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INTERIM REPORT # 2-AnaIvsis of Surface Water Samples STUDY TITLE Analysis of Perfluorobutanesulfonate (PFBS), Perfluorohexanesulfonate (PFHS), and Perfluorooctanesulfonate (PFOS) in Water, Soil, Sediment, Fish, Clams, Vegetation, Small Mammal Liver and Small Mammal Serum Using LC/MS/MS for the 3M Decatur Monitoring Program DATA REQUIREMENTS EPA TSCA Good Laboratory Practice Standards 40 CFR 792 STUDY DIRECTOR Jaisimha Kesari P.E., DEE Weston Solutions, Inc. 1400 Weston Way West Chester, PA 19380 Phone: 610-701-3761 INTERIM REPORT COMPLETION DATE March 22, 2005 PERFORMING LABORATORY Exygen Research 3058 Research Drive State College, PA 16801 Phone: 814-272-1039 STUDY SPONSOR 3M Company 3M Building 0236-01-B-10 St. Paul, MN 55144 Phone: 651-733-6374 PROJECT Protocol Number: P0001131 Exygen Study Number: P0001131 Total Pages: 100 Interim Report #2-Analysis of Surface 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 EPA TSCA Good Laboratory Practice Standards 40 CFR 792 by Exygen Research. Jonn Eianerty ' PP rriinnrcMipnaa ll ITnn\v/#estigator Exygen Research Jaisimha Ke . Study Director Weston Solutions, Inc. 3M Company Exygen Research Date Page 2 of 100 Interim Report #2-Analysis o f Surface Water Samples Exygen Study No.: P0001131 QUALITY ASSURANCE STATEMENT Exygen Research's Quality Assurance Unit reviewed Exygen Study Number P0001131, entitled, "Analysis of Perfluorobutanesulfonate (PFBS), Perfluorohexanesulfonate (PFHS), and Perfluorooctanesulfonate (PFOS) in Water, Soil, Sediment, Fish, Clams, Vegetation, Small Mammal Liver and Small Mammal Serum Using LC/MS/MS for the 3M Decatur Monitoring Program". All reviewed phases1 were inspected for conduct according to Exygen Research's Standard Operating Procedures, the Study Protocol, and all applicable Good Laboratory Practice Standards. All findings were reported to the Exygen Principal Investigator and Management and to the Study Director. Phase Date Inspected Date Reported to Date Reported to Principal Exygen Date Reported to Investigator Management Study Director 7. Raw Data Review 03/10,11/05 03/14/05 03/18/05 03/18/05 8. Interim Analytical Report Review 03/17/05 03/18/05 03/18/05 03/18/05 'Note: All in-lab inspections will be documented in the QA statement for the final analytical report at the conclusion o f the study. This QA statement involves only the review o f the interim report and associated raw data. Exygen Research Page 3 of 100 Interim Report #2-Analysis of Surface 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: Date Exygen Research Facility Management: 2 1 -A M rt--0 Date Study Director, Weston Solutions, Inc. Jaisimha i^esri P.E., DEE Weston Solutions, Inc. Sponsor Representative, 3M Company: iSi/uJA & Michael A. Santoro Director of Regulatory Affairs Exygen Research 3hh5 Date 3/ 3// PS Date Page 4 of 100 Interim Report #2-Analysis o f Surface Water Samples Exygen Study No.: P0001131 STUDY IDENTIFICATION Analysis of Perfluorobutanesulfonate (PFBS), Perfluorohexanesulfonate (PFHS), and Perfluorooctanesulfonate (PFOS) in Water, Soil, Sediment, Fish, Clams, Vegetation, Small Mammal Liver and Small Mammal Serum Using LC/MS/MS for the 3M Decatur Monitoring Program PROTOCOL NUMBER: P0001131 EXYGEN STUDY NUMBER: P0001131 TYPE OF STUDY: Residue SAMPLE MATRIX: Surface Water TEST SUBSTANCE: Perfluorobutanesulfonate (PFBS), Perfluorohexanesulfonate (PFHS), and Perfluorooctanesulfonate (PFOS) SPONSOR: 3M Company 3M Building 0236-01-B-10 St. Paul, MN 55144 STUDY DIRECTOR: Jaisimha Kesari P.E., DEE Weston Solutions, Inc. 1400 Weston Way West Chester, PA 19380 STUDY MONITOR: Michael A. Santoro 3M Company 3M Building 0236-01-B-10 St. Paul, MN 55144 PERFORMING LABORATORY: Exygen Research 3058 Research Drive State College, PA 16801 ANALYTICAL PHASE TIMETABLE: Study Initiation Date: 11/05/04 Interim Analytical Start Date: 02/14/05 Interim Analytical Termination Date: 02/17/05 Interim Report Completion Date: 03/22/05 Exygen Research Page 5 of 100 Interim Report #2-Analysis o f Surface 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 John Flaherty Karen Risha Paul Connolly Chrissy Edwards Mark Ammerman Amy Sheehan Title Vice President Scientist Technical Lead-LC/MS Technician Sample Custodian Associate Scientist Exygen Research Page 6 of 100 Interim Report #2-Analysis o f Surface 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 6.3 Chromatography.......................................................................................................14 6.4 Instrument Sensitivity...............................................................................................15 6.5 Description of LC/MS/MS Instrument and Operating Conditions...........................15 6.6 Quantitation and Example Calculation.....................................................................16 7.0 EXPERIMENTAL DESIGN......................................................................................17 8.0 RESULTS...................................................................................................................17 9.0 CONCLUSIONS.........................................................................................................17 10.0 RETENTION OF DATA AND SAMPLES.............................................................17 Exygen Research Page 7 of 100 Interim Report #2-Analysis o f Surface Water Samples Exygen Study No.: P0001131 Table I. Table II. LIST OF TABLES Page Summary of PFBS, PFHS and PFOS in Surface Water Samples.................. 19 Matrix Spike Recovery of PFBS, PFHS and PFOS in Surface Water Samples.......................................................................................................... 20 Exygen Research Page 8 o f 100 Interim Report #2-Analysis of Surface Water Samples Exygen Study No. : P0001131 Figure 1. LIST OF FIGURES Page Typical Calibration Curve for PFBS in Water............................................... 23 Figure 2. Extracted Standards of PFBS in Water, 50 ng/L and 100 ng/L, Respectively................................................................................................... 24 Figure 3. PFBS in Reagent Water, 500 ng/L Fortified Reagent Water, and 5000 ng/L (DF=10) Fortified Reagent Water, Respectively.......................... 25 Figure 4. Chromatogram Representing a Surface Water Sample Analyzed for PFBS (Exygen ID: C0052282, Data Set: 021405C)................................ 26 Figure 5. Typical Calibration Curve for PFHS in Water.............................................. 27 Figure 6. Extracted Standards of PFHS in Water, 50 ng/L and 100 ng/L, Respectively................................................................................................... 28 Figure 7. PFHS in Reagent Water, 500 ng/L Fortified Reagent Water, and 5000 ng/L (DF=10) Fortified Reagent Water, Respectively.......................... 29 Figure 8. Chromatogram Representing a Surface Water Sample Analyzed for PFHS (Exygen ID: C00522831, Data Set: 021405C).............................. 30 Figure 9. Typical Calibration Curve for PFOS in Water.............................................. 31 Figure 10. Extracted Standards of PFOS in Water, 50 ng/L and 100 ng/L, Respectively................................................................................................... 32 Figure 11. PFOS in Reagent Water, 500 ng/L Fortified Reagent Water, and 5000 ng/L (DF=10) Fortified Reagent Water, Respectively.......................... 33 Figure 12. Chromatogram Representing a Surface Water Sample Analyzed for PFOS (Exygen ID: C0052282, Data Set: 021405C)................................... 34 Exygen Research Page 9 of 100 Interim Report #2-Analysis o f Surface 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" .............................................................................. Page 35 Exygen Research Page 10 of 100 Interim Report #2-Analysis o f Surface Water Samples Exygen Study No.: P0001131 1.0 SUMMARY Exygen Research extracted and analyzed surface 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 water was 50 ng/L and the limit of detection was 25 ng/L. The PFBS, PFHS and PFOS residues found in surface water samples are summarized in Table 1. The average percent recoveries standard deviations for PFBS, PFHS, PFOS in surface water samples were 139% 21%, 95% 7%, and 96% 12%, 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 surface 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 surface water using the analytical method entitled, "V0001780: Method o f 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 February 14, 2005, and the analytical termination date for this interim report was February 17,2005. Exygen Research Page 11 o f 100 Interim Report #2-Analysis of Surface Water Samples Exygen Study No. : P0001131 4.0 ANALYTICAL TEST SAMPLES Forty-four surface water samples (Exygen ID C0052282-C0052299 and C0052310C0052335) were received on wet ice on December 04, 2004 from Charles Young 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 will be kept at Exygen Research until the conclusion of the study; they will then be located in the raw data package associated with this study. 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 July, 06, 2000. 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.. SP0000252 SP0002401 SP0002694 Lot# 101 SE036 430180-1 Purity (%) 96.7 98.6 101.2 Expiration Date 12/04/06 10/18/06 04/23/06 The molecular structures o f PFBS, PFHS and PFOS are given below: PFBS Chemical Name: Perfluorobutanesulfonate Molecular Weight: 338 supplied as the potassium salt ^FSO s'K '1') Transitions Monitored: 299 - 99 Structure: FF FF F S03 FF FF Exygen Research Page 12 of 100 Interim Report #2-Analysis o f Surface W ater Samples Exygen Study No.: P0001131 PFHS Chemical Name: Perfluorohexanesulfonate Molecular Weight: 438 supplied as the potassium salt (CeFnSCh'K*) Transitions Monitored: 399 --> 80 Structure: FFF FFF F S03 FFF FFF PFOS Chemical Name: Perfluorooctanesulfonate Molecular Weight: 538 supplied as the potassium salt (CgFnSCVK*) Transitions Monitored: 499 -> 80 Structure: FFFF FF F S03 FF FFFF 6.0 DESCRIPTION OF ANALYTICAL METHOD The analytical method "V0001780: Method of Analysis for the Determination of Perfluorooctanoic Acid (PFOA) in Water by LC/MS/MS" was used for this study. 6.1. Extraction Procedure A 40 mL aliquot of the water sample was used for the extraction procedure. After fortification of appropriate samples, the samples were loaded onto a Cig SPE cartridge conditioned with 10 mL of methanol and 5 mL of water. The eluate was discarded. Approximately five milliliters of methanol was added to the cartridge. Five milliliters of eluate was collected into a graduated 15 mL polypropylene centrifuge tube. Each sample was analyzed by LC/MS/MS electrospray. Exygen Research Page 13 o f 100 Interim Report #2-Analysis o f Surface Water Samples Exygen Study No. : P0001131 6.2 Preparation of Standards and Fortification Solutions Individual stock standard solutions of PFBS, PFHS and PFOS were prepared as specified in Exygen method V0001780. The stock standard solutions were prepared at a concentration of 100 pg/mL by dissolving 10 mg of each of the standards (corrected for purity and salt content) in methanol. From these solutions, mixed 1.0 pg/mL fortification standard solutions were prepared by taking 1 mL of each of the appropriate stock solutions and bringing the volume up to 100 mL with methanol. By taking 10 mL of the mixed 1.0 pg/mL fortification standard and bringing the volume up to 100 mL with methanol, a mixed 0.1 pg/mL fortification standard was prepared. By taking 10 mL of the mixed 0.1 pg/mL fortification standard and bringing the volume up to 100 mL with methanol, a mixed 0.01 pg/mL fortification standard was prepared. A set of standards containing PFBS, PFHS and PFOS was 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 (UL) 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 o f standard preparation will be kept at Exygen Research until the conclusion of the study; it will then be located in the raw data associated with this study. 6.3 Chromatography Quantification of PFBS, PFHS and PFOS was accomplished by LC/MS/MS electrospray. The retention times of PFBS, PFHS and PFOS were ~ 3.9 mins, ~ 10.4 mins, and ~ 12.6 mins, respectively. Peaks above the LOD were not detected in any of the reagent blank samples corresponding to the analyte retention time. Exygen Research Page 14 of 100 Interim Report #2-Analysis o f Surface Water Samples Exygen Study No.: P0001131 6.4 Instrument Sensitivity The smallest standard amount injected during the chromatographic run had a concentration of 25 ng/L of PFBS, PFHS and PFOS. 6.5 Description of LC/MS/MS Instrument and Operating Conditions Instrument: API 4000 Biomolecular Mass Analyzer Interface: Turbo Ion Spray Liquid Introduction Interface Computer: DELL OptiPlex GX400 Software: Windows NT, Analyst 1.4.1 HPLC: Hewlett Packard (HP) Series 1100 HP Quat Pump HP Vacuum Degasser HP Autosampler HP Column Oven HPLC Column: Thermo Fluophase RP, 50 mm x 2.1 mm Column Temp.: 35 C Injection Voi.: 15 pL Mobile Phase (A): 2 mM Ammonium Acetate in water Mobile Phase (B): Methanol Time ('min') 0.0 1.0 8.0 10.0 11.0 18.0 Total run time: ~18 min %A 65 65 25 25 65 65 %B 35 35 75 75 35 35 Flow Rate: 0.3 mL/min Ions monitored: Analvte PFBS PFHS PFOS Mode negative negative negative Transition Monitored 299 -> 99 399 -> 80 499->80 Approximate Retention Time ('mini ~3.9 min. ~10.4 min. ~12.6 min. Exygen Research Page 15 o f 100 Interim Report #2-Analysis o f Surface Water Samples Exygen Study No.: P0001131 6.6 Quantitation and Example Calculation Fifteen microliters of sample or calibration standard were injected into the LC/MS/MS. The peak area was measured and the standard curve was generated (using 1/x fit weighted linear regression) by Analyst software using six concentrations of standards. The concentration was determined from the equations below. Equation 1 calculated the amount of analyte found (in ng/L, based on peak area) using the standard curve (linear regression parameters) generated by the Analyst software program. Equation 1: Analyte found (ng/L) = (Peak area - intercept! x DF slope Where: DF = Dilution Factor, factor by which the final volume was diluted, if necessary. For samples fortified with known amounts of PFBS, PFHS and PFOS prior to extraction, Equation 2 calculated the percent recovery. Equation 2: Recovery (%) = (analyte found (ng/L) - analyte in control (ng/Ll) xl00% amount added (ng/L) An example of a calculation using an actual sample follows (calculation is for PFOS only): Water sample Exygen ID: C0052282 Spk C (Set: 021405C), fortified at 500 ng/L with where: peak area = 84394 intercept = 3070 slope = 162 dilution factor =1 ng/L PFOA added (fort level) = 500 amt in corresponding sample = 0 (Not Detected) From equation 1: Analyte found (ng/L) = f84394 - 30701 x 1 162 From equation 2: % Recovery = 502.0 ng/L = (502.0 - 0 ng/L) x 100% 500 = 100 % Exygen Research Page 16 of 100 Interim Report #2-Analysis o f Surface Water Samples Exygen Study No.: P0001131 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 two sets. Each set consisted of one reagent blank, two reagent blanks fortified at known concentrations. One set contained five sample sites and the second set contained four sample sites, along with all the field blanks and field blank spikes collected for the surface water samples. For each site, a sample, a field duplicate and two matrix field spikes were collected. For each site, a laboratory duplicate was extracted and two laboratory matrix spikes were also extracted. 8.0 RESULTS The PFBS, PFHS and PFOS found in the surface water samples are listed in Table I. Fortification recoveries for PFBS, PFHS and PFOS in the water samples are detailed in Table II. The average percent recoveries standard deviations for PFBS, PFHS, PFOS in water samples were 139% 21%, 95% 7%, and 96% 12%, respectively. 9.0 CONCLUSIONS The surface water samples were successfully extracted and analyzed for PFBS, PFHS and PFOS according to analytical method V0001780. 