7.0 EXPERIMENTAL DESIGN For samples designated as field matrix spikes, PFBS, PFHS and PFOS were added at a known concentration to the bottles in the laboratory before being shipped to the field. The samples were filled to a 200 mL volumetric fill line in the field. The samples were extracted in six sets, two of which contained re-extractions. Each set included one reagent blank and two reagent blanks fortified at known concentrations. Two sets contained two sample sites, one set contained three sample sites, and one set contained two sample sites along with the trip blank and trip blank spikes collected for the ground water samples. The first set that contained re-extractions contained three Exygen Research Page 16 o f 121 Interim Report #9-Analysis o f Ground Water Samples Exygen Study No.: P0001131 sample sites. The second set of re-extracted water samples (which was re-extracted much later) contained samples from two sample sites. For each site, a sample, a field duplicate and two-matrix field spikes were collected. For each site, a laboratory duplicate and two laboratory matrix spikes were extracted, in addition to the samples collected in the field. 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. Analytical results and assessed accuracies for the analysis of PFBS, PFHS and PFOS found in re-extracted ground water samples are summarized in Table II. 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 accuracy. In instances of failed laboratory or field spikes, recoveries associated with other spikes were used to assess sample accuracy. Fortification recoveries for PFBS, PFHS and PFOS in the ground water samples are detailed in Table III. The average percent recoveries standard deviations for PFBS, PFHS, PFOS in ground water samples were 110 27%, 106 20%, and 123 35%, respectively. Fortification recoveries for PFBS, PFHS and PFOS in the re-extracted ground water samples are detailed in Table IV. The average percent recoveries standard deviations for PFBS, PFHS, PFOS in re-extracted ground water samples were 99 33%, 81 14%, and 104 43% respectively. 9.0 CONCLUSIONS The ground water samples were successfully extracted and analyzed for PFBS, PFHS and PFOS according to analytical method V0001780. 10.0 RETENTION OF DATA AND SAMPLES All original paper data generated by Exygen Research that pertains to this interim report will be shipped to the study director. This does not include facility-specific raw data such as instrument or temperature logs. Exact copies of all raw data, as well as a signed copy of the final analytical report and all original facility-specific raw data, will be retained in the Exygen Research archives for the period of time specified in EPA TSCA Good Laboratory Practice Standards 40 CFR 792. Exygen Research Page 17 o f 121 Interim Report #9-Analysis of Ground Water Samples Exygen Study No.: P0001131 TABLES Exygen Research Page 18 o f 121 Interim Report #9-Analysis of Ground Water Samples Exygen Study No.: P0001131 Table I. Summary of PFBS, PFHS and PFOS in Ground Water Samples E xyg e n ID C0074404 C0074404 Rep C0074405 C0074408 C0074408 Rep C0074409 C0074412 C0074412 Rep C0074413 C0074416 C0074416 Rep C0074417 C0074420 C0074420 Rep C0074421 C0074424 C0074424 Rep C0074425 C0074428 C0074429 Rep C0074429 C0074432 C0074435 C0074436 Rep C0074436 C0074439 C0074440 Rep C0074440 C lie n t Sample ID C4 Sulfonate PFBS C6 Sulfonate PFHS C8 Sulfonate PFOS PsrfluorobuUnesulfonats______ Pertluorohaxsnasulfonata______ PsrfluorooctanasuHonete Analyte Found (PPt. ng/L) Assessed Accuracy </-%) Analyte Found (ppt, ng/L) Assessed Accuracy (+/-%> Analyte Found (ppt, ng/L) Assessed Accuracy <+/-%) D A L-LC H -M C LF-0-050609 D A L-LC H -M C LF-0-050609* D A L-LC H -M C LF-D B -050609 8800 9280 9420 30 16100 50 41600 30 30 17100 50 42900 30 30 17800 50 45100 30 GW -327R-Q2-Y05-CP-0 172000 30 268000 30 415000 30 GW -327R-Q2-Y05-CP-0* 155000 30 248000 30 356000 30 GW -327R-Q2-Y05-CP-DB 120000 30 207000 30 354000 30 GW -226R-Q2-Y05-LF-0 NR NR NR NR NR NR GW -226R-Q2-Y05-LF-0* NR NR NR NR NR NR GW -226R-Q2-Y05-LF-DB NR NR NR NR NR NR GW -226L-Q2-Y05-LF-0 ND 30 ND 30 38.9 30 GW -226L-Q2-Y05-LF-0* ND 30 ND 30 33.5 30 GW -226L-Q2-Y05-LF-DB ND 30 ND 30 38.4 30 GW -310R-Q2-Y05-CP-0 356000 50 501000 30 1130000 30 GW -310R-Q2-Y05-CP-0* 342000 50 460000 30 935000 30 GW -310R-Q2-Y05-CP-DB 263000 50 394000 30 879000 30 GW -317L-Q2-Y05-CP-0 32.2 40 125 40 756 30 GW -317L-Q2-Y05-CP-0* 47.6 40 221 40 1320 30 GW -317L-Q2-Y05-CP-DB 38.0 40 160 40 1090 30 GW -320L-Q2-Y05-CP-0 67.7 30 54.5 30 394 50 GW -320L-Q2-Y05-CP-DB*A 73.2 30 66.9 30 536 50 GW -320L-Q2-Y05-CP-DB 72.9 30 68.5 30 505 50 GW -TRIP-Q2-Y05-CP-0 ND 30 ND 30 ND 30 GW -220R-Q2-Y05-LF-0 GW -220R-Q2-Y05-LF-DB*A GW -220R-Q2-Y05-LF-DB 6270 6620 6500 30 34500 30 44600 30 30 37300 30 55300 30 30 36600 30 46300 30 GW -220L-Q2-Y05-LF-0 GW -220L-Q2-Y05-LF-DB*A GW -220L-Q2-Y05-LF-DB 8090 7970 7780 30 44000 30 62600 30 30 45300 30 84200 30 30 41500 30 63500 30 ` Laboratory Duplicate ASamples were re-extracted due to possible laboratory error. Due to lack of sam ple, field duplicate was extracted in replicate. ND = Not detected a t or above the Lim it o f Quantitation (LOQ), which is 25 ng/L. NR = Not reported due to quality control failure. For re-analysis see Table II. Exygen Research Page 19 o f 121 Interim Report #9-Analysis o f Ground Water Samples Exygen Study No.: P0001131 Table II. Summary of PFBS, PFHS and PFOS in Re-extracted Ground Water Samples Exygen ID C0074412 C0074413 C0074421A C lient SamDle ID C4 Sulfonate PFBS C6 Sulfonate PFHS C8 Sulfonate PFOS Perfluorobutanesulffonala______ Perfluorohtxan-- uifonate_______ Pertluorooctanesulfonata Analyte Found (PPt. ng/L) Assessed Accuracy (+/-% ) Analyte Found (PPL ng/L) Assessed A ccu racy <+/-%> Analyte Found (PPL no/L) Assessed Accuracy (/-% ) GW -226R-Q2-Y05-LF-0 GW -226R-Q2-Y05-LF-DB 2070 2180 30 3580 30 3500 30 14500 50 30 14100 50 GW -310R-Q2-Y05-CP-DB 212000 40 686000 30 1050000 40 ` Laboratory Duplicate A Due to low sam ple volum e, only the sam ple duplicate was re-extracted. ND = Not detected a t o r above the Lim it o f Q uantitation (LOQ), which is 25 ng/L. Exygen Research Page 20 o f 121 Interim Report #9-Analysis o f Ground Water Samples Exygen Study No. : P0001131 Table III. Matrix Spike Recovery of PFBS, PFHS and PFOS in Ground Water Samples Sam ple Description C 4 S u lfo n a te PFBS________________ C 6 S u lfo n a te P FH S ________________ CS S u lfo n a te PFO S Amount Am t Found Amount Am t Found Amount Am t Found Amount S piked in S am ple R ecovered Recovery In S am ple R ecovered Recovery In S em ple R ecovered Recovery (nfl/L) (no/L) w 1-)_____ m __ (nfl/L) -- (n g /L )_____ (%) (ng/L) ( " f l'i) _____ ! * ! _ DAL-LCH-MCLF-0-050609 (C0074404 Spk C. 10000 ng/L Lab Spike) DAL-LCH-MCLF-0-050609 (C0074404 Spk D, 100000 ng/L Lab Spike) DAL-LCH-MCLF-LS-050609 (C007440S, 10000 ng/L Field Spike) DAL-LCH-MCLF-HS-050609 (C0074407,100000 ng/L Field Spike) 10000 100000 10000 100000 8800 8800 8800 8800 20300 145000 21000 157000 115 136 122 148 16100 16100 16100 16100 27400 131000 31600 160000 113 115 155 144 41600 41600 41600 41600 51800 159000 63500 175000 117 . 133 GW-327R-Q2-Y05-CP-0 (C007440S Spk C, 100000 ng/L Lab 8pike) GW-327R-Q2-Y05-CP-0 (C007440S Spk D, 1000000 ng/L Lab Spike) GW-327R-Q2-Y05-CP-LS (C0074410,100000 ng/L Field Spike) GW-327R-Q2-Y05-CP-HS (C0074411, 1000000 ng/L Field Spike) 100000 1000000 100000 1000000 172000 172000 172000 172000 274000 1460000 273000 1190000 102 129 101 102 268000 347000 268000 1410000 268000 391000 268000 1210000 79 114 123 94 415000 458000 415000 1600000 415000 720000 415000 1640000 . 119 123 GW-226R-Q2-Y05-LF-0 (C0074412 Spk E, 1000 ng/L Lab Spike) G W -226R-Q 2-Y05-LF-0 (C0074412 Spk F. 10000 ng/L Lab Spike) GW-226R-Q2-Y05-LF-LS (C0074414,1000 ng/L Field Spike) GW-226R-Q2-Y05-LF-HS (C0074415,10000 ng/L Field Spike) 1000 10000 1000 10000 NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR GW -226L-Q 2-Y05-LF-0 (C007441I Spk C. 100 ng/L Lab Spike) 100 ND 62.1 62 ND 72.7 73 38.9 133 94 GW -226L-Q 2-Y05-LF-0 (C007441I Spk D, 1000 ng/L Lab Spike) 1000 ND 1140 114 ND 1120 112 38.9 1300 126 GW-226L-Q2-Y05-LF-LS (C007441S, 100 ng/L Field Spike) 100 ND 85.0 85 ND 83.1 83 38.9 178 139 GW-226L-Q2-Y05-LF-HS (C0074419,1000 ng/L Field Spike) 1000 ND 1230 123 ND 1180 118 38.9 1290 125 GW-310R-Q2-Y05-CP-0 (C0074420 Spk E, 100000 ng/L Lab Spike) GW-310R-Q2-Y05-CP-0 (C0O7442O Spk F. 1000000 ng/L Lab Sp/k} GW-310R-Q2-Y05-CP-LS (C0074422,100000 ng/L Field Spike) GW-310R-Q2-Y05-CP-HS (C007442S, 1000000 ng/L Field Spike) 100000 1000000 100000 1000000 356000 356000 356000 NR 427000 1770000 562000 NR 71 141 206 NR 501000 533000 501000 1710000 501000 778000 NR NR 121 NR 1130000 970000 1130000 2140000 1130000 1450000 NR NR 101 NR ` Sample residue exceeds the spiking level significantly; therefore, an accurate recovery value cannot be calculated ND Not detected at o r above 25 ng/L (ppt). NR = Not reported due to quality control failure. For re-analysis see table IV. Note: Since this summary table shows rounded results, recovery values may vary slightly from the values in die raw data. Exygen Research Page 21 o f 121 Interim Report #9-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 Sam ple Description GW-317L-Q2-Y05-CP-0 (C0074424 Spk C, 900 ng/L Lab Spike) GW -317L-Q 2-Y05-CP-0 (C0074424 Spk D. 9000 ng/L Lab Spike) GW-317L-Q2-Y05-CP-LS (C0074429,900 npA. Field Spike) GW-317L-Q2-Y05-CP-HS (C0074427,9000 ng/L Field Spike) GW-320L-Q2-Y05-CP-0 (C007442I Spk C, 100 ngfL U b Spike) GW-320L-Q2-Y05-CP-DBA (C0074429 Spk D, 1000 ng/L U b Spike) GW-320L-Q2-Y05-CP-LS (C0074430,100 ng/L Field Spike) GW-320L-Q2-Y05-CP-HS (C0074431,1000 ng/L Field Spike) GW-TRIP-Q2-Y05-CP-LS (C00744S3.100 ng/L Field Spike) GW -TRIP-Q2-Y05-C P-HS (C00744S4,1000 ng/L Field Spike) G W -220R-Q 2-Y05-LF-0 (C0074439 Spk E, 10000 ng/L U b Spike) GW-220R-Q2-Y05-LF-DBA (C0074499 Spk F. 100000 ng/L U b Spike) GW-220R-Q2-Y05-LF-LS (C00744S7.10000 ng/L Field Spike) GW-220R-Q2-Y05-LF-HS (C0074439,100000 ng/L Field Spike) GW-220L-Q2-Y05-LF-0 (C0074499 S pk 0 . 10000 ng/L U b Spike) GW-220L-Q2-Y05~LF-DBA (C0074440 Spk H. 100000 ng/L U b Spike) G W -220L-Q 2-Y05-LF-LS (C0074441,10000 ngA. Field Spike) GW-220L-Q2-Y05-LF-HS (C0O74442,100000 ngA. Field Spike) C 4 S u lfo n a te PFBS________________ CS S u lfo n a te P FH S ________________ C 8 S u lfo n a te PFO S Amount Am t Found Amount Amt Found Amount Am t Found Amount S p fte d In S am ple R ecovered R aco vaiy in Sam pla R ecovered Rocovory in S am pla R ecovered Recovery (n u n .) (nsrt.) (n/L )_____ m __ tng/L) (n fl/L ) (%) (nfl/L) (")_____ i * L _ 500 32 2 604 114 125 803 136 756 2090 267 5000 32.2 5200 103 125 5590 109 756 6590 117 500 32.2 302 54 125 452 65 756 1260 101 5000 32.2 5720 114 125 5910 116 756 7410 133 100 67.7 167 99 54.5 159 105 394 506 112 1000 67.7 1160 109 54.5 1080 103 394 1600 121 100 67.7 169 101 54.5 166 112 394 540 146 1000 67.7 1430 136 54.5 970 92 394 1320 93 100 1000 ND NO 10000 100000 10000 100000 6270 6270 6270 6270 10000 100000 10000 100000 8090 8090 8090 8090 122 1090 122 109 17400 118000 16300 92000 111 112 100 66 19800 113000 16900 97100 117 105 88 69 ND 114 114 ND 1120 112 34500 34500 34500 34500 44000 44000 44000 44000 44100 141000 43500 119000 54100 153000 49600 132000 96 107 90 85 * 109 * 88 ND 133 133 ND 1210 121 44600 44600 44600 44600 55500 162000 49100 134000 117 * 89 62600 62600 62600 62600 73500 175000 64600 159000 112 * 96 Average: Standard Deviation: 110 27 Average: Standard Deviation: ASamptea were re-extracted due to possible laboratory error. Due to lack o f ample, field duplicate was extracted in replicate. ND = Not detected at o r above 25 ng/L (ppt). Note: Since this summary table shows rounded results, recovery values may vary slightly from the values in the raw data. 106 20 Average: Standard Deviation: 123 36 Exygen Research Page 22 o f 121 Interim Report #9-Analysis o f Ground Water Samples Exygen Study No.: P0001131 Table IV. Matrix Spike Recovery of PFBS, PFHS and PFOS in Re-extracted Ground Water Samples Sam ple D e sc rip tio n GW-226R-Q2-Y05-LF-DBA (C0074413 Spk 0.1000 ng/L Lab Spfca) GW-226R-Q2-Y05-LF-DBA (C0074413 Spk H. 10000 ng/L Lab Spike) GW-226R-Q2-Y05-LF-LS (C0074414,1000 ng/L Field Spike) GW-226R-Q2-Y05-LF-HS (C0074415,10000 ng/L Field Spike) GW-310R-G2-Y05-CP-HS (C0074423.1000000 ng/L Field Spike) C 4 S u lfo n a te PFBS________________ C 6 S u lfo n a te P FH S ________________ CS S u lfo n a te P FO S Amount S p ike d ("fA J Am t Found in S am ple (nfl/L) Amount Recovered Recovery (nS/L) (*l Amt Found Amount In Sam ple Recovered Recovery ("9fl->______ ( S U ____ Am t Found In Sam ple Amount Recovered Recovery (ng/L) <*> 1000 2070 2820 75 3580 4320 74 ._ 10000 . .- - - 14500 25100 106 1000 2070 2930 86 3580 4300 72 -- 10000 - -- - - - 14500 29000 145 1000000 212000 1580000 137 666000 1660000 97 1050000 1650000 60 Average: Standard Deviation: 9 33 ND * Not detected a t o r above 25 ng/L (ppt). A Due to tow sample volume, the duplicate sample was used for laboratory spikes. Note: Since this summary table shows rounded results, recovery values may vary slightly from the values In the raw data. Average: Standard Deviation: 81 14 Average: Standard Deviation: 104 43 Exygen Research Page 23 o f 121 Interim Report #9-Analysis o f Ground Water Samples Exygen Study No.: P0001131 FIGURES Exygen Research Page 24 o f 121 Interim Report #9-Analysis o f Ground Water Samples Exygen Study No.: P0001131 Figure 1. Typical Calibration Curve for PFBS in Reagent Water 0 70705E W atai.idb (PFBS> "U n ta r1R a g its io n f 1 / x" iw tighting): y 00.1 x + 0.00171 (r 0.0057) Area, counts Exygen Research Page 25 o f 121 Interim Report #9-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 XC710S-*.PFBS (Staadard)2991*99.0aaia.aampl1of31from 7t70S.witf paat aotfot/ad) 11.02 A t* j : 34Q1 counts H eight: 64.1 cps RT: 5 .1 8 m in 5 .1 6 Intensity, cps Intensity, cps Exygen Research Page 26 o f 121 Interim Report #9-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 f t u g u t C ontrol - PFBS (Unknown) 29910*9.1 Mir -SMmph 9 o f 31 from *77*5.w iff t>*ak n o t foamd) Intensity, cps Intensity, cps Area: 5823 count* Height: 100. cps RT: 5.13 min | R t ig o n t Spk B - PFBS (Q 0 )2 8 9 .0 /8 8 .0 i m u - u m p l t 11 o f 31 fro m 0 7 0 7 0 3 E .i iff A it a : 4 7 8 3 7 counts H eight: 0 3 0 . ops RT: 3.10 m in 5.18 Tim e, min Intensity, cps Exygen Research Page 27 o f 121 Interim Report #9-Analysis o f Ground Water Samples Exygen Study No. : P0001131 Figure 4. Chromatogram Representing a Ground Water Sample Analyzed for PFBS (Exygen ID: C0074420 Rep, Data Set:070705E) C$074420R*p - PFBS (UMtmowm) 29& 49M am u 26 o f 31 fro m 070705E.wiff A m ; 179179S9count* 34500$. c p r RT: 5.15m la 0.15 8 0 10 13 ie Tim , min Intensity, cps Exygen Research Page 28 o f 121 Interim Report #9-Analysis of Ground Water Samples Exygen Study No.: P0001131 Figure 5. Typical Calibration Curve for PFHS in Reagent Water B 0 70705E W ittr.rd b (PFHS): "U n a f R tg rta tio n / * * vM ighting): y * 208 x -f 536 ( r * 0.0896) Area, counts Exygen Research Page 29 o f 121 Interim Report #9-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 xcmi05-0 - PFHS (Standard) 399.f t * * $ama -aampta 1o f 31 from $70?$SE.vnff A n a : S3$coaata Hatgte 36.7epa RT: 1k$m ia 10.01 Intensity, cps A rcs: 500 5 counts Height: 407. cps RT: 10.0 min 10.02 Intensity, cps Exygen Research Page 30 o f 121 Interim Report #9-Analysis of 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 I AM0M t Control PFHS (Unknown) 399UMML9ama ta m ph 9 o f 31 from l70W 5w ff p ta k not foaad) At*: 12002 counts Height: 835. c p i RT: 10.6 min Exygen Research Page 31 o f 121 Interim Report #9-Analysis of Ground Water Samples Exygen Study No.: P0001131 Figure 8. Chromatogram Representing a Ground Water Sample Analyzed for PFHS (Exygen ID: C0074404, Data Set: 070705E) I CWM404-PFHS (U>it*ow*)399.m .0am* -am pia 1*o f 31 from 707t 5m iff A ra : 2*27729 count* might: 197**. epa AT: 1<K*min 10.03 Intensity, cps Exygen Research Page 32 o f 121 Interim Report #9-Analysis o f Ground Water Samples Exygen Study No. : P0001131 Figure 9. Typical Calibration Curve for PFOS in Reagent Water > 0 7 0 7 0 5 E W lttr .r d b (P F O S * " U n i f R * g r io n C"1 / * tw ig M in g ): y - 8 4 .4 x + 7 1 2 ( i 0 .8 8 9 1 ) Area, counts Exygen Research Page 33 o f 121 Interim Report #9-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 XC$7$105-$ - PFOS (Standard) 499L&VM mir - t o m p k 1 o f 21 from 9707$5E.Wiff A n t : 712 c o tti* H o fg k t 222 cp* RT: 129 m it 12.77 Aio j: 2052 counta Hoight 152. cps RT: 12.8 min 12.83 Intensity, cps Intensity, cps Exygen Research Page 34 o f 121 Interim Report #9-Analysis of Ground Water Samples Exygen Study No.: P0001131 Figure 11. PFOS in Reagent Water, 50 ng/L Fortified Reagent Spk A, and 500 ng/L Fortified Reagent Spk B, Respectively I A ia g a a t C ontrol PFOS fiM ta o w a ) 4MLMILO am a -ta m p I t 9 o f 31 from OltTOSE.wiff A n a : m e o a a ta H tiftt: 23L0cpr AT: I Z tm ia 0 .7 2 I R e a g e n t Spk A * PFOS (QC)4QO.O/BO.O im u s im p le 10 o f 31 from 070 70 S E .w iff Area: 4855 count! Height: 204. cps RT: 12.8 min 8 0 10 Tim e, min I R e ig e n t Spk 8 - PFOS ( 0 0 )4 0 0 .0 /6 0 .0 im u - s im p le 11 o f 31 from 07Q 705E .w iff A re i: 41528 counts Height: 2550. cps RT: 12.8 min 11.08. >13.20 ,,1 4 .4 2 14 15 15 17 S' Exygen Research Page 35 o f 121 Interim Report #9-Analysis of Ground Water Samples Exygen Study No.: P0001131 Figure 12. Chromatogram Representing a Ground Water Sample Analyzed for PFOS (Exygen ID: C0074404, Data Set:070705 E) Intensity, cps C m 4494-PFOS (Unknown)499.9/9$,$ ama -tam pto 1%o f 31 from $7$7$5Em i f f A n a : 302M95 co n a ti H eigt: 7370*0, cpa RT: 1Z9 m in 1.3 #5* 1 .2*5- 1.1* 6 1.0*6- 9 .0 *4 - 8 .0 *4 - 7 .0 *4 - 0 .0 *4 - 5 .0 *4 - 4 .0 *4 - 3 .0 *4 - 2 .0 *4 1 .0 *4 - 12.00^ 0JO1 2 3 4 5 0 7 8 0 10 11 12 13 1 4 15 10 17 Exygen Research Page 36 o f 121 Interim Report #9-Analysis of Ground Water Samples Exygen Study No.: P0001131 APPENDIX A Study Protocol P0001131 (Exygen Study No. P0001131) with Analytical Methods, Protocol Amendments and Deviations Exygen Research Page 37 o f 121 Interim Report #9-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 Performing Laboratory: Exygen Research 3058 Research Drive State College, PA 16801 Phone: (814) 272-1039 Sponsor Representative: Michael A. Santoro Director o f Regulatory Affairs 3M Building 0236-01-B-10 St. Paul, M N 55144 Phone: (651) 733-6374 Page ! oj 6} Exygen Research Page 38 o f 121 Interim Report #9-Analysis o f Ground Water Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 DISTRIBUTION: 1) Jaisimha Kesari, Study Director, Weston Solutions 2) John M. Flaherty, Principal Investigator, Exygen Research 3) Michael A. Santoro, Sponsor Representative, 3M Company 4) Exygen Research Quality Assurance Unit Exygen Research Page 2 o f 65 Page 39 o f 121 Interim Report #9-Analysis of Ground Water Samples Exygen Study No.: P0001131 Exygen Protocol Nuniber: P0001131 PROTOCOL APPROVAL Study Title: Analysis o f Perfluorobutanesulfonate (PFBS), Perfluorohexanesulfonate (PFHS), and Perfluorooctanesulfonate (PFOS) in Water, Soil, Sediment, Fish, Clams, Vegetation, Small Mammal Livers and Small Mammal Serum Using LC/MS/MS for the 3M Decatur Monitoring Program Exygen Protocol Number: P0001131 APPROVALS JaisimhauCesan, St Weston Solutions 1 __________ Quality Assurance Unit Date T^-OcT-iC Date Page 3 of 65 Exygen Research Page 40 o f 121 Interim Report #9-Analysis of Ground Water Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 TABLE OF CONTENTS TITLE P A G E .....................................................................................................................................................................1 D IS T R IB U T IO N ...............................................................................................................................................................2 PROTOCOL APPROVAL..................................................... !...................................................................................... 