Document DMGaYyDa2JgGdbXEnoNvDeDQB

INTERIM REPORT # 12 - Analysis of Vegetation 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 November 6,2006 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: 111 Interim Report #12 - Analysis of Vegetation Samples Exygen Study No.: P0001131 GOOD LABORATORY PRACTICE COMPLIANCE STATEMENT Exygen Study Number P0001131, entitled "Analysis of Perfluorobutanesulfonate (PFBS), Perfluorohexanesulfonate (PFHS), and Perfluorooctanesulfonate (PFOS) in Water, Soil, Sediment, Fish, Clams, Vegetation, Small Mammal Liver and Small Mammal Serum Using LC/MS/MS for the 3M Decatur Monitoring Program," conducted for 3M Company, is being performed in compliance with EPA TSCA Good Laboratory Practice Standards 40 CFR 792 by Exygen Research. Study Director Weston Solutions, Inc. Michael A. Santoro Sponsor Representative 3M Company Exygen Research Date Page 2 of 111 Interim Report #12 - Analysis of Vegetation Samples Exygen Study No.: P0001131 QUALITY ASSURANCE STATEMENT Exygen Research's Quality Assurance Unit reviewed Exygen Study Number P0001131, entitled, "Analysis of Perfluorobutanesulfonate (PFBS), Perfluorohexanesulfonate (PFHS), and Perfluorooctanesulfonate (PFOS) in Water, Soil, Sediment, Fish, Clams, Vegetation, Small Mammal Liver and Small Mammal Serum Using LC/MS/MS for the 3M Decatur Monitoring Program". All reviewed phases1 were inspected for conduct according to Exygen Research's Standard Operating Procedures, the Study Protocol, and all applicable Good Laboratory Practice Standards. All findings were reported to the Exygen Principal Investigator and Management and to the Study Director. Phase Date Inspected Date Reported to Date Reported to Principal Exygen Date Reported to Investigator Management Studv Director 20. Raw Data Review and Interim Analytical Report Review 27. Raw Data Review and Interim Analytical Report Review 09/01-02, 0608/05 06/21/06 09/22/05 06/29/06 09/22/05 06/30/06 09/22/05 11/06/06 43. Final Interim Report Reviw 11/03/06 11/06/06 11/06/06 11/06/06 Lydia Shaffer Technical Lead, Quality Assurance Unit Date 'Note: All in-lab inspections will be documented in the QA statement for the final analytical report at the conclusion of the study. This QA statement involves only the review of the interim report and associated raw data. Exygen Research Page 3 of 111 Interim Report #12 - Analysis of Vegetation 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: Exygen Research Study Director, Weston Solutions, Inc. Jaisimha Kesari P.E., DEE Weston Solutions, Inc. Sponsor Representative, 3M Company: Michael A. Sapforo Director of Regulatory Affairs Exygen Research Date Date Page 4 of 111 Interim Report #12 - Analysis of Vegetation 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: Vegetation 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: 07/12/05 Interim Analytical Termination Date: 06/09/06 Interim Report Completion Date: 11/06/06 Exygen Research Page 5 of 111 Interim Report #12 - Analysis of Vegetation 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 Christine Edwards Mark Ammerman Amy Sheehan Brittany Kravets Scott Crain Frances Crespi Mindy Cressley Krista Gallant Kim Hall Cammy Graybill Carlyle Horrell Title Vice President Laboratory Supervisor Technician Sample Custodian Associate Scientist Technician Technician Technician Technician Technician Technician Technician Intern Technician Exygen Research Page 6 of 111 Interim Report #12 - Analysis of Vegetation 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........................................................................................13 6.0 DESCRIPTION OF ANALYTICAL METHOD........................................................14 6.1 Extraction Procedure...............................................................................................14 6.1.1 Sample Preparation.........................................................................................14 6.1.2 Glassware Preparation....................................................................................15 6.1.3 Column Preparation........................................................................................15 6.1.4 Sample Extraction..........................................................................................15 6.2 Preparation of Standards and Fortification Solutions.............................................. 16 6.3 Chromatography......................................................................................................16 6.4 Instrument Sensitivity..............................................................................................17 6.5 Description of LC/MS/MS Instrument and Operating Conditions..........................17 6.6 Quantitation and Example Calculation....................................................................18 7.0 EXPERIMENTAL DESIGN......................................................................................19 8.0 RESULTS.................................................................................................................. 20 9.0 CONCLUSIONS............................................................................................. 20 10.0 RETENTION OF DATA AND SAMPLES............................................................ 20 Exygen Research Page 7 of 111 Interim Report #12 - Analysis of Vegetation Samples Exygen Study No.: P0001131 Table I. LIST OF TABLES Page Summary of PFBS, PFHS and PFOS in Vegetation Samples....................... 22 Table II. Summary of PFBS, PFHS and PFOS in Re-extracted Vegetation Samples...24 Table HI. Matrix Spike Recovery of PFBS, PFHS and PFOS in Vegetation Samples ..25 Table IV. Matrix Spike Recovery of PFBS, PFHS and PFOS in Re-extracted Vegetation Samples....................................................................................... 29 Exygen Research Page 8 of 111 Interim Report #12 - Analysis of Vegetation Samples Exygen Study No.: P0001131 LIST OF FIGURES Page Figure 1. Typical Calibration Curve for PFBS in Methanol........................................... 32 Figure 2. Non-Extracted Standards of PFBS in Methanol, 0.5 ng/mL and 1.0 ng/mL, Respectively................................................................................................... 33 Figure 3. PFBS in Control, 6.25 ng/mL Fortified Spk A, and 25 ng/mL Fortified Spk B, Respectively........................................................................................34 Figure 4. Chromatogram Representing a Vegetation Sample Analyzed for PFBS (Exygen ID: C0049876, Data Set:071205D)..................................................35 Figure 5. Typical Calibration Curve for PFHS in Methanol...........................................36 Figure 6. Non-Extracted Standards of PFHS in Methanol, 0.5 ng/mL and 1.0 ng/mL, Respectively....................................................................................................37 Figure 7. PFHS in Control, 6.25 ng/mL Fortified Spk A, and 25 ng/mL Fortified Spk B, Respectively........................................................................................38 Figure 8. Chromatogram Representing a Vegetation Sample Analyzed for PFHS (Exygen ID: C0049876, Data Set: 071205D).................................................39 Figure 9. Typical Calibration Curve for PFOS in Methanol...........................................40 Figure 10. Non-Extracted Standards of PFOS in Methanol, 0.5 ng/mL and 1.0 ng/mL, Respectively................................................................................................... 41 Figure 11. PFOS in Control, 6.25 ng/mL Fortified Spk A, and 25 ng/mL Fortified Spk B, Respectively....................................................................................... 42 Figure 12. Chromatogram Representing a Vegetation Sample Analyzed for PFOS (Exygen ID: C0049876, Data Set:071205D )......................................................... 43 Exygen Research Page 9 of 111 Interim Report #12 - Analysis of Vegetation Samples Exygen Study No.: P0001131 LIST OF APPENDICES Page Appendix A Study Protocol P0001131 (Exygen Study No. P0001131) with Analytical Method V0001784: "Method of Analysis for the Determination of Perfluorooctanoic Acid (PFOA) in Vegetation by LC/MS/MS", and Protocol Amendments............................................ 44 Exygen Research Page 10 of 111 Interim Report #12 - Analysis of Vegetation Samples Exygen Study No.: P0001131 1.0 SUMMARY Exygen Research extracted and analyzed vegetation samples for the determination of perfluorobutanesulfonate (PFBS), perfluorohexanesulfonate (PFHS), and perfluorooctanesulfonate (PFOS) according to Exygen Method V0001784 (Appendix A). The limit of quantitation for PFBS, PFHS and PFOS in vegetation was 0.2 ng/g. Analytical results for the initial analysis of PFBS, PFHS and PFOS found in vegetation samples and individual assessed accuracies are summarized in Table I. Initial vegetation sample results that did not meet data quality objectives were designated as not reported (NR). Re-extraction and reanalyses were performed in an attempt to obtain quantitative results. Analytical results for the analysis of PFBS, PFHS and PFOS found in re extracted vegetation samples and individual assessed accuracies are summarized in Table II. Quantitative results were obtained for all samples and analytes except for PFHS in one sample and PFOS in one sample. Fortification recoveries for PFBS, PFHS and PFOS in the initial analyses of vegetation samples are detailed in Table III. The average percent recoveries standard deviations for PFBS, PFHS, PFOS in vegetation samples were 78 12%, 92 13%, and 89 14%, respectively. Fortification recoveries for PFBS, PFHS and PFOS in the re-extracted vegetation samples are detailed in Table IV. The average percent recoveries standard deviations for PFBS, PFHS, PFOS in re-extracted vegetation samples were 76%, 94 28%, and 100 23% 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 vegetation 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 vegetation using the analytical method entitled, "V0001784: Method of Analysis for the Determination of Perfluorooctanoic Acid (PFOA) in Vegetation by LC/MS/MS." The study was initiated on November 05, 2004, when the study director signed protocol number P0001131. The analytical start date for this interim report was July 12, 2005 and the analytical termination date for this interim report was June 9,2006. Exygen Research Page 11 of 111 Interim Report #12 - Analysis of Vegetation Samples Exygen Study No.: P0001131 4.0 ANALYTICAL TEST SAMPLES The vegetation sample used as the control was grass obtained from Exygen's facility on June 1, 2005. Nine vegetation samples (Exygen ID C0050021-C0050029) were received on dry ice on October 22, 2004 from Tim Frinak at Weston Solutions, Inc Twenty-two vegetation samples (Exygen ID C0049876-C0049897) were received on dry ice on October 23, 2004 from Tim Frinak at Weston Solutions, Inc. The samples were logged in by Exygen personnel and placed in refrigerated storage. The vegetation sample identification (ID) codes for the samples analyzed and reported in PI 131 Interim Report #12 are in the following format: Dxx-x0x-xxxxxx-x-04xxxx Where the first string begins with a D for Decatur, Alabama and the xxx defines the sampling location within the study area where: DF06 = Field 6 DF8b = Field 8b DF09 = Field 9 DF14 = Field 14 DBKG = Northwest comer reference location The second string defines the first and second vegetation samples of each species within the specific location where: V01 = First vegetation sample V02 = Second vegetation sample The third string defines the species (first two characters), the sample matrix (third character; P = plant), and a sample number (last three characters) where the species are coded as: AV = Andropogon virginicus = broomsedge PA = Phytolacca americana = pokeweed RC = Rumex crispas = curly dock SS = Solidago spp. = goldenrod species UG = unidentified grass species The fourth string (single character) additionally defines the sample type where: 0 = Primary field sample 1 = Field duplicate sample 2 = Equipment rinsate blank sample Exygen Research Page 12 of 111 Interim Report #12 - Analysis of Vegetation Samples Exygen Study No. : P0001131 The fifth string defines the sample preparation date in YYMMDD format. Examples of this sample ID system are: Sample DF8b - V01 - PAP001 - 0 - 041007 is the sample prepared on October 7, 2004 from the first pokeweed sample collected from the Field 8b sampling location. Sample DF14 -V02 - AVP001 - 0 - 041007 is the sample prepared on October 7, 2004 from the second broomsedge sample collected from the Field 14 sampling location. Sample login and chain of custody information is located in the raw data package associated with this interim report. Storage records will be kept at Exygen Research. 5.0 REFERENCE MATERIAL The analytical standards, PFBS and PFHS, were supplied by 3M. PFBS was received from 3M at Exygen on May 13, 2005. PFHS was received from 3M at Exygen on January 20, 2003. PFOS was purchased from Fluka Corporation and was received at Exygen on April 23,2003. The available information for the reference materials is listed below. PFBS and PFHS were stored frozen and PFOS was stored refrigerated. Compound PFBS PFHS PFOS Exygen Inventory No. SP0005726 SP0002401 SP0002694 Lot # 101 SE036 430180-1 Purity (%) 96.7 98.6 101.2 Expiration Date 12/04/06 10/18/06 10/31/07 The molecular structures of PFBS, PFHS and PFOS are given below: PFBS Chemical Name: Perfluorobutanesulfonate Molecular Weight: 338 supplied as the potassium salt ^FgSOaTC*) Transitions Monitored: 299 -> 99 Structure: F FF F SO 3 FF FF Exygen Research Page 13 of 111 Interim Report #12 - Analysis of Vegetation Samples Exygen Study No.: P0001131 PFHS Chemical Name: Perfluorohexanesulfonate Molecular Weight: 438 supplied as the potassium salt (CeFoSC^'K-1") Transitions Monitored: 399 - 80 Structure: FFF FFF F SO3 FFF FFF PFOS Chemical Name: Perfluorooctanesulfonate Molecular Weight: 538 supplied as the potassium salt (CsFnSOs'K*) Transitions Monitored: 499 -> 80 Structure: FFFF FFFF F SO3 FFFF FFFF 6.0 DESCRIPTION OF ANALYTICAL METHOD The analytical method "V0001784: Method of Analysis for the Determination of Perfluorooctanoic Acid (PFOA) in Vegetation by LC/MS/MS" was used for the vegetation samples in this study. 6.1 Extraction Procedure 6.1.1 Sample Preparation Before the samples could be weighed for the extraction, they had to be processed. To process, the frozen samples were placed into a food processor and homogenized with dry ice. The samples were transferred to one-gallon Ziploc bags and placed in frozen storage with bag left open to allow the dry ice to sublime. After sublimation, the sample bags were sealed and remained in frozen storage until time of analysis. Exygen Research Page 14 of 111 Interim Report #12 - Analysis of Vegetation Samples Exygen Study No.: P0001131 6.1.2 Glassware Preparation The 125 mL pear-shaped flasks were silanized by first rinsing the flask with 30% dimethyldichlorosilane in toluene solution. The flasks were rinsed once with toluene followed by three rinses with methanol. The flasks were allowed to air dry before use. 6.1.3 Column Preparation The 20 mL columns were packed at Exygen in sequence with 2 grams florisil, 2 grams silica gel, 2 grams of carbon, and 1 gram LC-NH2. The columns were conditioned on the day of extraction with 20 mL of methanol followed by 20 mL of acetonitrile. All washes were discarded. These clean-up columns were used to remove matrix interference and not to retain PFBS, PFHS, and PFOS. Additional details about the column packing materials can be found in the raw data package associated with this report. 6.1.4 Sample Extraction A 5-gram portion of vegetation sample was weighed into a fifty-milliliter centrifuge tube for the extraction. After fortification of appropriate samples, 30 mL of acetonitrile was added to the samples. The samples were allowed to shake on a wrist action shaker for ~15 minutes. The samples were centrifuged for ~10 minutes at ~2000 rpm. The supernatant was then loaded onto the conditioned clean-up column fitted inside the silanized pear shaped flask. The eluate was collected in the pear-shaped flask. Twenty milliliters of acetonitrile was added to the samples left in the centrifuge tube. The samples were allowed to shake on a wrist action shaker for another 10 minutes. The samples were centrifuged again for ~10 minutes at ~2000 rpm. The supernatant was then loaded onto the same clean-up column. The eluate was collected into the same pear shaped flask. Twenty milliliters of acetonitrile was again added to the samples left in the centrifuge tube. The samples were allowed to shake on a wrist action shaker for another 10 minutes. The samples were centrifuged again for ~10 minutes at ~2000 rpm. The supernatant was then loaded onto the same clean-up column. The eluate was collected into the same pear-shaped flask. Approximately 3 or 4 drops of octanol was added to the extracts in the flasks. The samples were evaporated using a rotary evaporator at reduced pressure. Two milliliters of 2% ascorbic acid in methanol was added to the flasks to make final volume. The flask was swirled to dissolve and mix the sample. The sample was transferred to a HPLC vial using a disposable pipet. Each sample was analyzed by LC/MS/MS electrospray. Exygen Research Page 15 of 111 Interim Report #12 - Analysis of Vegetation Samples Exygen Study No.: P0001131 6.2 Preparation of Standards and Fortification Solutions A mixed stock standard solution of PFBS, PFHS, and PFOS was prepared. The stock standard solution was prepared at a concentration of 1000 pg/mL by dissolving 100 mg of each of the standards (corrected for purity and salt content, if necessary) in methanol. From this solution, a 100 pg/mL fortification standard solution was prepared by taking 10 mL of the stock and bringing the volume up to 100 mL with methanol. By taking 10 mL of the 100 pg/mL fortification standard and bringing the volume up to 100 mL with methanol, a 10 pg/mL fortification standard was prepared. By taking 10 mL of the 10 pg/mL fortification standard and bringing the volume up to 100 mL with methanol, a 1.0 pg/mL fortification standard were prepared. By taking 10 mL of the 1.0 pg/mL fortification standard and bringing the volume up to 100 mL with methanol, a 0.1 pg/mL fortification standard was prepared. By taking 10 mL of the 0.1 pg/mL fortification standard and bringing the volume up to 100 mL with methanol, a 0.01 pg/mL fortification standard were prepared. A set of non-extracted standards containing PFBS, PFHS and PFOS was prepared in methanol. The following concentrations were prepared: Cone, of Fort Fort Solution Volume (pg/mL)1 (mL) 1.0 5.0 1.0 2.5 1.0 1.0 0.05 10 0.025 10 0.01 10 0.005 10 of PFBS, PFHS and PFOS Volume of Fortified Sample (mL) 100 100 100 100 100 100 100 Final Cone, of Calibration Std. (pg/mL) 0.05 0.025 0.01 0.005 0.0025 0.001 0.0005 The stock standard solution and all fortification and calibration standard solutions were stored in a refrigerator (4 2C) when not in use. Documentation of standard preparation is located in the raw data package associated with this interim report. 