10.0 RETENTION OF DATA AND SAMPLES When the final analytical report is complete, all original paper data generated by Exygen Research will be shipped to the sponsor. This does not include facility-specific raw data such as instrument or temperature logs. Exact copies of all raw data, as well as a signed copy of the final analytical report and all original facility-specific raw data, will be retained in the Exygen Research archives for the period of time specified in EPA TSCA Good Laboratory Practice Standards 40 CFR 792. Exygen Research Page 17 of 100 Interim Report #2-Analysis o f Surface Water Samples Exygen Study No.: P0001131 TABLES Exygen Research Page 18 of 100 Interim Report #2-Analysis o f Surface Water Samples Exygen Study No.: P0001131 Table I. Summary of PFBS, PFHS and PFOS in Surface Water Samples Client ExygenID Sample ID C0052282 DL3-SW-LOC001 -0-041201 C0052282 Rep DL3-SW-LOC001 -0-041201 * C0052283 DL3-SW-LOC001-0-041201 Dup C0052286 DL2-SW-LOC001 -0-041201 C0052286 Rep DL2-SW-LOC001 -0-041201 * C0052287 DL2-SW-LOC001-0-041201 Dup C0052290 DBC-SW-LOC001 -0-041202 C0052290 Rep DBC-SW-LOC001 -0-041202* C0052291 DBC-SW-LOC001-0-041202 Dup C0052294 DOU-SW-LOC001-0-041202 C0052294 Rep DOU-SW-LOC001 -0-041202* C0052295 DOU-SW-LOC001 -0-041202 Dup C0052298 DDO-SW-LOC001 -0-041202 C0052298 Rep DDO-SW-LOCO01-0-041202* C0052299 DDO-SW-LOC001-0-041202 Dup C0052312 DL1-SW-LOC001 -0-041202 C0052312 Rep DL1-SW-LOC001 -0-041202* C0052313 DL1-SW-LOC001-0-041202 Dup C0052316 DMC-SW-LOC001 -0-041202 C0052316 Rep DMC-SW-LOC001 -0-041202* C0052317 DMC-SW-LOC001-0-041202 Dup C0052322 DAA-SW-LOC005-0-041202 C0052322 Rep DAA-SW-LOC005-0-041202* C0052323 DAA-SW-LOC005-0-041202 Dup C0052326 DAA-SW-LOC002-0-041202 C0052326Rep DAA-SW-LOC002-0-041202* C0052327 DAA-SW-LOC002-0-041202 Dup C0052330 Field Blank C0052333 Field Blank C0052320 DMC-SW-LOC001 -2-041202 C0052321 DMC-SD-LOC001 -2-041202 Analyte Found (ppt, ng/L) C4 Sulfonate PFBS Perfluorobutanesulfonate C6 Sulfonate PFHS Perfluorohexanosu(fonate ND ND ND ND ND ND ND ND ND ND ND ND 93.8 96.2 97.5 14000 14700 12800 231 233 252 1620 1650 1600 ND ND ND ND ND ND 76.8 77.4 45.7 48.3 77.4 107 49.2 53.6 97.6 104 2460 3270 3100 379 49.5 54.0 7980 7350 7880 375 456 433 432 445 ND ND ND ND ND ND ND ND C8 Sulfonate PFOS Parfluorooctanesulfonate ND ND ND ND ND ND 3460 2880 3920 5710 5820 5640 ND ND ND 179 185 213 284 257 283 18800 18000 19600 2780 2930 3760 ND ND ND ND ` Laboratory Duplicate ND - Not detected at or above 25 ng/L. NQ - Not quantifiable = Measured concentration between 25 ng/L and the Limit of Quantitation (LOQ) which is 50 ng/L. Exygen Research Page 19 of 100 Interim Report #2-Analysis of Surface Water Samples Exygen Study No.: P0001131 Table II. Matrix Spike Recovery of PFBS, PFHS and PFOS in Surface Water Samples Sample DL3-SW-LOC001-0-041201 (C00S22I2 SpkC, 500 ngli. Spike) C4 Sulfonate PFBS_____________ C6 Sulfonate PFHS_____________ C8 Sulfonate PFOS Amount Amt Found Amount Amt Found Amount Amt Found Amount Spiked in Sample Recovered Recovery In Sample Recovered Recovery In Sample Recovered Recovery (ng/L) (ng/L) (ng/L)_____ <%) (ng/L) (ng/L)_____ (%) (ng/L) (ng/L)_____ (%> 500 ND 748 150 ND 485 97 ND 502 100 DL3-SW-LOC001-0-041201 (C00522I2 Spk D, 5000 ng/L Spike) 5000 ND 7090 142 ND 4560 91 ND 4450 89 DL3-SW-LOC001-0-041201 Low Spk (C0052254,500 ng/LSpike) 500 ND 694 139 ND 496 99 ND 508 102 DL3-SW-LOC001-0-041201 High Spk (C0052255, 5000ng/LSpike) 5000 ND 7320 146 ND 4580 92 ND 3840 77 DL2-SW-LOC001-0-041201 (C0052255 SpkE, 500 ng/L Spike) 500 ND 779 156 ND 516 103 ND 490 98 DL2-SW-LOC001-0-041201 (C00522SSSpkF. 5000ng/LSpike) 5000 ND 7200 144 ND 4630 93 ND 4680 94 DL2-SW-LOC001-0-041201 Low Spk (C00S22SS, 500ng/LSpike) 500 ND 734 147 ND 492 98 ND 515 103 DL2-SW-LOC001-0-041201 High Spk (C0052215, 5000ng/LSpike) 5000 ND 7200 144 ND 4660 93 ND 5440 109 DBC-SW-LOC001-0-041202 (C00522S0Spk0 ,500 ng/L Spike) 500 93.8 819 145 231 714 97 3460 3840 * DBC-SW-LOC001-0-041202 (C0052290SpkH, 5000 ng/L Spike) 5000 93.8 7330 145 231 5060 97 3460 7930 89 DBC-SW-LOC001-0-041202 Low Spk (C0052292,500 ng/LSpike) 500 93.8 742 130 231 667 87 3460 3800 . DBC-SW-LOC001-0-041202 High Spk (C0052295,5000 ng/L Spike) 5000 93.8 6830 135 231 4600 87 3460 7090 73 DOU-SW-LOC001-0-041202 (C0052294Spkt, 500 ng/L Spike) DOU-SW-LOC001 -0-041202 (C00522MSpkJ, 5000ngIL Spike) DOU-SW-LOC001-0-041202 Low Spk (C0052299,500 ng/LSpike) DOU-SW-LOCO01-0-041202 High Spk (C0052297,5000ng(l Spike) 500 5000 500 5000 14000 14000 14000 14000 12200 14800 15200 14100 16" * 2" 1620 2130 102 5710 5820 * 1620 6160 91 5710 10300 92 1620 2150 106 5710 6720 1620 5720 82 5710 9350 73 DOO-SW-LOCOOI-O-041202 (C005229I SpkK, 500 ng/LSpike) 500 ND 805 161 ND 482 96 ND 50 5 101 DDO-SW-LOC001-0-041202 (C005229I SpkL, 5000ng/LSpfce) 5000 ND 7240 145 ND 4590 92 ND 4600 92 DDO-SW-LOC001-0-041202 Low Spk (C00S2510,500 ng/LSpike) 500 ND 794 159 ND 517 103 ND 517 103 DDO-SW-LOC001-0-041202 High Spk (C0052311,5000ng/L Spike) 5000 ND 5310 106 ND 4680 94 ND 5000 100 ` Sam ple residue exceeds the spiking le vel sig nifican tly; therefore, an accurate recovery value cannot be calculated " P oint w as rem oved from the calculation o f average and standard deviation as p er em ail request dated 03/21/05. ND * N ot detected at o r above 25 ng/L (ppt). NQ = N ot q uantifiable * M easured concentration between 25 ng/L (ppt) and the L im it o f Q uantitation (LO Q ) w hich is 50 ng/L (ppt). Note: Since this summary table shows rounded results, recovery values may vary slightly from die values in the raw data. Exygen Research Page 20 of 100 Interim Report #2-Analysis o f Surface Water Samples Exygen Study No.: P0001131 Table II. Matrix Spike Recovery of PFBS, PFHS and PFOS in Surface Water Samples Continued Sample Description DL1-SW-LOC001-0-041202 (C0052312 SpkC. 500 ng/LSpike) C4 Sulfonate PFBS_____________ C6 Sulfonate PFHS_____________ C8 Sulfonate PFOS Amount Amt Found Amount Amt Found Amount Amt Found Amount Spiked in Sample Recovered Recovery In Sample Recovered Recovery In Sample Recovered Recovery (n o n -) (nfl/L) __ ("fl-)_____ !*1 __ (no/L) w u _____ e a __ (ng/L) w d _____ _____ 500 76.8 849 154 45.7 55 6 111 179 707 106 DL1-SW-LOC001-0-041202 (C0052312Spk 0.5000 ng/L Spike) 5000 76.8 7280 144 45.7 4810 96 179 4800 92 DL1-SW-LOC001-0-041202 Low Spk (C0052314.500 ng/L Spike) 500 76.8 620 149 45.7 566 113 179 726 109 DL1-SW-LOC001-0-041202 High Spk (C0Q52315,5000 ng/L Spike) 5000 76.8 7970 158 45.7 4700 94 179 4100 78 DMC-SW-LOC001 -0-041202 (C00S2315Spk E. 500 ng/L Spike) 500 107 893 157 53.6 556 100 284 764 96 DMC-SW-LOC001-0-041202 (C005231SSpk F. 5000ng/L Spike) 5000 107 7780 153 53.6 4740 94 264 5130 97 DMC-SW-LOC001-0-041202 Low Spk (C0052315,500ng/L Spike) 500 107 786 136 53.6 544 98 284 807 105 DMC-SW-LOC001-0-041202 High Spk (C0052319,5000ng/L Spike) 5000 107 6480 127 53.6 4560 90 284 3890 72 DAA-SW-LOC005-0-041202 (C0052322 Spk0 ,500 ngA Spike) DAA-SW-LOC005-0-041202 (C00S2322Spk H, 5000 ngA Spike) DAA-SW-LOC005-0-041202 Low Spk (C00S2324,500ng/L Spike) DAA-SW-LOC005-0-041202 High Spk (C005232S, 5000ng/L Spike) 500 5000 500 5000 2460 2460 2460 2460 4100 6840 3840 10600 * 88 167 7980 7980 7980 7980 9270 12200 8290 11900 * 84 78 18800 18800 18800 18800 23500 23600 20500 20200 * 96 28" DAA-SW-LOC002-0-041202 (C00S2320 SpkI, 500 ngA Spike) 500 379 1250 174 375 891 103 2780 3530 * DAA-SW-LOC002-0-041202 (C00S2320SpkJ. 5000ngA Spike) 5000 379 6430 121 375 5320 99 2780 8310 111 DAA-SW-LOC002-0-041202 Low Spk (C00S2320,500ng/L Spike) 500 379 762 77 375 860 97 2780 3440 DAA-SW-LOC002-0-041202 High Spk (C0052329.5000ngA Spike) 5000 379 7850 149 375 5180 96 2780 7740 99 Field Blank Low Spk (C0052331,500ng/L Spike) Field Blank High Spk (C0052332,5000ngA Spike) Field Blank Low Spk (C0082334, 90 0 ng/L Spike) Field Blank High Spk (C0052335, 5000ngA Spike) 500 5000 500 5000 ND 594 119 ND 6070 121 N D 625 125 ND 6550 Average: Standard Deviation: 131 139 21 ND 458 92 ND 4670 93 N D 475 95 ND 4450 Average: Standard Deviation: 89 95 7 ND 574 115 ND 5470 109 N D 541 108 ND 5100 Average: Standard Deviation: 102 96 12 ` Sam ple residue exceeds the spiking le vel sig n ifica n tly; therefore, an accurate recovery value cannot be calculated " P oint w as rem oved from the calculation o f average and standard deviation as p er em ail request dated 03/21/05. ND * N ot detected a t o r above 25 ng/L (ppt). NQ * N ot q uantifiable * M easured concentration between 25 ng/L (p p t) and th e L im it of Q uantitation (LO Q ) w hich is 50 ng/L (ppt). Note: Since this summary table shows rounded results, recovery values mayvary slightly from the values in the raw date. Exygen Research Page 21 of 100 Interim Report #2-Analysis of Surface Water Samples Exygen Study No.: P0001131 FIGURES Exygen Research Page 22 of 100 Interim Report #2-Analysis o f Surface Water Samples Exygen Study No.: P0001131 Figure 1. Typical Calibration Curve for PFBS in Water 021405C revl.ndb (PFBS): "Unear Regression ('1 t x" weighting): y * 52.7 x + 0.000802 (r 0.9864) Area, counts Exygen Research Page 23 of 100 Interim Report #2-Analysis o f Surface Water Samples Exygen Study No.: P0001131 Figure 2. Extracted Standards of PFBS in Water, 50 ng/L and 100 ng/L, Respectively I XC021105-2, 50ng/L Standard - PFBS (Standard) 299.W99.0anw -sam ple 3 o f 52 from 021405C.wiff Area: 3492counts Height: 05.2cps RJ: 3.93 min 3.83 Intensity, cps Time, min XC021105-3, 100 ng/L Standard - PFBS (Standard) 299.0/99.0 amu - sample 4 of 52 from 021405C.wiff Area: 5190 counts Height: 105. cps RT:4.00 min Intensity, cps Exygen Research Page 24 of 100 Interim Report #2-Analysis o f Surface Water Samples Exygen Study No.: P0001131 Figure 3. PFBS in Reagent Water, 500 ng/L Fortified Reagent Water, and 5000 ng/L (DF=10) Fortified Reagent Water, Respectively Reagent Control A - PFBS Jnknown) 299.9/99.9 amu -sample 9 o f 52 from 921495C.wiff (peak not found) CO CO Q. O `co(f>) c I Time, min Reagent Spike A, 500 ng/L - PFBS (Q0)299.0/99.0 amu - sample 10 of 52 from 021405C.wiiff Area: 26775 counts Height: 633. ops RT: 3.93 min 3.93 500 0.83 ,,1.49 0 -- 3.18 5.666.437.20 8.72-9.55^10.13 8 10 11.56^.11.95 12 14.06,14.37 >.15.64 17.36 -- I----------- I --------- 14 16 Time, min Reagent Spike B. 5000 ng/L, DF=10 - PFBS (Q C )299.0/99.0 amu - sample 11 of 52 from 021405C.wiff Area: 25449 counts Height: 596. ops RT: 3.94 min CO Q. o 500 CO 0 -- 3.94 9.00-^9.45 11.71^12.1013.04 ^13.72 15.41,15.77 17.55 8 10 12 14 16 Time, min Exygen Research Page 25 of 100 Interim Report #2-Analysis of Surface Water Samples Exygen Study No.: P0001131 Figure 4. Chromatogram Representing a Surface Water Sample Analyzed for PFBS (Exygen ID: C0052282, Data Set: 021405C) C0052282 - PFBS (Unknown) 299.0/99.0 amu sample 15 o f 52 from 021405C.wiff (peak not found) 0.58 Intensity, cps Exygen Research Page 26 of 100 Interim Report #2-Analysis o f Surface Water Samples Exygen Study No.: P0001131 Figure 5. Typical Calibration Curve for PFHS in Water 021405C rev1 .rdb (PFHS): "Unear'' Regression ('1 / x" weighting): y = 187 x + -0.000613 (r = 0.9994) Area, counts Exygen Research Page 27 of 100 Interim Report #2-Analysis o f Surface Water Samples Exygen Study No.: P0001131 Figure 6. Extracted Standards of PFHS in Water, 50 ng/L and 100 ng/L, Respectively I XC021105-2, 50 ng/L Standard PFHS (Standard) 399.0/80.0 amn -sample 3 o f 52 from 021405C.wiff Area: 9721 counts Height: 695. cps RT: 10.4min Time, min I XC021105-3, 100 ng/L Standard - PFHS (Standard)399.0/80.0 amu - sample 4 of 52 from 021405C.iruiff Area: 16628 counts Height: 1380. cps RT: 10.5 min 10.46 Exygen Research Page 28 of 100 Interim Report #2-Analysis o f Surface Water Samples Exygen Study No.: P0001131 Figure 7. PFHS in Reagent Water, 500 ng/L Fortified Reagent Water, and 5000 ng/L (DF=10) Fortified Reagent Water, Respectively I Reagent Contro/ A - PFHS (Uttnown) 399.0/9M amu -sample 9 o f 52 from 021405G.mff (peak not found) (ft CL O 20 10.45 (ft* 0.63-JD.se 2.90 -3.44>1.32 6.81-.7.2_8 8.23 10.08. c -4*_A . !a -- ^ /l-- tyS-- ft 12.98^ 13.5^.13.88 15,03 ^16.69 4c-* 10 12 14 16 Time, min I Reagent Spike A, 500 ng/L - PFHS (QC) 399.0/80.0 amu sample 10 of 52 from 021405C .wifi Area: 94857 counts Height: 7770. ops RT: 10.5 min (A Q. o 10.46 (0 JLwc 10.03. 8 10 12 14 16 Time, min I Reagent Spike B, 5000 ng/L. DF=10 PFHS (QC) 399.0/80.0 amu - sample 11 of 52 from 021405C.wiff Area: 80963 counts Height: 6530. ops RT: 10.4 min co CL 5000 4<c0c-}^o 0 10.43 X10.00. 8 10 12 14 16 Time, min Exygen Research Page 29 of 100 Interim Report #2-Analysis o f Surface Water Samples Exygen Study No.: P0001131 Figure 8. Chromatogram Representing a Surface Water Sample Analyzed for PFHS (Exygen ID: C00522831, Data Set: 021405C) I C0052282 - PFHS (Unknown) 399.0/89.0 amu -ample 15 o f 52 from 021405C.wiff A n a: 1974counts Height: 56.3 cps RT: 10.5 mitt 0.57 Intensity, cps Exygen Research Page 30 of 100 Interim Report #2-Analysis o f Surface Water Samples Exygen Study No.: P0001131 Figure 9. Typical Calibration Curve for PFOS in Water 021405C rev1 .rdb (PFOS): "Unear'' Regression ('1 / x" weighting): y * 162 x + 3.07e+003 (r = 0.9978) Area, counts Exygen Research Page 31 o f 100 Interim Report #2-Analysis of Surface Water Samples Exygen Study No. : P0001131 Figure 10. Extracted Standards of PFOS in Water, 50 ng/L and 100 ng/L, Respectively I XC021105-2, SOng/L Standard - PFOS (Standard) 499.M0.0 amn -sample 3 o f 52 from 02140SC.wiff A n a: 9931 counts Height: 505. cps RT: 12.6 min 12.60 Intensity, cps Time, min XC021105-3, 100 ng/L Standard - PFOS (Standard)409.0/80.0 amu - sample 4 of 52 from 021405C.wiff Area: 16106 counts Height: 925. cps RT: 12.6 min 12.62 Intensity, cps Exygen Research Page 32 of 100 Interim Report #2-Analysis o f Surface Water Samples Exygen Study No.: P0001131 Figure 11. PFOS in Reagent Water, 500 ng/L Fortified Reagent Water, and 5000 ng/L (DF=10) Fortified Reagent Water, Respectively Reagent Control A - PFOS {Unknown) 499.0/80.0 amu -sample 9 o f 52 from 021405C.wiff Area: 3107 counts Height: 5&6cps RT: 12.6min (O Qo. 12.64 &'CO c0) 50 0 5.35-1-.-17---r 3.55 >1.06 0.31 12.35^1 8.71 v 10.14^10.66 _ ^ 14.15 _ ,, 4 5 t-15.61^16.73 2 4 6 8 10 12 14 16 Time, min Reagent Spike A, 500 ng/L - PFOS (QC) 400.0/80.0 amu-sam ple 10 of 52 from 021405C.uviff I Area: 78386 counts Height: 5080. ops RT: 12.6 min o> Q. U 5000-1 &to 0- - I ------------- -------------- r 46 12.62 12.18.. I ---- -- i-,------------------, --------- ,-------------------- r- 8 10 12 14 16 Time, min I Reagent Spike B. 5000 ng/L, DF=10- PFOS (00 )4 6 9 .0 /8 0 .0 amu-sam ple 11 of 52 from 021405C.wiff Area: 72672 counts Height: 4660. cps RT: 12.6 min CO Q . 12.62 &<0 c0) o C. --r-- 8 10 12 14 16 Time, min Exygen Research Page 33 of 100 Interim Report #2-Analysis o f Surface Water Samples Exygen Study No.: P0001131 Figure 12. Chromatogram Representing a Surface Water Sample Analyzed for PFOS (Exygen ID: C0052282, Data Set: 021405C) I C0052282 - PFOS (Unknown) 499.0/90.0 ama -sample 15 o f 52 from 021405C.wiff Area: 3595 counts Height: 99.5 cps RT: 12,7 min Intensity, cps Time, min 17.9K A_aJ Exygen Research Page 34 of 100 Interim Report #2-Analysis o f Surface Water Samples Exygen Study No.: P0001131 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" Exygen Research Page 35 of 100 Interim Report #2-Analysis o f Surface Water Samples Exygen Study No.: P0001131 E x y g e n P ro to c o l N u m b e r: POOO1131 STUDY PROTOCOL Study Title: Analysis of Perfluorobutanesulfonate (PFBS), Perfluorohexanesulfonate (PFHS), and Perfluorooctanesulfonate (PFOS) in Water, Soil, Sediment, Fish, Clams, Vegetation, Small Mammal Liver and Small Mammal Serum Using LC/MS/MS for the 3M Decatur Monitoring Program Exygen Protocol Number: P0001131 Performing Laboratory: Exygen Research 3058 Research Drive State College, PA 16801 Phone:(814)272-1039 Sponsor Representative: M ichael A . Santoro Director o f Regulatory Affairs 3M Building 0236-01-B-10 St. Paul, M N 55144 Phone: (651) 733-6374 Page / oj 65 Exygen Research Page 36 of 100 Interim Report #2-Analysis of Surface Water Samples Exygen Study No.: P0001131 E x y g e n P ro to c o l N u m b e r: P 0 0 0 1131 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 37 of 100 Interim Report #2-Analysis of Surface Water Samples Exygen Study No.: P0001131 E x y g e n P ro to c o l N u m b e r: P 0 0 0 1 131 PROTOCOL APPROVAL Study Title: Analysis o f Perfluorobutanesulfonate (PFBS), Perfluorohexanesulfonate (PFHS), and Periluorooctanesulfonate (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 JaisimhaKesan, Study Director Weston Solutions Michael A. 3M Comparfy Sponsor Representative ohn M. Flaherty, Principal Investigator Exygen Research Richard A. Gi Exygen Resei ident. Facility Management U.Z LydfitShaffer, Technioaiead, Quality Assurance Unit Exgen Research m Date 2$ -Ccr-KZ^ Date / t Dwatj Page 3 o f 65 Exygen Research Page 38 of 100 Interim Report #2-Analysis of Surface Water Samples Exygen Study No.: P0001131 E x y g e n P ro to c o l N u m b e r: P 0 0 0 1131 TABLE OF CONTENTS TITLE PAGE...................................................................................................................................................... 1 DISTRIBUTION................................................................................................................................................. 2 PROTOCOL APPROVAL..................................................................................................................................3 TABLE OF CONTENTS....................................................................................................................................4 INTRODUCTION............................................................................................................................................... 5 TEST MATERIALS...........................................................................................................................................5 OBJECTIVE....................................................................................................................................................... 6 TESTING FACILITY.........................................................................................................................................6 STUDY DIRECTOR...........................................................................................................................................7 SPONSOR REPRESENTATIVE........................................................................................................................7 PRINCIPAL INVESTIGATOR..........................................................................................................................7 PROPOSED EXPERIMENTAL START AND TERMINATION DATES...................................................... 7 IDENTIFICATION AND JUSTIFICATION OF THE TEST SYSTEM.......................................................... 8 SAMPLE PROCUREMENT, RECEIPT AND RETENTION.......................................................................... 8 SAMPLE IDENTIFICATION............................................................................................................................9 ANALYTICAL PROCEDURE SUMMARY.................................................................................................... 9 VERIFICATION OF ANALYTICAL PROCEDURE....................................................................................... 9 METHOD FOR CONTROL OF BIAS...............................................................................................................11 STATISTICAL METHODS............................................................................................................................... 