3 TABLE OF CONTENTS................................................................................................................................................4 I N T R O D U C T IO N .............................................................................................................................................................5 TEST M ATERIA LS........................................................................................................................................................ 5 O B JECTIV E..................................................................................................................................................................... 6 TESTING FACILITY......................................................................................................................................................6 STUDY DIRECTOR........................................................................................................................................................7 SPONSOR REPRESENTATIVE................................................................................................................................... 7 PRINCIPAL INVESTIGATOR..................................................................................................................................... 7 PROPOSED EXPERIMENTAL START AND TERMINATION D A TE S........................................................... 7 IDENTIFICATION AND JUSTIFICATION OF THE TEST SY ST EM ................................................................8 SAMPLE PROCUREMENT, RECEIPT AND RETENTION................................................................................. 8 SAMPLE IDENTIFICATION....................................................................................................................................... 9 ANALYTICAL PROCEDURE SUMMARY.............................................................................................................. 9 VERIFICATION OF ANALYTICAL PROCEDURE................................................................................................9 METHOD FOR CONTROL OF B IA S......................................................................................................................... 11 STATISTICAL M ETHODS........................................................................................................................................... II GLP STA TEM EN T..........................................................................................................................................................11 R EPO R T............................................................................................................................................................................ 11 SAFETY AND H EA LTH ................................................................................................................................................12 AMENDMENTS TO PROTOCOL............................................................................................................................... 13 DATA RECORD K EE PIN G .......................................................................................................................................... 13 QUALITY A SSURANCE...............................................................................................................................................14 RETENTION OF DATA AND ARCHIVING............................................................................................................ 14 APPENDIX I, ANALYTICAL METHODS.................................................................................................................15 Page 4 of 6S Exygen Research Page 41 o f 121 Interim Report #9-Analysis o f Ground Water Samples Exygen Study No.: P0001131 Exygen Protocol Number: POOO1131 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 LC/MS/MS for the 3M Decatur Monitoring Program. The study will be audited for compliance with EPA TSCA Good Laboratory Practice Standards 40 CFR 792 by the Quality Assurance Unit o f Exygen Research. TEST MATERIALS The test materials are perfluorobutanesulfonate (PFBS), perfluorohexanesulfonate (PFHS) and perfluorooctanesulfonate (PFOS) and are all supplied by 3M. PFBS Chemical Name: Perfluorobutanesulfonate Molecular Weight: 338 supplied as the potassium salt (CFSOjT O Lot Number: 101 Purity: 96.7% Transitions Monitored: 299 -->99 Structure: PFHS Chemical Name: Perfluorohexanesulfonate Molecular Weight: 438 supplied as the potassium salt (CFuSOsTC*) Lot Number: SE036 Purity: 98.6% Transitions Monitored: 399 - 80 Structure: Page 5 o f 65 Exygen Research Page 42 o f 121 Interim Report #9-Analysis of Ground Water Samples Exygen Study No.: P0001131 Exygen Protocol Number: POOO1131 PFOS Chemical Name: Perfluorooctanesulfonate Molecular Weight: 538 supplied as the potassium salt (CsFnSOjX*) 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), periluorohexanesulfonate (PFHS) and perfluorooctanesulfonate (PFOS) in water, soil, sediment, fish, clams, vegetation, small mammal livers and small mammal serum for the 3M Decatur Monitoring Program using the current versions o f the following Exygen analytical methods: V0001780: "Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Water by LC/MS/MS" V0001781: "Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Soil by LC/MS/MS" V0001782: "Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Sediment by LC/MS/MS" V0001783: "Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Fish and Clams by LC/MS/MS" V0001784: "Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Vegetation by LC/MS/MS" V0001785: "Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Small Mammal Liver by LC/MS/MS" V0001786: "Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Small Mammal Serum by LC/MS/MS" TESTING FACILITY Exygen Research 3058 Research Drive State College, PA 16801 Phone: (814) 272-1039 Page 6 o f 65 Exygen Research Page 43 o f 121 Interim Report #9-Analysis o f Ground Water Samples Exygen Study No.: P0001131 Exygen Protocol Number: P 0001131 STUDY DIRECTOR Jaisimha Kesari P.E , DEB Weston Solutions, Inc. 1400 Weston Way West Chester, PA 19380 Phone: (610) 701-3761 Fax:(610)701-7401 j .kesari@westonsolutions.com SPONSOR REPRESENTATIVE Michael A. Santoro 3M Company Director o f Regulatory Affairs 3M Building 0236-0I-B-10 St. Paul, MN 55144 Phone: (651) 733-6374 PRINCIPAL INVESTIGATOR John M. Flaherty Exygen Research 3058 Research Drive State College, PA 16801 Phone: (814) 272-1039 j ohn.flaherty@exygen.com PROPOSED EXPERIMENTAL START AND TERMINATION DATES It is proposed that the analytical portion o f this study be conducted from October 01, 2004 to December 31, 2005. The actual experimental start and termination dates will be included in the final report. Page 7o f 65 Exygen Research Page 44 o f 121 Interim Report #9-Analysis o f Ground Water Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 roENTIFTCAHON AND JUSTIFICATION OF THE TEST SYSTEM The following are the test systems for this study: Water (groundwater and surface water) Soil Sediment Fish Clams Vegetation Small Mammal Liver Small Mammal Serum The samples will be collected by Weston Solutions. The control samples will be purchased and prepared by the testing facility. Purchase and processing details for the control samples will be included in the final report associated with this study. The test systems were chosen to access the environmental impact o f PFBS, PFHS and PFOS in the Decatur, Alabama area. SAMPLE PROCUREMENT, RECEIPT AND RETENTION Water, soil, sediment, fish, clam, vegetation, small mammal liver and small mammal serum samples will be received at Exygen directly from Weston Solutions. The details o f sample procurement for this study are outlined in the 3M work plan entitled "Phase 2 Work Plan for Sampling Environmental Media." The number and types o f samples collected will vary depending availability in the field. The total number o f samples received and analyzed for each matrix will be documented in the final report associated with this study. Water, soil, and sediment samples will be used as received without further processing at Exygen. These samples will be stored refrigerated at 2C-8C. Fish, clam, vegetation and small mammal liver samples will be processed according to the appropriate analytical method (see Appendix I). These samples will be stored frozen at -10C. Small mammal whole blood samples will be centrifuged in the field at the time o f collection and the serum fraction will be used for the study. Small mammal serum will be stored frozen at S -10C. The receipt and processing o f the samples will be documented in the final report and raw data associated with the study. Page 3 o f 63 Exygen Research Page 45 o f 121 Interim Report #9-Analysis o f Ground Water Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 SAMPLE IDENTIFICATION Prior to analysis, each sample will be assigned a laboratory sample reference number. The reference number will be unique and will distinguish each laboratory sample that is processed throughout the analytical procedure. Chromatographic data will be identified by the laboratory sample reference number. Sample storage conditions and locations will be documented throughout the study. ANALYTICAL PROCEDURE SUMMARY References: V0001780: "Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Water by LC/MS/MS" V0001781: "Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Soil by LC/MS/MS" V0001782: "Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Sediment by LC/MS/MS" V0001783: "Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Fish and Clams by LC/MS/MS" V0001784: "Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Vegetation by LC/MS/MS" V0001785: "Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Small Mammal Liver by LC/MS/MS" V0001786: "Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Small Mammal Serum by LC/MS/MS" The above methods use analytical conditions capable o f separating the isomers o f PFBS, PFHS and PFOS. The final report will include the isomers summed into total PFBS, total PFHS, and total PFOS found. VERIFICATION OF ANALYTICAL PROCEDURE A laboratory control sample will be used for the preparation o f fortified control samples. The test substance will be made into solutions as per the method, and added to the matrices via a micropipette. For water sampling, Exygen will supply one bottle per sample collected. The bottles will be 500 mL precleaned Sci/Spec Premier wide mouth HDPE bottles. These bottles have been routinely used for fluorochemical sample Page 9 of65 Exygen Research Page 46 o f 121 Interim Report #9-Analysis o f Ground Water Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 collection at the testing facility and have been shown to be free o f PFBS, PFHS and PFOS. Samples will be added to each container to a volumetric fill line at 200 mL. A field duplicate, a low field spike and a high field spike of each sample will be collected. The low and high field spike bottles will contain PFBS, PFHS and PFOS as well as perfluorooctanoic acid (PFOA) and 1.2-13C perfluorooctanoic acid (13C PFOA). PFOA and l3C PFOA are included in the solutions used to spike the samples. The results for PFOA and l3C PFOA will not be reported in this study. Exygen will supply one field blank (control water) and two field blank spikes (control water fortified with PFBS, PFHS and PFOS at a low and high level) for every twenty samples collected. At the testing facility, each water sample (excluding field duplicates and field spikes) will be extracted in duplicate and will also be fortified at a low and high concentration with PFBS, PFHS and PFOS and processed through the described procedure to determine method accuracy and to check for bias. For soil, sediment, clams, and vegetation, Exygen will supply one 500 mL precleaned Sci/Spec Premier wide mouth HDPE bottle per sample collected or a zip-seal bag. All containers/bags used for sample collection will be shipped to the sample location. Samples will be added to each container or bag in the field. At the testing facility, each sample will be extracted in duplicate and will also be fortified at a known concentration with PFBS, PFHS and PFOS at both a low and high level and processed through the described procedure to determine method accuracy and to check for bias. For small mammal liver, Exygen will supply a 50 mL polypropylene centrifuge tube. For small mammal serum, Exygen will supply a collection kit for each sample containing serum separator tubes (red top), vacutainers, needle holders and needles, transfer pipettes, and polypropylene tubes. At the testing facility, each liver and serum sample will be extracted in duplicate and will also be fortified at a known concentration with PFBS, PFHS and PFOS at both a low and high level and processed through the described procedure to determine method accuracy and to check for bias. Low and high spiking levels for each matrix are defined below: M atrix Low Spiking Level High Spiking Level Water 500 ng/L 5000 ng/L Soil Sediment 4 ng/g 4 ng/g 40 ng/g 40 ng/g Fish 10 ng/g 100 ng/g Clams 10 ng/g 100 ng/g Vegetation 10 ng/g 100 ng/g Small Mammal Liver 10 ng/g 100 ng/g Small Mammal Serum 10 ng/mL 100 ng/mL Page Oof65 Exygen Research Page 47 o f 121 Interim Report #9-Analysis o f Ground Water Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 Recoveries are anticipated to be between 70% and 130% o f the fortified levels; however, the exact precision and accuracy will be determined by the analysis o f the quality control samples described above. A statement o f accuracy will be included in the final report. METHOD FOR CONTROL OF BIAS Control o f bias will be addressed by taking representative sub-samples from a homogeneous mixture o f each matrix from untreated control samples, and by analyzing at least two levels o f fortifications. STATISTICAL METHODS Statistics will be limited to those specified in the subject methods and to the calculation o f average recoveries, as applicable. GLP STATEMENT All aspects o f this study shall be performed and reported in compliance with EPA TSCA Good Laboratory Practice Standards 40 CFR 792. The final report or data package (supplied to the Sponsor) shall contain a statement that the study was conducted in compliance with current and applicable GLP standards and will outline any deviations in the study from those standards. This statement will be signed by the Study Director and Sponsor Representative. REPORT A final report will be prepared by the principal investigator or their designee at the conclusion o f the study. The report will include, but will not be limited to, the following: The name and address o f die 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 II of 65 Exygen Research Page 48 o f 121 Interim Report #9-Analysis o f Ground Water Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 The signed and dated statement by the Exygen Research Quality Assurance Unit regarding dates o f study inspections and dates findings were reported to the Study Director and Management. A description o f the exact analytical conditions employed in the study. If the subject method was followed exactly, it is necessary to include only a copy o f the analytical method. Any modifications to this method will be incorporated into the report. If the method is photo-reduced, the project number and page number must be included on each page. Description o f the instrumentation used and operating conditions. All results from all sets analyzed. Control and fortified samples will be identified and the data table will include sample number and fortification level. Representative chromatograms for each analyte in each matrix, including chromatograms o f a standard and a control sample, and a chromatogram at a fortification level. The location o f the analyte peaks will be clearly identified in all chromatograms. All circumstances that may have