6.3 Chromatography Quantification of PFBS, PFHS and PFOS was accomplished by LC/MS/MS electrospray. The retention times of PFBS, PFHS and PFOS were ~ 3.9 mins, ~ 10.0 mins, and ~ 12.2 mins, respectively. Peaks above the LOQ were not detected in any of the control vegetation samples corresponding to the analyte retention times. Exygen Research Page 16 of 111 Interim Report #12 - Analysis of Vegetation Samples Exygen Study No.: P0001131 6.4 Instrument Sensitivity The smallest standard amount injected during the chromatographic run had a concentration of 0.5 ng/mL 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: HPLC: Windows NT, Analyst 1.4.1 Hewlett Packard (HP) Series 1100 HP Quat Pump HP Vacuum Degasser HP Autosampler HP Column Oven HPLC Column: Thermo Fluophase RP, 50 mm x 2.1 mm Column Temp.: -30 C Injection Voi.: 15 jxL Mobile Phase (A): 2 mM Ammonium Acetate in water Mobile Phase (B): Methanol Time (mini 0.0 1.0 8.0 10.0 11.0 18.0 Total run time: ~18 min Flow Rate: 0.3 mL/min Ions monitored: %A 65 65 25 25 65 65 Analvte Mode PFBS PFHS PFOS negative negative negative %B 35 35 75 75 35 35 Transition Monitored 299 99 399 80 499 80 Approximate Retention Time finin') ~3.9 min. -10.0 min. -12.2 min. Exygen Research Page 17 of 111 Interim Report #12 - Analysis of Vegetation 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 concentrations in unknown samples were determined from the equations below. Equation 1 calculated the amount of analyte found (in ng/mL, based on peak area) in the unknown samples using the standard curve (linear regression parameters) generated by the Analyst software program. Equation 1: Analyte found (ng/mL) = (Peak area - intercept) x DF Slope Where: DF = Dilution Factor, factor by which the final volume was diluted, if necessary. Equation 2 was used to convert the amount of PFBS, PFHS and PFOS found in ng/mL to ng/g (ppb) on a wet weight basis. Equation 2: Analyte found (ppb) wet weight = Analyte found (ng/mLl wet weight x final volume (2 m Q sample weight (5 g) For samples fortified with known amounts of PFBS, PFHS and PFOS prior to extraction, Equation 3 was used to calculate the percent recovery. Equation 3: Recovery (%) = (analvte found (ne/el - analyte in control (ng/g)) xl00% amount added (ng/g) Note: For the analyte recovery calculation, the "control" is the unspiked aliquot of the primary field sample. An example of a calculation using an actual sample follows (calculation is for PFBS only): Vegetation sample Exygen ID: C0049884 Spk J (Set: 071205E), fortified at 100 ng/g with where: Exygen Research Page 18 of 111 Interim Report #12 - Analysis of Vegetation Samples Exygen Study No.: P0001131 peak area intercept slope dilution factor ng/g PFBS added (fort level) amt in corresponding sample final volume (mL) sample weight (g) = = = = = = = = 625244 4560 27500 1 100 1.50 2 5 From equation 1: Analyte found (ng/mL) = i625244 - 45601 x 10 27500 226 ng/mL From equation 2: Analyte found ng/g (ppb) wet weight = (226 ng/mL x 2 mL) 5g = 90.4 ng/g (ppb) From equation 3: % Recovery = (90.4 n g /g - 1.50 ng/g) x 100% 100 ng/g = 89% 7.0 EXPERIMENTAL DESIGN For vegetation samples designated as laboratory matrix spikes, PFBS, PFHS, and PFOS were added at a known concentration to the samples in the laboratory after the samples were weighed for extraction. The vegetation samples were extracted in twelve sets. Each set included one control vegetation blank and two control vegetation samples fortified at known concentrations. This control was used to ensure that contamination was not occurring during extraction. PFBS, PFHS, and PFOS were not found in any recordable level in the control samples. Laboratory control spikes are used to assess method accuracy. Five sets of vegetation contained five samples, one set of vegetation consisted of four samples, and one set of vegetation contained two samples. Five sets consisted of re-extracted samples to verify results or to re-extract with a more appropriate spiking level. Exygen Research Page 19 of 111 Interim Report #12 - Analysis of Vegetation Samples Exygen Study No.: P0001131 8.0 RESULTS Analytical results for the initial analysis of PFBS, PFHS and PFOS found in vegetation samples and individual assessed accuracies are summarized in Table I. Initial vegetation sample results that did not meet data quality objectives were designated as not reported (NR). Re-extraction and reanalyses were performed in an attempt to obtain quantitative results. Analytical results for the analysis of PFBS, PFHS and PFOS found in re extracted vegetation samples and individual assessed accuracies are summarized in Table II. Quantitative results were obtained for all samples and analytes except for PFHS in one sample and PFOS in one sample. Fortification recoveries for PFBS, PFHS and PFOS in the initial analyses of vegetation samples are detailed in Table III. The average percent recoveries standard deviations for PFBS, PFHS, PFOS in vegetation samples were 78 12%, 92 13%, and 89 14%, respectively. Fortification recoveries for PFBS, PFHS and PFOS in the re-extracted vegetation samples are detailed in Table IV. The average percent recoveries standard deviations for PFBS, PFHS, PFOS in re-extracted vegetation samples were 76%, 94 28%, and 100 23% respectively. Accuracies were assessed for each sample by reviewing the individual QC results obtained for each sample site. In most cases, there were two laboratory spike recovery results available for each sample that were used to assess the accuracy. 9.0 CONCLUSIONS Except as noted above, the vegetation samples were successfully extracted and analyzed for PFBS, PFHS and PFOS according to analytical method V0001784. 10.0 RETENTION OF DATA AND SAMPLES All original paper data generated by Exygen Research that pertains to this interim report will be shipped to the study director. This does not include facility-specific raw data such as instrument or temperature logs. Exact copies of all raw data, as well as a signed copy of the final analytical report and all original facility-specific raw data, will be retained in the Exygen Research archives for the period of time specified in EPA TSCA Good Laboratory Practice Standards 40 CFR 792. Exygen Research Page 20 of 111 Interim Report #12 - Analysis of Vegetation Samples Exygen Study No.: P0001131 TABLES Exygen Research Page 21 of 111 Interim Report #12 - Analysis of Vegetation Samples Exygen Study No.: P0001131 Table I. Summary of PFBS, PFHS and PFOS in Vegetation Samples Exvaen ID C0049876 C0049876 Rep C0049877 C0049877 Rep C0049878 C0049878 Rep C0049879 C0049879 Rep C0049880 C0049880 Rep C0049881 C0049881 Rep C0049882 C0049882 Rep C0049883 C0049883 Rep C0049884 C0049884 Rep C0049685 C0049885 Rep C0049886 C0049886 Rep C0049887 C0049887 Rep C0049888 C0049888 Rep C0049889 C0049889 Rep C0049890 C0049890 Rep C0049891 C0049891 Rep C0049892 C0049892 Rep Client Sample ID C4 Sulfonate PFBS Perfluorobutanesulfonate Analyte Found (ppb, ng/g) Wet Weight Assessed Accuracy {+/- %) C6 Sulfonate PFHS Perfluorohaxanesulfonate Analyte Found (ppb, ng/g) Wet Weioht Assessed Accuracy (+/-%) C8 Sulfonate PFOS Perfluorooctanesulfonate Analyte Found (ppb, ng/g) Wet Weight Assessed Accuracy (+/-%) D F 0 6 -V 0 1-AV P O 0 1-0-0 4 1 00 7 DF06-V01-AVP001-0-041007* 11.7 11.1 30 41.2 30 35.8 30 30 NR NR NR NR DF06-V01 -PAP001-0-041007 32.6 30 NR NR NR NR DF06-V01-PAP001 -0-041007* 31.4 30 NR NR NR NR DF06-V01-RCP001-0-041007 DF06-V01-RCP001 -0-041007* 4.48 4.36 30 20.9 30 23.7 40 40 NR NR NR NR DF06-V02-AVP001-0-041007 11.7 50 18.5 30 D F 0 6 -V 0 2 -A V P 0 0 1-0 -04 1 0 07 * 10.5 50 16.3 30 NR NR NR NR DF06-V02-PAP001-0-041007 DF06-V02-PAPO01-0-041007* 8.68 8.72 30 55.6 30 56.4 30 30 NR NR NR NR DF06-V02-RCP001-0-041007 5.92 30 9.12 30 224 30 DF06-V02-RCP001 -0-041007* 5.96 30 9.28 30 252 30 DF09-V01-AVP001-0-041007 3.49 30 20.7 30 198 50 DF09-V01-AVP001-0-041007* 3.71 30 20.9 30 187 50 DF09-V01-PAP001-0-041007 5.04 30 13.0 30 DF09-V01-PAP001 -0-041007* 4.92 30 14.7 30 NR NR NR NR DF09-V01-RCP001-0-041007 1.50 30 11.1 30 99.6 30 DF09-V01-RCP001 -0-041007* 1.40 30 10.9 30 100 30 DF09-V02-RCP001-0-041007 1.38 30 35.3 30 DF09-V02-RCP001-0-041007* 1.37 30 39.3 30 NR NR NR NR DF09-V02-PAP001-0-041007 20.1 40 NR NR NR NR DF09-V02-PAP001-0-041007* 19.9 40 NR NR NR NR DF8b-V01 -PAP001-0-041007 30.9 30 188 30 NR NR D F8b-V01 -PAP001-0-041007* 30.3 30 190 30 NR NR DF8b-V01-RCP001-0-041007 DF8b-V01 -RCP001 -0-041007* 2.16 2.11 30 35.2 30 32.6 30 30 NR NR NR NR DF8b-V02-PAP001-0-041007 62.0 40 NR NR NR NR DF8b-V02-PAP001-0-041007* 56.8 40 NR NR NR NR DF8b-V02-RCP001-0-041007 2.53 30 36.3 30 DF8b-V02-RCP001 -0-041007* 2.24 30 37.0 30 NR NR NR NR DF14-V01-AVP001-0-041007 0.408 30 ND 30 3.94 30 DF14-V01-AVP001-0-041007* 0.390 30 ND 30 3.82 30 DF14-V01-PAP001-0-041007 DF14-V01-PAP001-0-041007* 2.05 2.15 30 0.632 30 30 0.636 30 NR NR NR NR Laboratory Duplicate ND = Not detected at or above the Limit of Quantitation (LOQ) of 0.2 ng/g. NR = Not reported due to quality control failure, see Table II for re-extract data. Exygen Research Page 22 of 111 Interim Report #12 - Analysis of Vegetation Samples Exygen Study No.: P0001131 Table I. Summary of PFBS, PFHS and PFOS in Vegetation Samples (continued) Exvaen ID C0049893 C0049893 Rep C0049894 C0049894 Rep C004989S C0049895 Rep C0049896 C0049896 Rep C0049897 C0049897Rep C0050021 C0050021 Rep C0050022 COO50022 Rep C0050023 C0050023 Rep C0050024 C0050024 Rep C0050025 C0050025 Rep COO50026 COO50026 Rep C0050027 C0050027 Rep C0050028 C0050028Rep C0050029 C0050029 Rep Client Sample ID C4 Sulfonate PFBS Perfluorobutan--utfonato Analyte Found (PPb. ng/g) Wet Weight Assessed Accuracy (+/-%> C6 Sulfonate PFHS C8 Sulfonate PFOS Perfluorohexan--ulfonate_______P ffluorooctansulfonate Analyte Found (ppb, ng/g) Assessed Accuracy Analyte Found (ppb, ng/g) Assessed Accuracy Wet Weight <+/-%) Wet Weight (+/-%) D F 14-V02-AVPO01-0-041007 0.393 30 ND 30 5.20 30 DF14-V02-AVP001-0-041007* 0.432 30 ND 30 5.76 30 DF14-V02-PAP001-0-041007 DF14-V02-PAP001 -0-041007* 1.55 1.45 30 0.944 30 30 0.864 30 9.08 8.76 30 30 D B K G -V 0 1-A V P 0 0 1 -0 -04 1 0 07 0.928 30 ND 30 5.48 30 DBKG-V01-AVP001-0-041007* 0.888 30 ND 30 5.00 30 D BKG-V01-PAP001-0-041007 D B K G -V 0 1-P A P 0 0 1-0 -04 1 0 07 * 6.28 6.04 30 0.608 30 0.564 30 30 18.7 19.3 30 30 DBKG-V01-RCPO01-0-041007 0.460 30 ND 30 1.64 30 DBKG-V01-RCP001-0-041007* 0.464 30 ND 30 1.93 30 DF09-V01-SSP001-0-041007 9.56 30 14.6 30 DF09-V01-SSP001-0-041007* 8.64 30 14.2 30 NR NR NR NR D F 0 9 -V 0 2 -U G P 0 0 1-0-041007 8.28 30 98.8 30 DF09-V02-UGP001-0-041007* 7.96 30 104 30 NR NR NR NR DF06-V01-SSP001-0-041007 DF06-V01-SSP001 -0-041007* NR NR NR 51.2 NR 50.8 30 30 NR NR NR NR DF06-V02-SSP001-0-041007 10.4 30 NR NR 101 30 DF06-V02-SSP001-0-041007* 9.80 30 NR NR 99.2 30 DF8D-V01-UGP001-0-041007 12.4 40 146 30 DF8b-V01 -UGP001 -0-041007* 11.6 40 149 30 NR NR NR NR DF8b-V02-UGP001-0-041007 9.88 30 183 30 DF8b-V02-UGP001-0-041007* 9.68 30 193 30 NR NR NR NR DF14-V01-SSP001-0-041007 3.16 30 NR NR NR NR DF14-V01-SSP001-0-041007* 3.33 30 NR NR NR NR D F14-V02-SSP001-0-041007 1.53 30 NR NR NR NR DF14-V02-SSP001-0-041007* 1.68 30 NR NR NR NR DBKG-V01-SSP001-0-041007 DBKG-V01-SSP001-0-041007* 7.12 7.72 30 0.236 40 30 0.267 40 NR NR NR NR ` Laboratory Duplicate ND * Not detected at or above the Limit of Quantitation (LOQ) of 0.2 ng/g. NR = Not reported due to quality control failure, see Table II for re-extract data. Exygen Research Page 23 of 111 Interim Report #12 - Analysis of Vegetation Samples Exygen Study No.: P0001131 Table II. Summary of PFBS, PFHS and PFOS in Re-extracted Vegetation Samples ExynenID C0049876 C0049877 C0049878 C0049879 C0049880 C0049883 C0049885 C0049886 C0049887 C0049888 C0049889 C0049890 C0049892 C0050021 C0050022 C0050023 C0050024 C0050025 C0050026 C0050027 C0050028 C0050029 Client Sample ID C4 Sulfonate PFBS P e rflu o ro b u ta n e su lfo n a te Analyte Found (ppb, ng/g) Dry Weight Assessed Accuracy (+/.%) C6 Sulfonate PFHS C8 Sulfonate PFOS Parfluorohaxan--ulfonate_______ P e riluorooctanesulfonate Analyte Found (ppb, ng/g) Drv Weight Assessed Accuracy (+/-%) Analyte Found (ppb, ng/g) Dry Weight Assessed Accuracy <+/-%> DF06-V01-AVP001 -0-041007 - - - 2400 30 DF06-V01-PAP001-0-041007 - - 608 30 56400 50 DF06-V01-RCP001-0-041007 - - - - 436 30 DF06-V02-AVP001-0-041007 - - - - 212 30 DF06-V02-PAP001-0-041007 - - - - 3000 30 DF09-V01-PAP001-0-041007 - - - - 532 30 D F 0 9-V 0 2 -R C P 0 0 1-0-041007 - - - - 1880 30 DF09-V02-PAP001-0-041007 - - 460 30 11000 30 DF8b-V01 -PAP001 -0-041007 - - - - 6480 40 DF8b-V01-RCP001-0-041007 - - - - 5960 30 DF8b-V02-PAP001 -0-041007 - - 1060 30 22100 30 DF8b-V02-RCP001-0-041007 - - - - 3510 30 DF14-V01-PAP001-0-041007 - - - - 14.4 30 DF09-V01-SSP001-0-041007 - - - - 584 40 DF09-V02-UGP001-0-041007 - - - - 2680 30 DF06-V01-SSP001-0-041007 16.2 30 - - 3440 30 DF06-V02-SSP001-0-041007 - - NR NR - - DF8b-V01-UGP001-0-041007 DF8b-V02-UGPO01-0-041007 DF14-V01-SSP001 -0-041007 D F 14-V02-SSP001-0-041007 DBKG-V01-SSP001-0-041007 - - - - NR NR - - - 7840 30 - 0.274 50 4.88 30 - 1.33 30 2.24 30 - - - 9.72 40 ` Laboratory Duplicate NR = Not reported due to quality control failure. Exygen Research Page 24 of 111 Interim Report #12 - Analysis of Vegetation Samples Exygen Study No.: P0001131 Table III. Matrix Spike Recovery of PFBS, PFHS and PFOS in Vegetation Samples Sample Description DF06-V01-AVP001-0-041007 (C0049876Spk C, 10ppb) DF06-V01 -AVP001-0-041007 (C0049676Spk D, 100ppb) DF06-V01-PAP001 -0-041007 (C0049877 Spk E, 10ppb) DF06-V01-PAP001-0-041007 (C0049877Spk P. 100 ppb) DF06-V01-RCP001 -0-041007 (C0049878 SpkG, 10ppb) DF06-V01-RCP001-0-041007 (C0049870Spk H, 100ppb) DF06-V02-AVP001-0-041007 <00049879Spk 1,10 ppb) DFO6-V02-AVP001-0-041007 (C0049879SpkJ, 100 ppb) DF06-V02-PAP001-0-041007 (C0049880SpkK. 10ppb) DF06-V02-PAP001-0-041007 (C0049880Spk L. 100ppb) DF06-V02-RCP001 -0-041007 (C0049881 Spk C, 10ppb) DF06-V02-RCP001-0-041007 (C0049881 Spk D, 100ppb) DF09-V01-AVP001-0-041007 (C0049882 Spk E. 10 ppb Spike) DF09-V01-AVP001-0-041007 (C0049682Spk F. 100 ppb Spite) DF09-V01-PAP001-0-041007 (C0049883 Spk G. 10 ppb Spite) DF09-V01-PAP001-0-041007 (C 0049883 Spk H , 100 ppb Spike) DF09-V01-RCP001-0-041007 (C0049884 Spk 1. 10ppb Spite) DF09-V01-RCP001-0-041007 (C0049884 SpkJ. 100ppb Spike) DF09-V02-RCP001-0-041007 (C0049885 Spk K. 10ppb Spite) DF09-V02-RCP001-0-041007 (C0049885 SpkL, 100ppb Spike) Amount Spiked (ng/g) C4 Sulfonate PFBS______________ C6 Sulfonate PFHS_______________C8 Sulfonate PFOS Amt Found Amount Amt Found Amount Amt Found Amount in Sample Recovered Recovery in Sample Recovered Recovery in Sample Recovered Recovery (ng/g) (na/a) (%) (ng/g) fno/a) (% ) (ng/g) (ng/g) <%> 10 11.7 19.5 78 41.2 55.2 100 11.7 81.6 70 41.2 118 77 NR NR NR NR NR NR 10 32.6 37.0 44 NR NR NR NR 100 32.6 109 76 NR NR NR NR NR NR NR NR 10 4.48 12.4 79 20.9 34.2 133 NR 100 4.48 86.8 82 20.9 120 99 NR NR NR NR NR 10 11.7 100 11.7 10 8.68 100 8.68 10 5.92 100 5.92 10 3.49 100 3.49 17.3 76.4 17.1 89.6 13.5 86.0 11.2 72.8 56 65 64 81 76 80 112 73 18.5 18.5 55.6 55.6 9.12 9.12 20.7 20.7 26.0 88.4 68.8 145 18.3 103 28.3 97.6 75 70 89 92 94 76 77 NR NR NR NR 224 224 198 198 NR NR NR NR NR NR NR NR 210 . 