11 GLP STATEMENT............................................................................................................................................ 11 REPORT............................................................................................................................................................. 11 SAFETY AND HEALTH................................................................................................................................... 12 AMENDMENTS TO PROTOCOL.................................................................................................................... 13 DATA RECORD KEEPING.............................................................................................................................. 13 QUALITY ASSURANCE.................................................................................................................................. 14 RETENTION OF DATA AND ARCHIVING................................................................................................... 14 APPENDIX I, ANALYTICAL METHODS....................................................................................................... 15 Page 4 o f 65 Exygen Research Page 39 of 100 Interim Report #2-Analysis o f Surface Water Samples Exygen Study N o.: P0001131 E x y g e n P ro to c o l N u m b e r: 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 (CiF^SOj'K*) Lot Number: 101 Purity: 96.7% Transitions Monitored: 299 - 99 Structure: PFHS Chemical Name: Perfluorohexanesulfonate Molecular Weight: 438 supplied as the potassium salt (CsFijSOjTi*) Lot Number: SE036 Purity: 98.6% Transitions Monitored: 399 - 80 Structure: Page 5 o f 6} Exygen Research Page 40 of 100 Interim Report #2-Analysis o f Surface Water Samples Exygen Study No. : P0001131 Exygen Protocol Number: P0001131 PFOS Chemical Name: Perfluorooctanesulfonate Molecular Weight: 538 supplied as the potassium salt (C8F|7S03TC+) 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), perfluorohexanesuifonate (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 of 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 41 of 100 Interim Report #2-Analysis o f Surface Water Samples Exygen Study No.: P0001131 E x y g e n P ro to c o l N u m b e r: P 0 0 0 1131 STUDY DIRECTOR Jaisimha Kesari P.E., DEE Weston Solutions, Inc. 1400 Weston Way West Chester, PA 19380 Phone: (610) 701-3761 F a x :(610) 701-7401 j .kesari@westonsolutions.com SPONSOR REPRESENTATIVE Michael A. Santoro 3M Company Director o f Regulatory Affairs 3M Building 0236-01-B-10 St. Paul, MN 55144 Phone: (651) 733-6374 PRINCIPAL INVESTIGATOR John M. Flaherty Exygen Research 3058 Research Drive State College, PA 16801 Phone: (814)272-1039 john.flaherty@exygen.com PROPOSED EXPERIMENTAL START AND TERMINATION DATES It is proposed that the analytical portion o f this study be conducted from October 01, 2004 to December 31, 2005. The actual experimental start and term ination dates w ill be included in the final report. Page 7 o f 65 Exygen Research Page 42 of 100 Interim Report #2-Analysis of Surface Water Samples Exygen Study No.: P0001131 E x y g e n P ro to c o l N u m b e r: P 0 0 0 1 131 IDENTIFICATION AND JUSTIFICATION OF THE TEST SYSTEM The following are the test systems for this study: Water (groundwater and surface water) Soil Sediment Fish Clams Vegetation Small Mammal Liver Small Mammal Serum The samples will be collected by Weston Solutions. The control samples will be purchased and prepared by the testing facility. Purchase and processing details for the control samples will be included in the final report associated with this study. The test systems were chosen to access the environmental impact 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 -10C. The receipt and processing o f the samples will be documented in the final report and raw data associated with the study. Page 8 o f 65 Exygen Research Page 43 of 100 Interim Report #2-Analysis o f Surface Water Samples Exygen Study No.: P0001131 E x y g e n P ro to c o l N u m b e r: P 0 0 0 1 131 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 preclean! Sci/Spec Premier wide mouth HDPE bottles. These bottles have been routinely used for fluorochemical sample Page 9 o f 65 Exygen Research Page 44 of 100 Interim Report #2-Analysis of Surface Water Samples Exygen Study No.: P0001131 E x y g e n P ro to c o l N u m b e r: P 0 0 0 1131 . 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 f i l i 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 (l3C 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 500ng/L 5000 ng/L Soil 4 ng/g 40 ng/g S edim ent 4 ng/g 40 ng/g Fish 10 ng/g 100 ng/g 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 lOng/mL 100 ng/mL Page 10 o f 65 Exygen Research Page 45 of 100 Interim Report #2-Analysis o f Surface Water Samples Exygen Study No.: P0001131 E x y g e n P ro to c o l N u m b e r: P 0 0 0 1 131 Recoveries are anticipated to be between 70% and 130% of the fortified levels; however, the exact precision and accuracy will be determined by the analysis o f the quality control samples described above. A statement of accuracy will be included in the final report. METHOD FOR CONTROL OF BIAS Control o f bias will be addressed by taking representative sub-samples from a homogeneous mixture o f each matrix from untreated control samples, and by analyzing at least two levels o f fortifications. STATISTICAL METHODS Statistics will be limited to those specified in the subject methods and to the calculation o f average recoveries, as applicable. 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 the Study Director, Sponsor Representative, and o f the testing facility. A statement o f GLP compliance (any related documentation, such as chain-of-custody records, must be in the study records). Page l o f 65 Exygen Research Page 46 of 100 Interim Report #2-Analysis of Surface Water Samples Exygen Study No.: P0001131 E x y g e n P ro to c o l N u m b e r: P 0 0 0 1 131 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 of personnel and the environment to the test or reference substance(s). Page 12 o f 62 Exygen Research Page 47 of 100 Interim Report #2-Analysis of Surface Water Samples Exygen Study No.: P0001131 E x y g e n P ro to c o l N u m b e r: P 0 0 0 1131 AMENDMENTS TO PROTOCOL All significant changes to the analytical protocol outlined here will be expressed in writing, signed and dated by the Study Director and Sponsor Representative. Amendments usually will be issued prior to initiation o f study plan change. However, when a change is required without sufficient time for the issue o f a written amendment, that change may be effected verbally with supporting documentation signed and dated by the Study Director and followed with a written amendment as soon as possible. In this case, the effective date o f the written amendment will be the date o f the documented change. Copies o f the signed amendments will be appended to all distributed study plan copies. The original amendment will be maintained with the original study plan. Any deviations from the study plan or from the analytical method as provided will be documented and reported promptly to the Sponsor Representative. DATA RECORD KEEPING Records to be maintained include the following (as appropriate): Sample tracking sheet(s) Sample receipt records, storage history, and chains o f custody History and preparation o f standards (stock, fortification, calibration) Description of any modifications to the method Instrument run sheets, bench-sheets or logs Analytical data tables All chromatographic and instrumental conditions Sample extraction and analysis dates A complete listing o f study personnel, signatures and initials Chronological presentation o f all study correspondence Any other documentation necessary for the reconstruction o f the study Chromatograms- All chromatograms will contain the following: Sample identification, injection date, arrow or other indication o f the area o f interest, and injection number corresponding to the run. Additionally, fortifications will include the amount o f analyte added and the sample number o f the sample that was fortified. Analytical standard chromatograms will additionally include the concentration (e.g., pg/mL). Page 13 o f 65 Exygen Research Page 48 of 100 Interim Report #2-Analysis of Surface Water Samples Exygen Study No.: P0001131 E x y g e n P ro to c o l N u m b e r: POOO1131 As part of the documentation the following sheets will be included in each analytical set: a run sheet listing the samples to be run in the set, and an instrument conditions sheet describing the instrument type and operating conditions. 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 49 of 100 Interim Report #2-Analysis o f Surface Water Samples Exygen Study No.: P0001131 E x y g e n P ro to c o l N u m b e r: P 0 0 0 1131 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: `M ethod 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: `M ethod o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Vegetation by LC/MS/MS" V0001785: "Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Small Mammal Liver by LC/MS/MS" V0001786: "Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Small Mammal Serum by LC/MS/MS" Exygen Research Page IS o f 65 Page 50 of 100 Interim Report #2-Analysis of Surface Water Samples Exygen Study No.: P0001131 E x y g e n P ro to c o l N u m b e r; POOOt 131 ANALYTICAL METHOD M ethod Num ber: V 0001780 Method o f Analysis for the Determination o f Ferflaorooctaooic A d d (PFOA) la Water by LC/MS/MS A n a lytica l Teeting F a c ility : Exygen Research 3058 Research D riv e State C ollege, P A 16801 A pproved B y: JT vU c O L ______ Paul C o nn o lly ' Technical Leader, LC -M S , E xygen Research ?l/r?/hL zf /J''Joohhnn F la hheorltyy / ' VViice* fPtrruisitidaanilt, OrkpueMrattiinonnsi , Exygen Research ioIx-wA)/ D a te Exygen Research T o ta l Pages: 7 Page 6 o f 65 Page 51 o f 100 Interim Report #2-Analysis of Surface Water Samples Exygen Study No.: P0001131 Exygen Protocol Number: P000113) Exygta Research Method Number V0001780 I A N A L Y T IC A L m e t h o d M ethod o f A na lysis fo r the D eterm ination o f P erfluorooctanoic A c id (P F O A ) in W ater by LC /M S /M S 1.0 Scope T h u m ethod is to be em ployed fo r the iso la tio n and q u a n tita tio n o f p erfluorooctanoic a dd b y H igh Perform ance L iq u id C hrom atography coupled to a tandem Mass S poctrom etric D etector (LC /M S /M S ) in w ater. 2.0 S afety 2.1 A lw ays observe safe laboratory practices. 2.2 C onsult the appropriate M SDS before h a n dlin g any chem ical fo r proper safety precautions. 3.0 Sam ple Requirem ent 3.1 A t least 40 m L o f test sample fo r e xtractio n . 3.2 N o sample processing is needed fo r w ater sam ples. 3.3 Samples stored refrigerated should be allo w ed to e qu ilib ra te to room tem perature. 3.4 A ll samples m ust be tho ro u gh ly m ixed before being sam pled fo r e xtraction 3.3 A n y samples containing p articles should be ce ntrifu g ed at -3 0 0 0 rpm fo r -5 m inutes and the supernatant used fo r the e xtractio n . 3.6 Sam ple co lle ctio n procedures w ill be sp ecifie d in the sam pling plan fo r this p ro je ct 4 .0 Reagents and Standards 4.1 W ater - H PLC grade 4.2 M ethanol - H PLC grade 4.3 A m m onium Acetate - A .C .S . Reagent G rade 4.4 P erfluorooctanoic A d d - Sigm a*A ld ric h 3.0 Instrum ent and E quipm ent j. l 3.2 3.3 3 .4 3.5 3 .6 5 .7 3 .8 5 .9 5 .1 0 5.11 A high perform ance liq u id chrom atograph capable o f pum ping up to 2 solventa equipped w ith a va ria ble volum e in je c to r capable o f in je ctin g 3*200 p L connected to a tandem Mass Spectrom eter (L C /M S /M S ). A device to co lle ct raw data fo r peak in te g ra tio n and q ua ntita tion . A n a lytica l balance capable o f reading to 0.00001 g. 30 m L disposable polypropylene eentrifU ge tubes. 15 m L disposable polypropylene ce ntrifu g e tubes. Disposable m icropipets (S 0*100uL, 100*200uL). 125*m L LDPE narrow *m outh bottles. 2 m L cle ar H P LC v ia l k it. Disposable pipettes. A u to p ip e tte t (100*1000 p L and 10-100 p L ), w ith disposable tip s. W aters Sep Pak Vac 6 cc (tg ) tC IS SPE cartridges. Pase2of? Page 7 o f 65 Exygen Research Page 52 of 100 Interim Report #2-Analysis o f Surface Water Samples Exygen Study No.: P0001131 E x y g e n P ro to c o l N u m b e r: POOO1131 Exygen Research Method Number V0001780 A N A L Y T IC A L m e t h o d M ethod o f A na lysis fo r the D eterm ination o fP e rflu o ro o cta n o ic A c id (P FO A ) in W ater by U V M S /M S 5.12 SPE vacuum m anifold. 5.13 C entrifU ge capable o f spinning 50 m L p olyp ro p ylen e tubes at 3000 rpm . 6.0 Chrom atographic System 6.1 A n a lytica l C olum n: Fluophase RP (K eystone S c ie n tific ), 2.1 mm x 50 mm , Sp (P /N : 82505-052130) 6.2 Tem perature: 30*C 6.3 M o b ile Phase (A ) : 2 m M A m m onium A cetate in W ater 6.4 M o b ile Phase < B ): M ethanol 6.5 G radient Program : Tim e (m in i 0.0 1.0 8 .0 20.0 22.5 %A 65 65 25 25 65 F lo w Rate & S fm L /m in t 35 0.3 35 0.3 75 0.3 75 0.3 35 0.3 6.6 In je ctio n V olum e: 15 p L (can be increased to as m uch as 50 p L ). 6.7 Q uantitation: Peak A rea - e xternal standard ca lib ra tio n curve. 6.8 R unT im e: - 2 3 m inutes. The above co nd itio ns are intended as a g uid e and m ay be changed in order to o ptim ize the H P LC system. 7.0 M S /M S System 7.1 M ode: E lectrospray N egative M R M mode, m o n ito rin g 413 - * 369 m /z. The above co nd itio ns are intended as g uid e and m ay be changed tn order to o ptim ize the M SM S system . 8.0 P reparation o f S olutions 8.1 M o b ile Phase 8.1.1 2 m M am m onium acetate in w ater is prepared b y adding 0.154 g o f am m onium acetate to 1000 m L o f w ater. A lternate volum es m ay be prepared. Page ui Page 8 o f 65 Exygen Research Page 53 of 100 Interim Report #2-Analysis o f Surface Water Samples Exygen Study No.: P0001131 E x y g e n P ro to c o l N u m b e r: POOO1131 Exygea lUtearch Method Number VOOOI7SO ANALYTICAL METHOD M ethod o f A na lysis fo r the D eterm ination o f P erfluorooctanoic A c id (PFO A ) in W ater by LC /M S /M S 9.0 Standard Preparation 9.1 Standard S to clc/F o rtifica tion SohUion 9.1.1 Prepare a stock so lutio n o f ~ 100 p g/m L o f PFO A b y w e ig hin g 10 mg o f a na lytica l standard (corrected fo r p u rity ) and d ilu te to 100 m L w ith m ethanol in a 12S-m L LD PE b o ttle . 9.1.2 A 10 fig /m L fo rtific a tio n so lu tio n o f PFO A is prepared b y b rin g in g 10 m L o f the 100 jig /m L so lu tio n to a fin a l volum e o f 100 w ith methanol in a 125 m L LD PE b o ttle . 9.1.3 A 1.0 p ^ m L fo rtific a tio n so lu tio n o f PFO A is prepared by b rin ging to m L o f the 10 p g/m L so lu tio n to a fin a l volum e o f 100 w ith methanol in a 125 m L LD P E b ottle . 9.1.4 A 0 .1 p g/m L fo rtific a tio n so lu tio n o f PFO A is prepared b y b rin g in g 10 m L o ftb e 1.0 p y fa L so lu tio n to a fin a l volum e o f 100 w ith methanol in a 125 m L LDPE b o ttle . 9.1.5 A 0.01 p g fa iL fo rtific a tio n so lu tio n o f P FO A is prepared b y b rin g in g 10 m L o f the 0.1 p g /m L so lu tio n to a fin a l volum e o f 100 w ith methanol in a 125 m L LD P E b o ttle . 9.1.6 The stock and fo rtific a tio n so lutio n s are to be stored in a re frig e ra to r at approxim ately 4C and are stable fo r a m axim um period o f 6 months fro m the date o f preparation. 9.2 Standard C a lib ra tion S olutions 9.2.1 9 .2 .2 L O M S /M S ca lib ra tio n standards are prepared in H P LC water. The ca lib ra tio n standards are processed through the e xtractio n procedure, id e n tica l to samples. The fo llo w in g is a ty p ic a l exam ple: a d d itio n a l concentrations m ay be prepared as needed. Concentration o f F ortification Solution ioob 0 10 10 10 100 100 100 F ortification Volume o f Volume F ortified Control (P L) Sanale (m L) 0 40 100 40 200 40 400 40 100 40 200 40 400 40 F in a l Concentration o f C alibration Standard (n o t)* 0 25 50 100 250 500 1000 C a lib ra tion Standard ID (exam ple) X C m raddyy-0 XC m m ddyy'l XC m m ddyy-2 XCm m ddyy-3 X C m m ddyy-4 X C m m ddyy-5 X C m m ddyy-6 * The extracted concentration o f the ca lib ra tio n standard is equal to 8x its in itia l concentration, due to the concentration o f the standard d uring the e xtractio n (SPE). X C extracted ca lib ra tio n standard. Page4 of ' Page 19 o f 65 Exygen Research Page 54 of 100 Interim Report #2-Analysis o f Surface Water Samples Exygen Study No.: P0001131 E x y g e n P ro to c o l N u m b : P 0 0 0 1131 Exygen Remrch Method Number V00017S0 ANALYTICAL METHOD M ethod o f A nalysis fo r the D eterm ination o fP e rflu o ro o ctsn o ic A c id (P FO A ) in W ater by LC /M S /M S 9 .2 .3 9 .2 .4 9 .2 .5 A z o o standard so lutio n (reagent b la n k) m ust be prepared w ith each set o f standards extracted. Store a ll extracted ca lib ra tio n standards in 15 -m L polypropylene tubes at 2*C to 6C , up to tw o weeks. A lternate volum es and concentrations o f standards m ay be prepared as needed. 10.0 B atch Set U p 10.1 Each batch o f camples extracted (ty p ic a lly 20 o r less) m ust include at least one reagent co n tro l (m ethod b lank using H P LC w ater) and tw o reagent controls fo rtifie d st know n concentrations (la b co n tro l spike) to v e rily procedural recovery fo r the batch. 10.2 Requirem ents fo r fie ld and laboratory duplicates and spikes w ill be specified in d ie q u a lity assurance p lan fo r th is p ro ject. 11.0 Sam ple E xtraction 11.1 M easure 40 m L o f sam ple o r a p o rtio n o f sam ple d ilu te d to 40 m L w ith w ater in to 50 m L polypropylene c e n trifiig e tubes (fo rtify as needed, replace lid and m ix w e ll). 