affected the quality or integrity o f the data will be documented in the report. Locations where raw data and the final report are to be archived. Additions or corrections to the final report shall be in the form o f an amendment signed by the Study Director. The amendment shall clearly identify that part o f the report that is being altered and the reasons for the alterations. The amendment will be signed and dated by the Study Director and the Sponsor Representative. All applicable requirements for reporting o f study results as per 40 CFR 792.185. SAFETY AND HEALTH Laboratory personnel 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 substancefs). Page 12 o f65 Exygen Research Page 49 o f 121 Interim Report #9-Analysis o f Ground Water Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 AMENDMENTS TO PROTOCOL All significant changes to the analytical protocol outlined here will be expressed in writing, signed and dated by file Study Director and Sponsor Representative. Amendments usually will be issued prior to initiation o f study plan change. However, when a change is required without sufficient time for the issue o f a written amendment, that change may be effected verbally with supporting documentation signed and dated by the Study Director and followed with a written amendment as soon as possible. In this case, the effective date o f the written amendment will be file date o f the documented change. Copies o f the signed amendments will be appended to all distributed study plan copies. The original amendment will be maintained with the original study plan. Any deviations from the study plan or from the analytical method as provided will be documented and reported promptly to the Sponsor Representative. DATA RECORD KEEPING Records to be maintained include the following (as appropriate): Sample tracking sheet(s) Sample receipt records, storage history, and chains o f custody History and preparation o f standards (stock, fortification, calibration) Description o f any modifications to the method Instrument run sheets, bench-sheets or logs Analytical data tables All chromatographic and instrumental conditions Sample extraction and analysis dates A complete listing o f study personnel, signatures and initials Chronological presentation o f all study correspondence Any other documentation necessary for the reconstruction o f the study Chromatograms- All chromatograms will contain the following: Sample identification, injection date, arrow or other indication o f the area o f interest, and injection number corresponding to the run. Additionally, fortifications will include the amount o f analyte added and the sample number o f the sample that was fortified. Analytical standard chromatograms will additionally include the concentration (e.g., pg/mL). Page IS o f 65 Exygen Research Page 50 o f 121 Interim Report #9-Analysis o f Ground Water Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 As part o f the documentation the following sheets will be included in each analytical set: a tun sheet listing the samples to be tun in the set, and an instrument conditions sheet describing the instrument type and operating conditions. QUALITY ASSURANCE The QA Unit o f Exygen Research will inspect the study at intervals adequate to assure compliance with GLP's, and will report the findings o f audits to the Study Director, Exygen Management, and the Sponsor Representative. RETENTION OF DATA AND ARCHIVING All hard copy raw data, including, but not limited to, the original chromatograms, worksheets, correspondence, and results shall be included with the data package submitted to the Study Director. These will be archived with the original study plan, amendments, final report, and all pertinent information from the Sponsor. The testing facility shall keep all electronic raw data and any instrument, equipment, and storage logs for the period o f time specified in 40 CFR 792.195. An exact copy o f the materials submitted to the study director will also be kept at Exygen Research. Exygen will obtain permission from the study director before discarding or returning samples. Exygen Research Page 4 o f 65 Page 51 o f 121 Interim Report #9-Analysis o f Ground Water Samples Exygen Study No.: P0001131 Exygcn Protocol Number: P0001131 APPENDIX I ANALYTICAL METHODS V0001780: "Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Water by LC/MS/MS" V0001781: "Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Soil by LC/MS/MS" V0001782: "Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Sediment by LC/MS/MS" V0001783: "Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Fish and Clams by LC/MS/MS" V0001784: "Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Vegetation by LC/MS/MS" V0001785: "Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Small Mammal Liver by LC/MS/MS" V0001786: "Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Small Mammal Serum by LC/MS/MS" Exygen Research Page 15 o f 65 Page 52 of 121 Interim Report #9-Analysis of Ground Water Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 ANALYTICAL METHOD Method Number V0001780 Method ofAnalyste for the DetermiaatloB ofPerfloorooctaaotc Acid (PFOA) lo Water byLC/MS/MS Analytic! Testing Facility. Exygen Research 3058 Research Drive State College, PA 16801 Approved By: _C-- __________ Paul Connolly 1 Technical Leader, LCMS, Exygen Research `la?y. Date / IJohn Flaherty Vic Prendali. Operation, Exygen Reteuch Exygen Research Total Paga: 7 Page 16 o f65 Page 53 o f 121 Interim Report #9-Analysis o f Ground Water Samples Exygen Study No.: P0001131 E x ygen P ro to co l N um ber: P 0 0 0 1131 xygM R m ttn b Method Number V000P80 I ANALYTICAL METHOD | Method o f Analysis for the Determination o f Pcrfluorooctanoic Acid (PFOA) in Water by LC/MS/MS 1.0 Scope Thie metfaod il to be employed for the isolation and quantitation o f pcrfluorooctanoic acid by Kigh Performance Liquid Chromatography coupled to a tandem Mass Spectromtrie Detector (LC/MS/MS) in water. 2.0 Safety 2.1 Always observe aafe laboratory practices. 2.2 Consult 0 appropriate MSDS before handling any chemical for proper safety precautions. 3.0 Sample Requirement 3.1 At least 40 mL of test sample for extraction. 3.2 No sample processing is needed for water samples. 3.3 Samples aimed refrigerated should be allowed to equilibrate to room temperature. 3.4 All samples must be thoroughly mixed before being sampled for extraction 3.5 Any samples containing particles should be centrifuged at -3000 rpm for ~S 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 Pcrfiuorooctmoic Acid - Sigma-Aldrich 5.0 Inatnunent 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 5-200 pL connected to a tandem Mast 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 tubee. 5.5 15 mL disposable polypropylene centrifuge tubes. 5.6 Disposable micropipe (50-100uL 100-200uL). 5.7 125-mL LOPE narrow-mouth bottles. 5.8 2 mL clear HPLC vial kit. 5.9 Disposable pipettes. 5.10 Autopipettes (100-1000 pL and 10-100 pL), with disposable tips. 5.11 Waters Sep Pak Vac 6 ec(lg)tC 18SPE cartridges. Pa|e2or? Page 17 of65 Exygen Research Page 54 o f 121 Interim Report #9-Analysis o f Ground Water Samples Exygen Study No.: P0001131 Exygen Protocol N um ber POOO1131 Exygroknesrdi Mttfaod Number V0001780 ________________________ ANALYTICAL iMETHOD Method o f Analysis for the Determination ofPerfluorooctanoic Acid (PFOA) in Water by LC/MS/MS 5.12 SPE vacuum manifold. 5.13 Ceotrifiige capable o f spuming 50 mL polypropylene tubee at 3000 rpm. 6.0 Chromatographic System 6.1 Analytical Column: Fluophaae RP (Keystone Scientific), 2.1 mm x 50 mm. 5 (P/N: 82505*052130) 6.2 Temperature: 30*C 6.3 Mobile Phase (A) : 2 mM Ammonium Acetate in Water Mobile Phase (B) : Methanol Gradient Program: Time fmin) 0.0 1.0 8.0 20.0 22.5 %A 65 65 25 25 65 Flow Rate S t f fmL/minl 35 0.3 35 0.3 75 0.3 75 0.3 35 0.3 6.6 Injection Voltane: 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 minute. The above conditions ate intended as a guide and may be changed in order to optimize the HPLC system. 7.0 MS/MS System 7.1 Mode: Electroepray Negative MRM mode, monitoring 413 -> 369 m/z. The above condition are intended as a guide and may be changed in order to optimize the MSMS system. 8.0 Preparation o f Solutions 8.1 Mobile Phase 8.1.1 2 mM ammonium acetate in water ia prepared by adding 0.IS4 g of ammonium acetate to 1000 mL o f water. Alternate volumes may be prepared. Page3ui ' Page 18 of 65 Exygen Research Page 55 o f 121 Interim Report #9-Analysis o f Ground Water Samples Exygen Study No. : P0001131 Exygen Protocol Number: P0001131 Exyg Ratearci Method Number V0001780 ANALYTICAL METHOD Method o f Analysis fix the Detennination o f Perfluarooctanoic Acid (PFOA) in Water by LC/MS/MS 9.0 Standard Preparation 9.1 Standard Stock/Fortificatxm Sofution 9.1.1 Prepare a stock solution of '*100 pg/mL o f PFOA by weighing 10 mg o f analytical standard (eometed for purity) and dilute to 100 mL with methanol in a 125-mL LOPE bottle. 9.1.2 A 10 pg/mL foitiflcation solution o f PFOA is prepared by bringing 10 mL o f the 100 pgfaL solution to a final volume of 100 with methanol in a 123 mL LDPE bottle. 9.1.3 A 1.0 pg/mL fortification solution o f PFOA ia prepared by bringing 10 mL o f the 10 pg/mL solution to a final volume o f 100 with methanol in a 123 mL LDPE bottle. 9.1.4 A 0.1 pgtoL fortification solution o f PFOA ie prepared by bringing 10 mL o ffoe 1.0 pgtaL solution to a final volume o f 100 with methanol in a 123 mL LOPE bottle. 9.1.5 A 0.01 pg/mL fortification solution o f PFOA is prepared by bringing 10 mL o f foe 0.1 pg/mL solution to a final volume of 100 with methanol in a 125 mL LDPE bottle. 9.1.6 The stock and fortification solutions are to be stored in a refrigerator at approximately 4C end we stable for a maximum period o f 6 months from the date o f preparation. 92 Standard Calibration Solutions 9.2.1 922 LC/MS/MS calibration standards are prepared in HPLC water. The calibration standards are processed through the extraction procedure, identical lo sanq>!ei. The following ia a typical example: additional concentrations may be Final Concentration Fortification Votums of Concentration of Calibration ofFottificatioa Volume Fortified Control Calibration Standard ID Solution (nob) 0 10 10 10 100 100 100 (jtL) 0 100 200 400 100 200 400 Santole imLi 40 40 40 40 40 40 40 Standard tonti* 0 23 30 100 230 500 1000 (example! XCmmddyy-0 XCmmddyy.] XCmmdyy.2 XCmmddyy-3 XCmmddyy-4 XCmmddyy*) XCmmddw-6 * The extracted concentration of the calibration atmdard is equal to 8 its initial concentration, due to the concentration o f the standard during the extraction (SPE). XC " extracted calibration standard. Page 4 of , Page 19 o f 6S Exygen Research Page 56 o f 121 Interim Report #9-Analysis o f Ground Water Samples Exygen Study No. : P0001131 Exygen Protocol Number: P0001131 Bxygaa Itim rc h M ethod Number V00017B0 ANALYTICAL METHOD Method o f Analysis for the Determination ofPerfhiorooctanoic Acid (PFOA) in Water by LC/MS/MS 9.2.3 A w o standard solution (reagent blank) must be prepared with each aet o f itanderds extracted. 9.2.4 Store all extracted calibration standards in 15>mL polypropylene tubes at 2"C to 6*C, up to two weeks. 9.2.5 Alternate volumes and concentrations o f standard! may be prepared as needed. 10.0 Batch Set Up 10.1 Each batch of samples extracted (typically 20 or (ess) must include at least ooe reagent control (method blank using HPLC water) and two reagent controls fortified at known concentrations (lab control spike) to verify pcocedural reoovery for the batch. 10.2 Requirementa for field and laboratory duplicates and spikes will be specified in the quality assurance plan for this project. 11.0 Simple Extraction 11.1 Measure 40 mL o f sample or a portion of sample diluted to 40 mL with water into 50 mL polypropylene eentriflige tubes (fortify as needed, replace lid and mix well). 11.2 Condition the C n SPE cartridges (l g, 6 mL) by passing 10 mL methanol followed by 5 mL o fHPLC water ( - 2 drop/bec). Do not let column run dry 11.3 Load sample on conditioned C n SPE cartridge. Discard eluate. 11.4 Elute with *5 mL 100% methanol Collect 3 mL o f eluate into graduated IS mL polypropylene centrifoge tubes (final volume S mL). 11.5 Analyze samples using electroapray LC/MS/MS. 12.0 Chromatography 12.1 Inject the same amount o f each standard, simple and fortified sample into the LOMS/MS system. A calibration standard must precede and follow all analyzed samples. 12.2 Standards o f PFOA corresponding to at least five or more concentration levels must be inchidod in an analytical set. 12.3 An entire set o f extracted calibration standards must be included m the beginning and at the end o f a sample set. Extracted standards must he interspersed between every 5-10 samples. As an alternative, an entire set of extracted calibration standards may be injected at the beginning of a set followed by extracted calibration standards interspersed every 5*10 samples (to account for s second act 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 of peak area Page 5 of 7 Page 20 o f 65 Exygen Research Page 57 o f 121 Interim Report #9-Analysis o f Ground Water Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 E xyfw R nM ich Method Number V00017S0 ANALYTICAL METHOD Method o f Analysis fordie Determination o f Perfluorooctanoic Add (PFOA) in Waier by LC/MS/MS versus calibration standard concentration wing M uiLynx 3.3 (or equivalent) software system. 12.5 Saddle response should not exceed standard responses. Any samples that exceed standard responses should be ftirther 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 entile set must be re-extracted. 13.3 Recoveries o f control spikes and matrix spikes must be between 70-130% of their known values. If a control spike foils outside the acceptable limits, the entire set o f samples should be re-extracted. Any matrix spike outside 70 130% should be evaluated by the analyst to determine if re-extraction is warranted. 13.4 Any calibration standard found to be a statistical outlier by using the Huge Error Test, may be excluded from the calculation o f the calibration curve. However, foe total number o f extracted calibration standards that could be excluded must not exceed 20% o f foe total number o f extracted standards injected. 13.5 The con-elation coefficient (R) for calibration curves generated must be 0.992 (R* 0.985). If calibration results foil outside these limits, then appropriate steps must be taken to adjust instrument operation, and the standards or foe relevant set o f ssmples should be reanalyzed. 13.6 Retention times between standards and samples must not drift more than 4 % within an analytical nm. If retention time drift exceeds this limit within an analytical nm then foe set must be reanalyzed. 14.0 Calculations 14.1 Use the following equation to calculate foe amount o f PFOA found (in ngfL. based on peak area) using foe standard curve (linear regresaion parameters) generated by foe M an Lynx software program: PFOA found (ng/L) - (Peak ares - intercept! x DF slope DF Actor by which the final volume waa diluted, if necessary. Page6of7 Page 21 o f 65 Exygen Research Page 58 o f 121 Interim Report #9-Analysis o f Ground Water Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 BxyfMRaMtreb MMbodNumber V0001780 I A N A tm C A L METHOD I Method o f Analyst for the Determination o f Perfluorooctanok A dd (PFOA) in Water by LC/MS/MS 142 For samples fortified with known amounts of PFOA prior to extraction, use the following equation to calculate the percent recovey. Recovery(H) - [ total analytefound (ng/L) analyte found in control (ng/L)] ^ analyteadded (ng/L) Exygen Research Page 7 of 7 Page 22 o f 65 Page 59 o f 121 Interim Report #9-Analysis o f Ground Water Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 ANALYTICAL METHOD Method Number V0001781 Method of Analyib tor the Detormtoaiton o f Perfluorooctuoic A dd (PFOA) hi Soil by LC/MS/MS Analytical Testing Facility: Exygen Research 3058 Research Drive Statt College, PA 10801 Approved By: T U . C J i ._____ Paul Connolly * Technical Leader, LC-MS, Bxygen Research lolu.fof Date D ato Exygen Research Total Pagai: 7 Page 23 o f 65 Page 60 o f 121 Interim Report #9-Analysis o f Ground Water Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 Bxyjn Raassrcfc Mttbod Number V00017S] ANALYTICAL METHOD J Method o f Analysis for die Determination ofPerfluorooctaooic Acid (PFOA) in Soil by LC/MS/MS 1.0 Scope This method is to be employed for the isolation and quantitation o f perfluorooctinoic acid by High Performance Liquid Chromatography coupled to a tandem Mass Spectrometric Detector (LC/MS/MS) in soil. 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 15 g o f test sample for extraction. 3.2 No sample processing is needed for soil samples. 3.3 Samples noted reftigerated should be allowed to equilibrate lo room temperature. 3.4 All samples must be thoroughly mixed before being sampled for extraction. 3.5 Sample oollection procedures will be specified in the sampling plan for this prqject. 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 - Sigmt-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 devioe to collect raw data for peak integration and quantitation. 5.3 Analytical balance capable o freading to 0.00001 g. 5.4 50 mL disposable polypropylene ccntrifoge tubes. 5.5 15 mL disposable polypropylene ccntrifoge tubes. 5.6 Disposable micropipets (50-100uL, 100-200uL). 5.7 125-mL LDPB narrow-mouth bottles. 5.8 2 mL clear HPLC vial kit. 5.9 Disposable pipettes. 5.10 Autopipetlea (100-1000 pL and 10-100 pL), with disposable tipe. 5.11 Waten Sep Pak Vac 6 cc (lg) tC18 SPE cartridges. 5.12 SPE vacuum manifold. 5.13 Ultrasonic bath. Page2of? Page 24 o f 65 Exygen Research Page 61 o f 121 Interim Report #9-Analysis o f Ground Water Samples Exygen Study No.: P0001131 Exygen Protocol N um ber P0001131 B x y a Rwewch Method NanbrVOOOI7Sl ANALYTICAL METHOD Method o f Analysis for the Determination o f PerfluoroocUnoic Acid (PFOA) in Soil by LC/MS/MS S. 14 Wriat-action shaker. 