301 77 203 * 250 52 10 5.04 12.2 72 100 5.04 82.0 77 13.0 232 102 13.0 105 92 NR NR 10 1.50 9.44 79 11.1 19.8 87 99.6 100 1.50 90.4 89 11.1 118 107 99.6 10 1.38 100 1.38 8.92 75 80.0 79 35.3 35.3 48.0 132 97 NR NR NR NR NR NR 108 210 110 NR NR NR NR *Sampie residue exceeds the spiking level significantly; therefore, an accurate recovery value cannot be calculated NR * Not reported due to quality control failure, see Table IV fo r re-extract data. Note: Since this summary table shows rounded results, recovery values may vary slightly from the values in the raw data. Exygen Research Page 25 of 111 Interim Report #12 - Analysis of Vegetation Samples Exygen Study No.: P0001131 Table III. Matrix Spike Recovery of PFBS, PFHS and PFOS in Vegetation Samples (continued) Sample Description DF09-V02-PAP001 -0-041007 (C0049M6 Spk C. 10 ppb Spike) DF09-V02-PAP001-0-041007 (C0049686 Spk D. 100ppb Spike) DF8b-V01 -PAP001-0-041007 (C0049887Spk E. 10 ppb Spike) DF8b-V01-PAP001 -0-041007 (C0049887Spk F. 100 ppb Spike) DF8b-V01 -RCP001 -0-041007 {COM9888Spk 6.10 ppb Spike) DF8b-V01-RCP001 -0-041007 (C0049888 SpkH. 100ppb Spike) DF8b-V02-PAP001-0-041007 (C0049889Spk 1.10 ppb Spike) DF8b-V02-PAP001-0-041007 (C0049889Spk J, 100ppbSpike) DF8b-V02-RCP001-0-041007 (C0049890 SpkK, 10ppb Spike) DF8b-V02-RCP001-0-041007 (C0049890 SpkL, 100 ppb Spice) DF14-V01-AVP001-0-041007 (C0049691 Spk C, 10ppb Spike) DF14-V01-AVP001-0-041007 (C0049691 Spk D. 100ppb Spike) DF14-V01-PAP001 -0-041007 (C0049692 Spk E, 10 ppb Spike) DF14-V01-PAP001-0-041007 (C0049892Spk F, 100 ppb Spike) DF14-V02-AVP001-0-041007 <00049693Spk G, 10ppbSpike) DF14-V02-AVP001-0-041007 (C0049693 SpkH, 100ppb Spike) DF14-V02-PAP001-0-041007 (C0049894 Spk 1,10ppbSpike) DF14-V02-PAP001-0-041007 (C0049694 SpkJ, 100ppbSpike) DBKG-V01-AVP001 -0-041007 (C0049895 SpkK. 10ppb Spike) DBKG-V01-AVP001 -0-041007 (C0049695 SpkL, 100 ppbSpike) Amount Spiked (no/a) C4 Sulfonate PFBS______________ C6 Sulfonate PFHS_______________C8 Sulfonate PFOS Amt Found Amount Amt Found Amount Amt Found in Sample Recovered Recovery in Sample Recovered Recovery in Sample (no/a) fna/a) <%> (no/a) (n fl/fl) (%> (ng/g) Amount Recovered Recovery (ng/g) <%) 10 20.1 100 20.1 33.4 133 110 90 NR NR NR NR NR NR NR NR NR NR NR NR 10 30.9 40.8 188 216 NR 100 30.9 105 74 188 295 107 NR NR NR NR NR 10 2.16 100 2.16 9.72 76 82.8 81 35.2 35.2 40.8 133 98 NR NR NR NR NR NR 10 62.0 692 NR NR NR NR 100 62.0 129 67 NR NR NR NR NR NR NR NR 10 2.53 102 77 36.3 46.0 * NR NR NR 100 2.53 87.6 85 36.3 146 110 NR NR NR 10 0.408 100 0.408 8.96 82.0 86 82 ND 10.0 100 3.94 ND 98.0 98 3.94 12.7 88 91.2 87 10 2.05 9.72 77 0.632 10.2 96 100 2.05 84.0 82 0.632 99.2 99 NR NR NR NR NR NR 10 0.393 6.00 76 ND 9.24 92 5.20 14.1 89 100 0.393 79.6 79 ND 91.6 92 5.20 91.6 86 10 1.55 9.72 82 0.944 10.4 95 100 1.55 84.8 83 0.944 97.2 96 9.08 9.08 17.6 85 100 91 10 0.928 9.12 82 ND 9.96 100 5.48 13.7 82 100 0.928 87.2 86 ND 102 102 5.48 98.4 93 ` Sample residue exceeds the spiking level significantly; therefore, an accurate recovery value cannot be calculated ND = Not detected at o r above the Limit o f Quantitation (LOQ) o f 0.2 ng/g. NR * Not reported due to quality control failure, see Table IV fo r re-extract data. Note: Since this summary table shows rounded results, recovery values may vary slightly from the values in the raw data. Exygen Research Page 26 of 111 Interim Report #12 - Analysis of Vegetation Samples Exygen Study No.: P0001131 Table III. Matrix Spike Recovery of PFBS, PFHS and PFOS in Vegetation Samples (continued) Sample Description DBKG-V01-PAP001-0-041007 (C0049896Spk C. 10ppb Spike) DBKG-V01-PAP001-0-041007 (C0049896Spk D, 100 ppb Spike) DBKG-V01-RCP001-0-041007 (C0049697Spk E. 10ppb Spike) DBKG-V01-RCP001-0-041007 (C0049897Spk F, 100 ppb Spike) DF09-V01-SSP001 -0-041007 (C0050021 SpkC, 10ppb) DF09-V01-SSP001 -0-041007 (C0050021 Spk D, 100ppb) DF09-V02-UG PO01-0-041007 (C0050022Spk E. 10ppb) DF09-V02-UGP001 -0-041007 (C0050022 SpkF, 100ppb) DF06-V01-SSP001-0-041007 (C0050023Spk 6,10 ppb) DF06-V01-SSP001-0-041007 (C00S0023 Spk H, 100ppb) DF06-V02-SSP001 -0-041007 (C0050024 Spk 1,10ppb) DF06-V02-SSP001 -0-041007 (C0050024 SpkJ, 100ppb) DF8b-V01-UGP001-0-041007 (C0050025 Spk K, 10ppb) DF8b-V01 -UGP001 -0-041007 (C005002SSpk L, 100ppb) DF8b-V02-UGP001 -0-041007 (C0050026Spk C, 10ppb) D F8b-V02-UG P001-0-041007 (C0050026 Spk D, 100 ppb) DF14-V01-SSP001-0-041007 (C0050027Spk E, 10ppb) DF14-V01-SSP001 -0-041007 (C0050027Spk F, 100 ppb) DF14-V02-SSP001-0-041007 (C0050028 SpkG, 10 ppb) DF14-V02-SSP001-0-041007 (C0050028 SpkH, 100ppb) Amount Spiked (ng/8> C4 Sulfonate PFBS______________ C6 Sulfonate PFHS_______________C8 Sulfonate PFOS Amt Found Amount Amt Found Amount Amt Found Amount in Sample Recovered Recovery In Sample Recovered Recovery in Sample Recovered Recovery (ng/g) (ng/g) (%) (ng/g) (ng/g) (%> (ng/g) (ng/g) (%) 10 6.28 13.8 75 0.608 9.56 90 18.7 29.4 107 100 6.28 83.2 77 0.608 68.4 88 18.7 103 84 10 0.460 8.32 79 ND 9.64 96 1.64 10.3 87 100 0.460 78.4 78 ND 96.4 96 1.64 87.6 86 10 9.56 100 9.56 10 8.28 100 8.28 10 NR 100 NR 10 10.4 100 10.4 10 12.4 100 12.4 10 9.88 100 9.88 16.7 87.2 15.6 79.2 NR NR 17.5 74.8 18.4 86.4 17.5 87.6 71 78 73 71 NR NR 71 64 60 74 76 78 14.6 14.6 98.8 98.8 51.2 51.2 NR NR 146 146 183 183 21.7 71 102 87 111 * 182 83 59.6 . 124 73 NR NR NR NR 153 * 248 102 210 * 274 91 NR NR NR NR NR NR 101 101 NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR 123 * 215 114 NR NR NR NR NR NR NR NR 10 3.16 10.3 71 NR NR NR NR 100 3.16 80.4 77 NR NR NR NR NR NR NR NR 10 1.53 8.88 74 NR NR NR NR 100 1.53 96.0 94 NR NR NR NR NR NR NR NR 'S am ple residue exceeds the spiking level significantly; therefore, an accurate recovery value cannot be calculated ND = Not detected at o r above the Limit of Quantitation (LOQ) o f 0.2 ng/g. NR = Not reported due to quality control failure, see Table IV fo r re-extract data. Note: Since this summary table shows rounded results, recovery values may vary slightly from the values in the raw data. Exygen Research Page 27 of 111 Interim Report #12 - Analysis of Vegetation Samples Exygen Study No.: P0001131 Table III. Matrix Spike Recovery of PFBS, PFHS and PFOS in Vegetation Samples (continued) Sample Description DBKG-V01-SSPO01-0-041007 (C0050029 Spk I.IO ppb) DBKG-V01-SSP001-0-041007 (C0050029 Spk J, 100 ppb) Amount Spiked (ng/a) C4 Sulfonate PFBS______________ C6 Sulfonate PFHS_______________C8 Sulfonate PFOS Amt Found Amount Amt Found Amount Amt Found in Sample Recovered Recovery in Sample Recovered Recovery in Sample (ng/g) (ng/g) (%) (ng/g) (ng/g) (%) (ng/g) Amount Recovered (ng/g) Recovery (%) 10 7.12 100 7.12 14.8 77 0.236 6.52 105 98 0.267 88.4 63 68 NR NR NR NR NR NR Average: Standard Deviation: 78 12 Average: Standard Deviation: NR Not reported due to quality control failure, see Table IV for re-extract data. Note: Since this summary table shows rounded results, recovery values may vary slightly from the values in the raw data. 92 13 Average: Standard Deviation: 89 14 Exygen Research Page 28 of 111 Interim Report #12 - Analysis of Vegetation Samples Exygen Study No.: P0001131 Table IV. Matrix Spike Recovery of PFBS, PFHS and PFOS in Re-extracted Vegetation Samples Sample Description DF06-V01-AVPO01-0-041007 (C0049876Spk E, 10000ppb) DF06-V01-PAP001-0-041007 (C0049877Spk G, 2000ppb) DF06-V01-PAP001 -0-041007 (C0049877SpkH, 100000ppb) DF06-V01-RCP001-0-041007 (C0049878Spk 1,2000ppb) DF06-V02-AVP001 -0-041007 (C0049870SpkK, 400ppb) DF06-V02-PAP001 -0-041007 (00049660 Spk M, 10000ppb) DF09-V01-PAP001-0-041007 (C0049663 Spk 1.1000ppb Spike) DF09-V02-RCP001 -0-041007 (C0049885 SpkM, 4000ppb Spike) DF09-V02-PAP001 -0-041007 (C0049866 SpkE, 1000ppb Spice) DF0-V02-PAP001 -0-041007 (C0049686 SpkF, 20000 ppb Spike) DF8b-V01 -PAP001-0-041007 (C0049667SpkG. 20000ppb Spike) DF8b-V01-RCP001-0-041007 (C0049888Spk 1.10000ppb Spike) DF8b-V02-PAP001-0-041007 (C0049889SpkK. 2000ppb Spice) DF8b-V02-PAP001-0-041007 (C004986SSpkL. 40000 ppb Spike) DF8b-V02-RCP001 -0-041007 (C0049690Spk M. 10000ppbSpice) DF14-V01-PAP001-0-041007 (C0049892SpkG. 10ppb Spike) DF09-V01-SSP001-0-041007 (C0050021 Spk E, 1000 ppb) DF09-V02-UGP001-0-041007 (C0050Q22SpkG, 8000ppb) DF06-V01-SSP001-0-041007 (C0050023 Spk1,10 ppb) DF06-V01-SSP001-0-041007 (C0050023 SpkJ, 6000ppb) DF06-V02-SSP001-0-041007 (C0050024 SpkK, 10ppb) Amount Spiked (nq/q) C4 Sulfonate PFBS______________ C6 Sulfonate PFHS_______________ C8 Sulfonate PFOS Amt Found Amount Amt Found Amount Amt Found in Sample Recovered Recovery in Sample Recovered Recovery in Sample (nq/q) (nq/q) <%) (nq/q) (nq/q) (%) (ng/g) Amount Recovered Recovery <%) 10000 2000 100000 . - .. - - 2400 9680 73 . . 608 2670 113 . -- - - - 56400 113000 57 2000 -- - - 436 2110 84 400 -- - - - 212 552 85 10000 -- - - - 3000 12400 94 1000 -- - - - 532 1480 95 4000 1000 20000 - -- - 1880 6480 115 . . 460 1480 102 - -- - - 11000 26200 76 20000 -- - - - 6480 33500 135 10000 2000 40000 - -- - - - 5960 15900 99 . . 1060 3480 121 .- -- - - - 22100 62400 101 10000 10 --- - - 3510 13000 95 - - 14.4 24.2 98 1000 - -- - - - 584 1920 134 8000 - -- - - - 2660 8960 79 10 16.2 23.8 76 8000 - -- . - - -- - - 3440 13500 126 10 - - - NR NR NR - - - Note: Since this summary table shows rounded results, recovery values may vary slightly from the values in the raw data. NR = Not reported due to quality control failure. Exygen Research Page 29 of 111 Interim Report #12 - Analysis of Vegetation Samples Exygen Study No.: P0001131 Table IV. Matrix Spike Recovery of PFBS, PFHS and PFOS in Re-extracted Vegetation Samples (continued) Sample Description DF8b-V01 -UGP001-0-041007 (C0050025 Spk M, 20000 ppb) DF8-V02-UGP001 -0-041007 (C0050026 Spk E. 20000 ppb) DF14-V01-SSP001 *0-041007 (C0050027 Spk 6 , 10 ppb) DF14-V02-SSP001-0-041007 (C0050026 Spk 1,10 ppb) DBKG-V01-SSPO01-0-041007 (C0050029 Spk K, 10 ppb) Amount Spiked (ng/g) C4 Sulfonate PFBS______________ C6 Sulfonate PFHS_______________ C8 Sulfonate PFOS Amt Found Amount Amt Found Amount Amt Found Amount in Sample Recovered Recovery in Sample Recovered Recovery in Sample Recovered Recovery (ng/g) (ng/g) <%) (ng/g) (ng/g) (%) (ng/g) (ng/g) <%> 20000 NR NR NR 20000 - . 7840 31300 117 10 - 0.274 5.52 52 4.88 13.7 88 10 . 1.33 9.44 81 2.24 15.2 130 10 - - - - - 9.72 22.8 131 Average: Standard Deviation: 7 NA Average: Standard Deviation: Note: Since this summary table shows rounded results, recovery values may vary slightly from the values in the raw data. NR = Not reported due to quality control failure. 94 28 Average: Standard Deviation: 100 23 Exygen Research Page 30 of 111 Interim Report #12 - Analysis of Vegetation Samples Exygen Study No.: P0001131 FIGURES Exygen Research Page 31 of 111 Interim Report #12 - Analysis of Vegetation Samples Exygen Study No.: P0001131 Figure 1. Typical Calibration Curve for PFBS in Methanol 0 71 20 5 D V E O .rd b(P F B S ): " L in e jr " Regression (*1 / x " w e ig htin g ): y * 2 .8 e + 0 0 4 x + 4 .2 4 e + 0 0 3 ( r * 0 .0 0 8 3 ) Area, counts Exygen Research Page 32 of 111 Interim Report #12 - Analysis of Vegetation Samples Exygen Study No.: P0001131 Figure 2. Non-Extracted Standards of PFBS in Methanol, 0.5 ng/mL and 1.0 ng/mL, Respectively SS7279-PFBS (S ta n d ard ) 2 99.9/9*0a m * -sam pfa 7 o f 39 fro m 071205D.wifF A n a : T7346 co u n ts H a ig t- 9.63a+002 cps R T: 0i 529 m in 0.53 Atj : 31055 counts Height: 1.70e+003 cps RT: 0.533 min 0.53 Intensity, cps Intensity, cps Exygen Research Page 33 of 111 Interim Report #12 - Analysis of Vegetation Samples Exygen Study No.: P0001131 Figure 3. PFBS in Control, 6.25 ng/mL Fortified Spk A, and 25 ng/mL Fortified Spk B, Respectively Raagaat C oatrot - PFBS (U ktaow a) 299Ltt9&f amu tam p/a 9 o f 3$ from $T12$Skwiff & aak a o t foaad) 4.43 9.09 3.55 T im t, min I R tjg tn tS p k B - PFBS(QC)209.0/99.0 amu-samplt 11 o f3 8 from0712050.vwff Arta: 750120 counts H tigh t 3.92t+004 ops RT: 0.529 min 0.53 11 12 15 Exygen Research Page 34 of 111 Interim Report #12 - Analysis of Vegetation Samples Exygen Study No.: P0001131 Figure 4. Chromatogram Representing a Vegetation Sample Analyzed for PFBS (Exygen ID: C0049876, Data Set:071205D) I C904M7C -PFBS (U H **ow *) 2991099.mu sampln 15o f 3 from 07125D.wiff A n a : tZ U T Ie o a a t* H aight: iH a + 9 *4 e p t RT: 3.92m i* 3.02 Intensity, cps Exygen Research Page 35 of 111 Interim Report #12 - Analysis of Vegetation Samples Exygen Study No.: P0001131 Figure 5. Typical Calibration Curve for PFHS in Methanol 071209b VEG.rdb (PFHS): " U n t il" Rgrsion C'1 / x " lig h t in g ) : y 4.03t+ 0 0 4 x + 5.001+003 (r 0.0088) Area, counts f Exygen Research Page 36 of 111 Interim Report #12 - Analysis of Vegetation Samples Exygen Study No.: P0001131 Figure 6. Non-Extracted Standards of PFHS in Methanol, 0.5 ng/mL and 1.0 ng/mL, Respectively SS7279 - PFHS (Standard) 399.&90.I ama sam pt* 1 o f 39 from Q71295D.wiff A n a : 29955 counts H sfgt: 1.32*+093 cps RT: 9.93 m in Intensity, cps A r tj : 53414 counts Height: 2.431+003 cps R T :0.04m in 0.04 Intensity, cps Exygen Research Page 37 of 111 Interim Report #12 - Analysis of Vegetation Samples Exygen Study No.: P0001131 Figure 7. PFHS in Control, 6.25 ng/mL Fortified Spk A, and 25 ng/mL Fortified Spk B, Respectively R M fM t C ontrol PFHS (l*lA*owir.)399.O/*&0a<ii -ta m p ll So f 3* from 0712SD.wiff n o t found) CO QO. 'Soe I Area: 375254 counts H eight 2.44e+004 eps RT: 10.0 min 10.01 10.02 Exygen Research Page 38 of 111 Interim Report #12 - Analysis of Vegetation Samples Exygen Study No.: P0001131 Figure 8. Chromatogram Representing a Vegetation Sample Analyzed for PFHS (Exygen ID: C0049876, Data Set: 071205D) CM49V7C - PFHS (Lkikaowa) 3 H L M M amu -ta m p ft is o f 38 from 07120SD.wiff A n a : 4412114conato H ttg t: 2.57*+005cpi KT: 10.0m ia 10JM Intensity, cps Exygen Research Page 39 of 111 Interim Report #12 - Analysis of Vegetation Samples Exygen Study No.: P0001131 Figure 9. Typical Calibration Curve for PFOS in Methanol i 071205D VEO.rdb (PFOS): ` U n t i l" R tgtM tio n C'1 /x " Knighting): y - 4.75+ 0 0 4 x+ 1 .8 7 t+ 0 0 4 ( r - 0.9870) Area, counts Exygen Research Page 40 of 111 Interim Report #12 - Analysis of Vegetation Samples Exygen Study No.: P0001131 Figure 10. Non-Extracted Standards of PFOS in Methanol, 0.5 ng/mL and 1.0 ng/mL, Respectively I SS7279 PFO S (standard) A t M t l amu sam ple 7 o f 3 from 077705D .w iff A n a : 371*7 counts Haight: 7.4fa * H 3 cps ITT: 77.7 m ia 12.10 Tim, min SS7278 - PFOS (Standard)488.0/80.0 amu - sampla 2 of 38 from 071205D.wiff A rta : 03738 counts Height: 229+003 cps RT: 12.1 min Intensity, cps Intensity, cps Exygen Research Page 41 of 111 Intensity, cps Interim Report #12 - Analysis of Vegetation Samples Exygen Study No.: P0001131 Figure 11. PFOS in Control, 6.25 ng/mL Fortified Spk A, and 25 ng/mL Fortified Spk B, Respectively Rnngnnt Control -PFOS (Unknown)499L*9 0 .9nmn -tnntpf* 9of 39 front 07f205D.wtff Ann: 4692 counts H tfgkt Z14+0P2cps T: 12.2ntin 12.18 Reagent Spk A * PFOS (QC) 400.0/80.0 am u-sam ple 10 of 38 from Q71205D.wiff Area: 338374 counts H eight 2.00e+004 cps RT: 12.2 min 12.18 Area: 1271720 counts Height: 5.00c+004 cps RT: 12.2 min 4.0e4* 2.0 e4 0.0-*---------- ,---------- ,---------- ,---------- ,---------- ,---------- ,----- ------1 2 3 4 0 7 Time, min 12.17 17 Intensity, cps Intensity, cps Exygen Research Page 42 of 111 Interim Report #12 - Analysis of Vegetation Samples Exygen Study No.: P0001131 Figure 12. Chromatogram Representing a Vegetation Sample Analyzed for PFOS (Exygen ID: C0049876, Data Set:071205D) C$04997* - P fO S (Unknown) 4991WML0 ama aampta 75 o f 39 from 9 7 in 5 D .w iff A n a : 51$41291coaat* H a ig h t 2.02+006cpa RT: 112 m ia 2.0*8 1.0*8 1.8*6 1.7*6 1.6*6 1.5*6 1.4*6 1.3*6 1.2 *6 1.1*6 1.0*6 0.0*6 8.0*6 7.0*5 6.0*5 5.0*5 4.0*6 3.0*5 2.0*5 1.0*5- 00 10 11 12 13 14 15 16 17 Intensity, cps Exygen Research Page 43 of 111 Interim Report #12 - Analysis of Vegetation Samples Exygen Study No.: P0001131 APPENDIX A Study Protocol P0001131 (Exygen Study No. P0001131) with Analytical Method and Protocol Amendments Exygen Research Page 44 of 111 Interim Report #12 - Analysis of Vegetation Samples Exygen Study No.: P0001131 Exygen Protocol Number: POOOU31 STUDY PROTOCOL Study Title: Analysis of Perfluorobutanesulfonate (PFBS), Perfluorohexanesulfonate (PFHS), and Perfiuorooctanesulfonate (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 Perfoiminc Laboratory: Exygen Research 3058 Research Drive State College, PA 16801 Phone: (814) 272-1039 Sponsor Representative: M ichael A. Santoro Director o f Regulatory Affairs 3M Building 0236-01-B-10 St. Paul, MN 55144 Phone: (651) 733-6374 Page I oj 65 Exygen Research Page 45 of 111 Interim Report #12 - Analysis of Vegetation Samples Exygen Study No.: P0001131 Exygen Protocol N um ber P0001131 DISTRIBUTION: 1) Jaisimha Kesari, Study Director, Weston Solutions 2) John M. Flaherty, Principal Investigator, Exygen Research 3) Michael A. Santoro, Sponsor Representative, 3M Company 4) Exygen Research Quality Assurance Unit Exygen Research Page 2 o f 65 Page 46 of 111 Interim Report #12 - Analysis of Vegetation Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 PROTOCOLAPPROVAL Study Title: Analysis o f Perfluorobutanesulfonate (PFBS), Perfluorohexanesulfonate (PFHS), and Perfluorooctanesulfonate (PFOS) in Water, Soil, Sediment, Fish, Clams, Vegetation, Small Mammal Livers and Small Mammal Serum Using LC/MS/MS for the 3M Decatur Monitoring Program Exygen Protocol Number: P0001131 APPROVALS JaisimhatKesan, Study Director Weston Solutions Michael A. Safitoro, Sponsor Representative 3M Comparty m Date Richard A. Gi Exygen Resi ident. Facility Management ead, Quality Assurance Unit Date - i Page 3 o f 63 Exygen Research Page 47 of 111 Interim Report #12 - Analysis of Vegetation Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 TABLE OFCONTENTS TITLE PA G E .....................................................................................................................................................................1 D IS T R IB U T IO N ............................................................................................................................................................... 2 PROTOCOL APPROVAL.............................................................................................................................................. 3 TABLE OF CONTENTS................................................................................................................................................ 