11.2 C o nd itio n the C u SPE cartridges (1 g, 6 m L ) b y passing 10 m L m ethanol fo llo w e d b y 5 m L o f H P LC w ater (~ 2 drop/sec). D o not le t colum n run dry 11.3 Load sam ple o n conditioned C u S P E cartridge. D iscard eluate. 11.4 E lu te w ith -5 m L 100% m ethanol. C o llo ct 5 m L o f eluate in to graduated 15 m L polypropylene ce n trifiig e tubes (fin a l volum e * 5 m L ). 11.5 A nalyze samples u sing electrospray LC /M S /M S . 12.0 Chrom atography 12.1 In je c t the same am ount o f each standard, sam ple and fo rtifie d sample in to the LC /M S /M S system . A ca lib ra tio n standard m ust precede and fo llo w a ll analyzed samples. 12.2 Standards o f PFO A corresponding to a t least fiv e o r m ore concentration levels m ust be included in an a na lytica l le t 12.3 A n e ntire set o f extracted c a lib ra tio n standards m ust be included at the beginning and at the end o f a sam ple set. E xtracted standards must he interspersed between every 5-10 sam ples. A s an a lte rn a tive , an e ntire set o f extracted ca lib ra tio n standards m ay be in je cte d at the b eginning o f a sei fo llo w e d b y extracted ca lib ra tio n standards interspersed every 5-10 samples (to account fo r a sooond set o f extracted standards). In e ith e r case, extracted ca lib ra tio n standards m ust be the firs t and last in je c tio n in a sam ple set. 12.4 Use lin e a r standard curves fo r q ua ntita tion . L ine a r standard curves are generated fo r the analyte by lin e a r regression u sing 1/x w e ig htin g o f peak area Pag $ of 7 Page 20 o f 65 Exygen Research Page 55 of 100 Interim Report #2-Analysis of Surface Water Samples Exygen Study No.: P0001131 E x y g e n P ro to c o l N u m b e r: P 0 0 0 1131 Exygea R m uch Method Number V0001780 | A N A L Y T IC A L M E T H O D ......... | M ethod o f A na lysis fo r the D eterm ination o fP e rflu o ro o cta n o ic A c id (P FO A ) in W ater by LC YM S/M S venue ca lib ra tio n standard concentration using M a tsL yn x 3.3 (o r equivalent) softw are system. 12.5 Sam ple response should n ot exceed standard responses. A n y samples that exceed standard responses should be A nther d ilu te d and reanalyzed. 13.0 Acceptance C rite ria 13.1 13.2 13.3 13.4 13.5 13.6 Chrom atogram m ust show a peak o f a daughter io n at 369 am u from a parent o f 413 amu. The 413 amu parent corresponds to the PFO A anion, w h ile the daughter io n (369 am u) represents the loss o f carbon d io xid e . M ethod blanks m ust not contain PFO A at levels greater than the LO Q . I f a blank contains PFO A s! levels p e s te r than 50 n g /L , then a new blank sample m ust be obtained and the e n tire set m ust be re-extracted. Recoveries o f co n tro l spikes and m a trix spikes m ust be between 70-130% o f th e ir know n values. I f a co n tro l spike fa lls outside the acceptable lim its , the e ntire set o f samples should be re-extracted. A n y m a trix spike outside 70 130% should be evaluated b y the analyst to determ ine i f re -e xtra ction is w a rranted. A ny ca lib ra tio n standard found to be a s ta tis tic a l o u tlie r b y using the Huge E rro r Test, m ay be excluded fro m the ca lcu la tio n o f the ca lib ra tio n curve. H ow ever, the to ta l num ber o f extracted ca lib ra tio n standards tha t could be excluded m ust not exceed 20% o f the to ta l num ber o f extracted standards iq je c te d . The co rre la tion c o e fficie n t (R ) fo r c a lib ra tio n curves generated m ust be 0.992 (R 2 0.985). I f ca lib ra tio n results fa ll o utside these lim its , then appropriate steps m ust be taken to adjust instrum ent operation, and the standards o r the relevsnt set o f sam ples should be reanalyzed. R etention tim es between standards and samples m ust not d rift more than 4 % w ith in an a na lytica l run. I f M e n tio n tim e d rift exceeds th is lim it w ith in an a na lytica l run then the set m ust be reanalyzed. 14.0 C alculations 14.1 Use the fo llo w in g equation to calculate the am ount o f P FO A found (in n g/L, based cm peak area) using the standard curve (lin e a r regression param eters) generated b y the Mass L yn x softw are program : PFO A found (n g /L ) - (Peak area - in te rce p t) x DF s lo p e DF fa cto r b y w hich the fin a l volum e was d ilu te d , i f necessary. Page 6 of 7 Page 21 o f 65 Exygen Research Page 56 of 100 Interim Report #2-Analysis o f Surface Water Samples Exygen Study No.: P0001131 E x y g e n P ro to c o l N u m b e r: P 0 0 0 1 13 1 Exygntowaieh Mttbod Number V0001780 | A N A L Y T IC A L M E T H O D ~| M ethod o f A na lysis fo r the D e te rm in itio n o f P erfluorooctanoic A c id (P FO A ) in W ater by LC /M S /M S 14.2 F o r samples fo rtifie d w ith know n am ounts o f PFO A p rio r to extraction, use the fo llo w in g equation to calculate the percent recovery. R ecovery (%) - [ to ta l analyte found (n g /L ) - analyte found in co n tro l (n g /L )] analyte added (n g /L ) 1 Exygen Research Page 7 of 7 Page 22 o f 65 Page 57 o f 1 0 0 Interim Report #2-Analysis o f Surface Water Samples Exygen Study No.: P0001131 E x y g e n P ro to c o l N u m b e r: P 0 0 0 1 131 ANALYTICAL METHOD M ethod N u m b er V0001781 M e tho d o f A n a lysis fo r d ie D e te rm in a tio n o f P e rflu o ro o cta n o fc A c id (P F O A ) in S o il by L C /M S /M S A n a lytica l T a tin g F a c ility : E xygen Research 3058 Research D rive State C ollege. P A 16801 A pproved B y: "v iA C -Ji. P aul C o nn o lly ' Technical Leader, L C -M S , Exygen Research D ate D ate Exygen Research T o ta l Pages: 7 Page 23 o f 65 Page 58 of 100 Interim Report #2-Analysis of Surface Water Samples Exygen Study No.: P0001131 E x y g e n P ro to c o l N u m b e r: POOO1131 Exygto ReMtrcli Method Number V0001781 ANALYTICAL METHOD M ethod o f A nalysis fo r the D eterm ination o f P erfluo ro octa no ic A c id (P FO A ) in S o il by LC /M S /M S 1.0 Scope T h u m ethod i i to be em ployed fo r the iso la tio n and q ua n tita tio n o f perfluorooctanoic a d d b y H ig h Perform ance liq u id C hrom atography coupled to a tandem Mass S pectrom etric D etector (L C /M S /M S ) in s o il. 2.0 S afety 2.1 A lw ays observe safe laboratory practices. 2.2 C onsult the appropriate M SDS before handling any chem ical fo r proper safety precautions. 3.0 Sam ple Requirem ent 3.1 A t least 15 g o f test sam ple fo r extraction. 3.2 N o sample processing is needed fo r s o il samples. 3.3 Samples stored refrigerated should be a llo w ed to e q u ilib ra te to room tem perature. 3.4 A ll samples m ust be thoroughly m ixed before being sam pled fo r extraction. 3.5 Sam ple co lle ctio n procedures w ill be sp ecifie d in the sam pling p lan fo r this p ro ject. 4.0 Reagents and Standards 4.1 W a te r-H P L C grade 4.2 M ethanol - H P LC grade 4.3 A m m onium A cetate -A .C .S . Reagent Grade 4.4 P erfluorooctanoic A cid - S igm a*A ldrich 5.0 Instrum ent and E quipm ent 5.1 5 .2 5.3 5 .4 5.5 5 .6 5.7 5.8 5 .9 5 .1 0 5.11 5.12 5.13 A h igh perform ance liq u id chrom atograph capable o f pum ping up to 2 solvents equipped w ith a variable volum e in je c to r capable o f in je ctin g 5-200 p L connected to a tandem M ia s Spectrom eter (L C /M S /M S ). A device to c o lle ct raw data fo r peak in te g ra tio n and q ua ntita tion . A n a lytica l balance capable o f reading to 0.00001 g. 50 m L disposable polypropylene c e n trifiig e tubes. 15 m L disposable polypropylene c e n trifiig e tubes. Disposable m icropipets (50-lO O uL, 100-200uL). 125-m L LD P E narrow -m outh b ottles. 2 m L cle ar H P LC v ia l k it. D isposable pipettes. A utopipettes (100-1000 jaL and 10*100 p L ), w ith disposable tip s. W aters Sep Pak Vac 6 ce (lg) tCIS SPB cartridges. SPE vacuum m a n ifold . U ltraso nic bath. Pag 2 or? Page 24 o f 65 Exygen Research Page 59 of 100 Interim Report #2-Analysis o f Surface Water Samples Exygen Study No.: P0001131 E x y g e n P ro to c o l N u m b e r: POOO1131 Exygea Rwcwch Method NumbnVOOO1781 ANALYTICAL METHOD M ethod o f A na lysis fo r the D eterm ination o fP e rflu o ro o cta n o ic A c id (P F O A ) in S o il by LC /M S /M S ] 5.14 W rist-a ction shaker. 5.15 C entrifuge capable o f apinning SOm L p olypropylene tubes at 5000 rpm . 6.0 Chrom atographic System 6.1 A n a lytica l C olum n: Fluophaae RP (K eystone S c ie n tific ), 2.1 m m x SO mm. 5p (P /N : 82305-052130) 6.2 Tem perature: 30*C 6.3 M o b ile Phase ( A ) : 2 m M A m m onium A cetate in W ater M o b ile Phase (B j : M ethanol G radient Program : Tim e fm in l 0.0 1.0 8.0 20.0 22.5 %A 65 65 25 25 65 F lo w Rate % B fm L /m in l 35 0.3 35 0.3 75 0.3 75 0.3 35 0.3 6.6 In je ctio n V olum e: 15 p L (can be increased to as m uch as SO p L ). 6.7 Q uantitation: Peak A rea -e x te rn a l standard c a lib ra tio n curve. 6.8 R unT im e: - 2 3 m inutes. The above conditions are intended as a g uide and m ay be changed in order to optim ize the H P LC system. 7.0 M S /M S System 7.1 M ode: E lectrospray N egative M R M m ode, m o n ito rin g 4 1 3 - 369 m /z fo r PFOA. The above conditions are intended as a guide and m ay be changed in order to optim ize the M SM S system . 8.0 Preparation o f S olutions 8.1 M o b ile Phase 8.1.1 2 m M am m onium acetate in w ater is prepared b y adding 0.154 g o f am m onium acetate to 1000 m L o f w ater. A lternate volum es m ay be prepared. Page 3 of 7 Page 25 o f 65 Exygen Research Page 60 of 100 Interim Report #2-Analysis of Surface Water Samples Exygen Study No. : P0001131 E x y g e n P ro to c o l N u m b e r: P 0 0 0 1 131 Exygen Research Method Number VOOOI781 ANALYTICAL METHOD M ethod o f A na lysis fo r the D eterm ination o fP e rflu o ro o cta n o ic A c id (P FO A ) in S o il by LC /M S /M S 9.0 Standard P reparation 9.1 Standard S to ck/F o rtifica tio n S olution 9.1.1 Prepare a stock so lutio n o f ~100 p g/m L o f PFO A by w eighing 10 mg o f a na lytica l standard (corrected fo r p u rity ) and d ilu te to 100 m L w ith m ethanol in l2 5*m L LD P E b o ttle . 9.1.2 A 10 pg/m L fo rtific a tio n so lu tio n o fP F O A is prepared b y b rin g in g 10 m L o f the 100 pg/m L so lutio n to a fin a l volum e o flO O w ith methanol in a 125 m L LO PE b o ttle . 9.1.3 A 1.0 pg/m L fo rtific a tio n so lu tio n o f P FO A is prepared b y b rin ging 10 m L o f the 10 pg/m L so lu tio n to fin a l volum e o f 100 w ith methanol in a 125 m L LD PE b ottle . 9.1.4 A 0 .1 pg/m L fo rtific a tio n so lu tio n o fP F O A is prepared b y b rin g in g 10 m L o f the 1.0 p g/m L s o lu tio n to a fin a l volum e o f 100 w ith methanol in a 125 m L LD P E b ottle . 9.1.5 A 0.01 pg/m L fo rtific a tio n so lu tio n o f P FO A is prepared b y b rin ging 10 m L o f tite 0.1 p g fa iL so lu tio n to a fin a l volum e o f 100 w ith methanol in 125 m L LD P E b o ttle . 9.1.6 The stock and fo rtific a tio n so lutio n s are to be stored in a re frig e ra to r at approxim ately 4*C and are stable fo r a m axim um p eriod o f 6 m onths fro m the date o f preparation. 9.2 Standard C a lib ra tio n S olutions 9.2.1 9 .2 .2 LC /M S /M S ca lib ra tio n standards are prepared in H P LC w ater The ca lib ra tio n standards are processed through the e xtractio n procedure, id en tica l to samples. The fo llo w in g is a ty p ic a l exam ple: a d d itio n a l concentrations may be prepared as needed. F in a l Concentration Fortification Volume o f Concentration o f Calibration o f Fortification Volume F ortified Control C alibration Standard ID Solution (m b) (tiL ) Santole (m L) Standard fo o t)* (examole) 00 10 100 10 200 10 400 100 100 100 200 100 400 40 40 40 40 40 40 40 0 25 50 100 250 500 1000 XCmmddyy*0 XCm m ddyy-1 X Cm m ddyy-2 X Cm m ddyy-3 XCmmddyy*4 X C m m ddyy-5 XCmmddvv6 * The extracted concentration o f the ca lib ra tio n standard is equal to 8x its in itia l concentration, due to the concentration o f the standard d uring th e e xtractio n (SPE). X C extracted c a lib ra tio n standard. Page 4 o f 7 Page 26 o f 65 Exygen Research Page 61 o f 100 Interim Report #2-Analysis o f Surface Water Samples Exygen Study No.: P0001131 E x y g e n P ro to c o l N u m b e r: P 0 0 0 1 131 ExygmfUwvcfe Method Number V00017BI | A N A L Y T IC A L M E T H O D M ethod o f A na lysis fo r the D eterm ination o f P erfluorooctanoic A c id (P FO A ) in S oil by LC /M S /M S 9 .2 .3 9 .2 .4 9 .2 .$ A zero standard so lu tio n (reagent b la n k) m ust be prepared w ith each set o f standards extracted. Store a ll a ttra cted ca lib ra tio n standards in 15-m L polypropylene lubes at 2C to 6*C , up to tw o weeks. A lterna te volum es and concentrations o f standards m ay be prepared as needed. 10.0 B atch Set U p 10.1 Each batch o f samples extracted (ty p ic a lly 20 o r less) m ust include at least one reagent co n tro l (m ethod b lan k using 5 m L o f m ethanol) and tw o reagent controls fo rtifie d at know n concentrations (la b co n tro l spike) to v e rify procedural recovery fo r the batch. 10.2 Requirem ents fo r fie ld and la bo ra tory duplicates and spikes w ill be specified in the q u a lity assurance p lan fo r th is p ro ject. 11.0 Sam ple E xtraction 1l. t 112 11.3 11.4 11.5 11.6 11.7 1l.S 1 1.9 W eigh 5 g o f sample in to 50 m L p olypropylene ce n trifu g e tubes (fo rtify as needed, replace lid and m ix w e ll). A dd 5 m L o f m ethanol and shake on a w ris t a ction shaker fo r -1 5 m inutes. T ransfer the tubes to in u ltra son ic bath and sonicate fo r ~ 15 m inutes. B rin g the volum e up to 40 m L w ith w a ter in the 50 m L polypropylene c e n trifiig e tube. C entrifuge fo r-1 0 m inutes at ~3000 ip m . C o nd itio n the C it SPE cartridges ( ! g , 6 m L ) b y passing 10 m L m ethanol fo llo w e d b y 5 m L o f H PLC w ater ( - 2 d ro p/tec). D o n ot le t colum n run d ry Load (decant) the sample on the conditioned C u SPE cartridge. Discard e lu tte . E lute w ith - 5 m L 100% m ethanol. C o lle ct S m L o f eluate in to graduated 1S m L p olypropylene c e n trifiig e tubes (fin a l volum e - S m L ). A nalyze samples using electrospray LC /M S /M S . 12.0 Chrom atography 12.1 In je c t the same am ount o f each standard, sam ple and fo rtifie d sample in to the LC /M S /M S system . A ca lib ra tio n standard m ust precede and fo llo w a ll analyzed samples. 12.2 Standards o f PFO A corresponding to at least fiv e o r m ore concentration levels m ust be included in an a n a lytica l a c t 12.3 A n e ntire set o f extracted ca lib ra tio n standards m ust be included at the beginning and at the end o f a sam ple s e t E xtracted standards m ust be interspersed between every 5*10 samples. A s an a lte rn a tive , an e ntire set o f pm * s of 7 Page 27 o f 65 Exygen Research Page 62 of 100 Interim Report #2-Analysis o f Surface Water Samples Exygen Study No.: P0001131 E x y g e n P ro to c o l N u m b e r: P 0 0 0 1 131 Exygen Research Method Number VQOO1781 ANALYTICAL METHOD M ethod o f A nalysis fo r the D eterm ination o f P erfluorooctanoic A c id (P FO A ) in S o il by LC /M S /M S extracted ca lib ra tio n atandarda m ay be in je cte d at the beginning o f a set follow ed b y extracted ca lib ra tio n standards interspersed every 5*10 samples (to account fo r a second set o f extracted standards). In e ith e r case, extracted ca lib ra tio n standards m ust be the firs t and la st in je c tio n in a sam ple set. 12.4 Use lin e a r standard curves fo r q ua ntita tion . L ine a r standard curves are generated fo r the analyte b y lin e a r regression u sing l/x w e ig h tin g o f peak area versus ca lib ra tio n standard concentration using M assLynx 3 3 (o r equivalent) softw are system. 12.5 Sam ple response should not exceed standard responses. A n y samples that exceed standard responses should be fu rth e r d ilu te d and reanalyzed. 13.0 Acceptance C rite ria 13.1 13.2 13.3 13.4 13.5 13.6 Chrom atogram m ust show a peak o f a daughter io n at 369 amu from a parent o f 413 am u. The 413 amu parent corresponds to the PFO A anion, w h ile the daughter ton (369 am u) represents the loss o f carbon d io xid e . M ethod blanks m ust not contain PFO A at le vels greater than the LO Q . I f a blank contains PFO A at levels greater than 50 n g /L , then a new blank sample m ust be obtained and the e ntire set m ust be re*extractod. R ecoveries o f co n tro l spikes and m a trix spikes m ust be between 70-130% o f th e ir know n values. I f a co n tro l spike fa lls o utside the acceptable lim its , the e ntire set o f samples should be re-extracted. A n y m a trix spike outside 70 130% should be evaluated b y the analyst to determ ine i f re -e xtra ction is w a rra n te d . A n y ca lib ra tio n standard found to be a s ta tis tic a l o u tlie r b y using the Huge E rro r Test, m ay be excluded fro m the ca lcu la tio n o f the ca lib ra tio n curve. H ow ever, the to ta l num ber o f extracted ca lib ra tio n standards that could be excluded m ust oot exceed 20% o f the to ta l num ber o f extracted standards in je c te d . The co rre la tion co e fficie n t (R ) fo r ca lib ra tio n curves generated must be 20.992 (R 1 20.985). I f ca lib ra tio n results fa ll outside these lim its , then appropriate steps m ust be taken to adjust instrum ent operation, and the standards o r the relevant set o f samples should be reanalyzed. R etention tim es between standards and sam ples m ust n o t d rift m ore than 4 % w ith in an a na lytica l run. I f re ten tio n tim e d rift exceeds th is lim it w ith in an a n a lytica l run then the set m ust be reanalyzed. Page 6 of 7 Page 28 o f 65 Exygen Research Page 63 of 100 Interim Report #2-Analysis o f Surface Water Samples Exygen Study No.: P0001131 E xyg en P rotoco l N u m b er: P 0 0 0 1 131 Exygci Rnearch Method N u n * V00017*I ANALYTICAL METHOD M ethod o f A na lysis fo r the D eterm ination o fP e rflu o ro o cta n o ic A c id (P FO A ) in S o il by LC /M S /M S | 14.0 C alculations 14.1 U ic the fo llo w in g equation to calculate the am ount o f P FO A found (in n g/L , baaed on peak area) uaing the standard curve (lin e a r regression param eters) generated b y the Mass L yn x softw are program : PFO A found (n g /L ) * (Peak area - in te rce p t) x DF slope DF * fa cto r b y w hich the fin a l volum e was d ilu te d , i f necessary. 