5.1$ Centrifuge capable of pinning 50 mL polypropylene tubes al 5000 rpm. 6.0 Chromatographic System 6.1 Analytic! Column: Fluophase RP (Keystone Scientific). 2 .1 mm x 50 nun. 5m (P/N: 82505*052130) 6.2 Temperature: 30*C 6.3 Mobile Pham (A ): 2 mM Ammonium Acetate in Water 6.4 Mobile Phase (B ): Methanol 6.5 Gradient Propam: X antom i 0.0 1.0 8.0 20.0 22.5 65 65 25 25 65 Flow Rate 2 i fmL/iwinl 35 0.3 35 0.3 75 0.3 75 0.3 35 0.3 6.6 Iqjecttat Volume: 15 pL (can be Increased to as much as 50 pL). 6.7 Quantitation: Peek A rea- external standard calibration curve. 6.8 RunTime: ~ 2 3 minutes. The above conditions are intended as a guide and may be changed in order to optimize the HPLC system. 7.0 MS/MS System 7.1 Mode: Electrospray Negative MRM mode, monitoring 4!3 -+ 369 m/z for PFOA. The above conditions are intended ae a guide and may be changed in order to optimize die MSMS system. 8.0 Preparation of Solutions 8.1 Mobile Phase 8.1.1 2 mM ammonium acetate in water ia prepared by adding 0.154 g or ammonium acetate to 1000 mL o f water. Alternate volumes may he prepared. PagJ of 7 Page 25 o f65 Exygen Research Page 62 o f 121 Interim Report #9-Analysis o f Ground Water Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 Exypn RMtarch Matbod Number V0001711 I ANALYTICAL METHOD Method o f Analysis for the Determination o f Perfiuorooctsnoic Acid (PFOA) in Soil by LC/MS/MS 9.0 Standard Preparation 9.1 Standard Stock/Fottiflcatioa Solution 9.1.1 Prepare a atock solution o f -100 pg/m Lof PFOA by weighing 10 mg of analytical standard (corrected for purity) and dilute to 100 mL with methanol in a 125-mL LDPE bottle. 9.1.2 A 10 pg/mL fortification solution o f PFOA is prepared by bringing 10 mL o f the 100 ng/mL solution to a final volume o f 100 with methanol in a 123 mL LDPfi bottle. 9.1.3 A 1.0 pg/mL fortification eolution o f PFOA is prepared by bringing 10 m Loftbe 10 p^m L eolution to a final volume of 100 with methanol in a 123 mL LDPE bottle. 9.1.4 A 0.1 pg/mL fortification solution o f PFOA ie prepend by bringing 10 mL o f foe 1.0 pgftnL solution to a final volume o f 100 with methanol in a 123 mL LIVE bottle. 9.1.5 A 0.01 p^m L fortification solution of PFOA is prepared by bringing 10 mL o f the 0.1 pg/mL solution to a final volume of 100 with in a 123 mL LDPE bottle. 9.1.6 The stock and fortification solutions are to be stored in s refrigerator at approximately 4*C sod ere 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 ere prepend in HPLC water The calibration standards are processed through the extraction procedure, identical to samples. The following ie a typical example: additional concentrations may be prepared as needed. Final runwiiimiffi Fortification Volume of Concentration of Calibration ofFortification Volume Fortified Control Calibration Standard ID Solution foott 0 10 10 10 100 100 100 .L) 0 100 200 400 100 200 400 SamclefmL) 40 40 40 40 40 40 40 Standard foot)4 0 23 SO 100 250 300 1000 (example) XCmmddyy-0 XCmmddyy-t XCmmddyy-2 XCmmddyy-3 XCmmddyy-4 XCmmddyy*5 XCmmddw-6 * The extracted concentration o f the calibration standard is equal to 8* its initial concentration, due to theconcentration o f the standard during the extraction fSPE). XC - extracted calibration standard. Pas*4 of? Page 26 o f 65 Exygen Research Page 63 of 121 Interim Report #9-Analysis o f Ground Water Samples Exygen Study No. : P0001131 E x y g e n P ro to co l N um ber: P 0 0 0 1 131 Exy|o ftaanrek M ette* M unter VOOO1781 ANALYTICAL METHOD Method o f Analysis for the Determination o f Porftuorooctanoic Acid (PFOA) in Soil by LC/MS/MS 9.2.3 9.2.4 9.2.5 A sera ctandard eolution (reagent blank) must be prepared with each set o f standards extracted. Store all extracted calibration standards in 15-mL polypropylene lubes at 2*C to 6*C, up to two weeks. Alternate volumes and concentrations o f standards may be prepared as needed. 10.0 Batch SetUp 10.1 Each batch o f samples extracted (typically 20 or leas) must include at least one reagent control (method blank using S mL o f methanol) and two reageni controls fortified as known concentrations (lab control spike) to verify procedural recovery for the batch. 10.2 Requirements for field and laboratory digriicatea and spikes will be specified in the quality assurance plan for this project. 11.0 Sample Extraction 11.1 Weigh S g o f sample into 50 mL polypropylene centrifuge tubes (fortify as 1U needed, replace lid and mix well). Add 5 mL o f methanol and shake on a wrist action shaker for ~15 minutes 11J Transfer the tubes to an ultrasonic bath and sonicate for 15 minutes. 11.4 Bring the volume up to 40 mL with water in the SO mL polypropylene ceotrifoge tube. 11.5 Centrifoge for 10 minutes at 3000 rpm. 11.6 Condition the Cis SPB cartridges (1 g. 6 mL) by passing 10 mL methanol followed by 5 mL o f HPLC water ( 2 drop/sec). Do not let column run dry 11.7 Load (decant) the sample on the conditioned Cti SPE cartridge. Discard 11.8 eluaie. Elute with 5 mL 100% methanol. Collect 5 mL o f eluate into graduated 15 mL polypropylene oentriftige tubes (final volume - 5 mL). 11.9 Analyze samples using eloctrospray LC/MS/MS. 12.0 Chromatography 12.1 Inject the same amount of 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 to at least five or more concentration levels must be included in an analytical set. 12.3 An entire set o f extracted calibration standards must be included at the beginning and at the end of a sample set. Extracted standards must be interspersed between every 5-10 samples. Ac an alternative, an entire set of Psg5or7 Page 27 o f 65 Exygen Research Page 64 o f 121 Interim Report #9-Analysis of Ground Water Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 ExyimReattick MethodNumber V00017$I | ANALYTICAL METHOD Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Soil by LC/MS/MS extracted calibration staodarda may be injected at the beginning o f a set followed by extracted calibration standards interspersed every 5-10 samples (to account for a second set o f extracted standards), hi either case, extracted calibration standards must be die 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 MaitLynx 3.3 (or equivalent) software system. 115 Sample response should not exceed standard responses. Any samples that exceed standard responses should be further diluted ami reanalyzed. 13.0 Acceptance Criteria 13.1 Chromatogram m ist 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 of carbon dioxide. 13.2 Method Mania must not contain PFOA at levels greater than the LOQ. If a blank contains PFOA as levels greater than 50 ng/L, then a new blank sample must be obtained and the tire set must be re-extracted. 133 Recoveries o f control spikes and matrix spikes must be between 70-130% of their known values. If a control spike falls outside the acceptable limits, the entire set o f samples should be re-extracted. Any matrix spike outside 70 130% should be evaluated by the analyst to determine if re-extraction is warranted. 13.4 Any calibration standard found to be a statistical outlier by using the Huge Error Test, may be excluded from the calculation o f the calibration curve. However, the total number o f extracted calibration standards that could be excluded must not exceed 20% o f the total number o f extracted standards injected. 13.5 The correlation coefficient (R) for calibration curves generated must be 20.992 (R3 20.985). If calibration results fall outside these limns, then appropriate steps must be taken to adjust instrument operation, and the staodarda or the relevant set of samples should be reanalyzed. 13.6 Retention times between standards and samples must not drift more than 4 % within an analytical run. If retention time drift exceeds this limit within an analytical run then the set must be reanalyzed. Page $of? Page 28 of 65 Exygen Research Page 65 o f 121 Interim Report #9-Analysis o f Ground Water Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 EaygeaRam ich Method N u o ta V0001781 ANALYTICAL METHOD J Method o f Analysis for the Determination of Perfluorooctanoic Acid (PFOA) in Soil by LC/MS/MS 14.0 Calculation! 14.1 Uie the following equation to calculate the amount o f PFOA found (in ng/L, baaed on peak ana) using the standard curve (linear regression parameters) generated by foe Mats Lynx software program: PFOA found (ng/L) - (Peak area - intercept! x DF slope DF Mfactor 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 foe percent recovery. Recovery (% ) [total analytefound (ng/L) - analyte found in control (ng/L)] ^ ^ analyte added (n^L) 14.3 (Jae foe following equation to convert foe amount o f PFOA found in ng/L to nrtippb). FFOA found (ppb) - fPFOA found fna/LI x volume extracted f0.04D1 sample weight (5 g) 14.4 Uae the following equation to calculate the amount of PFOA found in ppb baaed m dry weight. PFOA found (ppb) dry weight PFOA found (ppb) x [100% / total solids(%)] P*f*70f7 Page 29 o f 65 Exygen Research Page 66 o f 121 Interim Report #9-Analysis of Ground Water Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 ANALYTICAL METHOD Method Number V0001782 Method o f Analytic for the Determination of Perfluorooctanoic A dd (PFOA) io Sedinosi by LC/MS/MS Analytical Testing Facility: Exygen Research 3038 Reotorch Drive State College, PA 16801 Approved By: ^ _______________ Paul Connolly ( Technical Leader, LC-MS, Exygen Raeearch ____lofrfa/o*/ Date a* Aodhin Flaherty f Vice President, Operations, Exygen Research Date Exygen Research Total Pagee: 7 Page 30 o f 65 Page 67 o f 121 Interim Report #9-Analysis o f Ground Water Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 Ennnftam ick Mttbod Number VOOOI7B2 1 ANALYTICAL METHOD | Method o f Analysis for the Determination o fPerfluorooetanoic Acid (PFOA) in Sediment by LC/MS/MS 1.0 Scope Thia method it to be employed for the iaolalion and quantitation o f perfluorooetanoic acid by High Performance Liquid Chromatography coupled to a tandem Mass Spectrometric Detector (LC/MS/MS) in sediment 2.0 Safety 2.1 Always observe aafo laboratory practioee. 2.2 Coasuh the appropriate MSDS before handling any chemical for proper safety precautions. 3.0 Sample Requirement 3.1 At least 30 g o f test sen ile for extraction. 32 No sample processing is needed for sediment samples. 3.3 Samples stored refrigerated should be allowed to equilibrate io 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 4.2 Methanol - HPLC grade 4.3 Acetic Acid-Reagent grade 4.4 Ammonium Acetate - A.C.S. Reagent Grade 4.5 Perfluorooetanoic A dd - 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 centrifiige tubes. 5.5 15 mL dUposable polypropylene centrifuge tubes. 5.6 Disposable micropipets (50-100uL, 100-2D0uL). 5.7 125-mL LDPE narrow-mouth bottles. 5.8 2 mL clear HPLC vial kit. 5.9 Disposable pipettes. 5.10 Autopipettes (100-1000 pL and 10-100 pL), with disposable tips. 5.11 Waters Sep P*k Vac 6 oc (lg) tC18 SPE cartridges. 5.12 SPE vacuum manifold. Pag*2 of7 Page 31 o f65 Exygen Research Page 68 o f 121 Interim Report #9-Analysis of Ground Water Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 Exygra Rcmreli Mtffcod Number VOOOI7S2 1 ANALYTICAL METHOD | Method o f Analysis for the Determination of Periluorooctanoic Acid (PFOA) in Sediment by LC/MS/MS 5.13 Vortexer. 5.14 Wnat*actiaa ihaker. 5.15 CcntriAige capable of spinning 50 mL polypropylene tubes at 3000 rpm. 6.0 Chromatofraphic System 6.1 Analytical Column: Fluophase RP (Keystone Scientific), 2.1 mm a 50 mm, 5p (P/N: 92505*052130) 6.2 Temperature: 30*C 6.3 Mobile Pbias (A) : 2 mM Ammonium Acetate in Water 6.4 Mobile Phase (B) : Methanol 6.5 Gradient Program: Tima fminl 0.0 1.0 8.0 20.0 22.5 2 A 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 Iqjecdoo Volume: 15 pL (can be increased to a* much as 50 pL). 6.7 Quantitation: Peak Area - external standard calibration curve. 6.8 RunTime: - 2 3 minutes. The above eondithms are intended at a guide and may be changed in order to optimize the HPLC system. 7.0 MS/MS System 7.1 Mode: ElectrosprayNcgativeMRM mode, monitoring 413 -> 369 nv/z for PFOA. The above conditions are intended as a guide and may be changed in order to optimize the MSMS system. 8.0 Preparation o f Solutions 8.1 Mobile Phase 8.1.1 2 mM ammonium acetate in water ia prepared by adding 0.154 g of mmfgiium acetate to 1000 mL o f water. Page 3 o f7 Page 32 o f 65 Exygen Research Page 69 o f 121 Interim Report #9-Analysis of Ground Water Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 Buygea Rawerefc Method Number VQQ0I7I2 | ANALYTICAL METHOD 1 Method o fAnalysis for the Determination of PerQuorooctanoic Acid (PFOA) in Sedimem by LC/MS/MS 8.2 Extraction Solutions 8.2.1 1% acetic add in water is prepared by adding 10 mL o f acetic acid to 1000 mL o f water. Alternate volumes may be prepared. 9.0 Standard Preparation 9.1 Standard Stock/Fortification Solution 9.1.1 Prepare a stock solution o f "*100 pg/mL o f PFOA by weighing 10 mg o f analytical standard (corrected fix purity) and dilute to 100 mL with methanol in a 125-mL LDPE bottle. 9.12 A 10 pg/mL fortification solution o f PFOA i t prepared by bringing 10 mL o f the 100 p^m L solution to a final volume o f 100 with meihanot in a 125 mL LDPE bottle. 9.1.3 A l .0 pg/mL fortification solution of PFOA is prepared by bringing 10 mL o f file 10 jigfatL solution to a final volume of 100 with meihtnol in a 125 mL LDPE bottle. 9.1.4 A0.1 pgtaL fortification solution o f PFOA is prepared by bringing 10 mL o f the 1.0 pg/mL solution to a final volume o f 100 with methanol in a 125 mL LDPE bottle. 9.1.5 A 0.01 pg/mL fortification solution o f PFOA if prepared by bringing 10 mL o f the 0.1 pg/mL solution to a final volume o f 100 with methanol in a 125 mL LDPE bottle. 9.1.6 The stock and fortification solutions v e to be stored in a refrigerator it approximately 4*C and are stable for a maximum period o f 6 months from the date o f preparation. 92 Standard Calibration Solutions 9.2.1 LC/MS/MS calibration standards are prepared in methanol via dilution o f tite 0.1 pg/mL fortification solution. 9.22 The following is t typical example: additional concentrations may be Concentration ofFoctifiestion Solution faetaL) 100 100 100 10 5 2 Volume (mL) 10 5 2 10 10 10 Diluted to (mL) 100 100 too 100 too 100 Final Concentratimi (na/mL) 10.0 SO 2.0 1.0 0.5 0.2 Pag*4 of? Page 33 o f 63 Exygen Research Page 70 o f 121 Interim Report #9-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 ftw w lniw rt Method Number V0OO)712 | ANALYTICAL METHOD | Method o f Analysis fix'the Determination o f Periluorooctsnoic Acid (PFOA) in Sediment by LCVMS/MS 9.2.3 Store all calibration MandanU in 125-raL LDPE narrow-mouth bottles at 2"C to 6*C, up to six montha. 92.4 Alternate volumes and concentratioas o f standards may be prepared as needed. 10.0 Batch Set Up 10.1 Bach batch o f samples extracted (typically 20 or leas) must include at least one untreated control and two untreated controls fortified at known concentrations (lab control splice) to verily procedural recovery for the batch. 10.2 Requirements for Arid and laboratory duplicates and spikes will be specified in foe quality assurance plan for this project 11.0 Sample Extraction 11.1 Weigh S g o f sample into 50 mL polypropylene centrifoge tubes (fortify as needed, replace lid and mix well). 11.2 Add 35 mL of \% acetic acid, cap, vortex and shake on a wrist action shaker for'60 minutes. 11.3 Centrifoge the tubee at ~3000 rpm for ~20 minutes. 11.4 Condition foe Cti SPB cartridges (1 g, 6 mL) by passing 10 mL methanol followed by 20 mL ofHPLC water ( - 2 drop/sec). Do not let column nut dry 11.5 Load (decant) the sample on the conditioned C u SPE cartridge. Discard eluate. 11.6 Add 20 mL o f methanol to the left in foe bottom of the 50 mL centrifoge tube. Cap, vortex and shake on a wrist action shaker for -30 minute. 11.7 Centrifoge the tubes at "3000 rpm feu -2 0 minutes. 11.8 Decant the methanol onto the same SPE cartridge. Collect foe eluate. 11.9 Wash the column with 4 mL o f methanol Collect the eluate and add ii to the eluate oollected in step 11.8. 11.10 Condition a second Cu SPE cartridge (1 g, 6 mL) by passing 10 mL methanol followed by 20 mL ofHPLC water ( - 2 drop/sec). Do not let column run dry 11.11 Add tile methanol to -200 mL o f water and load on the second conditioned SPE cartridge. 11.12 Elute with -5 mL 100% methanol. Collect 5 mL o f eluate into graduated 15 mL polypropylene centrifoge tube* (final volume 5 mL). 11.13 Analyze samples using electroepray LC/MS/MS. PtttSoH Page 34 o f 65 Exygen Research Page 71 o f 121 Interim Report #9-Analysis of Ground Water Samples Exygen Study No. : P0001131 Exygen Protocol Number: P0001131 B iy y H iiMwt Method NuabtrVDOO 1782 I ANALYTICAL METHOD | Method o f Anilyni for the Determination o f Perfluovooctanoic Acid (PFOA) in Sediment by LC/MS/MS 12.0 Chromatograph? 12.1 Iq/oct the same amount o f each standard, utnple aod fortified sample into the LC/MS/MS system. A calibration standard must precede and follow all analyzed samples. 12.2 Standards o f PFOA cosreqtonding to at least five or more concentration levels must be included in an analytical aet. 12.3 An entire set o f extracted calibration standards must be included at (he beginning and it the end o f a sample set. Standards must be interspersed between every 5*10 samples. As an alternative, an entire set o f calibration standards may be injected at the beginning o f a act followed by calibration standards interspersed every 3-10 samples (to account for a second set of standards). In either case, calibration standards must be the first and last injection in a sample set. 12.4 Use linear standard curves for quantitation. Linear standard curves arc generated for the analyte by linear regression using l/x weighting o f peak area versus calibration standard concentration using MassLynx 3.3 (or equivalent) software system. 12.5 Sample response should not exceed standard responses. Any samples that exceed standard responses should be Anther diluted and reanalyzed. 13.0 Acceptance Criteria 13.1 Chromatogram must show a peak o f a daughter ion at 369 amu from a parent o f 413 amu. The 413 amu parent corresponds to the PFOA anion, while the daughter ion (369 amu) represents the loss o f carbon dioxide. 