4 I N T R O D U C T IO N .............................................................................................................................................................5 TEST M ATERIA LS........................................................................................................................................................ 5 O B JECTIV E..................................................................................................................................................................... 6 TESTING FACILITY......................................................................................................................................................6 STUDY DIRECTOR........................................................................................................................................................ 7 SPONSOR REPRESENTATIVE................................................................................................................................... 7 PRINCIPAL INVESTIGATOR..................................................................................................................................... 7 PROPOSED EXPERIMENTAL START AND TERMINATION D A TE S........................................................... 7 IDENTIFICATION AND JUSTIFICATION OF THE TEST SY ST EM ................................................................8 SAMPLE PROCUREMENT, RECEIPT AND RETENTION..................................................................................8 SAMPLE IDENTIFICATION....................................................................................................................................... 9 ANALYTICAL PROCEDURE SUMMARY.............................................................................................................. 9 VERIFICATION OF ANALYTICAL PROCEDURE................................................................................................9 METHOD FOR CONTROL OF B IA S..........................................................................................................................11 STATISTICAL M ETH O D S........................................................................................................................................... 11 GLP STA TEM EN T..........................................................................................................................................................11 REPO R T.............................................................................................................................................................................11 SAFETY AND H EA LTH ................................................................................................................................................12 AMENDMENTS TO PROTO CO L............................................................................................................................... 13 DATA RECORD K EE PIN G .......................................................................................................................................... 13 QUALITY ASSU RA N CE...............................................................................................................................................14 RETENTION OF DATA AND A RCHIVING............................................................................................................ 14 APPENDIX I, ANALYTICAL M ETHODS.................................................................................................................15 Page 4 of 65 Exygen Research Page 48 of 111 Interim Report #12 - Analysis of Vegetation Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 INTRODUCTION The purpose o f this study is to perform analysis for perfluorobutanesulfonate (PFBS), perfluorohexanesulfonate (PFHS) and perfluorooctanesulfonate (PFOS) in water, soil, sediment, fish, 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 (C4F9 SOjTC+) Lot Number: 101 Purity: 96.7% Transitions Monitored: 299 -> 99 Structure: PFHS Chemical Name: Perfluorohexanesulfonate Molecular Weight: 438 supplied as the potassium salt (CFb SCVK*) Lot Number: SE036 Purity: 98.6% Transitions Monitored: 399 -> 80 Structure: FFF FFF F S03 F Page 5 o f 65 Exygen Research Page 49 of 111 Interim Report #12 - Analysis of Vegetation Samples Exygen Study No. : P0001131 Exygen Protocol Number: POOO1131 PFOS Chemical Name: Periluorooctanesulfonate Molecular Weight: S38 supplied as the potassium salt (CjFpSO'K*) Lot Number: 217 Purity: 86.9% Transitions Monitored: 499 - 99 Structure: OBJECTIVE The purpose o f this study is to perform analysis for perfluorobutanesulfonate (PFBS), perfluorohexanesulfonate (PFHS) and perfluorooctanesulfonate (PFOS) in water, soil, sediment, fish, clams, vegetation, small mammal livers and small mammal serum for the 3M Decatur Monitoring Program using the current versions o f the following Exygen analytical methods: V0001780: "Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Water by LC/MS/MS" V0001781: "Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Soil by LC/MS/MS" V0001782: "Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Sediment by LC/MS/MS" V0001783: "Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Fish and Clams by LC/MS/MS" V0001784: "Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Vegetation by LC/MS/MS" V0001785: "Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Small Mammal Liver by LC/MS/MS" V0001786: "Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Small Mammal Serum by LC/MS/MS" TESTING FACILITY Exygen Research 3038 Research Drive State College, PA 16801 Phone: (814) 272-1039 Page 6 o f 65 Exygen Research Page 50 of 111 Interim Report #12 - Analysis of Vegetation Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 STUDYDIRECTOR Jaisimha Kesari P.E., DEE Weston Solutions, Inc. 1400 Weston Way West Chester, PA 19380 Phone: (610) 701-3761 Fax: (610) 701-7401 j .kesari@westonsolutions.com SPONSORREPRESENTATIVE Michael A. Santoro 3M Company Director o f Regulatory Affairs 3M Building 0236-01-B-10 St. Paul, MN 55144 Phone: (651) 733-6374 PRINCIPAL INVESTIGATOR John M. Flaherty Exygen Research 3058 Research Drive State College, PA 16801 Phone: (814) 272-1039 john.flaherty@exygen.com PROPOSED EXPERIMENTAL START AND TERMINATION DATES It is proposed that the analytical portion o f this study be conducted from October 01, 2004 to December 31, 2005. The actual experimental start and termination dates will be included in the final report. Page 7 o f 65 Exygen Research Page 51 of 111 Interim Report #12 - Analysis of Vegetation Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 IDENTIFICATION ANDJUSTIFICATION 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, RECEIPTANDRETENTION 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 p ita 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 -IC C . Small mammal whole blood samples will be centrifuged in the field at the time o f collection and the serum fraction will be used for the study. Small mammal serum will be stored frozen at S -10C. The receipt and processing 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 52 of 111 Interim Report #12 - Analysis of Vegetation Samples Exygen Study No.: P0001131 Exygen Protocol Number: POOOl 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 of Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Water by LC/MS/MS" V0001781: `M ethod 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 L C M S M S " V0001783: "Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Fish and Clams by L C M S M S " V0001784: "Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Vegetation by L C M S M S " V0001785: "Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Small Mammal Liver by L C M S M S " V0001786: `M ethod o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Small Mammal Serum by L C M S M S " The above methods use analytical conditions capable o f separating the isomers o f PFBS, PFHS and PFOS. The final report will include the isomers summed into total PFBS, total PFHS, and total PFOS found. VERIFICATION OF ANALYTICAL PROCEDURE A laboratory control sample will be used for the preparation o f fortified control samples. The test substance will be made into solutions as per the method, and added to the matrices via a micropipette. For water sampling, Exygen will supply one bottle per sample collected. The *** bottles will be 500 mL precleaned Sci/Spec Premier wide mouth HDPE bottles. These bottles have been routinely used for fluorochemical sample Page 9 oj 65 Exygen Research Page 53 of 111 Interim Report #12 - Analysis of Vegetation Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 collection at the testing facility and have been shown to be free o f PFBS, PFHS and PFOS. Samples will be added to each container to a volumetric fill line at 200 mL. A field duplicate, a low field spike and a high field spike o f each sample will be collected. The low and high field spike bottles will contain PFBS, PFHS and PFOS as well as perfluorooctanoic acid (PFOA) and 1.2-13C perfluorooctanoic acid (13C PFOA). PFOA and l3C PFOA are included in the solutions used to spike the samples. The results for PFOA and 13C PFOA will not be reported in this study. Exygen will supply one field blank (control water) and two field blank spikes (control water fortified with PFBS, PFHS and PFOS at a low and high level) for every twenty samples collected. At the testing facility, each water sample (excluding field duplicates and field spikes) will be extracted in duplicate and will also be fortified at a low and high concentration with PFBS, PFHS and PFOS and processed through the described procedure to determine method accuracy and to check for bias. For soil, sediment, clams, and vegetation, Exygen will supply one SOO 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 SO 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 S00 ng/L 5000 ng/L Soil Sediment 4 ng/g 4 ng/g 40 ng/g 40 ng/g Fish 10 ng/g 100 ng/g Clams 10 ng/g 100 ng/g Vegetation 10 ng/g 100 ng/g Small Mammal Liver 10 ng/g 100 ng/g Small Mammal Serum lOng/mL lOOng/mL Page 10 o f 65 Exygen Research Page 54 of 111 Interim Report #12 - Analysis of Vegetation Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 Recoveries are anticipated to be between 70% and 130% o f the fortified levels; however, the exact precision and accuracy will be determined by the analysis o f the quality control samples described above. A statement o f accuracy will be included in the final report. METHOD FOR CONTROL OF BIAS Control o f bias will be addressed by taking representative sub-samples from a homogeneous mixture o f each matrix from untreated control samples, and by analyzing at least two levels o f fortifications. STATISTICAL METHODS Statistics will be limited to those specified in the subject methods and to the calculation o f average recoveries, as applicable. GLP STATEMENT All aspects o f this study shall be performed and reported in compliance with EPA TSCA Good Laboratory Practice Standards 40 CFR 792. The final report or data package (supplied to the Sponsor) shall contain a statement that the study was conducted in compliance with current and applicable GLP standards and will outline any deviations in the study from those standards. This statement will be signed by the Study Director and Sponsor Representative. REPORT A final report will be prepared by the principal investigator or their designee at the conclusion o f the study. The report will include, but will not be limited to, the following: The name and address o f the Study Director, Sponsor Representative, and of the testing facility. A statement o f GLP compliance (any related documentation, such as chain-of-custody records, must be in the study records). Page I / o f fij Exygen Research Page 55 of 111 Interim Report #12 - Analysis of Vegetation Samples Exygen Study No.: P0001131 Exygen Protocol Number: POOO1131 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 of the analyte peaks will be clearly identified in all chromatograms. All circumstances that may have affected the quality or integrity o f the data will be documented in the report. Locations where raw data and the final report are to be archived. Additions or corrections to the final report shall be in the form o f an amendment signed by the Study Director. The amendment shall clearly identify that part o f the report that is being altered and the reasons for the alterations. The amendment will be signed and dated by the Study Director and the Sponsor Representative. All applicable requirements for reporting o f study results as per 40 CFR 792.185. SAFETY AND HEALTH Laboratory personnel will practice good sanitation and health habits. Every reasonable precaution shall be taken to prevent inadvertent exposure of personnel and the environment to the test or reference substance(s). Page 12 o f 65 Exygen Research Page 56 of 111 Interim Report #12 - Analysis of Vegetation Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 AMENDMENTS TO PROTOCOL AH 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, hi 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. DATARECORDKEEPING Records to be maintained include the following (as appropriate): Sample tracking sheet(s) Sample receipt records, storage history, and chains of 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. a Additionally, fortifications will include the amount o f analyte added and the sample number o f the sample that was fortified. a Analytical standard chromatograms will additionally include the concentration (e.g., pg/mL). Page 13 o f 65 Exygen Research Page 57 of 111 Interim Report #12 - Analysis of Vegetation Samples Exygen Study No.: P0001131 Exygen Protocol Number: POOOU31 As part o f the documentation the following sheets will be included in each analytical set: a run sheet listing the samples to be run in the set, and an instrument conditions sheet describing the instrument type and operating conditions. QUALITYASSURANCE 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 DATAAND 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 58 of 111 Interim Report #12 - Analysis of Vegetation Samples Exygen Study No.: P0001131 Exygen Protocol Number: POOOl 131 APPENDIX I ANALYTICAL METHODS V0001780: "Method o f Analysis for die Determination o f Perfluorooctanoic Acid (PFOA) in Water by LC/MS/MS" V0001781: "Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Soil by LC/MS/MS" V0001782: "Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Sediment by LC/MS/MS" V0001783: "Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Fish and Clams by LC/MS/MS" V0001784: "Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Vegetation by LC/MS/MS" V0001785: "Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Small Mammal Liver by LC/MS/MS" V0001786: "Method o f Analysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Small Mammal Serum by LC/MS/MS" Exygen Research Page 15 o f 65 Page 59 of 111 Interim Report #12 - Analysis of Vegetation Samples Exygen Study No.: P0001131 Exygen Protocol Number: POOO1131 ANALYTICAL METHOD Method Number: V0001780 Method o fA e tlyiU fo r the Determ luM iei o f Perfloerooctauolc Acid (PFOA) in Water byLC /M S /M S Analytical Testing Facility: Exygen Research 3058 Research Drive State College, PA 16801 Approved By: Paul Connolly ' Technical Leader, LC-MS, Exygen Research 2 IrtriL L cT ' /J''Joohhn Flaherty / VVicie PDrfeMsitdilemntt, O^ pioemraItiomnisi , Exygen Research Date Date Exygen Research Total Page*: 7 Page / 6 o f 65 Page 60 of 111 Interim Report #12 - Analysis of Vegetation Samples Exygen Study No.: P0001131 Exygen Protocol Number: POOOI131 ExygcaRttM Kh M ethod Num ber VOOO1780 ANALYTICAL m e t h o d Method o fAnalysis for the Determination ofPerfluorooctanoic Acid (PFOA) in Water by LC/MS/MS 1.0 Scope This method i i to be employed for the ieoladen and quantitation o f perfluorooctanoic add by High Performance Liquid Chromatography coupled to a tandem Mass Spectrometric Detector (LC/MS/MS) in water. 2.0 Safety 2.1 Always observe safe laboratory practices. 2.2 Consult the appropriate MSDS before handling any chemical for proper safety precautions. 3.0 Sample Requirement 3.1 A t least 40 mL o ftest sample for extraction. 3.2 No sample processing is needed for water samples. 3.3 Simples stored refrigerated should be allowed to equilibrate to room temperature. 3.4 A ll sample*must be thoroughly mixed before being sampled for extraction 3.5 Any samples containing particles should be centrifuged at -3000 rpm for -5 minutes andthe supernatant used for the extraction. 3.6 Sample collection procedures w ill be specified in the sampling plan for this project 4.0 Reagents andStandards 4.1 Water-HPLC grade 4.2 Methanol - HPLC grade 4.3 Ammonium Acetate - A.C.S. Reagent Grade 4.4 Perfluorooctanoic Acid - Sigma-Aldrich 5.0 Instrument and Equipment 5.1 A high performance liquid chromatograph capable o f pumping up to 2 solvents equipped w ith a variable volume injector eapable o f injecting S-200 pL connected to a tandem Maas Spectrometer (LC/MS/MS). 5.2 A device to collect raw data for peak integration and quantitation. 5.3 Analytical balance capable o freading to 0.00001 g. 5.4 50 mL disposable polypropylene eentrifoge tubes. 5.5 15 mL disposable polypropylene centrifuge tubes. 5.6 Disposable micropipets (50-100uL, 100-200uL). 5.7 125-raL LDPE narrow-mouth bottles. 5.8 2 mL clear HPLC vial kit. 5.9 Disposable pipettes. 