14.2 F or samples fo rtifie d w ith know n am ounts o f PFO A p rio r to e xtractio n , use the fo llo w in g equation to calculate the percent recovery. R ecovery (% ) [ to ta lanalyte found (n g /L ) analyte found in c o n tro l(n g /L )] . . . analyte added (n g /L ) 14.3 Uae the fo llo w in g equation to convert the am ount o f P FO A found in ng/L to n g /g (p pb ). PFO A found (ppb) - fP F O A found (n g /L ) x volum e extracted (Q.04U1 sam ple w e ig ht (5 g ) 14.4 Use foe fo llo w in g equation to calculate the am ount o f PFO A found in ppb based on d ry w e ig h t PFO A found (p p b) d ry w eight " PFO A found (p p b) x [100% / to ta l solid$(% )] Page 7 of 7 Page 29 o f 65 Exygen Research Page 64 of 100 Interim Report #2-Analysis o f Surface Water Samples Exygen Study No.: P0001131 E xyg en Protoco l N u m b er: P 0 0 0 1131 ANALYTICAL METHOD M ethod Number V0001782 Method o f Analysis for the DetersntioD o f Perfluerooctanoic A d d (PFOA) in Sediment by LC/MS/MS A n a lytica l Testing F a c ility : Exygen Research 3058 Research D rive State C ollege, P A 16801 A pproved B y: C JL P aul C o nn o lly i Technical Leader, LC -M S , Exygen Research &'J/>? d U S /Joioihnn Flaherty f V iecer P resident, O perations, Exygen Research __tokbl<yt D a te dr D ate Exygen Research T o ta l P a ^ a ; 7 Page 30 o f 65 Page 65 of 100 Interim Report #2-Analysis of Surface Water Samples Exygen Study No.: P0001131 E xyg en P rotocol N u m b er: P 0 0 0 1131 Exygen Rsaevnb Method Number VOOOI782 | A N A L Y T IC A L M E T H O D | M ethod o f A na lysis fo r the D eterm ination o f P erfluorooctanoic A c id (P FO A ) in Sedim ent bv LC /M S /M S 1.0 Scope T h is m ethod is to be em ployed fo r foe iso la tio n and q ua n tita tio n o f p erfluorooctanoic acid b y H ig h Perform ance L iq u id C hrom atography coupled to a tandem Mass S pectrom etric D etector (L C /M S /M S ) in sedim ent. 2.0 S afety 2.1 A lw ays observe safe laboratory practice. 2.2 C onsult the appropriate M SDS before h an dlin g any ebem ical fo r proper safety precautions. 3.0 Sam ple Requirem ent 3.1 A t least 30 g o f teat sam ple fo r extraction. 3.2 N o sam ple processing i t needed fo r sedim ent sam ples. 3.3 Sam ples stored refrigerated should be allo w ed to e q u ilib ra te to room tem perature. 3.4 A ll samples m ust be tho ro u gh ly m ixed before being sam pled fo r e xtraction. 3.5 Sam ple co lle ctio n procedures w ill be specified in the sam pling plan fo r this p ro je c t 4.0 Reagents and Standards 4.1 W a te r-H P L C grade 4.2 M e th a n o l-H P L C grade 4.3 A cetic A c id - Reagent grade 4.4 A m m onium Acetate - A .C .S . Reagent Grade 4.5 P erfluorooctanoic A c id - S ig m a-A ldrich 5.0 Instrum ent and E quipm ent 5.1 A h igh perform ance liq u id chrom atograph capable o f pum ping up to 2 solvents equipped w ith a va ria ble volum e iiy e c to r capable o f in je ctin g 5-200 p L connected to a tandem Mass Spectrom eter (L C /M S /M S ). 5.2 A device to c o lle ct raw data fo r peak in te g ra tio n and qua ntita tion . 5.3 A n a lytica l balance capable o f reading to 0.00001 g. 5.4 50 m L disposable p olypropylene c c n triftig e tubes. 5.5 15 m L disposable p olypropylene ce ntrifu g e tubes. 5.6 D isposable m icropipets (50-100uL, 100-200uL). $.7 125-m L LDPE narrow -m outh bottles. 5.8 2 m L clear H P LC v ia l k it. 5.9 D isposable pipettes. 5.10 A utopipettes (100-1000 p L and 10-100 p L ), w ith disposable tip i. S .l I W aters Sep Pak V ac 6 ec (Ig ) tC 18 SPE cartridges. 5.12 SPE vacuum m anifold. Page 2 of 7 Page 3! o f 65 Exygen Research Page 66 of 100 Interim Report #2-Analysis of Surface Water Samples Exygen Study No.: P0001131 E x y g e n P ro to c o l N u m b e r: P 0 0 0 1 131 Exygen Research Method Number VOOOi 782 | A N A L Y T IC A L M E T H O D ......... M ethod o f A na lysis fo r the D eterm ination o f P erfluorooctanoic A cid (P FO A ) in Sedim ent by LC /M S /M S 5.13 5.14 5.15 V ortexer. W rist-a ction shaker. C e n trifiig e capable o f apinning 50 m L p olyp ro p ylen e tubea at 3000 rpm . 6 0 Chrom atographic System 6.1 A n a ly tic a l C olum n: FluophaseR P (K eystone S cie ntific) 2.1 mm x 50 mm , 5m (P /N : 825054)52130) 6.2 Tem perature: 30*C 6.3 M o b ile Pbaae (A ) : 2 m M A m m onium A cetate in W ater 6.4 M o b ile Phase (B ): M ethanol 6.5 G radient Program : Tim e (m in ) 0 .0 1.0 8.0 20.0 22.5 65 65 25 25 65 2LB 35 35 75 75 35 F lo w Rate fm L /m in l 0.3 0.3 0.3 0.3 0.3 6.6 In je ctio n V olum e: 15 p L (can be increased to as m uch as 50 p L ). 6.7 Q uantitation: Peak A rea - external ita nd ard ca lib ra tio n curve. 6.8 R u nT im e : ~ 2 3 m inutes. The above conditions are intended aa a guide and m ay be changed in order to o ptim ize the H PLC system . 7.0 M S /M S System 7.1 M ode: E lectrO tpray N egative M R M m ode, m o n ito rin g 413 -> 369 m /z fo r PFOA. The above co nd itio ns are intended as a guide and m ay be changed in order to optim ize die M SM S system . 8.0 P reparation o f S olutions 8.1 M o b ile Phase 8.1.1 2 m M am m onium acetate in w ater la prepared b y adding 0.154 g o f am m onium acetate to 1000 m L o f w ater. Page 3 of 7 Page 22 o f 65 Exygen Research Page 67 of 100 Interim Report #2-Analysis o f Surface Water Samples Exygen Study No. : P0001131 Exygen Protocol Number: P0001131 Exygen Reteaich Method Number VQ001782 | A N A L Y T IC A L M E T H O D | M ethod o f A na lysis fo r the D eterm ination o f P erfluorooctanoic A c id (P FO A ) in Sedim ent by LC /M S /M S 8.2 E xtractio n S olutions 8.2.1 1% acetic acid in w ater is prepared b y adding 10 m L o f acetic acid to 1000 m L o f w ater. A lternate volum es m ay be prepared. 9.0 Standard P reparation 9.1 Standard S to ck/F o rtifica tio n S olutio n 9.1.1 Prepare a stock so lutio n o f-1 0 0 p g /c n L o f PFO A b y w eighing 10 mg o f a na lytica l standard (corrected fo r p u rity ) and d ilu te to 100 m L w ith m ethanol in a 125-m L LO PE b o ttle . 9.1.2 A 10 pg/m L fo rtific a tio n so lu tio n o f PFO A is prepared b y b rin g in g 10 m L o f the 100 p g /m L so lu tio n to fin a l volum e o f 100 w ith methanol in a 125 m L LOPE b o ttle . 9.1.3 A l .0 pg/m L fo rtific a tio n so lutio n o f PFO A is prepared by b rin g in g 10 m L o f the 10 p g/ra L so lutio n to a fin a l volum e o f 100 w ith methanol in a 125 m L LO PE bottle. 9.1.4 A 0.1 pg/m L fo rtific a tio n so lu tio n o f P FO A is prepared by b rin g in g 10 m L o fthe 1.0 p g/m L so lu tio n to a fin a l volum e o f 100 w ith methanol in a 125 m L L D P B b ottle . 9.1.5 A 0.01 p g/m L fo rtific a tio n so lu tio n o f P FO A is prepared by brin ging 10 m L o f tbe 0.1 p g fa iL so lu tio n to a fin a l volum e o f 100 w ith methanol in a 125 m L LD P E b o ttle . 9.1.6 The stock and fo rtific a tio n so lutio n s are to be stored in a re frig e ra to r at approxim ately 4 *C and are stable fo r a m axim um period o f 6 months fro m tbe date o f preparation. 9.2 Standard C a lib ra tio n S olutions 9.2.1 LC /M S /M S c a lib ra tio n standards are prepared in m ethanol v ia d ilu tio n o f tbe 0.1 pg/m L fo rtific a tio n so lu tio n . 9.2.2 The fo llo w in g is a ty p ic a l exam ple; a d d itio n a l concentrations may be Concentration o f Fortification Solutum ftix/m L ) too too too 10 5 2 V olum e (m L) 10 5 2 10 10 10 D iluted to (m L) 100 too 100 100 100 too Final Concentration (nn/m L) 10.0 50 20 10 05 0.2 Page 4 of 7 Page 33 o f 65 Exygen Research Page 68 of 100 Interim Report #2-Analysis o f Surface Water Samples Exygen Study No.: P0001131 E x y g e n P ro to c o l N u m b e r: P 0 0 0 1 131 Exyjm Rnetrch Method Number V0001782 | A N A L Y T IC A L M E T H O D | M ethod o f A na lysis fb rth e D e te n n in a tio n o fP e rflu o ro o cU n o ic A c id (P FO A ) in Sedim ent by LC /M S /M S 9.2.3 9 .2 .4 S tore t i l c ilib ra tio n e tandaidt in 125-m L LO PE narrow -m outh bottles a t 2*C to 6 *C , up to s ix m onth*. A lternate volum e* and concentration* o f standards m ay be prepared as needed. 10.0 B atch Set U p 10.1 Each batch o f am ple* extracted (ty p ic a lly 20 o r le s t) m ust include at least one untreated co n tro l and tw o untreated co n tro ls fo rtifie d at known concentration* (la b co n tro l spike) to v e rify procedural re co very fo r the batch. 10.2 Requirem ents fo r fie ld and laboratory duplicates and spikes w ill be specified in the q u a lify assurance plan fix ' th is p ro ject. 11.0 Sam ple E xtraction 1l . l W eigh 5 g o f sample in to 50 m L polypropylene ce m riftig e tubes (fo rtify us needed, replace lid and m ix w e ll). 11.2 A dd 35 m L o f 1% acetic acid, cap, vo rtex and shake on a w rist action shaker fo r -6 0 m inutes. 11.3 C entrifuge the tubes at -3 0 0 0 rpm fo r -2 0 m inutes. 11.4 C o nd itio n the C u SPE cartridges (1 g . 6 m L ) b y passing 10 m L m ethanol fo llo w e d b y 20 m L o f H PLC w a ter ( - 2 drop/sec). D o not le t colum n run dry 11.5 Load (decant) the sample on the co nd itio ne d C u SPE ca rtrid ge . Discard eluate. 11.6 A dd 20 m L o f m ethanol to the sedim ent le ft in the b ottom o f the 50 m L ccntrifU ge tube. Cap, vo rtex and shake on a w ris t a ction shaker fo r -3 0 m inutes. 11.7 C entrifuge the tubes at -3 0 0 0 rpm fo r -2 0 m inutes. 11.8 Decant the m ethanol onto the same SPE ca rtrid ge . C o lle ct the eluate. 11.9 W ash the colum n w ith 4 m L o f m e tha n ol C o lle ct the eluate and add it to the eluate co lle cte d in step 11.8. 11.10 C o nd itio n a second C u SPE cartridge (1 g , 6 m L ) b y passing 10 m L m ethanol follow ed b y 20 m L o f H P LC w a ter ( - 2 drop/sec). D o n o t le t colum n run dry 11.11 A dd the m ethanol to -2 0 0 m L o f w ater and load on d ie second conditioned SPE cartridge. 11.12 E lu te w ith -5 m L 100% m ethanol. C o lle ct 5 m L o f eluate in to graduated 15 m L polypropylene centrifu g e tubes (fin a l volum e 5 m L). 11.13 A nalyze samples using electrospray LC /M S /M S . Pag 5 of 7 Page 34 o f 65 Exygen Research Page 69 of 100 Interim Report #2-Analysis o f Surface Water Samples Exygen Study No.: P0001131 Exygen Protocol Number: POOO1131 Exygen Rffuarcfa Method Number VOOO1782 | A N A L Y T IC A L M E T H O D \ M ethod o f A n a ly rii fo r the D eterm ination o f P erfluorooctanoie A c id (P FO A ) in Sedim ent by LC /M S /M S 12.0 Chrom atography 12.1 12.2 12.3 12.4 12.5 In je ct the M ine am ount o f each standard, sam ple aod fo rtifie d sam ple in to the LC /M S /M S system . A c a lib ra tio n standard m ust precede and fo llo w a ll analyzed samples. Standards o f PFO A corresponding to at least fiv e o r m ore concentration levels m ust be included in an a n a lytica l set. A n e ntire set o f extracted ca lib ra tio n standards m ust be included at the beginning and at the end o f a sam ple aet. Standards m ust be interspersed between every 5-10 samples. A s an a lta n a n ve. an e ntire set o f ca lib ra tio n standards m ay be injected at the beg in ning o f a set fo llo w e d b y ca lib ra tio n standards interspersed every 5-10 sam ples (to account fo r second set o f standards). In e ithe r case, c a lib ra tio n standards m ust be the firs t and last in je ctio n in a sam ple set. Use lin e a r standard curves fo r q u a n tita tio n . L in e a r standard curves arc generated fo r the analyte b y lin e a r regression u sing 1/x w e ig h tin g o f peak area versus ca lib ra tio n standard concentration u sing M assLynx 3.3 (o r equivalent) softw are system . Sam ple response should not exceed standard responses. A n y samples that exceed standard responses should be ftu th e r d ilu te d and reanalyzed. 13.0 Acceptance C rite ria 13.1 13.2 13.3 13.4 13.5 Chrom atogram m ust show a peak o f a daughter io n at 369 amu fro m a parent o f 413 am u. The 413 am u parent corresponds to the P FO A anion, w h ile the daughter io n (369 am u) represents the lo s t o f carbon d io xid e . M ethod blanks m ust n o t contain P FO A a t levels greater than the LO Q . i f a b lin k w p fo in i PFO A at levels greater than 0 .2 n g fa iL , then a new blank sample m ust be obtained and the e n tire set m ust be re-extracted. Recoveries o f co n tro l spikes and m a trix spikes m ust be between 70-130% o f th e ir know n values. I f e co n tro l spike fo ils outside the acceptable lim its , the e ntire set o f samples should be re-extracted. A n y m a trix spike outside 70 130% should be evaluated b y the analyst to determ ine i f re-extraction is w a rra n te d . A n y ca lib ra tio n standard found to be a s ta tis tic a l o u tlie r b y using the Huge E rro r Test, m ay be excluded fro m the ca lcu la tio n o f d ie ca lib ra tio n curve. H ow ever, the to ta l num ber o f extracted ca lib ra tio n standards that could be excluded m ust n ot exceed 20% o f d ie to ta l num ber o f extracted standards in je c te d . The co rre la tion c o e fficie n t (R ) fo r c a lib ra tio n curves generated must be 0.992 (R 3 0.985). I f ca lib ra tio n re su lts fo il outside these lim its , then appropriate steps m ust be taken to adjust instrum ent operation, and the standards o r the relevant set o f samples should be reanalyzed. Page 6 of 7 Page 35 o f 65 Exygen Research Page 70 of 100 Interim Report #2-Analysis of Surface Water Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 Exygen ftMMieh Method Number V0001782 1 A N A L Y T IC A L M E T H O D | M ethod o f A nalysis fo r the D eterm ination o fP e rflu o ro o cta n o ic A c id (P F O A ) in Sedim ent by LC /M S /M S 13.6 R etention tim es between standards and sam ples m ust not d rift more than 4 % w ith in an a na lytica l run. I f re ten tio n tim e d rift exceeds th is lim it w ith in an a n a lytica l run then the set m ust be reanalyzed 14.0 C alculations 14.1 Use the fo llo w in g equation to calculate the am ount o f PFO A found (in ng/m L. baaed on peak area) uaing the standard curve (lin e a r regression param eters) generated b y foe Mass L yn x softw are program : PFO A found (n g /m L ) - (Peak area - in te rce p t) x DF slope D F - facto r b y w hich the fin a l volum e was d ilu te d , i f necessary. 14.2 F o r samples fo rtifie d w ith know n am ounts o f P FO A p rio r to e xtractio n , use the fo llo w in g equation to calculate the percent recovery. R ecovery (% ) - [to ta l analyte found (n g /m L) - analyte found in c o n tro l (n g /m L )] analyte added (n g /m L) 14.3 Use the fo llo w in g equation to co nvert the am ount o f P FO A found in ng/m L to ng/g (ppb). PFO A found (p p b) - fPFO A found fn e /m U x fin a l volum e (5 tn L )l sam ple w e ig ht (5 g ) 14.4 Use the fo llo w in g equation ( if necessary) to ca lculate the am ount o f PFOA found in ppb based on d ry w e ig h t PFO A found (ppb) d ry w e ig ht * PFO A found (p p b) x {100% / to ta l so lid s(% )] Page 7 of 7 Page 36 o f 65 Exygen Research Page 71 o f 100 Interim Report #2-Analysis o f Surface Water Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 ANALYTICAL METHOD M ethod N u m b er V0001783 Method o f Analysis for the Determination o f Perfluorooctaoole Acid (PFOA) lo Fish and C h u n by LC/MS/MS A n a lytica l Testing F a c ility . Exygen Research 305$ Research D rive State College PA 16801 Approved By. T U . c_j L Paul C o nn o lly 1 Technical Leader L C -M S , Exygen Research u'/# ? /f a r / ' Jowhin F laherty * V ice President, O perations, Exygen Research D ate D ate r Exygen Research T o ta l Paget: 8 Page 37 o f 65 Page 72 of 100 Interim Report #2-Analysis of Surface Water Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 Exygea Retouch Method Number VOOO1783 AM A L Y rtC A L M E TH O D I M ethod o f A nalysis fo r the D eterm ination o fP e rflu o ro o cta n o ic A c id (P FO A ) in Fish and Clam s b y LC /M S /M S 1.0 Scope T his m ethod is to be em ployed fo r the iso la tio n and q u a n tita tio n o f p erfluorooclanoic acid b y H igh Perform ance L iq u id C hrom atography coupled to a tandem M ass S pectrom trie D etector (L C /M S /M S ) in fis h and clam s. 2.0 Safety 2.1 A lw ays observe safe laboratory practices. 2.2 C onsult the appropriate MSDS before han dlin g any chem ical fo r proper safety precautions. 3.0 Sam ple Requirem ent 3.1 A t least 20 g o ftest sam ple fo r e xtractio n . 3.2 Sample# should be processed before e xtractio n . Place the frozen sample in a food procesaor and hom ogenize w ith d ry ice . Place the sam ples in containers and leave open in frozen storage o vernig h t to a llo w fo r carbon d ioxide sublim ation. Seal and place the sam ples in fro zen storage u n til tim e o f analysis. 3.3 Sam ple co lle ctio n procedures w ill be sp ecifie d in the sam pling plan fo r this p ro ject. Reagents and Standards 4 .1 4 .2 4 .3 4 .4 4 .5 4.6 4.7 4 .8 4 .9 4 .1 0 4.11 4.12 4 .1 3 W a te r-H P L C grade A ce to n itrile - H PLC grade Carbon (120-400 m esh) - Reagent grade M e th a n o l-H P L C grade S ilic a g el (60*200 m esh) - Reagent grade F lo ris il (60*100 m esh) - Reagent grade Superclean L C -N H j - Reagent grade 1 -O cta n o l- H PLC grade L 'A sco rb ic a dd - Reagent grade D im eth yld ichlorosilan e - Reagent grade Toluene - Reagent grade A m m onium A c e ta te -A .C .S . Reagent G rade P eriloorooctanoic A cid - S igm a-A ldrich 5.0 Instrum ent and Equipm ent 5.1 A h igh perform ance liq u id chrom atograph capable o f pum ping up to 2 solvents equipped w ith a va ria ble volum e in je c to r capable o f in je ctin g 5-200 p L connected to a tandem Maas Spectrom eter (L C /M S /M S ). 5.2 A devioe to co lle ct raw data fo r peak in te g ra tio n and q ua ntita tion . 