13.2 Method blanks must not contain PFOA at levels greater than the LOQ. If a blank contains PFOA at levels greater than 0.2 ng/mL, then a new blank sample must be obtained and the entire set must be re-extracted. 13.3 Roooveries o f control spikes and matrix spikes must be between 70-130% of their known vehies. If a control spike foils outside the acceptable limits, the entire set o f samples should be re-extracted. Any matrix spike outside 70 130% should be evaluated by file 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, foe total number o f extracted calibration standards that could be excluded must not exceed 20% o f the total number o f extracted standards injected. 13.5 The correlation coefficient (R) for calibration curvet generated must be 20.992 (R: 20.985). If calibration results foil 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. Ptgtiof? Page 35 o f 65 Exygen Research Page 72 o f 121 Interim Report #9-Analysis o f Ground Water Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 ExjrgaRMWch Mabo4NuabVOOOI7U I ANALYTICAL METHOD | Method o fAnal) for the Determination o fPerfluorooctanoic Acid (PFOA) in Sodimeni by LC/MS/MS 13.6 Retention time between standards and samples must not drift more than 4 % within an analytical run. If retention time drift exceeds this limit within an analytical run then the set must be reanalyzed. 14.0 Calculations 14.1 Use the following equation to calculate foe amount of PFOA found (in ng/mL. baeed mi peak area) using foe standard curve (linear regression parameters) generated by the Miss Lynx software program: PFOA bund fnnftnU - ffw k ran - intercept! x DF slope DF * factor by which foe final volume was diluted, if necessary. 14.2 For samples fortified with known amounts of PFOA prior to extraction, use foe following equation to calculate the percent recovery. Recovery (%) - f total analyte found (ng/tnL) - analyte found in control (ng/mL)l analyteadded (ng/mL) 14.3 Use the following equation to convert foe amount o f PFOA found in ng/mL to ntfi(ppt>)- PFOA bond (ppb) - rPFflA foimH fng/mLl x no! vftluma ft mi ll sample weight (5 g) 14.4 Use foe following equation (if necessary) to calculate the amount o f PFOA found in ppb based on dry weight. PFOA found (ppb) dry weight PFOA found (ppb) x [100% / total solidi(%l] Pace7of7 Page 36 of65 Exygen Research Page 73 of 121 Interim Report #9-Analysis of Ground Water Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 ANALYTICAL METHOD Method Number: V0001783 Method ofAaalyils forth Determination of Perftaoroocteaolc Add (PFOA) lo Fish ad Clami by LG/MS/MS Analytic] Testing Facility: Exygen Research 3058 Research Drive State College, PA 10801 Approved By. V J , C.J1L Paul Connolly I Technical Leader, LC-MS, Exygen Research Flaherty Vice President, Operations, Exygen Research Due _a 4 4 ; Due Exygen Research Total Pager 8 Page 37 o f65 Page 74 o f 121 Interim Report #9-Analysis of Ground Water Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 Exygen Research MethodNumber VOOOI7S3 I ANALYTICAL m e t h o p I Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Fish And CUms by LC/MS/MS 1.0 Scope This method is to be employed for the isolation and quantitation o f perfluorooctanoic acid by High Perfoimance Liquid Chromatography coupled to a tandem Mass Spectromtrie Detector (LC/MS/MS) in fish and clams. 2.0 Safoty 2.1 Always observe aafe 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 teat sample for extraction. 32 Samples should be processed before extraction. Place the frozen sample in a food processor and homogmize with dry ice. Place the simples in containers and leave open in frozen storage overnight to allow for carbon dioxide sublimation. Seal and place the samples in frozen storage until time of analysis. 3.3 Sample collection procedures will be specified in the sampling plan for this project 4.0 Reagents and Standards 4.1 W ater-HPLC grade 4.2 Acetonitrile - HPLC grade 4.3 Carbon (120-400 merit) - Reagent grade 4.4 Methanol - HPLC grade 4.3 Silica gel (40-200 merit) - Reagent p ade 4.6 Plorieil(60-100mesh)* Reagent grade 4.7 Superclean LC-NHj - Reagent grade 4.5 1-Octsno! - HPLC grade 4.9 L-Ascorbic acid - Reagent grade 4.10 DinMdiyidichlorosilane - Reagent grad 4.11 Toluene - Reagent grade 4.12 Ammonium Acetate - A C S . Reagent Grade 4.13 Perfluorooctanoic Acid - Sigma-Aldrich 5.0 Instrument and Equipment - 5.1 A high performance liquid chromatograph capable o f pumping up to 2 solvents equipped with a variable volume injector capable o f injecting 5-200 MLconnected to a tandem Mass Spectrometer (LC/MS/MS). 52 A device to collect raw data for peak integration and quantitation. 5.3 Analytical balance capable of reading to 0.00001 g. Page 2of t Paga 38 of 65 Exygen Research Page 75 o f 121 Interim Report #9-Analysis o f Ground Water Samples Exygen Study No.: P0001131 Exygen Protocol N um ber POOOl 131 ExypalUMttch Method Number V0001783 | ANALYTICAL METHOD | Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Fish and Clams by LC/MS/MS 5.4 Rotaiy evaporator. 5.5 Tissumizer. 3.6 123 raL pear-shaped flasks. 3.7 30 mL disposable polypropylene centrifuge tubes. 3.8 15 mL disposable polypropylene centriftige tubes. 5.9 Disposable micropipets (50-lOOuL, 100-200uL). 5.10 123-mL LDPE natrow-mouth bottles. 5.11 2 mL clear HPLC vial kit. 5.12 Disposable pipettes. 5.13 Auloptpettes (100*1000 |iL and 10-100 pL), with disposable tips. 5.14 SPE tubes (20mL) (Supelco cat. no. N057I77). 5.15 Wrist action taker. 5.16 Centrifuge capable o f spinning 50 mL polypropylene tubea at 2000 rpm. 6.0 Chromatographie System 6.1 Analytical Column: Fluopbaae RP (Keystone Scientific), 2.1 mm x 50 mm. 5*i (P/N: 82505-052130) 6.2 Temperature: 30*C 6.3 Mobile Phase (A): 2 mM Ammonium Acetate in Water Mobile Phase (B): Methanol Gradient Program: Flow Rate 2t A rinTTmifri 0.0 65 35 0.3 1.0 65 35 0.3 8.0 25 75 0.3 20.0 25 75 0.3 22.5 65 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 u a guide and may be changed in order to optimize the HPLC system. P isiiri Page 39 o f 65 Exygen Research Page 76 o f 121 Interim Report #9-Analysis of Ground Water Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 Exygea Romich Method Number V0Q017U ANALYTICAL METHOD Method o fAnalysis for the Determimtion o f Pcvfluorooetinoic Acid (PFOA) in Fish and Clams by LC/MS/MS 7.0 MS/MS System 7.1 Mode: Elsctrocpray Negative MRM mode, monitoring 413*- 369 mil for PFOA. The above conditions are intended as a guide and may be changed in order to optimize the MSMS system. 8.0 Preparation o f Solutions 8.1 Mobile Phase 8.1.1 2 mM ammonium acetate in water is prepared by adding 0.1 $4 g of ammonium acetate to 1000 mL o f water. 8.2 Extraction Solutions 8.2.1 2%ascorbic add in methanol is prepared by dissolving 2 g o f ascoibic acid in 100 mL o f methanol. 8.2.2 30% Dimethyldichk>rosi!ane in toluene is prepared by bringing 3 mL ofdimethyldiehlorosilaneto a final volume o f 10 mL with toluene. Alternate volumes may be prepared. 9.0 Standard Preparation 9.1 Standard Stock/Fortifiestion Solution 9.1.1 9.1.2 9.1.3 9.1.4 9.1.3 Prepare a Mock 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 LOPE bottle. A 1.0 pytaL fortification solution of PFOA ia prepared by bringing 1 mL o f the 100 p^m L solution to a final volume o f 100 with methanol in a 12S mL LOPE bottle. A 0.1 pgfaiL fortification solution o f PFOA is prepared by bringing 10 mLoftba 1.0 p^m L solution to a final volume o f 100 with methanol in a 123 mLLDPE botile. A 0.01 pg/mL fortification solution o f PFOA it prepared by bringing 10 mL o f the 0.1 pgftnL solution to a final volume o f 100 with methanol in a 125 mL LDPE bottle. The stock and fortification solutions are to be stored in a refrigerator at approximately 4*C and are stable for a maximum period o f 6 months from the date o f prepmtion. Page 4 o f8 Page 40 o f 65 Exygen Research Page 77 o f 121 Interim Report #9-Analysis of Ground Water Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 Exygea Research Method Number V0001783 ANALYTICAL METHOD Method o fAnalysis A r the Determination o f Perfluorooctanoic Acid (PFOA) in Fiih and Clams by LC/MS/MS 9.2 Standard Calibration Solutions 9.2.1 9.2.2 LC/MS/MS calibration standards are prepared in methanol via dilution of the 1.0 ug/mL fortification solution. The following is a typical example: additional concentrations may be prepared aa needed. Concentration Final of Fortification Volume Solution (uc/mL) (mL) Diluted to (mL) Concentration <u/mL) 1.0 5.0 100 0.05 1.0 23 100 0.025 IX) 1.0 100 0.01 0.05 10 100 0.005 0.025 10 100 0.002S 0.1 10 100 0.001 0.065 10 100 0.0003 9.23 Store all calibration standards in 125-mL LDPE narrow-mouth bottles at 2*C to 6*C, up to six months. 93.4 Alternate volumes and concentrations of standards may be prepared as needed. 10.0 Batch Set Up 10.1 Bach batch o f ample extracted (typically 20 or leas) must include at least one untreated control and two untreated controls fortified at known concentrations (lib control spike) to verily procedural recovery for the batch. 10.2 Requirements for field and laboratory duplicates and spikes will be specified in foe quality inuranco plan for this project. 11.0 Sample Extraction 11.1 Weigh S g o f frozen sample into 50 mL polypropylene centrifuge tubes (fortify as needed, replace lid and mix well). 11.2 Add 30 mL o f acetonitrile and shake on a wrist action shaker for - 1 S minutes 113 Place foe tubes in a freezer for -1 hour. 11.4 Pack and condition foe SPB tubes and silanize foe pear-shaped Basks. U.5 Pack the 20 mL SPE tubes in sequence with 2 g florinl, 2 g silica gel, 2 g carbon, and 1 g LC-NHj. Condition the columns with 20 mL o f methanol, then 20 mL o f acetonitrile. Discard all washes. Do not allow the column to dry. 11.6 Silanise the 125 mL pear-shaped flasks by rinsing with the 30% dimefoyldichlororilane is toluene solution. Rinae foe flask with toluene once, followed by methanol (three tiroes). Dry the flasks completely before use, either by air-drying or with a stream ofnitrogen. Peg* S o f X Page 4/ o f 65 Exygen Research Page 78 o f 121 Interim Report #9-Analysis of Ground Water Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 Exypa fUMareh Method Number VOOOP83 ANALYTICAL METHOD Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Fish and Clama by LC/MS/MS 11.7 Centrifiige the SO mL polypropylene tube* containing aamplc at *>2000 rpm for-1 0 minutes. 11.8 Decant the extract on to a conditioned SPE column fitted inside the mouth of the pear-shaped flask. Collect the eluate in the 125 mL silanized pear-shape flask. 11.9 Add 10 mL o f acetonitrile to the sample in the SO mL centrifuge tube. Homogenize die frozen fot phase using a tissumizer for -3 0 seconds and rinse the tiasumixer with -1 0 mL o f acetonitrile into the tube. 11.10 Shake the sample again for-1 0 minutes on a wrist-action shaker. 11.11 Knee die tubes in a freezer fin - 1 hour more. 11.12 Centrifuge the SO mL polypropylene tubes containing sample at -2000 rpm for-IOmioutes. 11.13 Decant the extras onto the same SPE column. Collect the eluate into the same pear-shaped flask and combine with die eluent from the initial extraction. 11.14 Pass 20 mL o f acetonitrile through the SPE column and combine the eluate in the seme pear-shaped flask. 11.15 Add 3-4 drops o f 1-octeool to the extract in the pear-shaped flask and evaporate at reduced pressure using e rotary evaporator (at < 4Q8C). 11.16 Make die final volume, by adding 2 mL o f 2% ascorbic acid in methanol io die pen-duped flask id swirl to mix/dissolve. 11.17 Transfer the extracts to HPLC vials using dispossble pipets. 11.18 Analyze samples using electrosptrayLC/MS/MS. 12.0 Chromatography 12.1 Inject die same amount o f each standard, sample and fortified sample into the LC/MS/MS system. A calibration standard must precede end follow all analyzed simples. 12.2 Standards ofPFOA corresponding to i t lead five or more concentration levels must be included in an analytical set 12.3 An entire set o f calibration standards must be included at the beginning and at the end o f 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 e set followed by calibration standards interspersed every 5-10 ample (to account for a second set of standards). In either ease, calibration standards must be the first and last injection in u sample set. 12.4 Use linear standard curves for quantitation. Linear standard curves are generated for the analyte by linear regression using l/x weighting of peak area versus calibration standard concentration using MassLynx 3.3 (or equivalent) software system. P igeofl Page 42 o f 65 Exygen Research Page 79 o f 121 Interim Report #9-Analysis o f Ground Water Samples Exygen Study No.: P0001131 E x ygen P ro to co l N um ber: P 0 0 0 1 131 ExygmlcMWcb Mated NwnbwV00017S3 [ ANALYTICAL m e t h o d J Method o f Analyiis for the Determination o f Perfluorooctanoic Acid (PFOA) in Fish and Clams by LC/MS/MS 12.5 Sample response should not exceed standard responses. Any samples that exceed standard responses should be Anther diluted and reanalyzed. 13.0 Acceptance Criteria 13.1 Chromatogram must show a peak o f a daughter ion at 369 amu from a parent of 413 amu. The 413 amu parent corresponds to the PFOA anion, while the daughter too (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 PFOA at levels paater than 0.5 ppb, then a new blank sample must be obtained and the entire set must be ro>extracted. 13.3 Recoveries o f control spikes and matrix spikes must be between 70-130% of their known values. If a control spike falls outside the acceptable limits, the entire set of samples should be re-extracted. 13.4 Any calibration standard found to be a statistical outlier by using the Huge Error Test, may be excluded from the calculation o f the calibration curve. However, the total number of calibration standards that could be excluded must not exceed 20% o f the total number o f standards injected. 13.5 The correlation coefficient (R.) for calibration curves generated must be 0.992 (R2 (L9S5). 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 mutt not drift more than i 4 % within an analytical run. If retention time drift exceeds this limit within an analytical run foot the set must be reanalyzed. 14.0 Calculations 14.1 Use the following equation to calculate the amount of PFOA found (in ng/mL based on peak area) using foe standard curve (linear regression parameters) generated by foe Mass Lynx software program: PFOA found (ng/mL) - (Peak area - intercept) slope 14.2 Use foe following equation to convert foe amount ofPFOA found in ng/mL to ng/gfoib). PFOA found fpnbl - fPFOA found f r f te f A final volume fmL) x DPI sample weight (g) DF * factor by which foe final volume was diluted, if necessary. P at*7 ofS Page 43 o f 65 Exygen Research Page 80 o f 121 Interim Report #9-Analysis of Ground Water Samples Exygen Study No. : P0001131 Exygen Protocol Number: P0001131 cExygee lUseeidi ANALYTIC.U , METHOD Mtthod Number V0001783 Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Fish and Clams by LC/MS/MS 14.3 For simples fortified with known amounts o f PFOA prior u> extraction, use foe following equation to calculate the percent recovery. Recovery (% ) [total analytefound W g ) snalyte found in control (ng/g)] ulpQ analyte added (ng/g) Exygen Research Pages ofs Page 44 o f 65 Page 81 o f 121 Interim Report #9-Analysis of Ground Water Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 ANALYTICAL METHOD Method Number V0001784 Method of Analysis for the Determination of Perflnorooctanolc Add (PFOA) in Vegetation by LC/MS/MS Analytical Testing Fecility: Exygen Research 3058 Research Drive State College, PA 16801 Approved By: T tA C JiL Paul Connolly Technical Leader, LC-MS, Exygen Research n / m f i U / __________ >John Flaherty ^ Vice President, Operations, Exygen Research ___ lqjm M Date Dat Exygen Research Total Pages: 7 Page 45 o f65 Page 82 o f 121 Interim Report #9-Analysis of Ground Water Samples Exygen Study No. : P0001131 Exygen Protocol Number; P0001131 BxyfMRmmicIi Method Number VQ00I7S4 ANALYTICAL METHOD Method o f Analysis for the Determination ofPerfluorooctinoic Acid (PFOA) in Vegetation by LC/MS/MS 1.0 Scope This method is to be employed for the isolation end quantitation o f perfluorooctenoic add by High Performance Liquid Chromatography coupled to a tandem Mass Spectrometric Detector (LC/MS/MS) in vegetation. 2.0 Safety 2.1 Always obeerve 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 temple for extraction. 3.2 Samples should be processed before extraction. Place the frozen sample in e food processor end homogenize with dry ice. Place the samples in containers and leave open in frozen storage overnight to allow for carbon dioxide sublimation. Seal and place the samples in frozen storage until time of analysis. 3.3 Sample collection procedures will be specified in the sampling plan for this project 4.0 Reagents end Standards 4.1 Water-HPLC grade 42 Acetonitrile-HPLC grade 4.3 Carbon (120-400 mesh) - Reagent grade 4.4 Methanol - HPLC grade 4.5 Silica gel (60-200 m esh)- Reagent grade 4.6 Florisit (60-100 mesh) - Reagent grade 4.7 Superclean LC-NHj - Reagent grade 4.8 1-Octanol-HPLC grade 4.9 L-Aecorbic add-R eagent grade 4.10 Dimethyldichlorosilane - Reagent grade 4.11 Toluene-Reagent grade 4.12 Ammonium Acetate - A.C.S. Reagent Grade 4.13 Perftuorooctaaoic Acid - Sigma-Aldrich 3.0 Instrument and Equipment 3.1 A high performance liquid chromatograph capable o f pumping up to 2 solvents equipped with a variable volume injector capable o f injecting 3-200 pL contacted to a indent Mess Spectrometer (LC/MS/MS). 3-2 A device to collect raw data for peak integration and quantitation. 5.3 Analytical balance capable o f reading to 0.00001 g. Page2ol7 Page 46 of65 Exygen Research Page 83 o f 121 Interim Report #9-Analysis of Ground Water Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 Exyga Research MmIkmSN uota V00017M ANALYTICAL METHOD Method o f Analysis for tbe Detennination ofPerfluorooclsnoic Acid (PFA) in Vegetation by LC/MS/MS 5.4 Rotary evapontor. 