5.10 Autopipettea (100*1000 pL and 10-100 pL), w ith disposable tips. 5.11 Waters Sep Pak Vac 6 cc (Ig ) tClS SPE cartridges. Pane 2 o f ? Page 17 o f 65 Exygen Research Page 61 of 111 Interim Report #12 - Analysis of Vegetation Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 E x yjea JUawch M M bod N u m b V O M IT O ANALYTICAL METHOD Method o fAn&lysil for the Determination o fPerfluorooctanoie Acid (PFOA) in Water by LC/MS/MS 5.12 SPEvacuum manifold. 5.13 Centiifoge enable o f ginning 50 mL polypropylene tubes at 3000 rpm. 6.0 Chromatographic System 6.1 Analytical Column: Flnophase RP (Keystone Scientific), 2.1 mm x SOmm, 5m (P/N: 82505-052130) 6.2 Temperature: 30C 6.3 Mobile Phase(A ): 2 mM Ammonium Acetate in Water Mobile Phase (B) : Methanol Gradient Program: Time (mini 0.0 1.0 8.0 20.0 22.5 &A 65 65 25 2$ 65 Flow Rate % B (mL/minl 35 0.3 35 0.3 75 0.3 75 0.3 35 0.3 6.6 Injection Vohane: 15 pL (can be increased to as much as 50 pL). 6.7 Quantitation: PeakArea - external standard calibration curve. 6.8 RunTime: -2 3 minutes. The above conditions are intended at guide and may be changed in order to optimize the HPLC system. 7.0 MS/MS System 7.1 Mode: Electrotpray Negative MRM mode, monitoring 413 - 369 ra/z. The above conditions an intended as a guide and may be changed in order to optimize foe MSMS system. 8.0 Preparation o fSolutions 8.1 Mobile Phase 8.1.1 2 mM ammonium acetate in water is prepared by adding 0.154 g of ammonium acetateto 1000 raL o f water. Alternate volumes may be prepared. P iy r 3al ' Page 18 o f 65 Exygen Research Page 62 of 111 Interim Report #12 - Analysis of Vegetation Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 E xy g eaR ttttfck M ethod Num ber VOGO! 780 I a n a l y t ic a l m e t h o d ~i Method o fAnalysis for the Determination o fPerfluorooctanoic Acid (PFOA) in Water by LC/MS/MS 9.0 Standard Preparation 9.1 Standard Stock/Fortificatian Solution 9.1.1 Prepare a stock solution o f- 100 pg/mL o f PFOA by weighing 10 mg o f analytical standard (corrected for purity) and dilute to 100 mL with methanol in a 125*mL LDPE bottle. 9.12 A 10 pg/mL fortification solution o f PFOA is prepared by bringing 10 mL o f the 100 pg/mL solution to a final volume o f 100 with methanol in a 125 mL LDPE bottle. 9.1.3 A 1.0 pg/mL fortification solution o fPFOA is prepared by bringing 10 mL o fthe 10 pg/mL solution to a final volume o f 100 with methanol in a 125 mL LDPE bottle. 9.1.4 A 0.1 pg/mL fortification solution o fPFOA is prepared by bringing 10 mL o fthe 1.0 pg/mL solution to a final volume o f 100with methanol in a 125 mL LDPE bottle. 9.1.5 A 0.01 pgfaiL fortification solution o f PFOA is prepared by bringing 10 mL o f die 0.1 pg/mL solution to a final volume o f 100 with HfrtiMirti in a 125 mL LDPE bottle. 9.1.6 The stock and fortification solutions are to be stored in a refrigerator at approximately 4*C and are stable for a maximum period o f 6 months from the date o fpreparation. 9.2 Standard Calibration Solutions 9.2.1 92.2 LC/MS/MS calibration standards are prepared in HPLC water. The calibration standards are processed through die extraction procedure, identical to samples. The following is a typical example: additional concentrations may be prepared asneeded. Final Concentration Fortification Volumeof Concentration of o fFortification Votums Fortified Control Calibration Solution (mb) (PL) Samel(mL) Standard(m t)* 0 0 40 0 10 100 40 25 10 200 40 50 10 400 40 100 100 100 40 250 100 200 40 500 100 400 40 1000 Calibration Standard ID (examnlel XCmroddyy-0 XCmmddyyl XCmmddyy-2 XCmraddyyO XCmmddyy-4 XCminddyy-5 XCmmddYY- * The extracted concentration o f the calibration itandard is equal to 8* its initial concentration, due to the concentration o f the standard during the extraction (SPE) XC " extracted calibration standard. Page 4 i` ' Page 19 o f 65 Exygen Research Page 63 of 111 Interim Report #12 - Analysis of Vegetation Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 B x y g tt R M M K h M ethod N um ber VOOO1780 [ ANALYTICAL METHOD Method o fAnalysis for the Determination ofPerfluorooctanoic Acid (PFOA) in Water by LC/MS/MS 9.2.3 A zero standard solution (reagent blank) must be prepared with each set o fstandards extracted. 9.2.4 Store all extracted calibration standards in 15-mL polypropylene tubes at 2*C to 6C, up to two weeks. 9.2.5 Alternate volumes and concentrations o fstandards may be prepared as needed. 10.0 Batch Set Up 10.1 Each batch o f samples extracted (typically 20 or less) must include at least one reagent control (method blank using HPLC water) and two reagent controls fortified at known concentrations (tab control spike) to verify procedural recovery for the batch. 10.2 Requirements for field and laboratory duplicates and spikes w ill be specified in the quality assuranceplan for this project 11.0 Sample Extraction 11.1 Measure 40 mL o f sample or a portion o fsample diluted to 40 mL with water into 50 mL polypropylene centrifiige tubes (fo rtify as needed, replace lid and mix well). 11.2 Condition the Cu SFE cartridges (1 g, 6 mL) by passing 10 mL methanol followed by 5 mL o fHPLC water (~ 2 drop/sec). Do not let column run dry 11.3 Load sampleon conditioned Cu SPE cartridge. Discard eluate. 11.4 Elute with 5 mL 100% methanol Collect 5 mL o f eluate into graduated 1SmL polypropylene centrifiige tubes (final volume 5 mL). 11.5 Analyze samplesusing eleetrosprsy LC/MS/MS. 12.0 Chromatography 12.1 Inject the same m ount o f each standard, sample and fortified sample into the LC/MS/MS system. A calibration standard must precede and follow all analyzed samples. 12.2 Standards o fPFOA corresponding to at least five or more concentration levels must be included in an analytical set. 12.3 An entire set o f extracted calibration standards must be included at the beginning and at the end o f a sample set. Extracted standards must be intenpersed between every 5-10 samples. As an alternative, an entire set of extracted calibration standards may be injected at the beginning o f s set followed by extracted calibration standards interspersed every 5-10 samples (to account for a second set o f extracted standards). In either case, extracted calibration standards mustbe the first and last injection in a sample set. 12.4 Use linear standard curves for quantitation. Linear standard curves are generated for the analyte by linear regression using 1/x weighting o fpeak area Pag 5 o f 7 Page 20 o f 65 Exygen Research Page 64 of 111 Interim Report #12 - Analysis of Vegetation Samples Exygen Study No.: P0001131 Exygen Protocol Number: P 0001131 ExygcaRMMKh M ethod Num ber V00O17S0 | ANALYTICAL M E T H O D .................... | Method o fAnalysis for the Determination ofPerfhiorooctanoic Acid (PFOA) in Water by LC/MS/MS versus calibration standard concentration using MatsLynx 3.3 (or equivalent) software system. 12.5 Sample response should not exceed standard responses. Any samples that exceed standard responses should be ftirther diluted and reanalyzed. 13.0 Acceptance Criteria 13.1 Chromatogram mu show a peak o f a daughter ion at 369 amu from a parent o f 413 amu. The 413 amu parent corresponds to the PFOA anion, while the daughter ion (369 amu) represents the loss o fcarbon dioxide. 13.2 Method blanks must not contain PFOA at levels greater than the LOQ. I f a blank contains PFOA at levels greater than 50 ng/L, then a new blank sample must beobtained and the entire act must be re-extracted. 13.3 Recoveries o f control spikes and matrix tikes must be between 70-130V of their known values. I f a control tike falls outside the acceptable lim its, the entire set o f samples should be re-extracted. Any matrix spike outside 70* 130% should be evaluated by the analyst to determine i f re-extraction is 13.4 Any calibration standard found to be a statistical outlier by using the Huge Error Test, may be excluded from the calculation o f the calibration curve. However, the total number o f extracted calibration standards that could be excluded must not exceed 20% o f foe total number o f extracted standards injected. 13.5 The correlation coefficient (R) for calibration curves generated must be 20.992 (R3 20.985). I f calibration resulta fa ll outside these lim its, then appropriate steps must be taken to adjust instrument operation, and the standards or the relevant act o fsamples should be reanalyzed. 13.6 Retention timet between standards and samples must not d rift more than 4 % within an analytical ran. I f retention time d rift exceeds this lim it within an analytical run then foe set mustbe reanalyzed. 14.0 Calculations 14.1 Use the following equation to calculate foe amount o f PFOA found (in ng/L. based on peak area) using the standard curve (linear regression parameters) generated by the Mass Lynx software program: PFOA found (ng/L) - (Peak area - intercept) x DF slope DF - Actor by which foe final volume was diluted, if necessary. Page 6 o f 7 Page 21 o f 65 Exygen Research Page 65 of 111 Interim Report #12 - Analysis of Vegetation Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 Exygra lUttarcb M bod N u n to r V00017B0 ANALYTICAL METHOD Method o fAnalysi* Jbr the Determination ofPeruorooctanoic A dd (PFOA) in Water by LC/MS/MS 14J2 For samples fortified with known amount* o f PFOA prior to extraction, use the following equation to calculate tbe percent recovery. Recovery (%) - [total analytefound (ng/L) analyte found in control (ng^L)] ^ analyteadded (ng/L) Exygen Research Page 7 o f7 Page 22 o f 65 Page 66 of 111 Interim Report #12 - Analysis of Vegetation Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 ANALYTICAL METHOD Method Number V0001781 Method o fAnalyais fo r the Determination o f Perflnorooctanok A dd (PFOA) in Soil by LC/MS/MS Analytical Testing Facility: Exygen Research 3038 Research Drive State College, PA 1680] Approved By: 1 U . C -Ji. Paul Connolly ` Technical Leader, LC-MS, Exygen Research __ io Im ./O '/ Date 6hn Flaherty r Vice Preeident, Operations, Exygen Research Date Exygen Research Total Pages: 7 Page 23 o f 65 Page 67 of 111 Interim Report #12 - Analysis of Vegetation Samples Exygen Study No.: P0001131 Exygen Protocol Number. P000U31 E x y y n fcwBMch Msthod Number V00017J1 ANALYTICAL METHOD Method o fAnalysis for the Determination ofperfluorooctanoic Acid (PFOA) in Soil by LC/MS/MS 1.0 Scope This method is to be employed for the isolation and quantitation ofperfluorooctanoic acid by High Performance Liquid Chromatography coupled to a tandem Mass Spectiometrie Detector (LC/MS/MS) in soil. 2.0 Safety 2.1 Always observe safe laboratory practices. 2.2 Consult tbs appropriate MSDS before handling any chemical for proper safety precautions. 3.0 Sample Requirement 3.1 A t least 15g o f testsample for extraction. 3.2 No sample processing is needed for soil samples. 3.3 Samples stored refrigerated should be allowed to equilibrate to room temperature. 3.4 A ll samples mustbe thoroughly mixed before being sampled for extraction. 3.3 Sample collection procedures w ill be specified in the sampling plan for this project. 4.0 Reagents and Standards 4.1 Water- HPLC grade 4.2 Methanol - HPLC grade 4.3 Ammonium Acetate-A.C.S. Reagent Grade 4.4 Perfiuorooctanoic Add - Sigma-Aldrich 5.0 Instrumentand Equipment 5.1 A high performance liquid chromatograph capable o f pumping up to 2 solvents equipped with a variable volume injector capable o f injecting 5-200 pL connected to atandem Maas Spectrometer (LC/MS/MS). 5.2 A device to collect raw data for peak integration and quantitation. 5.3 Analytical balance capable o freading to 0.00001 g. 5.4 50 mL disposable polypropylene centrifuge tubes. 5.5 15 mL disposable polypropylene centrifuge tubes. 5.6 Disposable micropipets (50-100uL, l00-200uL). 5.7 125-mL LDPE narrow-mouth bottles. 5.8 2 mL clear HPLC vial kit. 5.9 Disposable pipettes. 5.10 Autopipettes (100-1000 pL and 10-100 pL), with disposable tips. 5.11 Waters SepPak Vac 6 ec (lg ) tC18 SPE cartridges. 5.12 SPE vacuum manifold. 5.13 Ultrasonic bath. Page 2 o f? Page 24 o f 65 Exygen Research Page 68 of 111 Interim Report #12 - Analysis of Vegetation Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 Exygen Reecarch Method Ntunber V0001781 _____________________ ANALYTICAL METHOD J Method o fAnalysis for the Determination o fPerfluoroocta&oic Acid (PFOA) in Soil by LC/MS/MS 5.14 Wrist-action ihaker. 5.15 Centrifoge capable o fspinning 30 mL polypropylene tubes at 5000 rpm. 6.0 Chromatographic System 6.1 Analytical Colisnn: Fluophase RP (Keystone Scientific), 2.1 mm x 50 mm. 5p (EVN: 82505-052130) 6.2 Temperature: 30*C 6.3 Mobile Phase(A) : 2 mM Ammonium Acetate in Water 6.4 Mobile Phase(B) : Methanol 6.5 GradientProgram: Time fmirri 0.0 1.0 8.0 20.0 22.5 65 65 25 25 65 Flow Rate (m L/m inl 35 0.3 35 0.3 75 0.3 75 0.3 35 0.3 6.6 Injection Volume: IS jiL (can be increased to as much as 50 pL). 6.7 Quantitation: PeakArea- external standard calibration curve. 6.8 Run Tin: - 23 minutea. The aboveconditions are intended as a guide and may be changed in order to optimize the HPLC system. 7.0 MS/MS System 7.1 Mode: Electrospray Negative MRM mode, monitoring 4 13 -* 369 m/z for PFOA. The above conditions are intended asa guide and may be changed in order to optimize the MSMS system. 8.0 Preparation o fSolutions 8.1 Mobile Phase 8.1.1 2 mM ammonium acetate in water is prepared by adding 0.154 g o f ammonium acetate to 1000 mL o fwater. Alternate volumes may be prepared. Page 3 of 7 Page 25 o f 65 Exygen Research Page 69 of 111 Interim Report #12 - Analysis of Vegetation Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 Exyfea (Uaurefc Method N unfctr V000178I ANALYTICAL METHOD 1 Method o fAnalyaii for foe Determination o fPerfluorooctanoic Acid (PFOA) in Soil by LC/MS/MS 9.0 Standard Preparation 9.1 Standard Stock/Foctification Solution 9.1.1 Prepare a stock solution of-lO O pgi'raL o f PFOA by w e irin g 10 mg o f analytical standard (corrected for purity) and dilute to 100 mL with methanol in a 123-tnL LDPE bottle. 9.1.2 A 10 pg/mL fortification solution o f PFOA it prepared by bringing 1 0 mL o f the 100 pg/mL solution to a final volume o f 100 with methanol in a 12$mL LOPE bottle. 9.1.3 A 1.0 pgfaiL fortification solution o f PFOA is prepared by bringing 10 m L of the 10 pg/mL solution to a final volume o f 100 with methanol in a 12$ mL LDPE bottle. 9.1.4 A 0.1 p^m L fortification solution o f PFOA is prepared by bringing 10 m L of(he 1.0 pg^mLsolution to a final volume o f 100 w ith methanol in a 12$ mL LDPE bottle. 9.1.$ A 0.01 pg/mL fortification solution o f PFOA is prepared by bringing 10 mL o f the 0.1 pg/mL solution to a final volume o f 100 with methanolin a 12$ mL LDPE bottle. 9.1.6 The stock and fortification solutions are to be stored in a refrigerator at approximately 4*C and are stable for a maximum period o f 6 months from the date o fpreparation. 9.2 Standard Calibration Solutions 9.2.1 9.2.2 LC/MS/MS calibration standards ire prepared in HPLC water. The calibration standards are processed through the extraction procedure, identical to samples. The following is a typical example: additional concentrations may be prepared is needed. Fine! Concentration Fortification Volume of Concentration of Calibration ofFortification Volume Fortified Control Calibration Standard ID Solution(nob) (UL) SemoteimL) Standardfoot)* fexamolel 0 0 40 0 XCmmddyy-0 10 100 40 10 200 40 23 XCmmddyy-l so XCmmddyy-2 10 400 100 100 100 200 100 400 40 40 40 40 100 XCmmddyyO 250 XCmmddyy-4 500 XCmmddyy-5 1000 XCmmddvY'd * The extracted concentration o ffoe calibration standard is equal to 8x its initial concentration, due to the concentration o ffoe itandard during foe extraction (SPE). XC * extracted calibration standard. Page 4 o f7 Page 26 o f 65 Exygen Research Page 70 of 111 Interim Report #12 - Analysis of Vegetation Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 Exygi Raaurcb M ethod Num ber VOOO1781 ANALYTICAL METHOD Method o fAnalysis for the Determination o fPerfhiorooctanoic Acid (PFOA) in Soil by LC/MS/MS 9.2.3 92A 9.2.5 A aero standard solution (reagent blank) must be prepared with each set o f standards extracted. Store a ll extracted calibration standards in 15*mL polypropylene lubes at2C to 6*C. up to two weeks. Alternate volumes and concentrations o f standards may be prepared as 10.0 Batch Set Up 10.1 Each batch o f sample extracted (typically 20 or less) must include at least one reagent control (method blank using S mL o f methanol) and two reagent controls fortified at known concentrations (lab control spike) to verify procedural recovery for the batch. 10.2 Requirements for field and laboratory duplicates and spikes w ill be specified in tbe quality assuranceplan for this project. 11.0 Sample Extraction 11.1 Weigh 5 g o f sample into 50 mL polypropylene centrifuge tubes (fortify as needed, replace lid and mix well). 11.2 Add 5 mL o fmethanol and shake on a wrist action shaker fo r - l 5 minutes 11.3 Transfer the tube* to an ultrasonic bath and sonicate for ~ 13 minutes. 11.4 Bring the volume up to 40 mL with water in tbe 50 mL polypropylene centrifbge tube. 11.5 Centriftge for M 0 minutes at~3000rpm. 11.6 Condition the Cis SPB cartridges (1 g, 6 mL) by passing 10 mL methanol followed by 5 mL o f HPLC water (~ 2 drop/sec). Do not let column run dry 11.7 Load (decant) tbe sample on the conditioned Cn SPE cartridge. Discard eluate. 11.8 Elute with - 5 mL 100% methanol. Collect 5 mL o f eluate into graduated 15 mL polypropylene centrifttge tubes (final volume - 5 mL). 11.9 Analyze samples using electrospray LC/MS/MS. 12.0 Chromatography 12.1 Iqject the same amount o f each standard, sample and fortified sample into the LC/MS/MS system. A calibration standard must precede and follow all analyzed samples. 