5.3 A n a lytica l balance capable o f reading to 0.00001 g. P.ge2 of8 Page 38 o f 65 Exygen Research Page 73 of 100 Interim Report #2-Analysis o f Surface Water Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 Exygca Raaearch 1Method Number V000 783 A N A L Y T IC A L M E T H O D M ethod o f A na lysis fo r the D e te rm in ttio o o fP e rflu o ro o cta n o ic A c id (P F O A ) in Fish and Clam s b y LC /M S /M S 5.4 5 .5 5 .6 5 .7 5.8 5 .9 5.10 5.11 5.12 5.13 5.14 5 .1 5 5 .1 6 R otary evaporator. Tissum izer. 125 m L pear-shaped flasks. 50 m L disposable polypropylene ce n triftig e tubes. 15 m L disposable p olypropylene ce ntrifu g e tubes. D isposable m icropipets (50-1 OOuL, !0 0 -2 0 0u L). 125-m L LDPE narrow -m outh b ottles. 2 m L clear HPLC v ia l k it. D isposable pipettes. A utopipettes (100*1000 p L and 10*100 p L ), w ith disposable tip s. SPE tubes (2 0 m L) (Sapelco cat. no. N 057177). W rist action shaker. C entrifuge capable o f spinning 50 m L p olypropylene tubes at 2000 rpm . 6.0 C hrom atographic System 6.1 A n a ly tic a l C olum n: Fluopbase RP (K eystone S c ie n tific ), 2.1 m m x 50 mm. 5m (P /N : 82505*052130) 6 2 Tem perature: 30*C 6.3 M o b ile Phase ( A ) : 2 m M A m m onium A cetate in W ater M o b ile Phase (B ) : M ethanol G radient Program : Tim a ftn in t 0 .0 1.0 8.0 20.0 22.5 SU 65 65 25 25 65 F lo w Rate % B fm L /m in j 35 0.3 35 0.3 75 0.3 75 0.3 35 0.3 6.6 In je ctio n V olum e: 15 p L (can be increased to s s m uch as 50 p L ). 6.7 Q uantitation: Peak Area - external standard ca lib ra tio n curve. 6.8 R unT im e: - 2 3 m inutes. The above co nd itio ns an intended as guide and m ay be changed in o rd er to optim ize the H PLC system . Pag 3 rs Page 39 o f 65 Exygen Research Page 74 of 100 Interim Report #2-Analysis o f Surface W ater Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 Exygen Research Method Number V0001783 A N A L Y T IC A L M E T H O D M ethod o f A na lysis fo r the D eterm ination o fP e rflu o ro o cta n o ic A c id (P F O A ) in Fish and Clam s by LC /M S /M S 7.0 M S /M S System 7.1 M ode: E le ctro sp n y N egative M R M m ode, m o n ito rin g 413 - * 369 m/z fo r PFOA. The above conditions are intended as a guide and m ay be changed in o rd er to o ptim ize the M SM S system. 8.0 Preparation o f S olutions 8.1 M o b ile Phase 8.1.1 2 m M am m onium acetate in w a ter ia prepared b y adding 0 .IS 4 g o f am m onium acetate to 1000 m L o f w ater. 82 E xtractio n S olutions 8.2.1 8 .2 .2 2% ascorbic acid in m ethanol ia prepared b y d isso lvin g 2 g o f ascorbic a dd in 100 m L o f m ethanol. 30% D im ethyldichlorositane in toluene is prepared b y b rin g in g 3 mL o f d im e thyklich lo ro a ila n e to a fin a l volum e o f 10 m L w ith toluene. A lternate volum es m ay be prepared. 9.0 Standard P reparation 9.1 Standard S to ck/F o itifica tio n S olution 9.1.1 9 .1 .2 9 .1 .3 9 .1 .4 9 .1 .5 Prepare a stock so lutio n o f 100 p g /m L o f PFO A b y w e ig hin g 10 mg o f a n a lytica l standard (corrected fo r p u rity ) and d ilu te to 100 m L w ith m ethanol in a 125-m L LD P E b ottle . A 1.0 p g/m L fo rtific a tio n so lu tio n o f P FO A is prepared b y b rin g in g I m L o f the 100 p g ftn L so lu tio n to a fin a l volum e o f 100 w ith methanol in a 125 m L LD PE b o ttle . A 0.1 p ^ m L fo rtific a tio n so lu tio n o f P FO A is prepared b y b rin g in g 10 m L o f tiw 1.0 p g/m L so lutio n to a fin a l volum e o f 100 w ith methanol in a 125 m L LD P E b ottle . A 0.01 p g/m L fo rtific a tio n so lutio n o f P FO A is prepared by brin ging 10 m L o f the 0.1 p g /m L so lu tio n to a fin a l volum e o f 100 w ith methanol in a 125 m L LD P E b o ttle . The stock sod fo rtific a tio n solutions are to be stored in a re frig e ra to r at approxim ately 4*C and are stable fo r a m axim um p eriod o f 6 months fro m the date o f preparation. Page 4 of 8 Page 40 o f 65 Exygen Research Page 75 of 100 Interim Report #2-Analysis of Surface Water Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 Exygen Retetrch Method Number V000I783 A N A L Y T IC A L M E T H O D M ethod o f A na lysis fi the D eterm ination o fP e rflu o ro o cu n o ic A c id (P FO A ) in Fish and G am s b y LC /M S /M S 9.2 Standard C a lib ra tion S olutions 9.2.1 9 .2 .2 LC /M S /M S ca lib ra tio n standards are prepared in m ethanol via d ilu tio n o f the 1.0 pg/m L fo rtific a tio n so lu tio n . The fo llo w in g is s typ ica l exam ple: a d d itio n a l concentrations m ay be prepared as needed. Concentration o f Fortification Solution fua/raL) Volume (m L) D iluted to (m L) Final Concentration (/m L) 1.0 1.0 1.0 0.05 0.025 5.0 2.5 1.0 10 10 100 100 100 lo o 100 0.05 0.025 0.01 0.005 0.002S 0.1 0.005 10 10 100 100 0.001 0.0005 9.2.3 Store a ll ca lib ra tio n standards in 125-m L LD P E narrow -m outh bottles at 2*C to 6 *C , tv to fix m onths. 9.2.4 A lternate volum es and concentrations o f standards m ay be prepared as needed. 10.0 B atch Set U p 10.1 Each batch o f samples extracted (ty p ic a lly 20 o r less) m ust include at least one untreated co n tro l and tw o untreated co n tro ls fo rtifie d at known concentrations (la b co n tro l spike) to v e rity procedural re covery fo r the batch. 10.2 Requirem ents fo r fie ld and laboratory duplicates and spikes w ill be specified in the q u a lity assurance plan fo r th is p ro ject. 11.0 Sam ple E xtraction 11.1 11.2 11.3 11.4 11.5 11.6 W eigh 5 g o f frozen sam ple in to 50 m L p olyp ro p ylen e centrifu g e lubes (fo rtify as needed, replace lid and m ix w e ll). A dd 30 m L o f a ce to n itrile and shake on a w ris t a ctio n shaker fo r '- l S m inutes Place the tubes in a freezer fo r-1 hour. Pack and co nd itio n the SPE tubes and sila n ize the pear-shaped flasks. Pack the 20 m L SPE tubes in sequence w ith 2 g flo ris il, 2 g s ilic a g el, 2 g carbon, and 1 g L C -N H j. C o nd itio n the colum ns w ith 20 m L o f m ethanol, then 20 m L o f a ce to nitrile . D iscard a ll washes. D o n ot a llo w the colum n to d ry . S ilanize the 125 m L pear-shaped flasks b y rin sin g w ith the 30% dim e thyld ich lo ro sila ne in toluene so lu tio n . R inse the fla sk w ith toluene once, fo llo w e d b y m ethanol (three tim es). D ry the fla sks co m p lete ly before use. e ith e r b y a ir-d ry in g o r w ith a stream o f nitro ge n. Page 5 of 8 Page 4! o f 65 Exygen Research Page 76 of 100 Interim Report #2-Analysis o f Surface Water Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 Eitygm lUsMich Method Number VOOOI783 A N A L Y T IC A L M E T H O D M ethod o f A na lysis fo r the D eterm ination o f P e rflu o ro o ctin o ic A c id (P F O A ) in Fish and Clam s b y LC /M S /M S 11.7 C e n triftig e the 50 m L polypropylene tubes co nta ining sam ple at -2 0 0 0 rpm fo r-1 0 m inutes. 11.8 Decant the extract on to a conditioned SPE colum n fitte d in sid e the m outh o f the pear-shaped fla sk. C o lle ct the eluate in the 125 m L sila n ized pear-shape fla sk. 11.9 A dd 10 m L o f a ce to n itrile to the sam ple in the 50 m L centrifu g e tube. Hom ogenize the frozen fa t phase using a ria iu m iz e r fo r -3 0 seconds and rinse the tissum izer w ith -1 0 m L o f a ce to n itrile in to the tube. 11.10 Shake the sam ple again fo r-1 0 m inutes on a w ris t-a c tio n shaker. 11.11 Place the tubes in a freezer fo r - 1 h ou r m ore. 11.12 C entrifuge the 50 m L polypropylene tubes co nta ining sam ple at -2 0 0 0 rpm fo r -1 0 m inutes. 11.13 Decant the extract onto the a n SPE colum n. C o lle ct the eluate in to the tam e pear-shaped fla sk and com bine w ith the eluent fro m the in itia l e x tra c tio n . 11.14 Pass 20 m L o f a ce to n itrile through the SPE co lum n and com bine the eluate in the same pear-shaped fla sk. 11.15 A dd 3-4 drops o f 1-octanol to d ie extract in the pear-shaped flask and evaporate at reduced pressure using a ro ta ry evaporator (a t < 40C ). 11.16 M ake the fin a l volum e, b y adding 2 m L o f 2% ascorbic a cid in m ethanol to the pear-shaped fla sk and s w irl to m ix/d isso lve . 11.17 Transfer the e xtracts to H P LC v ia ls u sing disposable pipets. 11.18 A nalyze samples using eloctrosprsy LC /M S /M S . 12.0 Chrom atography 12.1 In je ct the tam e am ount o f each standard, sam ple and fo rtifie d sample in to the LC /M S /M S system . A c a lib ra tio n standard m ust precede and fo llo w a ll analyzed samples. 12.2 Standards o f PFO A corresponding to at least fiv e o r m ore concentration levels m ust be included in an a na lytica l set. 12.3 A n e ntire set o f ca lib ra tio n standards m u tt be in clu de d at the beginning and at foe end o f a sam ple set. Standards m ust be interspersed between every 5* 10 samples. A s an alternative, an e ntire set o f c a lib ra tio n standards may be injected at foe beginning o f a set fo llo w e d b y c a lib ra tio n standards interspersed every 5-10 samples (to account fo r a second set o f standards), in e ithe r case, ca lib ra tio n standards m ust be foe firs t end la st in je ctio n in a sam ple se t 12.4 Use lin e a r standard curves fo r q u a ntita tion . L ine a r standard curves are generated fo r the analyte b y lin e a r regression u sing 1 /x w e ig h tin g o f peek area versus ca lib ra tio n standard concentration using M assLynx 3-3 (o r equivalent) softw are system . Pag 6 of 8 Page 42 o f 65 Exygen Research Page 77 of 100 Interim Report #2-Analysis o f Surface Water Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 Exygen lUMsrch Method Number VOOOI783 ANALYTICAL METHOD M ethod o f A na lysis fo r the D eterm ination o fP e rilu o ro o cta n o ic A c id (P FO A ) in Fish and Clam s b y LC /M S /M S 12.5 Sam ple response should not exceed standard responses. A n y samples that exceed standard responses should be A nther d ilu te d and reanalyzed. 13.0 Acceptance C rite ria 13.1 13.2 13.3 13.4 13.5 13.6 Chrom atogram m ust show a peak o f a daughter io n at 369 amu from a parent o f 413 amu. The 413 amu parent corresponds to the PFO A anion, w h ile the daughter io n (369 am u) represents the loss o f c a rto n d io xid e . M ethod blanks m ust n o t contain PFO A at le vels greater than the LO Q I f a b lank contains PFO A at levels greater than O.S ppb, then a new blank sample m ust be obtained and the e ntire set m ust be re-extracted. Recoveries o f o o n tro l spikes and m a trix spikes m ust be between 70-130% o f th e ir know n values. I f a co n tro l spike Adis outside the acceptable lim its , the e ntire set o f samples should be re-extracted. A ny ca lib ra tio n standard found to be a s ta tis tic a l o u tlie r b y using the Huge E rro r Test m ay be excluded fro m the ca lcu la tio n o f the ca lib ra tio n curve. H owever, the to ta l num ber o f ca lib ra tio n standards th a t could be excluded m ust n ot exceed 20% o f the to ta l num ber o f standards inje cte d . The co rre la tion co e fficie n t (R ) fo r c a lib ra tio n curves generated m ust be 20.992 (R * 20.985). I f ca lib ra tio n results fo il outside these lim its , then appropriate steps m ust be taken to adjust instrum ent operation, and the standards o r the relevant set o f sam ples should be reanalyzed. R etention tim es between standards and sam ples m ust n o t d rift m ore than 4 % w ith in an a na lytica l run. I f re ten tio n tim e d rift exceeds th is lim it w ith in an a na lytica l run then the set m ust be reanalyzed. 14.0 C alculations 14.1 Use the fo llo w in g equation to calculate the am ount o f P FO A found (in ng/m L, based on peak area) using the standard curve (lin e a r regression param eters) generated b y the M ass L yn x softw are program : PFO A found (n g /m L ) - (P k ITP I in tercep t) slope 14.2 Use the fo lk w in g equation to convert the am ount o f P FO A found in ng/m L to n g /g (p pb ). PFO A found (p p b ) fP F Q A found fn g /m L ) x fin a l volum e fm L ) x DF1 sam ple w e ig ht (g ) DF fa cto r b y w hich the fin a l volum e was d ilu te d , i f necessary. Pigs'? ofS Page 43 o f 65 Exygen Research Page 78 of 100 Interim Report #2-Analysis o f Surface Water Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 Exygen Research Method Number VOOOIW A N A L Y T IC ,V L M E T H O D M ethod o f A na lysis fo r the D eterm ination o f P erfluo ro octa no ic A c id (P F O A ) in F ish and Clam s b y LC /M S /M S 14.3 F o r samples fo rtifie d w ith know n am ounts o f PFO A p rio r to e xtractio n , use the fo llo w in g equation to calculate the percent recovery. R ecovery (V) - [ to ta l analyte found (n g /g ) . analyte found in c o n tro l (n g /g )] analyte added (n g /g ) Exygen Research Page S o f8 Page 44 o f 65 Page 79 of 100 Interim Report #2-Analysis o f Surface Water Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 ANALYTICAL METHOD M ethod N um ber: V0001784 M ethod o f A na lysis fo r d ie D e te rm in a tio n o f P e rfln o ro o c ta n o ic A c id (P FO A ) io V eg e ta tio n b y L C /M S /M S A n a lytica l Testing F a c ility : E xygen Research 3058 Research D rive State C ollege, P A 16801 A pproved B y: caL,_ Paul C o nn o lly Technical Leader, L C -M S , Exygen Research a / m f i U / ________ J o h n Flaherty ^ V ice President, O perations, Exygen Research __lOfc&lsi D a te Exygen Research T o ta l Pages: 7 Page 45 o f 65 Page 80 of 100 Interim Report #2-Analysis o f Surface Water Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 Exygen Reeetrcti Method Number V000I7W ANALYTICAL METHOD M ethod o f A nalysis fo r the D eterm ination o f P erfluorooctanoic A cid (P FO A ) in Vegetation by LC /M S /M S 1.0 Scope ThU m ethod i i to be em ployed fo r foe iso la tio n and q ua n tita tio n o f p erfluorooctanoic a d d b y H ig h Perform ance L iq u id C hrom atography coupled to a tandem Mass S pectrom etric D etector (LC /M S /M S ) in vegetation. 2.0 S afety 2.1 A lw ays observe safe laboratory practices. 2.2 C onsult the appropriate MSDS before handling any chem ical fo r proper safety precautions. 3.0 Sam ple Requirem ent 3.1 A t least 20 g o f test sam ple fo r e xtractio n . 3.2 Samples should be processed before e xtractio n . Place foe frozen sample in a food processor and hom ogenize w ith d ry ice . Place the sam ples in containers and leave open in frozen storage o vernig h t to a llo w fo r carbon d ioxide sublim ation. Seal and place foe samples in frozen storage u n til tim e o f analysis. 3.3 Sam ple co lle ctio n procedures w ill be specified in the sam pling plan fo r this p ro je c t 4.0 Reagents and Standards 4.1 4 .2 4.3 4 .4 4.5 4 .6 4 .7 4 .8 4 .9 4 .1 0 4.11 4.12 4.13 W a te r-H P L C grade A ce to n itrile - H PLC grade Carbon (120*400 m esh) - Reagent grade M ethanol - HPLC grade S ilica gel (60-200 m esh) - Reagent grade F lo ris il (60-100 m esh) -R ea ge n t grade Superclean LC -N H * - Reagent grade l-O cta n o l - H PLC grade L -A scorbic a d d - Reagent grade D im eth yid ichlorosilsne - Reagent grade T olu en e-R e ag en t grade A m m onium A cetate - A .C .S . Reagent Grade P erfluorooctanoic A c id - S ig raa -A ld rich 5.0 Instrum ent and Equipm ent 3.1 A h igh perform ance liq u id chrom atograph capable o f pum ping up to 2 solvents equipped w ith a variable volum e in je c to r capable o f in je ctin g 5-200 p L connected to a tandem M ass Spectrom eter (L C /M S /M S ). 5.2 A device to co lle ct raw data fo r peak in te g ra tio n and q ua ntita tion . 5.3 A nalyticalbalancecapablcofreiK lm gtoO .O O O O l g. Page 2 of 7 Page 46 o f 65 Exygen Research Page 81 of 100 Interim Report #2-Analysis of Surface Water Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 Exygea Research Method Number V00017M A N A L Y T IC A L M E T H O D I M ethod o f A na lysis fo r the D eterm ination o f P e rflu o ro o cttn o ic A c id (P F O A ) in Vegetation b y LC /M S /M S 5 .4 5.5 5.6 5.7 5.S 5 .9 5 .1 0 5.11 5.12 5.13 5.14 5.15 R otary ev^>orator. 125 m L pear-shaped flasks. 50 m L disposable polypropylene ce n trifu g e tdbea. 15 m L disposable polypropylene ce n trifu g e tubes. D isposable m icropipets (50-1 QOuL, 100*200uL). 125-m L LDPE narrow -m outh bottles. 2 m L cle ar H PLC v ia l k it Disposable pipettes. A utopipettes (100*1000 p L and 10*100 p L ), w ith disposable tip s. SPE tubes (20m L) (Supelco c a t no. N0S7177). W rist action shaker. C entrifbge capable o f spinning 50 m L p olyp ro p ylen e tubes at 2000 rpm . 6.0 Chrom atographic System 6.1 A n a lytica l C olum n: Fluophase RP (K eystone S c ie n tific ), 2.1 mm x 50 m m . 5p (P /N : 82505*052130) 6.2 Tem perature: 30*C 6.3 M o b ile Pharo ( A ) : 2 m M A m m onium A cetate in W ater 6.4 M o b ile Pharo (B ): M ethanol 6.5 G radient Program : Tim e (mini 0 .0 1.0 8.0 20.0 22.5 2kA 65 65 25 25 65 J ft 35 35 75 75 35 F lo w Rate fmL/minl 0.3 0.3 0.3 0.3 0.3 6.6 In je ctio n V olum e: 15 p L (can be increased to as m uch as 50 p L ). 6.7 Q uantitation: Peak A re a -e x te rn a l standard ca lib ra tio n curve. 6.8 R unT im e: - 2 3 m inutes. The above conditions a n intended u a guide and m ay be changed in o rd er to optim ize the H PLC system. 7.0 M S /M S System 7.1 M ode: E lectrospray N egative M R M m ode, m o n ito rin g 413 369 m /z fo r PFOA. Page 3 of 7 Page 47 o f 65 Exygen Research Page 82 of 100 Interim Report #2-Analysis o f Surface Water Samples Exygen Study No.: P0001131 E x y g e n P ro to c o l N u m b e r: P 0 0 0 1 131 Exygea Reswcb Method Number V0001784 ANALYTICAL METHOD M ethod o f A na lysis fo r the D eterm ination o fP e rflu o ro o c tin o ic A c id (P FO A ) in Vegetation b y LC /M S /M S The above co nd itio ns are intended u a guide and m ay be changed in order to o ptim ize the M SM S system . 8.0 P reparation o f S olutions 8.1 M o b ile Phase 8.1.1 2 m M am m onium acetate in w ater i t prepared b y adding 0.154 g o f am m onium acetate to 1000 m L o f w ater. 8.2 E xtractio n S olutions 8.2.1 8.2.2 2% ascorbic acid in m ethanol is prepared b y d isso lvin g 2 g o f ascorbic acid in 100 m L o f m ethanol. 