5.5 125 mL pear-shaped flasks. 5.6 SOmL disposable polypropylene centrifUge tubes. 5.7 15 mL disposable polypropylene centriibge tubes. 5.8 Disposable mkropipeta (50-1OOuL, 100>200uL). 5.9 125-mL LDPE narrow-mouth bottles. 5.10 2 mL clear HPLC vial kit. 5.11 Diipoaable pipettes. 5.12 Autopipettes (100-1000 pL and 10-100 pL), with disposable tips. 5.13 SPB tubes (20mL) (Supdco cat no. N057177). 5.14 Wrist action shaker. 5.15 Ceotriftige capable o f spinning 50 mL polypropylene tubes at 2000 rpm. 6.0 Chromatographic System 6.1 Analytical Column: FhiophaseRP (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 Acetste in Water Mobile Phase (B): Methanol Gradient Program: Tim ftnint 0.0 1.0 8.0 20.0 22.5 SiA 65 65 25 25 65 Flow Rate S ifi fmL/min) 35 0.3 35 0.3 75 0.3 75 0.3 35 0.3 6.6 Iiyectioo Volume: 15 pL (can be increased to as much as 50 pL). 6.7 Quantitation: Peak Aren-external standard calibration curve. 6.8 R unTim e:- 2 3 minutes. The above are intended u a guide and may be changed in order to optimize the HPLC system. 7.0 MS/MS System 7.1 Mode: ElectroaprayNegativeMRMmod6l n>omtoring4l3-369TiVzfor PFOA. Page 3of? Page 47 o f65 Exygen Research Page 84 o f 121 Interim Report #9-Analysis of Ground Water Samples Exygen Study No.: P0001131 Exygen Protocol Number: POOOU31 Exygen lUsMieb Method Number V00017S4 ANALYTICAL METHOD Method o f Analysis for the Determination o f Perfiuorooctanotc Acid (PFOA) in Vegetation byLC/MS/MS The above conditiona are intended as a guide and may be changed in order to optimize die MSMS system. 8.0 Preparation o f Solutions 8.1 Mobile Phase 8.1.1 2 mM ammonium sedate in water ia prepared by adding 0.154 g of ammonium acetate to 1000 mL o f water. 8.2 Extraction Solutions 8.2.1 8.2.2 2% ascorbic add in methanol ia prepared by diasolving 2 g o f ascorbic ad d in 100 a L o f mathanol. 30% Dimathytdichlofodlane hi toluene ia prepared by bringing 3 mL o f dimcthyldkhlorosilane to a final volume o f 10 mL with toluene. Alternate volumes may be prepared. 9.0 Standard Preparation 9.1 Standard StocttFortification Solution 9.1.1 Prepare a stock solution of~10 0 y ^m L o fP F O A by weighing 10 mg o f analytical standard (corrected for purity) and dilute to 100 mL with mediano] in a 125-mL LDPE bottle. 9.1.2 A 1.0 yg/mL fortification solution o f PFOA is prepared by bringing 1 mL of die 100 ygfaiL solution to a final volume o f 100 with methanol in a 125 mL LDPE bottle. 9.1.3 A0.1 yg/mL fortification solution ofPFOA is prepared by bringing 10 mL o f the 1.0 yg/mL solution to a final volume o f 100 with methanol in a 125 mL LDPE bottle. 9.1.4 A 0.01 yg/mL fortification soluticm of PFOA is prepared by bringing 10 mL o f the 0.1 yg/mL solution to final volume of 100 with methanol io a 125 mL LDPE bottle. 9.1.5 The stock and fortification solutions are to be stored in s refrigerator si approximately 4*C sod are stable for a maximum period o f 6 months from the date o f preparation. 9.2 Standard Calibratimi Solutions 9.2.1 LC/MS/MS calibratimi standards are prepared in methanol via dilution o f die 1.0 yg/mL fortification solution. Page 4 ul ' Page 48 o f 65 Exygen Research Page 85 o f 121 Interim Report #9-Analysis of Ground Water Samples Exygen Study No. : P0001131 Exygen P ro to co l N um ber: P 0 0 0 1131 Exygac lUicuch Method Number VOOOI784 ASIYTICALMETHOD | Method o f Analysis forth Determination o f Pcrfluorooctanoic Acid (PFOA) in Vegetation byLC/MS/MS 92.2 The following ii a typical example: additional concentrations may be prepared as needed. Concentration Final of Fortification Volume Diluted to Concentration Solution fuafaiL) (mL) (mL) (MfifolL) 1.0 5.0 100 1.0 2.5 100 0.05 0.025 1.0 0.05 0.025 1.0 10 10 100 100 100 0.01 0.005 0.0025 0.1 0.005 10 10 100 100 0.001 0.0005 9.2.3 Store all calibratioa standards in 125-mL LDPE narrow-mouth bottles at 2*C to 6*C, up to six months. 9.2.4 Alternate volumes and concentrations o f standards may be prepared as needed. 10.0 Batch Set Up 10.1 Bach batch o f samples extracted (typically 20 or less) must include at least one untreated control and two untreated controls fortified at known concentrations (lab control spike) to verify procedural recovery for the batch. 10.2 Requirements fin field and laboratory duplicates and spikes will be specified in the quality assurance plan for this project. 11.0 Sample Extraction 11.1 Weigh 5 g o f frozen sample into 50 mL polypropylene centrifuge tubes (fortify as needed, replace lid end mix well). 11.2 Add 30 mL of acetonitrile and shake on a wrist action shaker for - l 5 minutes. 11.3 Centrifoge the 50 mL polypropylene tubes containing sample at -2000 rpm for-10 minutes. 11.4 Pack and condition the SPB tu b a and silanize the pear-shaped flasks. 11.5 Pack foe 20 mL SPE tu b a in sequence with 2 g florisil. 2 g silica get. 2 g carbon, and 1 g LC-NHi. Condition the columns with 20 mL o f methanol, than 20 mL o f acetonitrile. Discard all washes. Do not allow the column to dry. 11.6 Silanize foe 125 mL pear-shaped flasks by muting with the 30% dimefoyldichloroailaae in toluene solution. Rinse the fla k with toluene once, followed by methanol (fo ra tim a). Dry foe flasks completely before use. either by air-drying or with a stream o f nitrogen. 11.7 Decant foe extract on to a conditioned SPE column fitted inside the mouth of tiie pear-shaped flask. Collect foe eluate in foe 125 mL silanized pear-shape flask. Page 5of' Page 49 o f 65 Exygen Research Page 86 of 121 Interim Report #9-Analysis of Ground Water Samples Exygen Study No.: P0001131 Exygen Protocol Number: PO001131 Exygen Rasench Mxthod Number V000I7M | A N A LYTICAL M S T H O P ~| Method o f Analysis for the Determination o f Perfiuorooctanoic A dd (PFOA) in Vegetation by LC/MS/MS 11.8 Add 20 mL of acetonitrile to the sample in the 50 mL centrifuge tube. 11.9 Shake die temple again for 10 minutes on a wrist-action shako'. 11.10 CcstrifUge the SO mL polypropylene tubes containing sample at 2000 rpm for 5 minutes. 11.11 Deeant the extract onto the same SPE column. Collect the eluste into the same pear-ahaped flask and combine with the eluent from the initial extraction. 11.12 Repeat steps 11.8 through 11.11 again. 11.13 Add 3-4 drops o f 1-octanol to die extract in the pear-ahaped flask and evaporate at reduced pressure using a rotary evaporator (at < 40C). 11.14 Maim the final volume by adding 2 mL o f 2% ascorbic acid in methanol to the pear-shaped flask and swirl to mix/diseolve. 11.15 Transfer the extracts to HPLC vials using disposable pipeti. 11.16 Analyze simples using electrospray LC/MS/MS. 12.0 Chromatography 12.1 Inject die same amount of each standard sample and fortified sample into the LC/MS/MS system. A calibration standard must precede and follow all analyzed samples. 12.2 Standards o f PFOA corresponding to at least five or more concentration levels must be included in an analytical set. 12.3 An entire set o f extracted calibration standards must be included at the beginning and at the end o f a sample set. Extracted standards must be interspersed between every 5-10 sampler. As an alternative, an entire set of extracted calibration standards may be injected at the beginning of 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 die first and last injection in a sample set. 12.4 Use Hnear standard curves for quantitation. Linear standard curves are generated for the analyte by linear regression using 1/x weighting of peek area versus calibration standard concentration using MaaaLynx 3.3 (or equivalent) software system. 12.5 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 a peak o f a daughter ion at 369 smu from a parent o f 413 amu. The 413 smu parent corresponds to the PFOA anion, while the daughter urn (369 amu) represents the loss o f carbon dioxide. Hge6or? Page 50 o f 65 Exygen Research Page 87 o f 121 Interim Report #9-Analysis of Ground Water Samples Exygen Study No.: P0001131 Exygen Protocol Number: P000113I ExygmUMtarch Method N uocer VOOOI7M ANALYTICAL m e t h o d Method o f Analyst! for the Determination o f Perfluorooctanoic Acid (PFOA) in Vegetation by LC/MS/MS 13.2 Method blanks must not contain PFOA at levels greater than the LOQ. If a blank contains PFOA at levels greater thin 0.3 ppb, then a new blank sample must be obtained and the entire set must be re-extracted. 13.3 Recoveries o f control spikes and matrix spikes must be between 70-130% of their known values. If a control spike falls outside the acceptable limits, the entire set o f samples should be re s tric te d . 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 20H o f die total number o f standards injected. 13.5 The correlation coefficient (R) for calibration curves generated must be 20.992 (R* 0.985), If calibration results foil outside these limits, then appropriate steps must be taken to adjust instrument operation, and the standards or die relevant set o f samptoe should be reanalyzed. 13.6 Retention times between standards and samples must not drift more than i 4 % within an analytical run. If retention time drift exceeds this limit within an analytical run then the set must be reanalyzed. 14.0 Calculations 14.1 Use die following equation to calculate the amount o f PFOA found fin ng/mL, based on peak area) using the standard curve (linear regression parameters) generated by the Mass Lynx software program; PFOA found (ng/mL) * fPcak area - intercept) slope 142 Use the following equation to convert the amount o f PFOA found in ng/mL to ng/g(ppb). PFOA found faobl - fPFOA found /ng/mL) * final volume fmLl x DF1 sample weight (g) DP - factor by which die final volume was diluted, if necessary. 14J For samples fortified with knows amounts o f PFOA prior to extraction, use the following equation to calculate foe percent recovery. Recovery (%) (total analyte found (ng/g) analyte found in control (ng/g)] n ^ analyteadded (ng/g) Page ? ui 7 Page 51 o f 65 Exygen Research Page 88 o f 121 Interim Report #9-Analysis o f Ground Water Samples Exygen Study No.: P0001131 Exygen Protocol Nuiriber: P0001131 ANALYTICAL METHOD Method Number. V0001785 Method of Analysis for the Dstermlaatien of Perfinarooctaaote Add (PFOA) I Small Mammal Liver by LG/MS/MS Analytical Teating Facility: Exygen Reaearch 3058 Research Drive State College, PA 16801 Approved By: C-JlL ____ Paul Connolly I Technical Leader, LC-MS, Exygen Research Due 4L John Flaherty / Vice President, Operations, Exygen Research Date Exygen Research Total Pages: 7 Page 52 o f 65 Page 89 o f 121 Interim Report #9-Analysis o f Ground Water Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 Exy fra Research MvUwd Number V00017S I ....... ANALYTICAL METHOD Method o f Analysis fbr (be Detenninstioa o f Perfluorooctanoic Acid (PFOA) in Smell .Marame] Liver by LC/MS/MS 1.0 Scope 11111method U to be employed fbr the isolation end quantitation o f perfluorooctanoic acid by High Performance Liquid Chromatography coupled to a tandem Mass Spectroraetric Detector (LC/MS/MS) in small mammal liver. 3.0 Safety 3.1 Always observe safe laboratory practices. 3.2 Consult die appropriate MSDS before handling any chemical for proper safety precautions. 3.0 Sample Requirement 3.1 At least Sg o f teat sample for extraction. 3.2 Samples should be processed before extraction. Place the frozen sample in a food process snd 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. Alternately, if there is an insufficient amount of 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 aid Standards 4.1 Water-HPLC grade 4.2 Methanol - HPLC grade 4.3 Acetonitrile - HPLC grade 4.4 Ammonium Acetate - AC.S. Reagent Grade 4.5 Perfluorooctanoic Acid - Sigma-Aldrich 5.0 Instrument and Equipment 5.1 A Ugh performance liquid chromatograph capable o f pumping up to 3 solvents equipped with a variable volume injector capable o f injecting 5-200 pL connected to a tandem Mass Spectrometer (LC/MS/MS). 52 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 centriAige tubes. 5.5 15 mL disposable polypropylene centriAige tubes. 5.6 Disposable micropipets (50-lOOuL, 100>200uL). 5.7 125-mL LDPE narrow-mouth bottles. 5.8 2 mL clear HPLC vial kit. Page 2 of? Page 53 o f65 Exygen Research Page 90 o f 121 Interim Report #9-Analysis o f Ground Water Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 BnyyaKwcMdi MsftodNuabor VOOOI7I5 | ANALYTICAL METHOD Method of Analysis fix:the Determination ofPerfluorooctanoic Acid (PFOA) in Small Mammal Liver by LC/MS/MS I 5.9 Diapoeable pipettes. 5.10 Autopipettee (100*1000 pL and 10*100 pL), with disposable tipi. 5.11 Water* Sep Pak Vac 6 e c (lg ) tCISSPE cartridge*. 5.12 SPE vacuum manifold. 5.13 Tiasuemiser. 5.14 Wrist-action ahaker. 5.15 Centriftige capable of apinning 15 mL polypropylene tubea at 3000 rpm. 6.0 Chromatographic Syetean 6.1 Analytical Coburn; Fluophaie RP (Keystone Scientific), 2.1 mm x 50 mm. 5p (P/N: 82505*052130) 62 Temperature: 30*C 6.3 Mobile Phase (A ): 2 raM Ammonium Acetate in Water Mobile Phase (B): Methanol Gradient Program: Tima (mint 0.0 1.0 8.0 20.0 22.5 %A 65 65 25 25 65 Flow Rale B fmL/minl 35 03 35 0.3 75 0.3 75 0.3 35 0.3 6.6 Injection Volume: 15 pL (can be increased to aa much as 50 pL). 6.7 Quantitation: Peak Area - external standard calibration curve. 6.8 RunTime: - 2 3 minutes. The above conditions are intended as a guide and may be changed in order to optimize the HPLC system. 7.0 MS/MS System 7.1 Mode: Electrospray Negative MRM mode, monitoring 413 - 369 m/z for PFOA. The above rewdfrymf are intended as a guide and may be changed in order to optimize the MSMS system. P a p i of? Page 54 o f 65 Exygen Research Page 91 o f 121 Interim Report #9-Analysis of Ground Water Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 Exyfta RMMfch Method Number VOOOl' 85 I AN/avTicAL M rm o p Method o f Analysis far the Determination o f Perfluorooctanoic Acid (PFOA) in Small Mammal Liver by LC/MS/MS 8.0 Preparation o f Solutions 8.1 Mobil* Phase 8.1.1 2 mM ammonium acetate in water is prepared by adding 0 154 g of ammonium acetate to 1000 mL o f water. Alternate volumes may be prepared. 9.0 Standard Preparation 9.1 Standard Stoclc/Foctification Solatimi 9.1.1 Pnpareastoclcsolutionof'-IOOjig/mLofPFOAby 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 yg/mL fortification solution of PFOA is prepared by bringing I mL o f the 100 pg/mL solution to a final volume of 100 with methanol in a 125 mL LDPE bottle. 9.1.3 A 0.1 pg/mL fortification solution o f PFOA is prepared by bringing 10 mLofthe 1.0 pg/mL solution to a final volume o f 100 with methanol in a 125 mL LDPE bottle. 9.1.4 Tbe stock and fortification solutions are to be stored in a refrigerator at approximately 4C and are stable for a maximum period of 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 methanol via dilution o f the 0.1 pg/mL fortification solution. The following is a typical example: additional concentrations may be prepend as needed. Concentration of Fortification Solution fni/tnL) 100 100 100 5.0 2.0 IX) Volume (m 5.0 2.0 1.0 to 10 10 Diluted to (mL) too to o 100 to o 100 100 Final Concentration (na/mL) 5.0 2.0 1.0 0.5 OJ 0.1 at 2*C to 6*C, up to six months. 9.2.4 Alternate volumes and concentrations o f standards may be prepared as Pag*4 o f7 Page 55 o f 65 Exygen Research Page 92 o f 121 Interim Report #9-Analysis of Ground Water Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 ExyieaRMHich Mnbod Numbet V0001?S I ANALYTICAL METHOD Method o f Aiulyeit for the Dotermlnitioa o f PerlhioioocUnoic Acid (PFOA) in Smeli Mm unti Liver by LC/MS/MS 10.0 Batch SetUp 10.1 Each batch o f samples extracted (typically 20 or less) must include at least one untreated control and two untreated contrala fortified at known concentrations (lab oontral spike) to verify procedural recovery for the batch. 10.2 Requirements for field and laboratory duplicates and spikes will be specified in the qualify assurance plan for this project. 11.0 Sample Extraction 1l.l Weigh 1 1 o f sample into a 50 mL polypropylene centrifuge tubes (fortify as needed, replace lid and mix well). Note that alternate weights of liver may be measured depending on the sample size available for use. 11.2 Add water to the sample for a final volume o f 10 mL. 11.3 Homogenize sample using atiasuemixer for -1 minute. 11.4 Transfer 1 mL o f the sample using a disposable pipette into a IS mL disposable centriftige tube. 11.5 Add 5 mL o f acetonitrile and shake for 20 minutes on a wriil-action shaker. 11.6 Centrifuge the tubes at 3000 rpm for ~S minutes. 11.7 Decant die supernatant into a 30 mL disposable centriftige cube and add 35 mL o f water. 11.8 Condition the C u SPE cartridges (1 g, 6 mL) by passing 10 mL methanol followed by S mL o f HPLC water (~ 2 drop/sec). Do not let column run dry 11.9 Load the sample on conditioned Cu SPE cartridge. Discard eluate. 11.tO Elute wife 2 mL of methanoL Collect 2 mL o f eluate into a graduated IS mL polypropylene centriftige tube (final volume 2 mL). 11.11 Analyze samples using electroepray LC/MS/MS, 12.0 Chromatography 12.1 Iryect the same m ount o f each standard, sample and fortified sample into the LC/MS/MS system. A calibration standard must precede and follow all analyzed samples. 12.2 Standards o f PFOA corresponding to at least five or more concentration levels must be included in an analytical set. 12.3 An attire set o f calibration standards must be included at the beginning snd at the end o f a sample set. Standards must be interspersed between every 5*1V samples. As an alternative^ an entire set o f calibration standards may he iiyected at the beginning o f a set followed by calibration standards interspersed every S>10 samples (to account for a second set of standards). In either case, calibration standards must be the first and last injection in a sample set. 12.4 Use linear standard curves for quantitation. Linear standard curves are generated for the analyte by linear regression using 1/x weighting o fpeak area PtgB9of7 Page 56 o f 65 Exygen Research Page 93 o f 121 M erini Report #9-Analysis o f Ground Water Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 ExygacRsm rch M#iiod Number V0Q017S3 | ANALYTICAL METHOD Method o f Analysis for the Determiutioa o f Perfluocoootanoic Acid (PFOA) in Small Mammal Liver by LC/MS/MS venue cattwation standard concentration using MaseLynx 3.3 (or equivalent) software system. 