12.2 Standards o fPFOA corresponding to at least five or more concentration levels must be included in an analytical set. 12.3 An entire set o f extracted calibration standards must be included at the beguning and at the end o f a sample set Extracted standards must be interspersed between every 5*10 samples. As an alternative, an entire set of Page 5 of7 Page 27 o f 65 Exygen Research Page 71 of 111 Interim Report #12 - Analysis of Vegetation Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 ExypaXMcarch Mwhod Number V0001781 I ANALYTICAL MKTHOD Method o fAnalysis for the Determination o fPerfluorooctanoic Acid (PFOA) in Soil by LC/MS/MS extracted calibration standards may be injected at the beginning o f a set followed by extracted calibration standards interspersed every 5*10 samples (to account for a second set o f extracted standards). In either case, extracted calibration standards must bethe first and tact injection in a sample set. 12.4 Use linear standard curves for quantitation. Linear standard curves are generated for the analyte by linear regression using 1/x weighting o fpeak area versus calibration standard concentration using MassLynx 3.3 (or equivalent) software system. 12.5 Sample response should not exceed standard responses. Any samples that exceed standard responses should be further diluted and reanalyzed. 13.0 Acceptance Criteria 13.1 Chromatogram must show a peak o f a daughter ion at 369 amu from a parent o f 413 amu. The 413 amu parent corresponds to the PFOA anion, while the daughter ion (369 amu) represents the loss o fcarbon dioxide. 13.2 Method blanks must not contain PFOA at levels greater than the LOQ. If a blank contains PFOA at levels greater than 50 ng/L, then a new blank sample must be ohtsined and the entire set must be re-extracted. 13.3 Recoveries o fcontrol spikes and matrix spikes must be between 70- 130% of their known values. I f a control spike falls outside the acceptable lim its, the entire set o f samples should be re-extracted. Any matrix spike outside 70 130% should be evaluated by the analyst to determine if re-extraction is warranted. 13.4 Any calibration standard found to be a statistical outlier by using the Huge Brrr Test, may be excluded from the calculation o f the calibration curve. However, the total number o f extracted calibration standards that could be excluded must not exceed 20% o f the total number o f extracted standards injected. 13.3 The correlation coefficient (R) for calibration curves generated must be 20.992 (R2 20.985). I f calibration results fall outside these limns, then appropriate steps must be taken to adjust instrument operation, and the standards or the relevant set o fsamples should be reanalyzed. 13.6 Retention times between standard and samples must not d rift more than i 4 % within an analytical run. I f retention time d rift exceeds this lim it within an analytical run then the set must be rewialyzed. ps* sof7 Page 28 o f 65 Exygen Research Page 72 of 111 Interim Report #12 - Analysis of Vegetation Samples Exygen Study No.: P0001131 Exygen P rotocol N um ber P0001131 B xygcaR M M ich M ethod Num ber V00017S1 I ANALYTICAL METHOD Method o fAnalysis for the Determination o fPerfluorooctanoic Acid (PFOA) in Soil by LC/MS/MS 14.0 Calculations 14.1 Use the following equation to calculate toe amount o f PFOA found (in ng/L, baaed on peak area) using toe standard curve (linear regression parameters) generated by toe Mass Lynx software program: PFOA found (ng/L) slope xDF DF factor by which toe final volume waa diluted, i f necessary. 14.2 P ot samples fortified w ith known amounts o f PFOA prior to extraction, use the following equation to calculate toe percent recovery. Recovery (%) - [ totalanalyte found(ng/L) analyte found in control(ng/L)] analyte addod(n ^L ) 14.3 Use the following equation to convert the amount o f PFOA found in ng/L to ug/g (ppb). PFOA found (ppb) - fPFOA found fna/L) x volume extracted (0.04L11 sample weight (5 g) 14.4 Use the following equation to calculate the amount o f PFOA found in ppb baaedon dry weight. PFOA found (ppb) dry weight PFOA found (ppb) x [ 100% / total solids(%)] Pag*7o(7 Page 29 o f 65 Exygen Research Page 73 of 111 Interim Report #12 - Analysis of Vegetation Samples Exygen Study No.: P0001131 Exygen Protocol Number: POOO1131 ANALYTICAL METHOD Method Number V0001782 Method o f Axotyils fo r the Determination o f Perfluoreoctanoic Acid (PFOA) in Sedlmeot by LC/MS/MS Analytical Testing Facility: Exygen Research 3038 Research Drive State College, PA 16801 Approved By: VwA c jL Paul Connolly I Technical Leader, LC-MS, Exygen Research '/m / u s A lcohhtn Flaherty / Viice President, Operations, Exygen Research ___ l o h . h M Date Date Exygen Research Total Pages: 7 Page 30 o f 65 Page 74 of 111 Interim Report #12 - Analysis of Vegetation Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 Exygm Rcaeaicb M ethod N umber VOOOI782 [ ANALYTICAL METHOD { Method o fAnalysis for foe Determination ofPerfluorooctanoic Acid (PFOA) in Sediment by LC/MS/MS 1.0 Scope This method is to be employed for foe isolation and quantitation o f perfluorooctanoic acid by High Performance Liquid Chromatography coupled to a tandem M ass Spectrometric Detector (LC/MS/MS) in sediment 2.0 Ssfoty 2.1 Always observe safo laboratory practioea. 2.2 Consult the appropriate MSDS before handling any chemical for proper safety precautions. 3.0 Sample Requirement 3.1 At least 30 g o ftest sample for extraction. 3 2 No sample processing is needed for sediment samples. 3.3 Samples stored refrigerated should be allowed to equilibrate to room temperature. 3.4 A ll samples must be thoroughly mixed before being sampled for extraction. 3.3 Semple collection procedures w ill be specified in foe sampling plan for ibis project 4.0 Reagents and Standards 4.1 Water-HPLC grade 4.2 Methanol-HPLC grade 4.3 Acetic Acid - Reagent grade 4.4 Ammonium Acetate - A.C.S. Reagent Grade 4.5 Perfluorooctanoic Acid - Sigma-Aldrich 5.0 Instrument and Equipment 5.1 A high performance liquid chromatograph capable o f pumping up to 2 solvents equipped w ith a variable volume iqjector capable o f injecting 5-200 pL connected to atandem Maas Spectrometer(LC/MS/MS). 5.2 A device to collect raw dais for peak integration and quantitation. 5.3 Analytical balance capable o freading to 0.00001 g. 5.4 50 mL disposablepolypropylene centrifuge tubes. 5.5 IS mL di^ossblepolypropylene centrifuge tubes. 5.6 Disposable micropipets (50*100uL, 100-200uL). 5.7 125-mL LDPE narrow-mouth bottles. 5.8 2 mL clear HPLC visild t. 5.9 Disposablepipettes. 5.10 Autopipettes (100*1000 pL and 10*100 pL), with disposable tips. 5.11 Waters SepPak Vac 6 cc (lg ) (C18 SPEcartridges. 5.12 SPE vacuum manifold. Pas2o f7 Page 31 o f 65 Exygen Research Page 75 of 111 Interim Report #12 - Analysis of Vegetation Samples Exygen Study No.: P0001131 Exygen Protocol Number: POOO1131 Exygra Research Method Number V00017S2 ANALYTICAL METHOD Method o fAnalysis for the Determination o fPerfluorooctanoic Acid (PFOA) in Sediment bv LC/MS/MS * 5.13 Vortexer. 5.14 Wrist-action shaker. 5.15 CentriAige capable o fspinning 50 mL polypropylene tubes at 3000 rpm. 6.0 Chromatographic System 6.1 Analytical Column: Fluophase RP (Keystone Scientific), 2.1mm x 30 mm. 5p (P/N: 82505*052130) 6.2 Temperature: 30*C 6.3 Mobile Phase (A ): 2 mM Anunooium Acetate in Water 6.4 Mobile Phase(B ): Methanol 6.5 Gradient Program: Tima fm inl 0.0 1.0 8.0 20.0 22.5 65 65 25 25 65 Flow Rate U fl (mL/minl 35 0.3 35 0.3 75 0.3 75 0.3 35 0.3 6.6 Injection Volume: 15 pL (can be increased to as much as 50 pL). 6.7 Quantitation: PeakAna - external standard calibration curve. 6.8 RunTime: -2 3 minutes. The shove conditions an intended as a guide and may be changed in order to optimize the HPLC system. 7.0 MS/MS System 7.1 Mode: Electidspny Negative MRM mode, monitoring 413 -+ 369 m/z for PFOA. The above conditions are intended asa guide and may be changed in order to optimize (heMSMS system. 8.0 Preparation o fSolutions 8.1 Mobile Phase 8.1.1 2 mM ammonium acetate in water is prepared by adding 0.154 g of ammonium acetate to 1000 mL o fwater. Page 3 o f 7 P age 32 o f 65 Exygen Research Page 76 of 111 Interim Report #12 - Analysis of Vegetation Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 Exygea R im itii M ethod Number VOOOI7B2 I ANALYTICAL METHOD | Method o fAnalysis for the Determination o fPerfluorooctanoic Acid (PFOA) in Sediment by LC/MS/MS 8.2 Extraction Solutions 8.2.1 1% acetic acid in water is prepared by adding 10 mL o f acetic acid to 1000 mL o fwater. Alternate volumes maybeprepared. 9.0 Standard Preparation 9.1 Standard Stock/Fortification Solution 9.1.1 Prepare a stock solution o f >100 pg/mL o f PFOA by weighing 10 mg o f analytical standard (corrected for purity) and dilute to 100 m l with methanol in a 125-mL LDPE bottle. 9.1.2 A 10 pg/mL fortification solution o f PFOA is prepared by bringing to mL o f the 100 pg/raL solution to e final volume o f 100 with methanol in a 125 mL LDPE bottle. 9.1.3 A 1.0 ngtaL fortification solution o f PFOA is prepared by bringing 10 mL o f tire 10 (igfaiL solution to a final volume o f 100 with methanol in 125 mL LDPE bottle. 9.1.4 A 0.1 pg/mL fortification solution o f PFOA is prepared by bringing 10 m Loftbe 1.0 pg/mL solution to final volume o f 100 with methanol in a 125 mL LDPE bottle. 9.1.5 A 0.01 jig/m L fortification solution o f PFOA is prepared by bringing 10 mL o f the 0.1 pgfaiL solution to a final volume o f 100 with methanol in a 125 mL LDPE bottle. 9.1.6 The stock and fortification solutions are to be stored in a refrigerator at approximately 4*C and are stable for a maximum period o f 6 months from the date o fpreparation. 9.2 Standard Calibration Solutions 9.2.1 LC/MS/MS calibration standards are prepared in methanol via dilution o fthe 0.1 pg/mL fortification solution. 9.2.2 The following is typical example: additional concentrations may be o f Fortification Solution fasfaiL) 100 100 100 10 5 2 Volume (mL> 10 5 2 10 10 10 Dihticdto (mL) 100 100 \0Q 100 100 100 Final Concentration ino/mL) 100 5.0 20 1.0 0.5 0.2 Pag*4 of 7 P age 33 o f 6$ Exygen Research Page 77 of 111 Interim Report #12 - Analysis of Vegetation Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 Exyffn JUwsrck Method Number V0001782 < ANALYTICAL METHOD | Method o fAnalysis for the Determination ofPerfluorooctanoic Acid (PFOA) in Sediment by LC/MS/MS 9.2.3 Store a ll calibration atandarda in 125-mL LDPE narrow-mouth bottles at 2*C to 6*C, up to six months. 9.2.4 Alternate volume! and concentrations o f standards may be prepared as needed. 10.0 Batch SetUp 10.1 Each batch o f aamplea extracted (typically 20 or le u ) must include at least one untreated control and two untreated controls fortified at known concentrations (lab control spike) to verity procedural recovery for the batch. 10.2 Requirements for field end laboratory duplicates and spikes w ilt be specified in foe quality assuranceplan for this project. 11.0 Sample Extraction 11.1 Weigh 5 g o f sample into 50 mL polypropylene centrifUge tubes (fortify as needed, replace lid and mix well). 11.2 Add 35 mL o f 1% acetic acid, cap, vortex and shake on a wrist action shaker fo r-60 minutes. 11.3 CentrifUge the tubes at -3000 rpm for *-20 minutes. 11.4 Condition foe Cn SPE cartridges (1 g, 6 mL) by passing 10 mL methanol followed by 20 mL ofHPLC water(~ 2 drop/soc). Do not let column run dry 11.5 Load (decent) foe sample on foe conditioned Cu SPE cartridge. Discard 11.6 Add 20 mL o f methanol to the eedimeot left in the bottom o f the 50 mL centrifUge tube. Cap, vortex and shake on a wrist action shaker for -30 minutes. 11.7 CentrifUge foe tube at-3000 rpm fo r-2 0 minute. 11.8 Decantthe methanol onto the same SPE cartridge. Collect the eluate. 11.9 Wash the column w ith 4 mL o fmethanol. Collect the eluate and add it to the eluate collected in step 11.8. 11.10 Condition a second Cu SPEcartridge (1 g, 6 mL) by passing 10 mL methanol followed by 20 mL ofHPLC water ( - 2 drop/sec). Do not let column run dry 11.11 Add the methanol to -200 mL o f water and load on the second conditioned SPEcartridge. 11.12 Elute with -5 mL 100K methanol. Collect 5 mL o f eluate into graduated 15 mL polypropylene centrifUge tubes (final volume 5 mL). 11.13 Analyze samples using electrosptray LC/MS/MS. Pag 5 of 7 P age 34 o f 65 Exygen Research Page 78 of 111 Interim Report #12 - Analysis of Vegetation Samples Exygen Study No.: P0001131 Exygen Protocol Number: POOO1131 Exygea Rawocfa Method Number V0001762 | ANALYTICAL METHOD | Method o fAnalyst! for the Determination of Perfluorooctanoic Acid (PFOA) in Sediment by LC/MS/MS 12.0 Chromatography 12.1 Inject tbe same amount o f each standard, sample and fortified simple into the LC/MS/MS system. A calibration standard must precede and follow all analysed samples. 12.2 Standards o fPFOA corresponding to at least five or more concentration levels mustbe included in an analytical set. 12.3 An entire set o f extracted calibration standards must be included at the beginning and at the end o f sample set. Standards must be interspersed between every 3-10 samples. As an alternative, an entire set o f calibration standards may be injected at the beginning o f a set followed by calibration standards interspersed every 3-10 samples (to account for a second set of standards). In either case, calibration standards must be the first and last injection in asample set. 12.4 Use linear standard curves for quantitation. Linear standard curves arc generated for the analyte by linear regression using 1/x weighting o f peak area versus calibration standard concentration using MassLynx 3.3 (or equivalent) software system. 12.3 Sample response should not exceed standard responses. Any samples that exceed standard responses should be ftuther diluted and reanalyzed. 13.0 Acceptance Criteria 13.1 Chromatogram must show a peak o f a daughter ion at 369 amu from a parent o f 413 amu. The 413 amu parent corresponds to the PFOA anion, while the daughterion (369 amu) represents tbe lots o fcarbon dioxide. 13.2 Method blanks must not contain PFOA at levels greater than the LOQ. If a blank PFOA at levels pester than 0.2 ng/mL, then a new blank sample must be obtained and the entire set must be re-extracted. 13.3 Recoveries o f control spikes and matrix spikes must be between 70-130% of their known values. I f a control spike foils outside the acceptable lim its, the entire set o f f t 1** should be re-extracted. Any matrix spike outside 70 130% should be evaluated by foe analyst to determine if re-extraction is warranted. 13.4 Any calibration standard found to be a statistical outlier by using the Huge Error Teat, may be excluded from tbe calculation o f the calibration curve However, foe total number o f extracted calibration standards that could be ^eluded must not exceed 20% o f the total number o f extracted standards injected. 13.3 The correlation coefficient (R) for calibration curves generated must be 20.992 (R3 0.985). I f calibration results fo il outside these lim its, then appropriate steps must be taken to adjust instrument operation, and the standards or the relevant set o f samples should be reanalyzed. Pigt 6 of 7 Page 35 o f 65 Exygen Research Page 79 of 111 Interim Report #12 - Analysis of Vegetation Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 B x y g n R --Mic h M faod Number V00017S2 I ANALYTICAL METHOD | Method o fAnalysis for the Determination ofPerfluorooctanoic Acid (PFOA) in Sediment by LC/MS/MS 13.6 Retention timet between etandarde and samples must not d rift more then 4 % within an analytical ran. I f retention time d rift exceeds this lim it within ananalytical tun then the set mustbereanalyzed. W.O Calculations 14.1 Use the following equation to calculate the amount o f PFOA found (in ng/mL, based on peak ana) using the standard curve (linear regression parameters) generated by the MassLynx software program: PFOA found (ng/mL) - (Peak area - intercept! x OF slope DF factor by which the final volume was diluted, i f necessary. 14.2 For samples fortified with known amounts o f PFOA prior to extraction, use the following equation to calculate the percent recovery. Recovery (% ) [ total analyte found (ng/mL) - analyte found in control (ng/mL)] ^ } analyte added(ng/mL) 14.3 Use the following equation to convert the amount o f PFOA found in ng/mL to n rtfa ib ). PFOA found fm b) - fPFQA (bind InaJmU x n il volume (5 mL>) sample weight (3 g) 14.4 Use the following equation ( if necessary) to calculate the amount o f PFOA found in ppb basedon dry weight. PFOA found (ppb) dry weight " PFOA found (ppb) x [100% / total solids(%)] Paje 7 of 7 Page 36 o f 65 Exygen Research Page 80 of 111 Interim Report #12 - Analysis of Vegetation Samples Exygen Study No.: P0001131 Exygen Protocol Number: POOOl 131 ANALYTICAL METHOD Method Number VOOOI783 Method of Analysis for the Detarmlaotloe of Pufliorooctasolc Acid (PFOA) io Fish a d C lans by LC/MS/MS Analytic] Testing Facility: Exygen Research 3058 Research Drive State College, PA 16801 Approved By: iL.(~ -- ____________ Paul Connolly ' Technical Leader. LC-MS, Exygen Resesrch Flaherty Vice President, Operations, Exygen Research m iw m Dale M r Date Exygen Research Total Paget: 8 Page 37 o f 65 Page 81 of 111 Interim Report #12 - Analysis of Vegetation Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 Exyges RoM icb Method Number VQQ01783 ANA LY TICA L m e t h o d Method o f Analysis for die Determination o fPerfluorooctanoic Acid (PFOA) in Fish and Clams by LC/MS/MS 1.0 Scope This method is to be employed for the isolation and quantitation o fperfluorooctanoic add by High Performance Liquid Chromatography coupled to a tandem Maas Spectrometric Detector (LC/MS/MS) in fid i and clams. 