30% D im eth yid ichlorosilan e in toluene is prepared b y b rin g in g 3 m L o fd im e th yklich lo ro sila a e to a fin a l volum e o f 10 m L w ith toluene. A lterna te volum es m ay be prepared. 9.0 Standard Preparation 9.1 Standard S to ck/F o rtifica tio n S olution 9.1.1 9.1.2 9.1.3 9 .1 .4 9.1.5 Prepare a stock so lu tio n o f -1 0 0 pg/mL o f P FO A b y w eighing 10 mg o f a na lytica l standard (corrected fo r p u rity ) and d ilu te to 100 m L w ith m ethanol in a 125-m L LD P E b o ttle . A 1.0 p g/m L fo rtific a tio n so lu tio n o f PFO A is prepared by b rin g in g I m L o f d ie 100 p g frn L so lu tio n to a fin a l volum e o f 100 w ith methanol in a 125 m L LDPE b ottle . A 0 .1 pg/m L fo rtific a tio n so lutio n o fP F O A is prepared b y b rin g in g 10 m L o f the 1.0 p g/ra L so lu tio n to a fin a l volum e o f 100 w ith methanol in a 125 m L LDPE b o ttle . A 0.01 p g/m L fo rtific a tio n so lu tio n o f PFO A is prepared b y brin ging 10 ra L o f the 0.1 p g/m L so lu tio n to a fin a l volum e o f 100 w ith methanol in a 125 m L LD P E b ottle . The stock sad fo rtific a tio n so lutio n s are to be stored in a re frig e ra to r at approxim ately 4 "C and are stable fo r a m axim um period o f 6 months fro m the date o f preparation. 9.2 Standard C a lib ra tion S olutions 9.2.1 LC /M S /M S ca lib ra tio n standards are prepared in m ethanol via d ilu tio n o f the 1.0 pg/m L fo rtific a tio n so lu tio n . P**e 4 ul Page 48 o f 65 Exygen Research Page 83 of 100 Interim Report #2-Analysis of Surface Water Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 Exygen JUteaich Method Number V0001784 A T S L Y IIC A L M E T H O D M ethod o f A nalysis fo r the D eterm ination o f P erfluo ro octsn oic A c id (P FO A ) in Vegetation b y LC /M S /M S 9.2.2 The fo llo w in g ic a ty p ic a l exam ple: a d d itio n a l concentrations m ay be prepared as needed. Concentration Fine! o f Fortification Volume D iluted to Concentration Solution fu tfm U (nO.) (m L) (ll fa lL ) 1.0 5.0 100 0.05 1.0 2.5 100 0.025 1.0 1.0 100 0 .0 ! 0.05 0.025 10 10 100 100 0.005 0.0025 0.1 10 100 0.001 0.005 10 100 0.0005 9.2.3 Store a ll ca lib ra tio n standards in 125-m L LD P E narrow -m outh bottles st 2*C to 6*C , up to s ix m onths. 9.2.4 A lterna te volum es and concentrations o f standards m ay be prepared as needed. 10.0 B atch Set U p 10.1 Each batch o f samples extracted (ty p ic a lly 20 o r less) m ust include at least one untreated co n tro l end tw o untreated co n tro ls fo rtifie d at known concentrations (la b co n tro l sp ike) to v e rify procedural re covery fo r the batch. 10.2 Requirem ents fo r fie ld and la bo ra tory duplicates and spikes w ill be specified in the q u a lity assurance plan fo r th is project. 11.0 Sam ple E xtraction 11.1 11.2 11.3 11.4 11.5 11.6 11.7 W eigh 5 g o f frozen sam ple in to 50 m L p olypropylene ce ntrifu g e tubes (fo rtify as needed, replace lid and m ix w e ll). A dd 3 0 m L o f a ce to n itrile and shake on a w ris t a ction shaker fo r -IS m inuies. C entrifuge the SO m L polypropylene tubes co n ta in in g sam ple at -2 0 0 0 rpm fo r- 1 0 m inutes. Pack and co nd itio n the SPE tubes and sila n ize the pear-shaped fiasks. Pack the 20 m L SPE tubes in sequence w ith 2 g flo ris il. 2 g s ilic a g e l. 2 g carbon, and 1 g L C -N H j. C o nd itio n the colum ns w ith 20 m L o f m ethanol, then 20 m L o f a ce to n itrile . D iscard a ll washes. D o n ot a llo w the colum n to d ry. S ilanize the 12$ m L pear-shaped flasks b y m u tin g w ith the 30% dim etbyldichloroslU ne in toluene so lu tio n . R inse the fla sk w ith toluene once, fo llo w e d b y m ethanol (three tim e t). D ry the fla sks co m p lete ly before use. e ithe r b y sir-d ry in g o r w ith a stream o f nitro ge n. Decant the extract m i to a conditioned SPE colum n fitte d in sid e the m outh o f the pear-shaped fla sk. C o lle ct the eluate in the 125 m L sila n ized pear-shape fla sk. Page 5 o f Page 49 o f 65 Exygen Research Page 84 of 100 Interim Report #2-Analysis of Surface Water Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 Exygen Research Method Number VQO0I7S4 ANALYTICAL m e t h o d M ethod o f A na lysis fo r the D eterm ination o f P erfluorooctaaoic A d d (P F O A ) in Vegetation b y LC /M S /M S 11.8 A dd 20 m L o f a ce to n itrile to the sam ple in the 50 m L ce ntrifu g e tube. 11.9 Shake d ie sample again fo r 10 m inutes on a w rist-a ctio n shaker. 11.10 C e ntrifttg e the 50 m L polypropylene tubes co nta ining sam ple at 2000 rpm fo r 5 m inutes. 11.11 Decant the extract onto the same SPE colum n. C o lle ct the eluate in to the same pear-shaped fla sk and com bine w ith the eluent from the in itia l e x tra c tio n . 11.12 R e p e a ts te p s ll.8 d tro u g h U .il again. 11.13 A dd 3-4 drops o f 1-octanol to the extract in the pear-shaped fla sk and evaporate at reduced pressure using a ro ta ry evaporator (a t < 40C ). 11.14 M a in the fin a l volum e, b y adding 2 m L o f 2% ascorbic acid in m ethanol to the pear-shaped fla sk and tw ir l to m ix/d ia solve. 11.15 T ransfer the extracts to H P LC via ls u sing disposable pipets. 11.16 A nalyze samples using electrospray LC /M S /M S . 12.0 Chrom atography 12.1 122 12.3 12.4 12.5 In je c t the same am ount o f each standard, sam ple and fo rtifie d sam ple in to the LC /M S /M S system . A ca lib ra tio n standard m ust precede and fo llo w a ll analyzed samples. Standards o f PFO A corresponding to st least fiv e o r m ore concentration levels m ust be included in an a na lytica l set. A n entire eet o f extracted ca lib ra tio n standards m ust be included at the beginning and st the end o f a sam ple set. E xtracted standards m ust be interspersed between every 5-10 sam ples. A s an a lte rn a tive , an e ntire set o f extracted ca lib ra tio n standards m ay be in je cte d at the b eginning o f a set fo llo w e d b y extracted ca lib ra tio n standards interspersed every 5-10 samples (to account fo r a second set o f extracted standards), b e ith e r case, extracted ca lib ra tio n standards m ust be the firs t and la st in je c tio n in t sam ple set. Use lin e a r standard curves fo r q ua ntita tion . L in e a r standard curves are generated fo r the analyte b y lin e a r regression using I/x w e ig h tin g o f peak area versus ca lib ra tio n standard concentration using M assLynx 3.3 (o r equivalent) softw are system . Sam ple response should not exceed standard responses. A ny samples that exceed standard responses should be ftirth e r d ilu te d and reanalyzed. 13.0 Acceptance Criteria 13.1 Chrom atogram m ust show a peak o f a daughter io n at 369 amu from a parent o f 413 am u. The 413 amu parent corresponds to the P FO A anion, w h ile the daughter io n (369 am u) represents the loss o f carbon d io xid e . Page 6 of 7 Page SOo f 65 Exygen Research Page 85 of 100 Interim Report #2-Analysis of Surface Water Samples Exygen Study No. : P0001131 Exygen Protocol Number: P0001131 Exyjw lU itnh Method Number V000I7S4 a n a l y t ic a l m e t h o d M ethod o f A na lysis fo r the D etennination o fP e rflu o ro o cta n o ic A c id (P FO A ) in Vegetation b y LC /M S /M S 13.2 13.3 13.4 13.5 13.6 M ethod b lan k* m ust not contain P FO A at le vels greater than the LO Q . I f a blank contain* PFO A at levels greater than 0.5 ppb, then a new blank sample m ust be obtained s k i the e ntire set m ust be re-extracted. Recoveries o f co n tro l spikes and m a trix spikes m ust be between 70-130/ o f th e ir know n values. I f a co n tro l spike fa ll* outside the acceptable lim its , the e ntire set o f sam ples should be re-extracted. A n y ca lib ra tio n standard found to be a s ta tis tic a l o u tlie r b y using the Huge E rro r Test, m ay be excluded fro m the ca lcu la tio n o f the ca lib ra tio n curve. H ow ever, the to ta l num ber o f ca lib ra tio n standards tha t co uld be excluded m ust not exceed 20% o f the to ta l num ber o f standards inje cte d . The co rre la tion co e fficie n t (R ) fo r c a lib ra tio n curves generated must be 0.992 (R J 0.985). I f ca lib ra tio n results fa ll outside these lim its , then appropriate steps m ust be taken to adjust instrum ent operation, and the standards o r d ie relevant set o f sam ple* abould be reanalyzed. R etention tim e * between standards and sam ples m ust n o t d rift m ore than i 4 % w ith in an a na lytica l run. I f retention tim e d rift exceeds th is lim it w ith in an a na lytica l ra n then foe set m ust be reanalyzed. 14.0 C alculations 14.1 Use foe fo llo w in g equation to calculate the am ount o f P FO A found (in ng/m L, baaed on peak area) using foe standard curve (lin e a r regression param eters) generated b y foe Mass L ynx softw are program : PFOA found (n g /m L ) - (P d ( t f t l - ifllg tr o l) slope 14.2 Use foe fo llo w in g equation to convert the am ount o f P FO A found in ng/m L to n g /g (p pb ). PFO A found lo o b ) - (PFO A found In g /m D x fin a l volum e (m L ) x DF1 sam ple w e ig ht (g ) D F " fa cto r b y w hich foe fin a l volum e was d ilu te d , i f necessary. 14.3 F o r samples fo rtifie d w ith know n am ounts o f P FO A p rio r to e xtractio n , use foe fo llo w in g equation to calculate foe percent recovery. R ecovery (%) [ to ta l analyte found (n g /g ) - analyte found in c o n tro l (n g /g )] ^ ^ analyte added (n g /g ) Page 7 oi 7 Page 51 o f 65 Exygen Research Page 86 of 100 Interim Report #2-Analysis of Surface Water Samples Exygen Study No.: P0001131 Exygen Protocol Number: P000! 131 ANALYTICAL METHOD M ethod N u m b er V0001785 Method o f Analysis for the Determination o f Perflnorooctanolc A d d (P F O A ) in S m a ll Mammal Liver by LG/MS/MS A n a lytica l Testing F a c ility : E xygen Research 3058 Research D rive State C ollege, P A 16801 A pproved B y: C O L ,____ Paul C o nn o lly l Technical Leader, L C -M S , Exygen Research D ate John F laherty ' V ice Pressidideenntt, O perations, Exygen Research D ate Exygen Research T o ta l Pages: 7 Page 52 o f 65 Page 87 of 100 Interim Report #2-Analysis of Surface Water Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 ExygM R m u c h Method Number VQ0017SJ I A N A L Y T IC A L M E T H O D M ethod o f A na lysis fo r the D eterm ination o f P erfluorooctanoic A c id (P FO A ) in S m all M am m al L iv e r b y LC /M S /M S 1.0 Scope T h is m ethod U to be em ployed fo r d ie iso la tio n and q u a n tita tio n o f p erfluorooctanoic acid b y H ig h Perform ance L iq u id C hrom atography coupled to a tandem Mass S pectrom etric D etector (LC /M S /M S ) in sm all m am m al liv e r. 2.0 Safety 2.1 A lw ays observe safe laboratory practices. 2.2 C onsult the appropriate M SDS before h a n dlin g any chem ical fo r proper safety precautions. 3.0 Sam ple Requirem ent 3.1 A t least 5 g o f test sample fo r e xtractio n . 3.2 Sam ples should he processed before e xtractio n . Place the frozen sample in a food processo r and hom ogenize w ith d ry ice . P lace the sam ples in containers and leave open in frozen storage o ve rn ig h t to a llo w fo r carbon d ioxide sublim a tion . Seal and place the samples in frozen storage u n til tim e o f analysis. A lte rn a te ly, i f there is an in s u ffic ie n t am ount o f sam ple (~less than 5 g ), then no processing is necessary and the sam ple can be used as supplied. 3.3 Sam ple co lle ctio n procedures w ill be sp ecifie d in the sam pling plan fo r this p roject. 4.0 Reagents and Standards 4.1 W ater -H P L C grade 4.2 M ethanol - H P LC grade 4.3 A ce to n itrile - H PLC grade 4.4 A m m onium A cetate - A .C .S . Reagent G rade 4.5 P erfluorooctanoic A c id - S igm a-A ldrich $.0 Instrum ent and E quipm ent 5.1 A h igh perform ance liq u id chrom atograph capable o f pum ping up to 2 solvents equipped w ith a variable volum e in je c to r capable o f in je ctin g 5*20<J p L connected to a tandem Maas S pectrom eter (L C /M S /M S ). 5.2 A device to co lle ct raw data fo r peak in te g ra tio n and q u a n tita tio n . 5.3 A n a lytica l balance capable o f re v tin g to 0.00001 g. 5.4 50 m L disposable polypropylene c e n trifrig e tubes. 5.5 15 m L disposable polypropylene c e n triftig e tubes. 5.6 Disposable micropipets (50-100uL, 100-200uL). 5.7 125-m L LDPE narrow *m outh bottles. S.S 2 m L cle ar H PLC v ia l k it Page 2 o f ' Page 53 o f 65 Exygen Research Page 88 of 100 Interim Report #2-Analysis of Surface Water Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 Exygca Research Mctbod Number VOOCI78S I A N A L Y T IC A L M E T H O D M ethod o f A na lysis fo r the D eterm ination o fP e rflu o ro o c tin o ic A c id (P FO A ) in S m ell M am m al U v e r b y LC /M S /M S 5 .9 5 .1 0 5.11 5.12 5.13 5.14 5.15 Disposable pipettes. A utopipettes (100*1000 p L and 10-100 p L ), w ith disposable tip s. W aters Sep Pak V ac 6 cc (Ig ) tC l 8 SPE cartridges. SPE vacuum m anifold. TiM uem izcr. W rist-a ction shaker. C entrifuge capable o f spinning 15 m L p olypropylene tubes at 3000 rpm . 6.0 Chrom atographic System 6.1 A n a lytica l C olum n: Fluophase RP (K eystone S c ie n tific ), 2.1 mm x $0 m m . 5p (P /N : 82505-052130) 6.2 Tem perature: 30*C 6.3 M o b ile Phase (A ) : 2 m M A m m onium A cetate in W ater 6.4 M o b ile Phase (B ): M ethanol 6.5 G radient P rogram : Time (mini 0.0 1.0 8.0 20.0 22.5 65 65 25 25 65 Flow Rate Z M ImL/minl 35 0.3 35 0.3 75 0.3 75 0.3 35 0.3 6.6 In je ctio n V olum e: 15 p L (can be increased to as m uch as 50 p L ). 6.7 Q uantitation: Peak A re a -e x te rn a l standard ca lib ra tio n curve. 6.8 R unT im e: - 2 3 m inutes. The above co nd itio ns are intended a t a guide and m ay be changed in o rd er to o ptim ize the H PLC system . 7.0 M S /M S System 7.1 M ode: E lectrospray N egative M R M m ode, m o n ito rin g 413 -* 369 m /z fo r PFOA. The above conditions ere intended as a guide and m ay be changed in order to o ptim ize the M SM S system . - Pag3 of? Page 54 o f 65 Exygen Research Page 89 of 100 Interim Report #2-Analysis o f Surface Water Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 Exygm Raaearch Method Number VOOOPgj I A N A L Y T IC A L M E T H O D M ethod o f A na lysis fo r the D eterm ination o f P erfhtorooctanoic A c id (P FO A ) in Sm all M am m al L iv e r b y LC /M S /M S 8.0 Preparation o f S olutions 8.1 M o b ile Pbaae 8.1.1 2 m M am m onium acetate in w ater ia prepared b y adding 0 .154 g o f am m onium acetate to 1000 m L o f w ater. A lternate volum e* m ay be prepared. 9.0 Standard P reparation 9.1 Standard S to ck/F o rtifica tio n S olutio n 9.1.1 Prepare a stock so lutio n o f-1 0 0 p g ftn L o f P FO A b y w eighing 10 mg o f a na lytica l standard (corrected fo r p u rity ) and d ilu te to 100 m L w ith m ethanol in a 125-m L LD P E b o ttle . 9.1.2 A 1.0 pg/m L fo rtific a tio n so lu tio n o f P FO A ia prepared by b rin g in g I m L o f the 100 p g/m L so lu tio n to a fin a l volum e o f 100 w ith methanol in a 125 m L LDPE b ottle . 9.1.3 A 0.1 p g ta L fo rtific a tio n so lu tio n o f PFO A is prepared b y b rin g in g 10 m L o fth e t.O p g 'm L so lu tio n to a fin a l volum e o f 100 w ith methanol in a 125 m L LD P E b o ttle . 9.1.4 The stock and fo rtific a tio n so lutio n s are to b e stored in a re frig e ra to r at approxim ately 4C and are stable fo r a m axim um p eriod o f 6 months fro m the date o f preparation. 9.2 Standard C a lib ra tio n S olutions 9.2.1 922 LC /M S /M S ca lib ra tio n standards are prepared in m ethanol via d ilu tio n o f the 0.1 p g/m L fo rtific a tio n so lutio n . The fo llo w in g is a typ ica l exam ple: a d d itio n a l concentrations may be prepared as needed. Concentration of Fortification Volume Solution fne/mL) (mL) Diluted to (mL) Final Concentration (ne/mL) 100 5.0 100 100 1.0 100 100 1.0 100 5X1 to 100 2.0 10 100 1.0 10 100 5.0 2.0 1.0 OS 0.2 0.1 9.2.3 Store a ll ca lib ra tio n standards in 125-m L LD P E narrow -m outh bottles at 2C to 6*C , up to s ix m onths. 9.2.4 A lterna te volum es and concentrations o f standards m ay be prepared as needed. Page 4 of 7 P age 55 o f 65 Exygen Research Page 90 of 100 Interim Report #2-Analysis o f Surface Water Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 Exyjen Rcaearch Method Number VOOOI785 [ A N A L Y T IC A L M E T H O D M ethod o f A nalysis fo r the D eterm ination o f P erfluorooctanoic A c id (P FO A ) in Sm ell M am m al L iv e r b y LC /M S /M S 10.0 Batch Set U p 10.1 Each batch o f samples extracted (ty p ic a lly 20 o r lees) m ust include at least one untreated co n tro l and tw o untreated co n tro ls fo rtifie d at known concentrations (la b c o n tro l spike) to v e rify p rocedural recovery fo r the batch 10.2 Requirem ents fo r fie ld and la bo ra tory duplicates and spikes w ill be specified in the q u a lity assurance p lan fo r th is p ro ject. 11.0 Sam ple E xtraction 11.) W eigh 1 g o f aample in to a 50 m L polypropylene ce ntrifu g e tubes (fo rtify u needed, replace lid and m ix w e ll). N ote th a t alternate w eights o f liv e r may be measured depending on the sample size a vailab le fo r use. 11.2 A dd w ater to the sam ple fo r a fin a l volum e o f 10 m L. 11.3 Hom ogenize sample using a tissuem izer fo r ~1 m inute. 11.4 T ransfer 1 m L o f the sam ple using a disposable p ipe tte in to a 15 m L disposable ce n triftig e tube. 11.5 A dd 5 m L o f a ce to n itrile and shake fo r -2 0 m inutes on a w rist-a ctio n shaker. 11.6 C entrifuge foe tubes at -3 0 0 0 rpm fo r -5 m inutes. 11.7 Decant the supernatant in to a SO m L disposable ce ntrifu g e tube and add 35 m L o f water. 11.8 C o nd itio n foe Cia SPE ca rtrid ge * (1 g, 6 m L ) b y passing 10 m L m ethanol fo llo w e d b y 5 m L o f H P LC w ater (~ 2 drqp/soc). D o n o t le t colum n run d ry 11.9 Load the sam ple on conditioned C n SPE ca rtrid ge . D iscard eluate. 11.10 E lu te w ith -2 m L o f m ethanol C o lle ct 2 m L o f eluate in to a graduated 15 m L polypropylene ce n triftig e tube (fin a l volum e 2 m L ). 