12.5 Sample response should not exceed standard responses. Any samples that exceed standard responses should be Anther diluted and reanalyzed. 13.0 Acceptance Criteria 13.1 Chromatogram must show a peak of 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 foe 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 foe entire set must be re-extraded. 13.3 Recoveries o f control spikes and matrix spikes must be between 70-130% of their known values. If a control spike foils outside the acceptable limits, the entire set o f samples should be re-extracted. Any matrix spike outside 70 130% should be evaluated by foe analyst to determine if re-extraction is warranted. 13.4 Any calibration standard found to be a statistical outlier by using foe Huge Error Test, may be excluded A on foe calculation o f foe calibration curve However, foe total number o f calibration standards that could be excluded must not exoeed 20% o f foe total number o f standards injected. 13.3 The correlation coefficient (R) for calibration curves generated must be 20.992 (R* 20.985). If calibration results foil outside these limits, then appropriate steps mum be taken to adjust instrument operation, and the standards or the relevant set o f samples should be reanalyzed. 13.6 Retention times between standards and samples must not drift more than 4 % within an analytical tun. If 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 lo calculate the amount o f PFOA found (in ng/mL. based on peak area) using foe standard curve (linear regression parameters) generated by foe Mass Lynx software program: PFOA found (ng/mL) slope x OF x aliquot factor DF footerby which foe Anal volume was diluted, if necessary. Aliquot factor* 10 Ptje 6 of 7 Page 57 o f 65 Exygen Research Page 94 o f 121 Interim Report #9-Analysis o f Ground Water Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 ExygwILMMRh Method Number V00017IJ | ANALYTICAL METHOD Method o fAnalysis for the Determination o f Periluorooctanolc Acid (PFOA) in Small Mammal Liver by LC/MS/MS 14.2 For eamplei fortified with known amount o f PFOA prior to extraction, use the following equation to calculate the percent recovery. Recovery (%) - [ total analyte found (ngifrnL) - analytefound in control (ngftnL)] ^ analyteadded (ng/mL) 14.3 Use the following equation to convert the amount o f PFOA found in ng/m L 10 ngfg(ppb). PFOA found fppbl - rPFOA found fne/mLl x final volume fmUl sample weight <g) Exygen Research Page 7 o f7 Page 18 o f 6S Page 95 o f 121 Interim Report #9-Analysis of Ground Water Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 ANALYTICAL METHOD Method Number V0001786 Method of Analysis for the Determlsidoo of Psfltuorooctanoic A dd (PFOA) to Small Mammal Serum by LC/MS/MS Analytical Testing Facility: Exygen Research 3098 Research Drive State College, PA 16801 Approved By: C -iL , Paul Connolly I Technical Leader, LC-MS, Exygen Rotatici Date __________ John Flaherty / Vice President, Operations, Bxygen Research Date Exygen Research Total Paget: 7 Page 59 o f 65 Page 96 o f 121 Interim Report #9-Analysis o f Ground Water Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 ExytonlUMiieb MttbodNunbtr VOOOnSe ANAL'iTlOU. METHOD Method o f Analysis for the Determination o f Perfluoroocttnoic Acid (PFOA) in Small Mammal Senno by LC/MS/MS 1.0 Scope This method it to be employed for the isolation and quantitation o f perfluorooctanoic acid by High Performance Liquid Chromatography coupled to a tandem Mass Spectrotnctric Detector (LC/MS/MS) in email mammal aerum. 2.0 Safety 2.1 Always observe safe laboratory practices. 2.2 Cooeult the appropriate MSDS before handling any chemical for proper safety precautions. 3.0 Simple Requirement 3.1 At least 1 mL o f teat sample for extraction. 3.2 No single processing is needed for aerum 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 Mdthanol - HPLC grade 4.3 Acetonitrile HPLC grade 4.4 Ammonium Acetate - A.C-S. Reagent Grade 4.5 Perfluorooctanoic Acid - Sigma-Aldrich 5.0 Instrument and Equipment 5.1 A high performance liquid chromatograph capable o f pumping up to 2 solvents equipped with a variable volume iqjector capable o f injecting 5-200 jiL connected to a tandem Mass Spectrometer (LC/MS/MS). 5.2 A device to collect raw data for peak integration and quantitation. 5.3 Analytical balance capable o f reading to 0.00001 g. 5.4 50 mL disposable polypropylene centrifoge tubes. 5.5 15 mL disposable polypropylene centrifoge tubes. 5.6 Disposable micropipets (50-lOOuL, 100-200uL). 5.7 125-raL LDPE narrow-mouth bottles. 5.8 2 mL clear HPLC vial Idt. 5.9 DUpotable pipettes. 5.10 Autopipettes (100-1000 pLand 10-100 pL), with disposable tips. 5.11 Waters Sep Pak Vac 6 cc <lg) IC18 SPE cartridges. 5.12 SPE vacuum manifold. 5.13 Vortexcr. Page 2 o P Page 60 o f 65 Exygen Research Page 97 o f 121 Interim Report #9-Analysis of Ground Water Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 ExygeaReaMich Method Number V00U17K6 I A )W -Y T1CA L M ETH O D Method o f Analysis far the Detenninatioo o f Perfluorooctanoic Acid (PFOA) in Smell Mammal Serum by LCMS/MS 5.14 Wrist-action shaker. 5.15 Centrifuge capable o f spinning 15 raL polypropylene tubes at 3000 rpm. 6.0 Chromatographic System 6.1 Analytical Column: Pluopbaae 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 Aoetate in Water 6.4 Mobile Phase (B ): Methanol 6.5 Gradient Program: Time (mini 0.0 1.0 8.0 20.0 22.5 65 65 25 25 65 Flow Rate 5k (mL/minl 35 0.3 35 0.3 75 0.3 75 0.3 35 0.3 6.6 liyeetion Volume: 15 jtL (can be increaaed to as much as 50 pL). 6.7 Quantitation: Peak Area - external atandard calibration curve. 6.8 RunTime: - 2 3 minutes. . The above conditions are tended as a guide and may be changed in order to optimize the HPLC system. 7.0 MS/MS System 7.1 Mode: Electrospxay Negative MRM mode, monitoring 413 --369 m/z for PFOA. The above conditions are intended is a guide and may ba changed in order to optimize the MSMS system. 8.0 Preparati o f Solutions 8.1 Mobile Phase 8.1.1 2 mM ammonium acetate in water is prepared by adding 0.154 g of ammonium acetate to 1000 mL o f water. Alternate volumes may be prepared. Pag* 3 of 7 Page 6J o f 65 Exygen Research Page 98 o f 121 Interim Report #9-Analysis of Ground Water Samples Exygen Study No.: P0001131 Exygen Protocol Number: POOOl 131 Exyyc gassarci! Malted Number VOQOPU | ANALYTICAL METHOD Mettimi o f Analysis for the Determination ofPerfluorooctanoic Acid (PFOA) in Small Mammal Serum by LC/MS/MS | 9.0 Standard Preparation 9.1 Standard Stock/Fortificadon Solution 9.1.t Prepare a stock solution o f -100 pg/mLofPFOA by weighing 10 mg o f analytical ataadard (corrected for purity) and dilute to 100 mL with methanol in a 125-raL LDPE bottle. 9.1.2 A 1.0 pg/mL fortificatici eolution o f PFOA is prepared by bringing I mL o f the 100 pgfaL solution to a final volume of 100 with methanol in a 123 mL LDPE bottle. 9.1.3 A0.1 pg/mL fortification solution o f PFOA is prepared by bringing 10 mL o f tbe 1.0 p^m L eolution to a final volume o f 100 with methanol in a 125 mL LDPE bottle. 9.1.4 Tbe stock and fortification eohitiana 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 fpreparation. 9.2 Standard Calibratimi 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 ofFortification Volume Solution fai/mL) (mL) Diluted to (mL) Final Concentration (na/mL) 100 5.0 100 2.0 100 1.0 5.0 10 2.0 10 1.0 10 100 100 100 100 100 100 5.0 2.0 1.0 0.5 0.2 0.1 9.2.3 Store all calibration standards in 125-mL LDPE narrow-mouth bodies at 2*C to 6*C, up to six months. 9.2.4 Alternate volumes and concentrations o f standards may be prepared as needed. 10.0 Batch Set Up 10.1 Each batch o f camples extracted (typically 20 or leas) must include at least one untreated control Mid two untreated controls fortified at known concentrations (leb 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. Peje 4 of? Page 62 o f 65 Exygen Research Page 99 o f 121 Interim Report #9-Analysis o f Ground Water Samples Exygen Study No. : P0001131 Exygen Protocol Number: P0001131 Exygm lluNicb Method Number VOOOI7Sb 1 ........... .... ANALYTICAL METHOD Method o fAnalysis forthe Determination o f Perfluorooctonoie Acid (PFOA) in Small Mammal Serum by LC/MS/MS 11.0 Sample Extraction 11.1 Measure 1mL o f sample into a 50 mL polypropylene centrifuge tubes (fortify as needed, replace lid and mix well). Note that alternate volumes o f serum may be measured depending on tire sample size available for use. 11.2 Add water to the sample for a final volume o f 20 m L Cap tightly 11.3 Vortex for-1 minute. 11.4 Transfer 1 mL 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 minutes on s wrist-action shaker. 11.6 Centrifuge the tubes at -3000 rpm for - 5 minutes. 11.7 Decant the supernatant into a 50 mL disposable centrifbge tube and add 35 mL o f water. 11.8 Condition the Cis SPE cartridges (1 g. 6 mL) by passing 10 mL methanol followed by 5 mL ofHPLC water (~ 2 drop/sec). Do not let column run dry 11.9 Load tbe sample on conditioned C u SPE cartridge. Discard eluate. 11.10 Elute with - 2 mL o f methanol. Collect 2 mL o f eluate into a graduated 15 mL polypropylene centrifbge tube (final volume 2 mL). 11.11 Analyze samples using electrocpray LC/MS/MS. 12.0 Chromatography 12.1 Inject tbe same amount o f each standard, sample and fortified sample into the LC/MS/MS system. A calibration standard must precede and follow all analyzed samples. 12.2 Standards o f PFOA corresponding to at least five or more concentration levels must be included in on analytical set. 12.3 An entire set o f calibration standards must be included at the beginning and at the end of a sample set Standards must be interspersed between every 5-1<> samples. As an alternative, an entire set o f calibration standards may be injected at the beginning o f a set followed by calibration standards interspersed every 5-10 samples (to account for a second set o f standards), in either ease, calibration standads 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 reqmnae should not exceed standard responses. Any samples that exceed standard responses should be ftirtber diluted and reanalyzed. P ip ) uf? Page 63 of 65 Exygen Research Page 100 o f 121 Interim Report #9-Analysis of Ground Water Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 Exygen Ram rch Method Number VOOO1786 | ..................ANALYTICALMKTHOD Method o f Analysis for the Determination ofPerfluorooctanoic Acid (PFOA) in Small Mammal Serum by LC/MS/MS 13.0 Acceptance Ciiteria 13.1 Chromatogram must ahow a peak of a daughter ion at 369 amu from a parent o f 413 amu. The 413 amu parent corresponds to the PFOA anion, while Uw daughter km (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 of control spikes and matrix spikes must be between 70-130% of their known values. If a control spike falls outside the acceptable limits, the entire set o f --mpt-- 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 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 (Ra 20.985). If calibration results foil outside these limits, then appropriate scape must be taken to adjust instrument operation, and the standards or foe relevant sec of samples should be reanalyzed. 13.6 Retention times between standards and samples must not drift more than 1 4 % within in analytical run. If retention time drift exceeds this limit within an analytical run then the set must be reanalyzed. 14.0 Calculations 14.1 Use the following equation to calculate the amount of PFOA found (in ng/mL. based on peak area) using the standard curve (linear regression parameters) generated by the Mass Lynx software program: PFOA found (ng/mL) * (Peak area - intercept) x DF x aliquot factor slope DF fhetor by which foe final volume was diluted, if necessary. Aliquot factor 2 0 14.2 For samples fortified with known amounts o f PFOA prior to extraction, use the following equation to calculate foe percent recovery. Recovery (% )- [ total snalytefound (ng/mL) - analyte found in control (ng/mL)] ]|0Q analyte added (ng/mL) . Pag6 o f7 Page 64 o f 65 Exygen Research Page 101 o f 121 Interim Report #9-Analysis of Ground Water Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 Bxy|a ItMMfch Method Number VOOO1786 1 ...... ANa L YTICAL METHOD ...... ......... Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Small Mammal Seium by LC7MS/MS 14.3 Use the following equation to convert the noun! of PFOA found in ng/m L to ppb. PFOA found (ppb) rPFOA found (noimLl fine! volume (m il) sample volume (mL) Exygen Research P*|e 7 of 7 Page 65 o f 65 Page 102 o f 121 Interim Report #9-Analysis of 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 D ESC R IPTIO N OF AM ENDED SEC TIO N 1) Analytical Procedure Summary V0001780:Sectton 9.1 2) Verification o f Analytical Procedure None AMENDED TO 1) Add to Section 9.1 : Section 9.1 .6, Alternate weights of standards m ay 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 m atrix m ay be altered depending on sam ple 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 sam ple size available for small mammal liver and serum was sm aller than expected. Spiking a t the pre-determ ined levels in the protocol puts the spiked concentration lower than the detection limit. Also, the analyte levels in the ground w ater samples are expected to greatly exceed the pre-determ ined spiking levels listed in the protocol. W hen the levels in the samples greatly exceed the spiking levels, an accurate recovery value cannot be calculated for the Q C sample. Higher spiking levels in the bottles will cover the analyte concentrations expected In the w ater sam ples. IM PACT O N STUDY The LOQ is 100 ng/g for a 0.1 g sample o f small mammal liver and is 1000 ng/m L for a 0.01 mL sam ple of small mammal serum. Higher levels of spiking for the w ater samples will ensure that m ore Q C recovery data can be used. LIBRARY IO: W0001226-6 ` . ADMINISTRATIVE FORM Exygen Research Page 103 o f 121 Interim Report #9-Analysis of Ground Water Samples Exygen Study No.: P0001131 E RESEARCH 3058 Research D rive Phone: 814-272-1039 S tate C ollege, PA 16801 Fax: 814-231-1580 Am endm ent Number: Effective Date: Exygen Study Number PROTOCOL AMENDMENT 2 0 3 /0 7 /0 5 P 0001131 Client Study N um ber Page 1 of 1 None D E S C R IP T IO N O F A M E N D E D S E C T IO N 1) R ep ort, page 11 o f 6 5 2 ) T e s t M aterials, page 6 o f 65: P F O S transition m onitored 4 9 9 -> 9 9 . AM ENDEQ TQ 1) Instead o f one fin al report, interim reports w ill b e issued. 2 ) P F O S transition m onitored m ay also be 4 9 9 -> 80. R A TIO N A LE 1) D ue to the excessive sizes o f the data sets, interim reports w ill b e issued to allow the client to receive d a ta in a tim elier a n e r-. * * * * ' e s fa /w - W 2 ) T h e A P I 4 0 0 0 L C /M S /M S system s d etect the 4 9 9 -> 80 P F O S transition w ith g rea ter sensitivity than the 4 9 9 -> 9 9 transition. IM P A C T O N S T U D Y 1) T h e client w ill be ab le to receive and review th e d ata m ore quickly. 2 ) T h e 4 9 9 -> 8 0 transition can be detected w ith g re a te r sensitivity; th erefo re, giving b etter chrom atography. LIBRARY IO. V0Q01226-8 Exygen Research ADMINISTRATIVE FORM Page 104 o f 121 Interim Report #9-Analysis of Ground Water Samples Exygen Study No.: P0001131 (SL . JLL.ZS.ZB0S 8:S6W EXYGEN RESEARCH NO. FM P .3 n RESEARCH 3068 Research Drive Phone: 814*272*1039 State College, PA 16801 Fax: 814-231-1580 Amendment Number. Effective Date: Exygen Study Num ber PROTOCOL AMENDMENT 3 0 7 /1 8 /0 5 P1131 Client Study Number: Pag io t i NA J22 a i iR K K s E fia i r i i.M i R Verification ofAnalytical Procedure, page lO o f protocol. ^ AMENDED TO The field duplicate can be used for the laboratory spates and replicate when the primary sample volume is limited. P ffflQ N IA H f The sample size for a water sample is 200 m l- V a sample site requires re-extraction for any reason, there would not be enough of the primary sample to repeat two laboratory spikes and a replicate. The field duplicate is technically the same sample es the primary sample and therefore, can be used for laboratory spikes and replicates es needed. [M P A C TbN sttjD y No negative Impact on the study. Using the duplicate sample allows for the fo l QC of the sample site to be completed. QMal StujddyOOJ(r<tSignature . a i. l M /h k r Mnelpel ktvesttgaurp iia y w Cite Date U - j t n - *S Data 'In k\OS Date E x m en QAU R eview v/aaJtiT LIBRARY ID:V000122SS ADMINISTRATIVE FORM Exygen Research Page 105 o f 121 Interim Report #9-Analysis of Ground Water Samples Exygen Study No.: P0001131 r 11-23-2005 04:22 Fron-WESTOM SOLUTIONS - NOVi.22.2005 4:5a m EWGEN RESEPRCH T-677 P.003/003 F-T13 NU.S1N r.J3 n RESEARCH 30SS Research Drive Phoiwi 814-272-10)* State College, PA 16801 Pax: 814-231-1510 Amendment Number. Effective Date: Exygen Study Number PROTOCOL AMENDMENT 4 libare ~ P1131 Client Study Number. Pagai eM N A __ Ia i l O I ' J 1 L 5 ! ] 1( ii Anaytical Procedure Summary: VOCOl7W:'Methcd o fAnalysis torm e DehMisnation or Parfluoroooctanoio Add (PPOAl in Water by LOM S/M S' Section 11.0 tfttw method. AMENDED TO Section 11.0. Samples may I (fluted before going through the extraction procedure. W 3Q !If a 40 mL portion of sample wfl not toed onto the Ct* SPE cartridge, a predilution can be prepared and extai 1 1MB&SL9NSTVB 7 T T No negative impact on the study. More usable data may be obtained. study DMenrcMgnan bwaogatar! STBCT ExygsnMan SfrneHw SpenserConsto OfiwjutmdT Oak O s wr LIBRARY ID: V0001228-1 RECEIVED TIME NOV.23. 5:40PM ADMINISTRATIVE FORM PRINT TIME NOV.23. S:41R1 Exygen Research Page 106 o f 121 Interim Report #9-Analysis of Ground Water Samples Exygen Study No.: P0001131 `H CHEN EHSR 236 1B 651 733 1958 JULM-*4j--P2BBB03 8a s5:1i s2 FRO l:3K ENU. U S 651 778 6176 Z Z .2 0 B 4I5BPPI EXYStN RESEflRtH 11/23 '05 14:12 NO.979 03/03 . 