2.0 Safety 2.1 Always observe safe laboratory practices. 2.2 Consult the appropriate MSDS before handling any chemical for proper safety precautions. 3.0 Sample Requirement 3.1 A t least 20 g o ftest sample for extractioa 3.2 Simples should be processed before extraction. Place the frozen sample in a food processorand homogenize w ith dry ice. Place the samples in containers and leave open in frozen storage overnight to allow for carbon dioxide sublimation. Seal and place the samples in frozen storage until time o f analysis. 3.3 Sample collection procedures w in be specified in the sampling plan for this project Reagents and Standards 4.1 Water-HPLC grade 4.2 Acetonitrile - HPLC grade 4.3 Carbon (120-400 mesh) - Reagent grade 4.4 Methanol - HPLC grade 4.3 Silica gel (60-200 mesh)- Reagent grade 4.6 Florisil (60-100mesh)- Reagent grade 4.7 Superclean LC-NHi - Reagent grade 4.8 1-Octanol - HPLC grade 4.9 L-Aacorbic acid - Reagent grade 4.10 DimethyldichloTOsilane - Reagent grade 4.11 Toluene - Reagent grade 4.12 Ammonium Acetate - AC.S. Reagent Grade 4.13 Perfluorooctanoic Acid - Sigma-Aldrich 3.0 Instrument and Equipment 5.1 A high performance liquid chromatograph capable o f pumping up to 2 solvents equipped with a variable volume iqjoctor capable o f injecting 5-200 pL connectedto a Mast Spectrometer (LC/MS/MS). 5.2 A device to collect raw data for peak integration and quantitation. 5.3 Analytical balance capable o freading to 0.00001 g. Page 2 o/S Page 38 o f 65 Exygen Research Page 82 of 111 Interim Report #12 - Analysis of Vegetation Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 Exyt*aR*M*rch M ethod N umber VQ001783 a n a l y t ic a l m e t h o d Method o fAnalysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Fish and Ciams by LC/MS/MS 5.4 Rotaryevaporator. 5.6 125 mL pear-shaped flasks. 5.7 50 mL disposable polypropylene centrifuge tubes. 5.8 IS mL disposable polypropylene centrifuge tubes. 5.9 Disposable micropipets (5<M00uL, 100-200uL). 5-10 125-mL LDPB narrow-mouth bottles. 5.11 2 mL clear HPLC vial IdL 5.12 Disposable pipettes. 5.13 Autopipettes (KMMOOOpLand 10-100 pL), w ith disposable tips. 5.14 SPE tubes (20mL) (Supelco cat. no. N057I77). 5.15 Wrist action shaker. 5.16 Centrifuge enable o fspinning 50 mL polypropylene tubes at 2000 rpm. 6.0 Chromatographie System 6.1 Analytical Column: Fluophase RP (Keystone Scientific), 2.1 mm x 50 mm. 5m (P/N: 82505-052130) 6.2 Temperature: 30*C 6.3 Mobile Phase(A ): 2 mM Ammonium Acetate in Water Mobile Phase(B ): Methanol Gradient Program: Flow Rate & A 2LB fm L/m inl 0.0 65 35 0.3 1.0 65 35 0.3 8.0 25 75 0.3 20.0 25 75 0.3 22.5 65 35 0.3 6.6 Injection Volume: 15 pL (can be increased to as much as 50 pL). 6.7 Quantitation: Peak Area-external standard calibration curve. 6.8 Run Time: - 23 minutes. The above conditions are intended as aguide and may be changed in order to optimize the HPLC system. Pi*e3of8 Page 39 o f 65 Exygen Research Page 83 of 111 Interim Report #12 - Analysis of Vegetation Samples Exygen Study No.: P0001131 Exygen Protocol Nuniber: P0001131 Hxyf KMMich MtChod Number V0001783 ANALYTICAL METHOD Method o fAnalysis for the Determination ofPerfluorooctinoic Acid (PFOA) in Fish and Clams by LC/MS/MS 7.0 MS/MS System 7.1 Mode: Electrospray Negative MRM mode, monitoring 413 - 369 m/z for PFOA. The aboveconditions ate intended asa guide and may be changed in order to optimize the MSMS system. 8.0 Preparation o fSolutions 8.1 Mobile Phase 8.1.1 2 mM ammonium acetate in water it prepared by adding 0.134 g o f ammonium acetate to 1000 mL o fwater. 8.2 Extraction Solution 8.2.1 8.2.2 2% ascorbic acid in methanol is prepared by dissolving 2 g o f accorine acid in 100 mL o fmethanol. 30% Dimethyldichforesilane in toluene is prepared by bringing 3 mL o fdimethyldichloioaUane to a final volume o f 10 mL with toluene. Alternate volumes may be prepared. 9.0 Standard Preparation 9.1 Standard Stock/Fortificatioo Solution 9.1.1 9.1.2 9.1.3 9.1.4 9.1.5 Prepare a stock solution o f-100 pg/raL o f PFOA by weighing 10 mg o f analytical standard (corrected for purity) and dilute to 100 mL with methanol in a 123-mL LDPE bottle. A 1.0 pgfaiL fortification solution o f PFOA is prepared by bringing 1 mL o f foe 100 pgfmL solution to a final volume o f 100 with methanol in a 123 mL LDPB bottle. A 0.1 pgfaiL fortification solution o f PFOA is prepared by bringing 10 mL o fthe 1.0 pgfaiL solution to a final volume o f 100 with methanol in a 123 mL LOPE bottle. A 0.01 p^m L fortification solution o f PFOA is prepared by bringing 10 mL o f the 0.1 pgtoL solution to a final volume o f 100 with methanol In a 123 mL LDPE bottle. The stock and fortification solutions are to be stored in a refrigerator at approximately 4*C and are stable for a maximum period o f 6 months from the dale o fpreparation. Page 4 of 8 Page 40 o f 65 Exygen Research Page 84 of 111 Interim Report #12 - Analysis of Vegetation Samples Exygen Study No.: P0001131 Exygen Protocol Number: POOO1131 Exygca Research Method Number VOOO1783 | .................... ANALYTICAL METHOD | Method o fAnalysis for die Determination ofPcrfhiorooctanoic Acid (PFOA) in Fish and Cams by LC/MS/MS 9.2 StandardCalibration Solutions 9.2.1 9.2.2 LC/MS/MS calibration standards are prepared in methanol via dilution o fdie 1.0 pg/mL fortification solution. The following is a typical example: additions! concentrations may be prepared asneeded. Concentration o fFortification Volume Diluted to Final Concentration Solution (u rttiL ) (mL) (mL) (ua/mL) 1JO 5.0 100 0.05 1.0 2.5 too 0.025 1JO 1.0 100 0.01 0.0S 10 too 0.005 0.025 10 100 0.0025 0.1 0.005 10 10 100 100 0.001 0.0005 9.2.3 Store a ll calibration standards in 125-mL LDPE narrow-mouth bottles at 2*C to 6C, up to fix months. 9.2.4 Alternate volumes and concentrations o fstandards may be prepared as needed. 10.0 Batch Set Up 10.1 Each batch o f samples extracted (typically 20 or less) must include at least one untreated control and two untreated controls fortified at known concentrations (lab control spike) to verify procedural recovery for the batch. 10.2 Requirements for field and laboratory duplicates and spikes w ill be specified in foe quality -- plan for this project. 11.0 Sample Extraction 11.1 Weigh 5 g o f frozen sample into SO mL polypropylene centrifuge tubes (fortify s i needed, replace lid and mix well). 11.2 Add 30 mL o facetonitrile and shake on a wrist action shaker for -13 minutes. 11.3 Place foe tubes in a freezer fo r-1 hour. 11.4 Pack and condition foe SPE tubes and silsnize the pear-shaped flasks. 11.5 Pack the 20 mL SPE tubes in sequence w ith 2 g flo risil, 2 g silica gel, 2 g carbon, and 1 g LC-NHj. Condition the columns with 20 mL o f methanol, then 20 mL o f acetonitrile. Discard e ll washes. Do not allow foe column to dry. 11.6 Silaniza the 125 mL pear-shaped flasks by rinsing with the 30% dimetbyidichloforilane in toluene solution. Rinse foe flask with toluene once, followed by methanol (three times). Dry foe flasks completely before use, either by air-drying or w ith a stream o fnitrogen. Page 5 of# Page 41 o f 65 Exygen Research Page 85 of 111 Interim Report #12 - Analysis of Vegetation Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 ExygnRMMich Mtbod Number VOOOH83 | .................. ANALYTICAL METHOD | Methodo f Analysis for the Determination o fPerfluorooctanoic Acid (PFOA) in Fish and Clams by LC/MS/MS 11.7 Centriftigc the 50 mL polypropylene tubes containing sample at -2000 rpm fo r-10 minutes. 11.8 Decant the extract on to a conditioned SPE column fitted inside the mouth of the pear-shaped flask. Collect the cluate in the 125 mL srianized pear-shape flask. 11.9 Add 10 mL o f acetonitrile to the sample in the SO mL centrifuge tube. Homogenize the frozen fat phase using a tiasumizer for -30 seconds and rinse the rissum tocrwith-IOm L o facetonitrile into the tube. 11.10 Shakethe sample again for - 10 minutes on a wrist-action shaker. 11.11 Place the tubes in a freezer for - 1 hour more. 11.12 Centrifuge the SOmL polypropylene tubes containing sample at -2000 rpm fo r-10 minutes. 11.13 Decant the extract onto the same SPE column. Collect the eluste into the same pear-shaped flask and combine w ith die eluent from the initial extraction. 11.14 Pass 20 mL o facetonitrile through the SPE column and combine the eluate in the tame peaMhapcd flask. 11.15 Add 3-4 chops o f L-octanol to the extract in the pear-shaped flask and evaporate at reduced pressure usings rotary evaporator (at < 40C). 11.16 Make the final volume, by adding 2 mL o f 2% ascorbic acid in methanol to die pear-shaped flask and swirl to mix/diisolve. 11.17 Transfer the extracts to HPLC vials using disposable pipets. 11.18 Analyze samplesusing electrospray LC/MS/MS. 12.0 Chromatography 12.1 Inject die same amount o f each standard, sample and fortified sample into the LC/MS/MS system. A calibration standard must precede and follow all analyzed samples. 12.2 Standards o fPFOA corresponding to at least five or more concentration levels must be included in an analytical set 12.3 An entire set o fcalibration standards must be included at the beginning and at the end o f sample set. Standards must be interspersed between every 5-10 samples. As an alternative, an entire set o f calibration standards may be injected at the beginning o f a set followed by calibration standards interspersed every 5-10 samples (to account for a second set o f standards). In either case, calibration standards must be the first and last injection in a sample set. 12.4 Use linear standard curves for quantitation. Linear standard curves are generated for the analyte by linear regression using 1/x weighting o fpeak area versus calibration standard concentration using MastLynx 3.3 (or equivalent) software system. Page 6 o f 8 Page 42 o f 65 Exygen Research Page 86 of 111 Interim Report #12 - Analysis of Vegetation Samples Exygen Study No.: P0001131 Exygen P rotocol N um ber: P0001131 ExyfaaKaMHcfa Md Number VOOOI7 [ ANALYTICAL METHOD J Method o fAnalysis for the Determinition o fPerfluorooctanolc Acid (PFOA) in Fish and Clams by LC/MS/MS 12.S Sample response should not exceed standard responses. Any samples that exceedstandard responses should be Anther diluted and reanalyzed. 13.0 Acceptance Criteria 13.1 Chromatogram must show s peak o f a daughter ion at 369 amu from a parent o f 413 amu. The 413 amu parent corresponds to the PFOA anion while the daughter ion (369 amu) represents the loss o fcarbon dioxide. 13.2 Method blanks must not contain PFOA at levels greater than the LOQ. If a blank contains PFOA at levels greater than 0.5 ppb then a new blank sample mutt be obtained andthe entire setmustbe re-extracted. 13.3 Reooveriea o f control spikes and matrix spikes must be between 70-130V of their known values. I f a control spike ftU i outside the acceptable lim its, the entire set o fsamples should be re-extracted. 13.4 Any calibration standard found to be a statistical outlier by using the Huge Error Test, may be excluded from the calculation o f the calibration curve. However, the total number o f calibration standards that could be excluded must not exceed20% o fthe total numbero fstandards injected. 13.5 The correlation coefficient (R) for calibration curves generated must be 20.992 (R2 20.985). I f calibration results ft]] outside these lim its, then appropriate steps must be taken to adjust instrument operation, and the standards or the relevant set o fsamples should be reanalyzed. 13.6 Retention times between standards and samples must not drift more than A % w ithin an analytical run. I f retention time d rift exceeds this lim it within an analytical run then foe set must be reanalyzed. 14.0 Calculations 14.1 Use the following equation to calculate the amount o f PFOA found (in ng/mL, based on peak area) using the standard curve (linear regression parameters) generated by die M an Lynx software program: PFOA found (ng/ral.) - fP w li I f t l - ialCTWBl) slope 14.2 Use the following equation to convert the amount o fPFOA found in ng/mL to n'g(ppb). PFOA bund (ppb) - 1PFOA found fa ./n in fin.1 volume fm Ll x DF1 sample weight (g) DF " factor by which the final volume w u diluted, if necessary. Pag*7 of 8 Page 43 o f 65 Exygen Research Page 87 of 111 Interim Report #12 - Analysis of Vegetation Samples Exygen Study No.: P0001131 Exygen Protocol Number. P0Q0U31 ExygvoResearch MMhodNumber V0001783 I a n a l y t ic a l m e t h o d ' Method o fAnalyst* for the Determination ofPerfluorooctanoic Acid (PFOA) in Fish and Clams by LC/MS/MS 14.3 For samples fortified with known amounts o f PFOA prior to extraction, use foe following equation to calculate the percent recovery. Recovery (%) - [ totalanalytefound(ng/g) snalytefound in control (ng/g)l clQ0 analyte added(ng/g) Exygen Research Page 8 o f8 Page 44 o f 65 Page 88 of 111 Interim Report #12 - Analysis of Vegetation Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 ANALYTICAL METHOD Method Number: V0001784 Method o fAnalysis fo r the DetermiiietJon o f Perfluorooctanolc Add (PFOA) In Vogetotlos by LC/MS/MS Analytical Testing Facility: Exygen Research 3058 Reaearch Drive State College, PA 16801 Approved By: T l A c j L ___ P uil Connolly Technical Leader, LC-MS, Exygen Reaearch a / n / i U / __________ John Flaherty ^ Vice President, Operations, Exygen Reaearch Date 4 Date Exygen Research Total Pages: 7 Page 45 o f 65 Page 89 of 111 Interim Report #12 - Analysis of Vegetation Samples Exygen Study No. : P0001131 Exygen Protocol Number: POOO1131 Exygen R eaw ch M ethod Number V000 1784 ANALYTICAL m e t h o d Method o fAnalyst* for the Determination o f Perfluorooctanoic Acid (PFOA) in Vegetation by LC/MS/MS 1.0 Scope This method is to be employed for the isolation and quantitation o fperfluorooctanoic acid by High Performance Liquid Chromatography coupled to a tandem Mass Spectrometric Detector (LC/MS/MS) in vegetation. 2.0 Safety 2.1 Always obeerve safe laboratorypractices. 2.2 Consultthe appropriate MSDS before handling any chemical for proper safety precautions. 3.0 Sample Requirement 3.1 A t least 20 g o fteet temple for extraction. 3.2 Samples should bo processed before extraction. Place foe frozen sample in a food processor and homogenize with dry ice. Place the samples in containers and leave open in frozen storage overnight to allow for carbon dioxide sublimation. Seal and place the samples in frozen storage until time of analysis. 3.3 Ssmple collection procedures w ill be specified in the sampling plan for this project. Reagents and Standards 4.1 W ater-HPLC grade 4.2 Acetonitrile-HPLC grade 4.3 Carbon (120*400 mesh) - Reagent grade 4.4 Methanol - HPLC grade 4.5 Silica gel (60*200 mesh)* Reagent grade 4.6 F lo riril (60-100 roesh) - Reagent grade 4.7 Superclean LC-NHj - Reagent grade 4.8 1-Octanol-HPLC grade 4.9 L-Ascorbic add - Reagent grade 4.10 DimcfoyldiehlofOiiUne - Reagent grade 4.11 Toluene - Reagent grade 4.12 Ammonium Acetate - A.C.S. Reagent Grade 4.13 Perfluorooctanoic Acid - Sigma-Aldrich 5.0 Instrument and Equipment 5.1 A high performance liquid chromatograph capable o f pumping up to 2 solvents equipped w ith a variable volume injector capable o f injecting 5*200 jjL connected to a tandem Mass Spectrometer(LC/MS/MS). 5.2 A device to collect raw data for peak integration and quantitation. SJ Analytical balance capableo freiding to 0.00001 g. Page 2 o f 7 Page 46 o f 65 Exygen Research Page 90 of 111 Interim Report #12 - Analysis of Vegetation Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 Exygen JUaeaich Method Number V00017M ANALYTICAL METHOD ........ 1 Method o fAnalysis for the Determination o fPeriluorooctanoic Acid (PFOA) in Vegetation by LCVMS/MS 5.4 Rotaiy evaporator. 5.5 125 mL pearehaped flasks. 5.6 SOmL diaposable polypropylene centrifuge tUbea. 5.7 15 mL disposable polypropylenecentrifuge tubes. Disposable micropipets (30100uL, 100-200uL). 125-mL LDPE nanow-mouth bottles. 5.10 2 mL clearHPLC vial h it S.1 Disposable pipettes. 5.12 Autopipettes (100-1000 pL and 10-100 pL), w ith disposable tips. 5.13 SPEtubes (20mL) (Supelco eat. no. N057177). 5.14 Wrist action shaker. 5.15 Centrifiige capable o fspinning 50 mL polypropylene tubes at 2000 rpm 6.0 Chromatographic System 6.1 Analytical Column: Fhiophase RP (Keystone Scientific), 2.1 mm x 50 mm. 5p (P/N: 82505-052130) 6.2 Temperature: 30*C 6.3 Mobile Phase(A ): 2 mM Ammonium Acetate in Water 6.4 Mobile Phase(B ): Methanol 6.5 Gradient Program: Time frnint 0.0 1.0 8.0 20.0 22.5 k A 65 65 25 25 65 Flow Rate 2LB fm L/m inl 35 0.3 35 0.3 75 0.3 75 0.3 35 0.3 6.6 Injection Volume: IS pL (can be increased to u much as 50 pL). 6.7 Quantitation: Peak Area - external standard calibration curve. 6.8 Run Time: ~ 23 minutes. The aboveconditions are intended as a guide and may be changed in order to optimize the HPLC system. 7.0 MS/MS System 7.1 Mode: Electrospray Negative MRM mode, monitoring 413 - 369 m/z for PFOA. Page 3 o f7 Page 47 o f 65 Exygen Research Page 91 of 111 Interim Report #12 - Analysis of Vegetation Samples Exygen Study No.: P0001131 Exygen Protocol Number: POTO1131 Exygen fUseirch M ethod Number VOO 1784 ANALYTICAL METHOD Method o fAnalysis for the Determination ofPerfluorooctanoic Add (PFOA) in Vegetation by LC/MS/MS The aboveconditions are intended as a guide and may be changed in order to optimize die MSMS system. 8.0 Preparation o fSolutions 8.1 Mobile Phase 8.1.1 2 mM ammonium acetate in water ia prepared by adding 0.154 g o f ammonium acetate to 1000 mL o fwater. 