11.11 A nalyze samples using electrospray LC /M S /M S . 12.0 Chrom atography 12.1 12.2 12.3 12.4 In je ct the same am ount o f each standard, a m p le and fo rtifie d sam ple in to the LC /M S /M S system . A c a lib ra tio n standard m ust precede and fo llo w a ll analyzed samples. Standards o f PFO A corresponding to a t least fiv e o r m ore concentration levels m ust be included in in a na lytica l s e t A n entire set o f ca lib ra tio n standards m ust be in clu de d at the beginning and at the end o f a sam ple set. Standards m ust be interspersed between every 5-iO samples. A s an a lterna tive, an e ntire set o f ca lib ra tio n standards may be injected at the beginning o f a set fo llo w e d b y ca lib ra tio n standards interspersed every $-10 samples (to account fo r a second set o f standards). In e ith e r case, ca lib ra tio n standards m ust be foe firs t and last in je ctio n in a sam ple s e t Use lin e a r standard curves fo r q u a n tita tio n . L in e a r standard curves are generated fo r foe analyte b y lin e a r regression u sing 1 /x w e ig h tin g o f peak area Psge 5 of? Page 56 o f 65 Exygen Research Page 91 of 100 Interim Report #2-Analysis o f Surface W ater Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 Exygen Research Method Number VOOOI7IS I ANALYTICAL METHOD M ethod o f A na lysis for the D eterm ination o f P erfluorooctanoic A c id (P F O A ) in S m all M am m al L iv e r b y LC /M S /M S versus ca lib ra tio n standard concentration using M assLynx 3.3 (o r equivalent) softw are system . 12.$ Sam ple response should n o t exceed standard responses. A n y samples that exceed standard responses should be Am ber d ilu te d and reanalyzed. 13.0 Acceptance C rite ria 13.1 13.2 13.3 13.4 13.5 13.6 Chrom atogram m ust show a peak o f a daughter io n at 369 amu from a parent o f 413 amu. The 413 amu parent corresponds to the PFO A anion, w h ile the daughter io n (369 am u) represents the loss o f carbon d io xid e . M ethod blanks m ust not contain PFO A at le vels greater than the LO Q . I f a b lank contains PFO A a t levels greater than 10 n g/g, then new blank sample m ust be obtained and the e ntire set m ust be re-extracted. Recoveries o f c o n tro l spikes and m a trix spikes m ust be between 70-130% o f th e ir know n values. I f a co n tro l spike ta ils outside the acceptable lim its , the entire set o f samples should be re-extracted. A n y m a trix spike outside 70 130% should be evaluated b y the analyst to determ ine i f re-extraction is w a rra n te d . A n y ca lib ra tio n standard found to be a s ta tis tic a l o u tlie r b y using the Huge E rro r Test, m ay be excluded fro m the ca lcu la tio n o f the ca lib ra tio n curve. H ow ever, the to ta l num ber o f ca lib ra tio n standards tha t co uld be excluded m ust n o t exceed 20% o f the to ta l num ber o f standards inje cte d . The co rre la tion co e fficie n t (R ) fo r ca lib ra tio n curves generated must be 20.992 (R 1 20.985). I f c a lib ra tio n results fa ll outside these lim its , then appropriate steps m ust be taken to adjust instrum ent operation, and the standards o r the relevant set o f samples should be reanalyzed. R etention tiroes between standards and sam ples m ust n ot d rift m ore than 4 % w ith in an a na lytica l run. I f retention tim e d rift exceeds th is lim it w ith in an a n a lytica l run then the set m ust be reanalyzed. 14.0 C alculations 14.1 Use the fo llo w in g equation to calculate the am ount o f PFO A found (in ng/m L, based on peak area) using the standard curve (lin e a r regression param eters) generated b y the M ass L yn x softw are program : P FO A found (n g /m L) - (Peak area - in te rce p t! x D F x a liq u o t facto r slope D F fa cto r b y w hich the fin a l volum e was d ilu te d , i f necessary. A liq u o t fa cto r " 10 Page 6 o f7 Page 57 o f 65 Exygen Research Page 92 of 100 Interim Report #2-Analysis of Surface Water Samples Exygen Study No.: P0001131 Exygen Protocol Number: POOOI131 BxygenResweb Method Number VOOOPIS I A N A 1.Y 1K .A L M E TH O D M ethod o f A nalyaia fo r the D eterm ination o fP e rflu o ro o cta n o ic A c id (P F O A ) in S m all M am m al L iv e r b y LC /M S /M S 14.2 F o r u m p le s fo rtifie d w ith know n amount o f PFO A p rio r to extraction, use the fo llo w in g equation to calculate the percent recovery. R ecovery (Vo) - [ to ta l analyte found (n g /m L) - analyte found in co n tro l (n g /m L )] analyte added (n g /m L) 14.3 Use the fo llo w in g equation to co nvert the am ount o f PFO A found in ng/m L lu n g /g (p pb ). PFO A found fp p b l - fPFQ A found fn a /m D x fin a l volum e fm U l sam ple w e ig ht (g ) Exygen Research Page 7 of? Page 58 o f 65 Page 93 of 100 Interim Report #2-Analysis o f Surface Water Samples Exygen Study No. : P0001131 Exygen Protocol Number: P0001131 ANALYTICAL METHOD M ethod N um ber V 0001786 Method o f Analysis for the Determination o f Perfluorooctaoofc A d d (PFOA) ia Smalt Mammal Serum by LC/MS/MS A n a lytica l Testing F a c ility : E xygen Research 3058 Research D rive State C ollege, P A 16801 A pproved B y: "v~*--V_C-- __ Paul C o nn o lly I Technical Leader, LC -M S , Exygen Research a / #>4 ^ / Jo h n F laherty / V ice Pressidideianit, O perations, Exygen Research __iain i.? '! D ate /' J r Due Exygen Research T o ttI Pages: 7 Page 59 o f 65 Page 94 of 100 Interim Report #2-Analysis o f Surface Water Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 E xygen R esearch Method Number V00017$6 A N A LYTICAL m e t h o d M ethod o f A nalysis fo r the D eterm ination o f P erfluorooctanoic A c id (P FO A ) in Sm all M am m al Serum b y LC /M S /M S 1.0 Scope T h is m ethod is to be em ployed fo r the is o la tio n and q u a n tita tio n o f perfluorooctanoic acid b y H igh Perform ance L iq u id C hrom atography coupled to a tandem Mass S pectrom etric D etector (L C /M S /M S ) in sm all m am m al serum. 2.0 S afety 2.1 A lw ays observe safe laboratory practices. 2.2 C onsult the appropriate M SDS before han dlin g any chem ical fo r proper safety precautions. 3.0 Sam ple Requirem ent 3.1 A t least 1 m L o f test sample fo r e xtraction. 3.2 N o sample processing is needed fo r serum sam ples. H ow ever, frozen serum samples m ust to allow ed to com pletely thaw to room tem perature before use. 3J Sam ple co lle ctio n procedures w ill be sp ecifie d in the sam pling plan fo r this p ro je c t 4.0 Reagents and Standards 4.1 W a te r-H P L C grade 4.2 M ethanol - H PLC grade 4.3 A ce to n itrile - H PLC grade 4.4 A m m onium Acetate - A .C .S . Reagent Grade 4.5 P erfluorooctanoic A cid - S igm a*A ldrich 5.0 Instrum ent and Equipm ent 5.1 5.2 5.3 5.4 5.5 5 .6 5.7 5.8 5 .9 5 .1 0 5.11 5 .1 2 5.13 A high perform ance liq u id chrom atograph capable o f pum ping up to 2 solvents equipped w ith a variable volum e iq je c to r capable o f in je ctin g 5-200 p L connected to a tandem Mass S pectrom eter (L C /M S /M S ) A device to c o lle ct raw data fo r peak in te g ra tio n and qua ntita tion . A n a lytica l balance capable o f reading to 0.00001 g. 50 m L disposable polypropylene c e n triftig e tubes. 15 m L disposable polypropylene c e n triftig e tubes. D isposable m icropipets (50-100uL, 100*200uL). 125-m L LD PE narrow -m outh b ottles. 2 m L clear H PLC v ia l ld t. D isposable pipettes. A utopipettes (100*1000 |tL and 10*100 p L ), w ith disposable tip s. W aters Sep Pak V ac 6 c c (lg )tC 1 8 S P E cartridges. SPE vacuum m a n ifold . V ortcxe r. Page 2 o f f Page 60 o f 65 Exygen Research Page 95 of 100 Interim Report #2-Analysis o f Surface Water Samples Exygen Study No.: P0001131 Exygen Protocol Number: POOO1131 Exygea ReaMfcb Method Number VOOO178b I ....................... ANALYTICAL METHOD M ethod o f A na lysis fo r the D eterm ination o f P erfluorooctanoic A c id (P FO A ) in S m all M am m al Serum b y LC^M S/M S 5.14 W rist-a ction shaker. 5.15 C entrifuge capable o f spinning 15 m L p olypropylene tubes at 3000 rpm . 6.0 Chrom atographic System 6.1 A n a lytica l C olum n: Fluophase RP (K eystone S c ie n tific ), 2.1 mm x 50 mm. 5p (P /N : 82505-052130) 6.2 Tem perature: 30*C 6.3 M o b ile Phase (A ) : 2 m M A m m onium A cetate in W ater 6.4 M o b ile Phase (B ) : M ethanol 6.5 G radient Program : Tim e (m in i 0 .0 1.0 8 .0 20.0 22.5 2L& 65 65 25 25 65 F lo w Rate 5 kB (m L /m in l 35 0.3 35 0.3 75 0.3 75 0.3 35 0.3 6.6 In fe ctio n V olum e: 15 jiL (can be increased to as m uch as 50 p L ). 6.7 Q uantitation: Peak A rea - external standard c a lib ra tio n curve. 6.8 R u nT im e : ~ 23 m inutes. The above conditions are intended as a guide and m ay be changed in order to optim ize the HPLC system . 7.0 M S /M S System 7.1 M ode: E lectrospray N egative M R M m ode, m o n ito rin g 413 - * 369 m /z fo r PFOA. The above co nd itio ns are intended as e guide and m ay be changed in order to o ptim ize the M SM S system. 8.0 P reparation o f S olutions 8.1 M o b ile Phase 8.1.1 2 m M am m onium acetate in w ater is prepared b y adding 0.154 g o f am m onium acetate to 1000 m L o f w ater. A lterna te volum es m ay be prepared. Page 3 of 7 Page 6 / o f 65 Exygen Research Page 96 of 100 Interim Report #2-Analysis o f Surface Water Samples Exygen Study No.: P0001131 E x y g e n P ro to c o l N u m b e r: POCO 1131 ExygmRMMKk Method Number V000I7S6 I ANALYTICAL METHOD M ethod o f A na lysis fo r the D eterm ination o fP e rflu o ro o c tin o ic A c id (P FO A ) in S m all M am m al Serum b y LC /M S /M S 9.0 Standard Preparation 9.1 Standard S to ck/F o rtifica tio n S olution 9.1.1 Prepare a stock so lutio n o f -1 0 0 p g /m L o f PFO A b y w eighing 10 mg o f a n a lytica l standard (corrected fo r p u rity ) and d ilu te to 100 m l u n it m ethanol in a 125-m L LD P E b ottle . 9.1.2 A 1.0 pg/m L fo rtific a tio n so lu tio n o f P FO A is prepared b y b rin ging I ra L o f the 100 p g /ra L so lu tio n to a fin a l volum e o f 100 w ith methanol in a 125 m L LDPE b o ttle . 9.1.3 A 0 .1 pg/m L fo rtific a tio n so lutio n o fP F O A is prepared b y b rin g in g 10 m L o f the 1.0 p g/m L so lu tio n to a fin a l volum e o f 100 w ith methanol in a 125 m L LD P E b ottle . 9.1.4 The stock and fo rtific a tio n so lutio n s are to be stored in a re frig e ra to r at approxim ately 4*C and are stable fo r a m axim um p eriod o f 6 months from the date o f preparation. 9.2 Standard C a lib ra tion S olutions 9.2.1 9 .2 .2 LC /M S /M S ca lib ra tio n standards are prepared in m ethanol v ia d ilu tio n o f the 0.1 p g /m L fo rtific a tio n so lutio n . The fo llo w in g is a ty p ic a l exam ple: a d d itio n a l concentrations m ay be prepared as needed. C oncentriti! Final o fF o itifica tio n Volume D ilu te d Concentration S o lu ti ! fns/m U (raL) (m L) (n i/m L ) 100 5.0 100 100 2.0 too 100 1.0 100 5.0 2.0 1.0 5.0 10 100 2.0 10 100 1.0 10 100 0.5 0.2 0.1 9.2.3 Store a ll ca lib ra tio n standards in 125-m L LD P E narrow -m outh bottles at 2C to 6" C , up to s ix m onths. 9.2.4 A lternate volum es and concentrations o f standards m ay be prepared as needed. 10.0 B atch Set U p 10.1 Each batch o f samples extracted (ty p ic a lly 20 o r less) m ust include at least one untreated co n tro l and tw o untreated co n tro ls fo rtifie d at known concentrations (la b co n tro l spike) to v e rify procedural re covery fo r the batch. 10.2 Requirem ents fo r fie ld and laboratory duplicates and spikes w ill be specified in the q u a lity assurance plan fo r th is p ro ject. Page 4 o f7 P age 62 o f 65 Exygen Research Page 97 of 100 Interim Report #2-Analysis of Surface Water Samples Exygen Study No.: P0001131 E x y g e n P ro to c o l N u n ib e r: POOO1131 Exygn Rmarcfc Method Number V000l78e I ANALYTICAL m e t h o d M ethod o f A na lysis fo r the D eterm ination o f P erfluorooctanoic A c id (P F O A ) in S m all M am m al S o w n b y LC /M S /M S 11.0 Sam ple E xtraction 11.1 Measure 1 m L o f sam ple in to a 50 m L p olypropylene ce ntrifu g e tubes (fo rtify as needed , replace lid and m ix w e ll). N ote tha t alternate volum es o f serum m ay be measured depending on the sam ple size a vailab le fo r use. 11.2 A dd w ater to the sam ple fo r a fin a l volum e o f 20 m L Cap tig h tly 11.3 V ortex f o r - 1 m inute. 11.4 T ransfer 1 m L o f the sam ple using a disposable p ipe tte in to a IS m L disposable centrifU ge tube. 11.5 A dd S m L o f a ce to n itrile and shake fo r -2 0 m inutes on a w rist-a ctio n shaker. 11.6 C entrifU ge the tubes at -3 0 0 0 ip m fo r -5 m inutes. 11.7 Decant the supernatant in to a 50 m L disposable centrifU ge tube and add 35 m L o f water. 11.8 C o nd itio n the C u SPE cartridges (1 g, 6 m L ) b y passing 10 m L m ethanol fo llo w e d b y 5 m L o fH P L C w ater (~ 2 drop/sec). D o n ot le t colum n run d ry 11.9 Load the sam ple o n conditioned C u SPE ca rtrid ge . D isca rd eluate. 11.10 E lu te w ith -2 m L o f m ethanol. C o lle ct 2 m L o f eluate in to a graduated 1S m L polypropylene centrifU ge tube (fin a l volum e - 2 m L ). 11.11 A nalyze samples using electroepray LC /M S /M S . 12.0 Chrom atography 12.1 12.2 12.3 12.4 12.5 In je ct the same am ount o f each standard, sam ple and fo rtifie d sam ple in to the LC /M S /M S system . A ca lib ra tio n standard m ust precede and fo llo w a ll analyzed samples. Standards o f P FO A corresponding to a t least fiv e o r m ore concentration levels m ust be included in an a na lytica l set. A n entire set o f ca lib ra tio n standards m ust be in cluded at the beginning and at the end o f a sample set. Standards m ust be interspersed between every 5 -1u samples. A s an a lterna tive, an e ntire set o f c a lib ra tio n standards may be injected a t the beginning o f a set fo llo w e d b y ca lib ra tio n standards interspersed every 5-10 sam ples (to account fo r a second set o f standards). In e ithe r case, ca lib ra tio n standards m ust be the firs t and la st in je ctio n in a sam ple s e t Use lin e a r standard curves fo r q u a n tita tio n . L in e a r standard curves are generated fo r the analyte b y lin e a r regression using I /x w e ig h tin g o f peak area versus ca lib ra tio n standard concentration using M assLynx 3 3 (o r equivalent) softw are system . Sam ple response should n ot exceed standard responses. A n y samples that exceed standard responses should be fu rth e r d ilu te d and reanalyzed. PageJ of 7 Page 63 o f 65 Exygen Research Page 98 of 100 Interim Report #2-Analysis of Surface Water Samples Exygen Study No.: P0001131 Exygen Protocol Num ber: POOOU31 Exygen Reaetreh______________________ Method Number VOOOI7&6 | ANALYTICAL METHOD M ethod o f A na lysis fo r the D eterm ination o f P erfluorooctanoic A c id (P FO A ) in Sm all M am m al Seram b y LC /M S /M S 13.0 Acceptance C rite ria 13.1 13.2 13.3 13.4 13.3 13.6 Chrom atogram m ust show a peak o f a daughter io n at 369 amu from a parent o f 413 amu. The 413 amu parent corresponds to the PFO A anion, w h ile the daughter io n (369 am u) represents the loss o f carbon d io xid e . M ethod blanks m ust n ot contain PFO A at le vels greater than the LOQ . I f a blank contains PFO A s t levels greater than 10 n g/m L . then a new blank sample m ust be obtained and the e n tire set m ust be re-extracted. Recoveries o f co n tro l spikes and m a trix spikes m ust be between 70-130% o f th e ir know n values. I f a co n tro l spike fa lls outside the acceptable lim its , the e ntire set o f samples should be re-extracted. A n y m a trix spike outside 70 130% fo o u ld be evalusted b y the analyst to determ ine i f re -e xtra ction is w a rra n te d . A n y ca lib ra tio n standard found to be a s ta tis tic a l o u tlie r b y using the Huge E rro r Test m ay be excluded fro m the ca lcu la tio n o f the ca lib ra tio n curve However foe to ta l num ber o f ca lib ra tio n standards th a t could be excluded m ust not exceed 20% o fthe to ta l num ber o f standards injected. The co rre la tion co e fficie n t (R ) fo r c a lib ra tio n curves generated must be 20.992 (R 3 20.985). I f ca lib ra tio n results fa ll outside these lim its , then appropriate steps m u tt be taken to adjust in stru m en t operation, and the standards o r the relevant set o f samples should be reanalyzed. R etention tim es between standards and sam ples m ust not d rift more than 1 4 % w ith in an ana lytics] run. I f retention tim e d rift exceeds th is lim it w ith in an analytics) ran then the set m ust be reanalyzed. 14.0 C alculations 14.1 Use foe fo llo w in g equation to calculate the am ount o f P FO A found (in ng/m L. based on peak ares) using the standard curve (lin e a r regression param eters) generated b y foe M ass L ynx softw are program : PFO A found (n g /m L ) (Peak area - in te rce p t) x D F x a liq u o t factor slope D F fa cto r b y w h ich foe fin a l volum e w as d ilu te d , i f necessary. A liq u o t fa cto r 20 14.2 Foe samples fo rtifie d w ith know n am ounts o f P FO A p rio r to e xtractio n , use the fo llo w in g equation to calculate the percent recovery. Recovery (% ) * [ to ta l analyte found (n g /m L) - analyte found in c o n tro l (n g /m L )] analyte added (n g /m L) Page 6 o f7 Page 64 o f 65 Exygen Research Page 99 of 100 Interim Report #2-Analysis of Surface Water Samples Exygen Study No.: P0001131 E x y g e n P rotoco l N u m b er: POOO1131 Exyjaa Raearch Method Number VOOO1786 I AWa L yTICAI. METHOD M ethod o f A n ily iii fo r the D eterm ination o f P erfluorooctanoic A cid (P FO A ) in Sm all M am m al Seram b y LC /M S /M S 14.3 U ie the fo llo w in g equation to convert the am ount o f PFO A found in ng/m L to ppb. PFOA found (ppb) - fP F O A found (na/mlA x final volum e fm L sam ple volum e (m L ) Exygen Research Page 7 ul ? Page 65 o f 65 Page 100 of 100
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