70:96517331938 NO.914 P .3 3058 Rm areh Drive Phan 814-272-1039 a te College, PA 16801 Fax: 814-23M S B Amendment Number Effective Data: Exygen Study Number PROTOCOL AMEM3M5NT 4 lia Z O B P1131 Client Study Number; Fag*1 oM NA ba^RIgOOMJF ^DEDifcTIQfl Analytical Procedure Summary: VQ09t7Sfr*Merhod erfAnalysis for Die M am ttw Son of Parfluoroooctanoic Acid (PFOA) in Waler by IC?MS/M8r Section 110 ofthe method. A rib v D E o T ft " 8eo8on11.0. Bamptesmeybe dflutsdbeforegoingthroughme oxtracCon procedure. m am----------------------- ----------------------- ITa 4 0 m L portion or sample will not load onto the C u S P E c e r M e e , a p re-d lu tio n can b e prepared and extracted. No negaOve impact on the study. More usatile Cala may be obtained. Study D M M r sanatare DM ss*irA f c . DM 8 r4 t7 9 r DM Mm . U . w>S Earw n QAU Review LIBRARY IO: V000183S-S a d m in is t r a t iv e f o r m RECEIVED TIME NOV.23. 3:32PM PRINT TIME N0V.23. 3:33PM /* N Exygen Research Page 107 o f 121 Interim Report #9-Analysis of Ground Water Samples Exygen Study No.: P0001131 '-200t 04:09m FraHtSTON SOLUTIONS T-I5 F.00|/D0; p - ir i II . f f S S m w "T K S IS yni ncm f: Amendment Nu> ibec Effective Dete: Exygen Study Numbsr VO CO L AMENDMENT j 12AJ1J pdoBfi5Sf~ Client Study Number 1 S t1 N A ' pi& ghfllftO N QF AMENDED S S S l M T t MMm M b, page & of Pratoeoi. AMENDED TO~ An temete lot of PFO Sm v used torthisaftriy. The PFOSueed prariouety in the s t^ lS h m m e r 217 from SM) neioutandKw ae neaeeeaiyta m e e ntwr tat No negaSMoknpeet on sbidy. m t'iS*V?T ii'.-V SEEL mmmf am J l W p ;3^nsin.y j]zi/Q b ygsn qaU Init/nu W g - /.a fe Gaie UBRARYID: VOIW 'OT RECEIVED TIME IRR. il :28PM , A M w iem A TivE eraw PRINT TIME I MfiR.21. 5:29PM Exygen Research Page 108 o f 121 Interim Report #9-Analysis o f Ground Water Samples Exygen Study No. : P0001131 3058 Research Drive Phone: 814-272-1039 S tate College, PA 16801 Fax: 814-231 -1580 Amendment Number. Effective Date: Exygen Study Number PROTOCOL AMENDMENT 6 . 05/17/06 ' P0001131 Client Study Number Paga 1 oM NA D ESCRIPTIO N O F AMENDED SECTION Appendix I, Analytical Method V 0001782 AMENDED TO . *` As per client request samples in login L4026 that have been tagged for re-extraction by the sponsor win be re-extracted using the foBowing method: D irect In jectio n M ethod: Before the samples are weighed for the extraction, they are mixed thoroughly by vigorously shaking the container. A one-gram portion o f sedim ent is weighed into a 15-miHiiiter centrifuge tube for the extraction. Ten m illllters o f 1% acetic add in methanol is added to each sam ple. The samples are then shaken by hand, vortexed, and sonicated fo r 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 TIO N A L E More usable data will be obtained by using an alternate method. IMPACT ON STUDY LIBRARY ID: V0001228- ' ADMINISTRATIVE FORM Exygen Research Page 109 of 121 Interim Report #9-Analysis o f Ground Water Samples Exygen Study No.: P0001131 3058 Research D rive Phone: 814-272-1039 S tate C ollege, PA 16801 Fax: 814-231-1580 Am endm ent Number: Effective Date: Exygen Study Number PROTOCOL AMENDMENT 8 0 7 /0 6 /0 6 P0001131 Client Study N um ber Page 1 of 1 NA D E S C R IP T IO N O F A M E N D E D S E C T IO N A ppendix I, A nalytical M ethod V 00 0 1 7 8 2 . AMENDED TO A s per client request, sedim ent sam ples re-tagged fo r re-extraction by th e sponsor will be re-extracted using th e follow ing m ethod: D ire c t In je c tio n M e th o d : B efore the sam ples a re w eighed fo r the extractio n, th ey a re m ixed thoroughly by vigorously shaking th e container. A one-gram portion o f sedim ent is w eighed into a 15-m illiliter centrifuge tu be fo r the extraction. T e n m illiliters o f 1% acetic a d d in m ethanol is added to each sam ple. T h e sam ples a re then shaken by hand, vortexed, and sonicated fo r thirty m inutes. T h e sam ples are then centrifuged for -1 0 m inutes a t -3 0 0 0 rpm . Each sam ple is analyzed by L C /M S /M S electrospray. R A TIO N A LE M ore usable d ata w ill be obtained by using an alternate m ethod. IM P A C T O N S T U D Y N o negative im pact on study. Study Director Signature a / m / A S - .________________ principal Investigator Signature Date ___ Date Study Director Management Signature Date Exygen Management Signature Date Sponsor Signature (if required) Date Exygen Q A U In it/D a te / T io ld f LIBRARYID: V0001226-9 ADMINISTRATIVE FORM Exygen Research Page 110 o f 121 Interim Report #9-Analysis of Ground Water Samples Exygen Study No.: P0001131 CHEM EHSR 236 1% 651 733 1958 07-1K0M 03:S7p* Frnr*E*TW I0LTI0KS 07/11 '06 1A:09 NO.142 02/02 + T -K i P-OOt/OOZ M 4 " research 3 0 5 8 R o c i r h O fW W? r,' j? lS o State CsUe, FA1M01 Fk ,. W E d flrb ^ A M 0 ^ E * lT M w n d iih M lN u rtb w : EcvDRte: -- ,M - g. Appirtx lt Analytical Mattiod *001782. b r i*b e d e d m ino tha folldiMnq mathod: -10 minutaat- 3QOOrpm. EachampiJsnalyzfdbyLC/MS/M8 alpetrtrspray. ..., . . . * ' -sj . ----------1 " " ----------------- ' N A T IO N A L ' Moni liaaUa data will ba obtainad try using,an altamkla mfthod. ------- --- --------------------------------------------- i M P A d - O N f f U g ? - No nsgattw impact on study. . tz - ju 'u n r ' & 6 : USHAflYID: VQ00122M RECEIVED TIME JUL.ll. 4:27PM PRINT TIME JUL. 11. 4:28PM Exygen Research Page 111 o f 121 Interim Report #9-Analysis of Ground Water Samples Exygen Study No.: P0001131 01-01-2007 03:50m FrurlESTON SOLUTIONS T-021 P.002/005 f-058 Exyn ^^R ESEA R C I BOSSResearch Drive Phone: 814272-1039 Stale College, PA 16801 Fax: 814-231-IS M . Amendment Number Effective Date: Exygen Study Number PROTOCOL AMENDMENT 11 12/21/06 POPP1131 ClientStudy Number ^ o *irfi P0001131 DESCRIPTIONOFAhifeNDEP SECTTOjT ProtocolDistribution Section: . 2) John M. Flaherty. Principal Investigator, Exygen Research ' AMP*PJ5LTQ 2) Charts*Simon*, PrincipalInvestigator,ExygenResearch John Flaherty retired and Chas Simons in s taken over John'S rate as Principal Investigatnrfbr the study. Nonegativeimped IMPACTONSTUPy SUdyOkfcfrfSSutuM rCL- A ^ mnopai , .___ NA DQ|SnMHuPHew{HnP^^ U toA tZ d & k J SpanierS^neknipfequInM) // 0*1 ( ' ae* on* S-c^J'li at* TT) oste f ExygenQAUIniL/Data U S t aijllrt LIBRARYID:VODOM2W RECEIVED TIME JfiN. 0. 4:02PM ADMINISTRATIVEPORM PRINT TIME JON. 8. 4:04PM Exygen Research Page 112 o f 121 Interim Report #9-Analysis of Ground Water Samples Exygen Study No.: P0001131 SEP.2 0 .2 0 0 5 1=52PM EXYGEN RESEARCH NO.377 P .2 3058 Research O rive Phone: 814-272-1039 S tate C ollege, PA 16801 Fax: 814-231-1580 DEVIATIO N FORM X Project Specific Deviation ____ Facility Deviation Exygen P ro ject#: P760/P1131 Client Project # : NA D ate o f Occurrence: 03/16/05 D eviatio n#: 1/1 Reference # : 05-122 R aoulatorv P rivar: Dviation TVRP flhdude \M fa r methods and SOPs) (3MP X <3LP Other None am ale D escription: Protocol Vfc 0001658-3 Notebook reference: NA Method X SOP L o g in #: NA C ontainer#: NA L o t# : NA Sum m ary o f D eviation: 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 (> 1 0g ). Causa: ____ P reparation_____ A nalysis_____ Instrum ent_____ _ Client Request X Other There has bean no negative impact on the study. All of the percent solid values that were determined during the time period In question are considered valid, although the SOP was not followed. In the newly revised version of the SOP blanks and duplicates are no longer required. Also, in the new SOP, the allowable amount of sample to be used is < 20 g. All of the samples in question in this deviation weighed (ess than 20 g. The technician analyzing the samples for percent solids was following the new procedure before it was formally approved. Corrective Action: A new version of the SOP has been issued and approved (V0000427-3). Qualhity Assurance LIBRARYID: V0001640-8 trees' Sponsor Representative . Sponsor Management D ate D ate D ate ADMINISTRATIVE FORM Exygen Research Page 113 o f 121 Interim Report #9-Analysis of Ground Water Samples Exygen Study No.: P0001131 SEP.20.2005 1:52PM EXYGEN RESERRCH NO.377 P.3 3058 Research D rive Phone; 814-272-1039 S tate C ollege, PA 16801 Fax: 814-231-15B 0 DEVIATION FORM General: X Prelect Specific Deviation ____ Facility Deviation P a n 1 of 1 D ale o f Occurrence: 06/29/05 __ Exygen P ro ject#: P760/P1131 D eviatio n#: 2/2 _ Client P ro ject#: NA R eference#: o f r - t d i Reoidstory D river: D eviation Tvae: /Include Vtt tbrm athoda and SOPs) _____ X _____ _____ GMP GLP Other None Semole Deecrlotlon: X Protocol _____ Method VS: NA Notebook reference: NA _____ SOP Lo gin #: L4264/L4256 C ontainer#: 0)086480-85 L o t# : _________ NA Summary of Deviation: The protocol states diet control and fortified control samples of each matrix will be analyzed; however. control dam was not obtained. Fish was used as the control for the analysis of these six dam samples. Cause: ____ P reparation_____ A nalysis_____ Instrum ent_____ O ient Request X Other Im pact: No negative impact on this study. LIBRARYID: VD001640-6 Exygen Research ADMINISTRATIVE FORM Page 114 o f 121 Interim Report #9-Analysis o f Ground Water Samples Exygen Study No.: P0001131 SEP.20.2005 52PM EXYGEN RESEARCH Exgen RESEARCH 3058 Research D rive S tate C ollege, PA 16801 NO.377 P.4 Phone: 814-272-1039 Fax: 814-231-1580 G en eral: X Project Specific Deviation DEVIATIO N FORM Facility Deviation P ao e lo f 1 Date o f Occurrence: 12/27/04 Exygen P ro ject#: P760/P1131 _ Deviation # : 3/3 O ient P ro ject#: NA____ Reference# : O S '- f i r R eouH torv D river: Deviation Tvna: (Include W for methods end SOPb) OMP GLP Other None Sam ple Description: _____ Protocol ______ Method V#: 202-20 Section 5.2.3 Notebook reference: NA X SOP Lo gin #: NA Container# : NA L o t# : NA Sum m ary o f D eviation; SL2114 (30% DimethyWichlorosllarie in Toluene) was given the expiration date of 02/13/05, but RE544 (Toluene) used to make SL2114 expired on 03/28/04. SL2114 was used to silanlzed glassware prepared for the fish extraction from 12/27/04 through 01/07/05. Cause: ____ P reparation_____ A nalysis_____ Instrum ent_____ Client Request X Other Im pact: No negative impact on the study. Toluene was used only as a solvent for the glassware preparation. Dimethyldlchlorosilane, which is the coating agent, was not expired. CorreoUva Action: Deviation Issued. S ig n a tu re : j ^PrfnoipaMHvestigat6r IC o A ftjO / Study Director O yC *. S k tJ L r K Quality Assurance ' Exygen tynggdment ' e ta / f/W o r D ate Sponsor Representative Sponsor Management Date D ate D e te LIBRARYID: V0001640-6 ADMINISTRATIVE FORM Exygen Research Page 115 o f 121 Interim Report #9-Analysis o f Ground Water Samples Exygen Study No.: P0001131 RESEARCH 3058 Research Drive Phone: 814-272-1039 SUte College, PA 16801 Fax: 814-231-1580 D EVIA TIO N FO RM Paqa1of2 X Project Specific Deviation ____Faculty Deviation Date of Occurrence: 04/26/06 Exygen Project : P760/P1131 D eviation#: S Oient Project # : ______ NA R e fe re n ce # : 06-076 Regulatory D rive r Deviation Type: (In clud e V# fo r m e th o d ! a n d SO Ps) _____ X _____ _____ GMP GLP Other None Semole D escription: X Protocol ____ Method V#:NA Notebook reference: NA _____ SOP L o g in # : L0008191 C ontainer#: 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 to r PFOS, PFOA and UC PFOA Because of this, the study sponsor requested the use of an allematlve extraction for these compounds, as follows: D irect Injection Method: Before the samples ware weighed for the extraction, they were mixed thoroughly by vigorously shaking the container. A one-gram portion of sediment was weighed Into a 15-mMiter centrifuge tube to r the extraction. Ten milliliters of 1% acetic acid in methanol was added to each sample. The samples were then shaken by hand, vortexed, and sonicated for thirty minutes. The samples were then centrifuged for -1 0 minutes at -3000 rpm. Each sample was analyzed by LC/MS/MS electrospray. Using this method acceptable data was obtainedfbr PFOS, but the recoveries for PFOA and UC PFOA were still poor. Another alternative method was then used for PFOA and ,JC PFOA, as follows: Alternative SPE Method: The samples that were prepared In 1% acetic add for the direct injection method were used for this extraction. Five mMliters of each sample was aliquoted into a 50-mL polypropylene centrifuge tube and the volume was taken to 40 mL with water. The samples were then centrifuged for -1 0 minutes at -3000 rpm. The supernatant was than loaded onto a C ,, SPE cartridge conditioned with 10 mL of methanol and 5 mL ofwater. The eluate was discarded. Approximately five m illiters of methanol was added to the cartridge. Five m illiters of eluate was collected into a g ra d u ated 1 6 m L p o lyp ro p ylen e cen trifu g e tu b e . E ach s am p le w as a n a ly ze d b y L C /M S /M S electrospray. Cause: ___ Preparation____ A nalysis____ Instrum ent____ Client Request X Other Im pact: More usable data was obtained. LIBRARY ID: V0001640-8 ADMINISTRATIVEFORM Exygen Research Page 116 o f 121 Interim Report #9-Analysis of Ground Water Samples Exygen Study No.: P0001131 CHEH EHSR 236 1B 6S1 733 1958 DM IW IN N :llx P rfrH n O I UTICW * 05/01 '06 13:46 N0.106 03/03 i M H P.M V W H H . , > . _\ ^ **1, .y ' lO M ta a a rtiB rtv i Statt Catlap, M lH P I STAUCH .. FW S lf !- * \ ' I . . . -, ' > . u m M tn o sv a u im * *' :> *.i ,* s . '< '.i ' .M 'i i* Exygen Research Page 117 o f 121 Interim Report #9-Analysis of Ground Water Samples Exygen Study No.: P0001131 EHS OPNS ENVIRONMENTAL LAB @ 651 778 4226 07/11/06 11:46 0 :04/07 N0:681 RESEARCH 3058 Research D rive Phone: 14-272-10S 9 s ta te C ollege, PA 16801 F ix : 814-2J1-15M . .O V A T IO N FORM sharal: '. X Project Specific Deviation _ _ Fecllty Deviation J B fc i& L Data ofOocurrn#: 0W W W Exygen P ro je c t*: P760/P1131 Deviation#: ' . 8it''" ; . Client P ro ject#: NA ! Wemititoiv Driver DavtaHpn fy n e : fln ckid t V# far methods and SOP t) .. ' ~W ~ '' -,__ QMP OLP Other None aemntePeeerlnttan: _____ 'P ro to co l ' X Method V#VWQ427-3 Notebook ference: NA ____ !;S P : Login#: NA Container#: C00fS9446: Lot: H SummeryofDeviation: ' = , , ^ i:' On# aoNsample (C00159446) wet weighed at -Bg tor pereant soRd analyeik, rether.thanM -20g which le etted In th method. ' '" . Cetiaa: ___ Preparation____ Analysis____ Instrument ___Client Request Other No negative Impact. An accurate percent eolld number wee obtained by ellghtly altering the calculation ueed. . ' ' ' Coicpotlvp Actions: Deviation laeued. d i ro f L L tf . PrincIpeUbiveatlgator s / ' ira u .j. - ? ! /ik Date . / tlv k Exygen g a n fig e m e n l^ Sponsorf ... 1Date < n tm __ ith iJ tt? j f e ____ Date Sponsor Management P a te " Exygen Research Page 118 of 121 Interim Report #9-Analysis of Ground Water Samples Exygen Study No.: P0001131 EHS OPNS ENVIRONMENTAL LAB '651 778 4226 07/11/06 11:46 0 :06/07 NO:681 RESEARCH 3058 Research Drive Phone: 814-272-f03> State College, P16801 Fax: 814-231-1580 DEVIATIO N FORM J itc istL X . Project Specific Deviation ___ _ Facility Deviation O ita ofOccurrsnoie: 68/2WDB Exvoan Project : P760ff1131 ; ' D eviatkin : V ' Client Project # : MA , ; ' N aferen o a*: RmUlalflrr Piftir ; Deviation TVae: (Includi V#for/nettate and SOPa) . ____ _ X _____ __ . GMP QLP other None im M e P tw flP to L o g in : _______ MA _____ Protocol ______ Method W . 1800 Notebook reference: - X -' i 6 :- Container#: NA Lot*:' i4 m ihihr^fP rvfttfgn; Peerreview of row date w as not documented per SOP V1800 prior 10 8/28/D6. Cause: ____ P reparation_____ A nalysis____ _ Instrum ent____ Client Request V " Other lipaict: '" ., Maw data w ll be more thoroughly evaluated and reviewed before QA Inspection ggfTnaiytetlani A note to fie wM be Issud to a i subsequent reports statino dial overall summaries have been peer reviewed. ir M a# .. ..Data; * X l M tH Sponsor Representative 0)A Sponsor Managem ent Exygen Research Page 119 o f 121 Interim Report #9-Analysis o f Ground Water Samples Exygen Study No.: P0001131 EHS OPNS ENVIRONMENTAL LAB 651 778 4226 0 7 /1 1 /0 6 1 1 :46 0 :0 7 /0 7 NO:681 If RESEARCH 3058 Research Drive State College, PA 16801 .' Phone: 8 4 -$ -1 0 3 9 Fax: 8 t4-231-1580 . pEVIATION FRM. Panerai: . X Prelect Spedile Deviation Facility Deviation Pjfcftfi. beta of O ccurence; m ttl Exygen Project#: P761PH31 ''D e v ia tio n # : ; ft' ' Cileni Project # : NA'. ItapiiUtenr Driver: DawtaHen Tyna: (laduda V# formethods and SOP; ___OMP . X. OLP ... Other ___None Semole Daacrtetlon: X Protocol V#; N A '. Notebook reference: Method SOP L o g in #: - L00O8121 UXXftOei C ontainer#: '. C 0f5#347. O1.60354 Lot#: ' .' 130171066 : __________ M n m ify o fP ttiiE g n i : Sample* ware filled to 250 mL Inatead.of 200 mL. NA ' babae:'- X Preparation ___ ;"AhiWy*i ;___ hitrumerit _ _ _ Client Reouest Impact: .: . .. Sample* could not be extracted according to the protocol. Qttter fiomslfti.Agiltto; SpMngTevela were adjusted to accommodate the alternative volume. Ipal Investigator y / 8tudy.DI l A -- Sponsor Management Data Exygen Research Page 120 o f 121 Interim Report #9-Analysis o f Ground Water Samples Exygen Study No.: P0001131 0MH-2007 D3:Slp* Froe-SESTO SOLUTIONS T-021 P.005/005 F-050 E ^ yaeiT ^JRESEA RCH 30S8 Research D rive Phone: 814-272*10 S ta te CoUege, PA 16801 Fax: 814*231*1580 D E V IA T IO N FO R M Peas l e f t X Project Specific Deviation Fae f% Deviation Exygen Project#: Client ftc ja e # : P00Q1131 P0001131 Date eTOccurrence: Nov. 20 06JS U m - D eviatio n #: 10 R eference#: f il- Q Q L D e v te tln n fv iie rfln c to fg v e ftrw a ffx x te a n rf S O ftl SMP OLP Other Snoot fteacrloBen; X Metiod VA V0001780 NOVDOQK I H B I V U i _____O P Login#: L00101S6 L0010169 M iP t- C o n ta in tr*: L o tt: Stack sunttanfc end feitfieetion standard# used In the adrac&on far m ete logine were prepared in ecetanhrte Indeed ctmothanoL C ew e: X Propnrallon _ Andysis _ _ _ t n trumant CHantRequest ___ Other o negaUve Imp eri. Compounds era completely soluble InaceiunM eastheyareInm ethinoL Cosrectfve A ction?" Deviation issued. No acbontaken. 4 LIBRARYID: VOOO1G40-7 RECEIVED TIME JAN. 8. 4102PM ADMINISTRATIV FORM PRINT TIME JAM. 8. 4:04PM '. \ I Exygen Research Page 121 o f 121