8.2 Extraction Solutions 8.2.1 8.2.2 2HafloosMc acid in metiianol is prepared by dissolving 2 g o f ascorbic add in 100 mL o fmathanoL 30% Dimethyldichlorosilane in toluene i t prepared by bringing 3 mL ofdiiM thyIdidiloroailaneto a final volume o f 10 mL with toluene. Alternate volumes may be prepared. 9.0 Standard Preparation 9.1 Standard Stock/Fortification Solution 9.1.1 Prepare a took solution of~100 pg/mL o f PFOA by weighing 10 mg o f analytical standard (corrected for purity) and dilute to 100 mL with methanol in a 125-mL LDPE bottle. 9.1.2 A 1.0 jif/m L fortification aolutian o f PFOA is prepared by bringing 1 mL o f the 100 pg/mL solution to a Anal volume o f 100 with methanol in a 125 mL LDPE bottle. 9.1.3 A 0.1 pgtoL fortification solution o fPFOA is prepared by bringing 10 m Lofthe 1.0 jjgAnL solution to a final volume o f 100 with methanol in a 125mL LDPE bottle. 9.1.4 A 0.01 pg/mL fortification solution o f PFOA is prepared by bringing 10 mL o f the 0.1 pg/raL solution to a final volume o f 100 with methanol in a 125 mL LDPE bottle. 9.1.5 The dock and fortification solutions are to be stored in a refrigerator at approximately 4*C and are stable for a maximum period o f 6 months from the date o fpreparation. 9.2 Standard Calibration Solutions 9.2.1 LC/MS/MS calibration standards are prepared in methanol via dilution o fthe 1.0 pg/mL fortification solution. Paye4ol * Page 48 o f 65 Exygen Research Page 92 of 111 Interim Report #12 - Analysis of Vegetation Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 Exy|taJUseareh M ethod Number V 0001784 AN ALYTIC AL m e t h o d Method o fAnalyst for the Determination o fPerfhiorooctanoic Acid (PFOA) in Vegetation by LC/MS/MS 9 2 .2 The following i i a typical example: additional concentrations may be prepared at needed. Concanfcation o fFortification Volume Diluted to Final Concentration Solution fui/m U (mL) (mL) (lis/m L) 1.0 3.0 100 1.0 2.S 100 1.0 1.0 100 0.05 0.025 0.01 0.05 0.02$ 0.1 10 10 10 100 100 100 0.005 0.0025 0.001 0.005 10 100 0.0005 9.2.3 Store a ll calibration standards in 123-mL LDPE narrow-mouth bottles at 2*C to 6C, up to six months. 9 2 A Alternate volumes and concentrations o f standards may be prepared as needed. 10.0 Batch Set Up 10.1 Each batch o f samples extracted (typically 20 or less) must include at least one untreated control end two untreated controls fortified at known concentrations (lab control spike) to verify procedural recovery for the batch. 10.2 Requirements for field and laboratory duplicates and spikes w ill be specified in the quality assurance plan for this project. 11.0 Sample Extraction 11.1 Weigh 5 g o f frozen sample into SO mL polypropylene centrifuge tubes (fortify es needed, replace lid and mix well). 11.2 Add 30 mL o facetonitrile end shake on a w rist action shaker for -1$ minutes. 11.3 Centrifuge the SO mL polypropylene tubes containing sample at -2000 rpm fo r-IQ minutes. 11.4 Pack and condition the SPBtubes and silanize the pear-shaped flasks. 11.5 Pack foe 20 mL SPE tubes in sequence with 2 g flo risil, 2 g silica gel, 2 g carbon, and 1 g LC-NHj. Condition the columns w ith 20 mL o f methanol, then 20 mL o f acetonitrile. Discard all washes. Do not allow the column to dry. 11.6 Silanize the 12S mL pear-shaped flasks by nnsing with the 30% dimethyldlchkuosilsne in toluene solution. Rinse the flask with toluene once, followed by methanol (three times). Dry the flasks completely before use, eitherby air-drying or with astream o fnitrogen. 11.7 Decant die extract m i to e conditioned SPE column fitted inside the mouth of the pear-shaped flask. Collect the eluate in the 125 mL silanized pear-shape Page 5 o f * Page 49 o f 65 Exygen Research Page 93 of 111 Interim Report #12 - Analysis of Vegetation Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 Exygen Research M ethod Num ber VOOO1784 AISa LVTICAL METHOD Method o fAnalysis for the Determination o fPerfluoiooctanoic Acid (PFOA) in Vegetation by LC/MS/MS 11.8 Add 20 mL o facetonitrile to the sample in die 50 mL centrifuge tube. 11.9 Shake die sample again for ~10 minutes on a wrist-action shaker. 11.10 Centriflige the 50 mL polypropylene tubec containing sample at -2000 rpm fo r-5 minutes. 11.11 Decant the extract onto die sane SPE column. Collect the eiuste into the sume pear-shaped flask and combine with the eluent from the initial extraction. 11.12 Repeatsteps 11.8 through 11.11 again. 11.13 Add 3-4 drops o f 1-octanol to the extract in the pear-shaped flask and evaporate at reducedpressure using arotary evaporator (at < 40C). 11.14 Make the final volume, by adding 2 mL o f 2% ascorbic acid in methanol to the pear-shaped flask and swirl to mix/disaolve. 11.15 Transfer the extracts to HPLC vials using disposable pipeta. 11.16 Analyze samples using electrospray LC/MS/MS. 12.0 Chromatography 12.1 Inject the same amount o feach standard, sample and fortified sample into the LC/MS/MS system. A calibration standard must precede and follow all analyzedsamples. 12.2 Standards o fPFOA corresponding to at least five or more concentration levels mustbe included in an analytical set 12.3 An entire set o f extracted calibration standards must be included at the beginning and at the end o f a sample set Extracted standards must be interspersed between every 5-10 samples. As an alternative, an entire set o f extracted calibration standards may be iqjected at the beginning o f a set followed by extracted calibration standards intenpersed every 5-10 samples (to account for a second set o f extracted standards). In either case, extracted calibration standards mustbe die first and last injection in a sample set. 12.4 Use linear standard curves for quantitation. Linear standard curves are generated for die analyte by linear regression using 1/x weighting o f peak area versus calibration standard concentration using MasaLynx 3.3 (or equivalent) software system. 12.5 Sample response should not exceed standard responses. Any samples that exceed standardresponses should be further diluted and reanalyzed. 13.0 Acceptance Criteria 13.1 Chromatogram must show a peak o f a daughter ion at 369 amu from a parent o f 413 amu. The 413 amu parent corresponds to the PFOA anion, while the daughter ion (369 amu) represents the loss o f carbon dioxide. Page 6 o f 7 Page 50 o f 65 Exygen Research Page 94 of 111 Interim Report #12 - Analysis of Vegetation Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 ExygenRw--ich MethodNumberV00017M A N A L Y T IC A L METHOD Method o fAnalysis for the Determination o fPerfluorooctanoic Acid (PFOA) in Vegetation by LC/MS/MS 13.2 Method blank* must not contain PFOA at levels greater than the LOQ. If a blank contains PFOA at levels greater than O.S ppb, then a new blank sample must be obtained and the entire setmust be re-extracted. 13.3 Recoveries o fcontrol spikes and matrix spikes must be between 70*130% of their known value*. I f a control spike A lls outside the acceptable lim its, the entire set o f samples shouldbe re-extracted. 13.4 Any calibration standard found to be a statistical outlier by using the Huge Error Test, may be excluded from the calculation o f the calibration curve. However, the total number o f calibration standards that could be excluded must not exceed 20% o f the total numbero f standards injected. 13.5 The correlation coefficient (R) for calibration curvet generated must be 20.992 (R3 20.985). I f calibration results A ll outside these lim its, then appropriate steps must be taken to adjust instrument operation, and (he standards or the relevant set o fsamples should be reanalyzed. 13.6 Retention times between standards and samples must not d rift more than 4 % within an analytical run. I f retention time d rift exceeds this lim it within in analytical run then foe setmust be reanalyzed. 14.0 Calculations 14.1 Use foe following equation to calculate foe amount o f PFOA found (in ng/mL, baaed on peak area) using foe standard curve (linear regression parameters) generated by the Mass Lynx software program: PFOA found (ng/mL) - (Peak area - intercept) slope 142 Uaefoe following equation to convert foe amount o f PFOA found in ng/mL to ng/g(ppb). W O A found (noM - ffPOA found InutaiD x final volume (m LI x DF1 sample weight (g) DF factor by which the final volume was diluted, i f necessary. 14.3 For samples fortified w ith known amounts o f PFOA prior to extraction, use foe following equation to calculate the percent recovery, Recovery (%) - [ totalanalyte found (n^g) analyte found in control (ng/g)] ^ ^ cnalym added (ng/g) Page 7 tit' 7 Page 51 o f 65 Exygen Research Page 95 of 111 Interim Report #12 - Analysis of Vegetation Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 ANALYTICAL METHOD Method Number V0001785 Method o f AnalyaU fo r the D otoru lu tlea o f Ferfleorooctasok Acid (PFOA) in Small Mammal Liver by LC/MS/MS Analytical Tearing Facility: Exygen Reaoarch 3058 Research Drive State College. PA 16801 Approved By: V,_C-- ____ Paul Connolly I Technical Leader, LC-MS, Exygen Research D ate Date Exygen Research Total Pages: 7 Page 52 o f 65 Page 96 of 111 Interim Report #12 - Analysis of Vegetation Samples Exygen Study No.: P0001131 Exygen Protocol Number: POOO1131 E xy|M lU w trck M ethod N umber VQQOI785 I ANALYTICAL M THO D Method o fAnalysis for the Determination o fPerfluorooctanoic Acid (PFOA) in Small Mamma] Liver by LC/MS/MS 1.0 Scope This method is to be employed for foe isolation and quantitation o fperfluorooctanoic acid by High Performance Liquid Chromatography coupled to a tandem Mass Spectrometric Detector (LC/MS/MS) in email mammal liver. 2.0 Safety 2.1 Always observe aafe laboratory practices. 2.2 Consult the appropriate MSDS before handling any chemical for proper safety precautions. 3.0 Sample Requirement 3.1 At least 5 g o fteet sample for extraction. 3.2 Samples should be processed before extraction. Place the frozen sample in a food processor and homogenize with dry ice. Place the samples in containers and leave open in frozen storage overnight to allow for carbon dioxide sublimation. Seal and place the samples in frozen storage until lime o f analysis. Alternately, i f there is an insufficient amount o f sample Mess than 5 g), then no processing is necessary and the sample can be used as supplied 3.3 Sample collection procedures w ill be specified in foe sampling plan for this project. 4.0 Reagents and Standwda 4.1 Water-HPLC grade 4.2 Methanol-HPLC grade 4.3 Acetonitrile - HPLC grade 4.4 Ammonium Acetate - A.C.S. Reagent Grade 4.5 Perfluorooctanoic Acid - Sigma-Aldrich 3.0 Instrument and Equipment 3.1 A Ugh performance liquid chromatograph capable o f pumping up to 2 solvents equipped with variable volume injector capable o f injecting 5-200 pL connected to a tandem Mass Spectrometer (LC/MS/MS). 52 A device to collect raw data for peak integration and quantitation. 5.3 Analytical balance capable o freading to 0.00001 g. 5.4 50 mL disposable polypropylene centriftige tubes. 5.5 15 mL disposable polypropylene cemrifoge tubes. 5.6 Disposable micropipets (50-lOOuL, 100-200uL). 5.7 125-mL LDPE narrow-mouth bottles. 5.8 2 mL clear HPLC vial k it Page 2 ufV Page 53 o f 65 Exygen Research Page 97 of 111 Interim Report #12 - Analysis of Vegetation Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 ExyteallMmicIi MethodNumberV00017S5 1 ANALYTICAL METHOD Method o fAnalysis for the Determination o f Perfluorooctanoic Add (PFOA) in Small Mammal Liver by LC/MS/MS 3.9 3.10 5.1 1 3.12 3.13 3.14 5.13 Disposable pipettes. Autopipettes (100*1000 pL and 10*100 pL), with disposable tips. Waters Sep Pak Vac 6 cc (lg ) tC18 SPE cartridges. SPEvacuum manifold. Tissuemizer. Wrist*action shikar. Centrifoge capable o fspinning 15 mL polypropylene tubes at 3000 rpm. 6.0 Chromatographic System 6.1 Analytical Column: Fluophase RP (Keystone Scientific), 2.1 mm x 30 mm. 5p (P/N: 82303-032130) 6.2 Temperature: 30*C 6.3 Mobile Phase(A ): 2 mM Ammonium Acetate in Water Mobile Phase(B ): Methanol Gradient Program: Time (mini 0.0 1.0 8.0 20.0 22.3 5kA 65 65 25 25 63 Flow Rate % B fmL/min^ 35 0.3 35 0.3 75. 0.3 75 0.3 35 0.3 6.6 Injection Volume: 13 pL (can be increased to as much as 30 pL). 6.7 Quantitation: PeakArea-external standard calibration curve. 6.8 Run Time: - 23 minutes. The above oooditions are intended is a guide sod may be changed in order to optimize the HPLC system. 7.0 MS/MS System 7.1 Mode: Electrospray Negative MRM mode, monitoring 413 - * 369 m/z for PFOA. The above conditions ire intended as a guide and may be changed in order to optimize the MSMS system. Pate 5 of? Page 54 o f 65 Exygen Research Page 98 of 111 Interim Report #12 - Analysis of Vegetation Samples Exygen Study No. : P0001131 Exygen Protocol Number: POOO1131 Bxygu Rucucli Method Number V0001785 | .......... ... ANALYTICAL M ETH O D Method o fAnalysis for the Determination ofPerfluorooctanoic Acid (PFOA) in Small Mammal Liver by LC/MS/MS 6.0 Preparation o fSolutions 8.1 Mobile Phase 8.1.1 2 mM ammonium acetate in water is prepared by adding 0.154 g of ammonium acetate to 1000 m L of weter. Alternate volumes may be prepared. 9.0 StandardPreparation 9.1 Standard Stock/Fortification Solution 9.1.1 Prepare a stock solution o f-100 pg/mL o f PFOA by weighing 10 mg o fanalytical standard (corrected for purity) and dilute to 100 mL with methanol in a 125-mL LDPE bottle. 9.1.2 A 1.0 pgAnL fortification solution o f PFOA il prepared by bringing I mL o f foe 100 p^m L solution to a final volume o f 100 with methanol in a 125 mL LDPE bottle. 9.1.3 A 0.1 pg/mL fortification solution o f PFOA ia prepared by bringing 10 mL o fdie 1.0 pg/mL solution to a final volume o f 100 with methanol in a 125 mL LDPE bottle. 9.1.4 The stock and fortification solutions are to be stored in a refrigerator at approximately 4C and are stable for maximum period o f 6 months from die date o fpreparation. 9.2 StandardCalibration Solutions 9.2.1 9.2.2 LC/MS/MS calibration standard* are prepared in methanol via dilution o fthe 0.1 p^m L fortification solution. The following is a typical example: additional concentrations msy be prepared asneeded. Concentration Final o fFortification Volume Diluted to Concentration Solution fns/raL) (mL) (mL) (na/mU 100 5.0 100 100 2.0 100 5.0 2.0 100 14 100 S.0 10 100 1.0 0.5 2.0 10 100 1.0 10 100 0.2 0.1 9.2J Store all calibration standards in 125-mL LDPE narrow-mouth bottles it 2*C to $*C, up to six months. 9.2.4 Alternate volumes and concentrations o f standards may be prepared as needed. Page 4 o f7 Page 55 o f 65 Exygen Research Page 99 of 111 Interim Report #12 - Analysis of Vegetation Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 ExyfealtMMfck Matbod Number V0001785 I ANALYTICAL METHOD Method o fAnalysii for the Determination o fPerfluorooctanoic Acid (PFOA) in Small Mammal Liver by LC/MS/MS 10.0 Batch Set Up 10.1 Each batch o f sample# extracted (typically 20 or leas) must include at least one untreated control and two untreated controls fortified at known concentrations (lab control spike) to verify procedural recovery for the batch. 10.2 Requirements for field and laboratory duplicates and spikes w ill be specified in the quality assuranceplan for this project. 11.0 Sample Extraction 11.1 Weigh 1 g o f sample into s 50 mL polypropylene centrifuge tubes (fortify as needed, replace lid and mix well). Note that alternate weights o f liver may be measured depending on the sample size available for use. 112 Add water to die sample for afinal volume o f 10 mL. . 11.3 Homogenize sample using atisiuemizer for ~1 minute. 11.4 Transfer 1 mL o f the sample using a disposable pipette into a IS mL disposable centrifiige tube. 11.5 Add 5 mL o facetonitrile and shake for20 minutes on a w risi-iction shaker. 11.6 Centrifiige die tubes at3000 rptn for 5 minutes. 11.7 Decant die supernatant into a SOmL disposable centrifiige tube and add 35 mL o fwater. 11.8 Condition the Cia SPE cartridges (1 g. 6 mL) by passing 10 mL methanol followed by Sm LofKPLC water ( 2 drpp/sec). Do not let column run dry 11.9 Load the aanple on conditioned Cn SPE cartridge. Discard eluate. 11.10 Elute with 2 mL o f methanol Collect 2 mL o f eluate into a graduated IS mL polypropylene oentriftjge tube (final volume 2 mL). 11.11 Analyze aamplca using eloctrospray LC/MS/MS. 12.0 Chromatography 12.1 Iqject the same amount o f each standard, sample and fortified sample into the LC/MS/MS system. A calibration standard must precede and follow all analyzed samples. 122 Standardso fPFOA corresponding to at least five or more concentration levels must be included in an analytical set 12.3 An entire set o f calibration standards must be included at the beginning and at the end o f a sample set. Standards must be interspersed between every 5-1u samples. As an alternative, an entire set o f calibration standards may be iryected at the beginning o f set followed by calibration standards interspersed every 5-10 simples (to account for a second set o f standards). In either case, calibration standards must be the first and last injection in a sample set. 12.4 Use linear standard curves for quantitation. Linear standard curves are generated for the analyte by linearregression using l/x weighting o fpeak area rip 5 of7 Page 56 o f 65 Exygen Research Page 100 of 111 Interim Report #12 - Analysis of Vegetation Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 ExygacAamrcfc Meted NumberV0001785 I ANALYTICAL METHOD ....... Method o fAnalysis for the Determination ofPerfhiorooctanoic Acid (PFOA) in Small MammalLiver by LC/MS/MS versus calibration standard concentration using MasaLynx 3.3 (or equivalent) software system. 12.5 Sample response should not exceed standard responses. Any samples that exceed standard responses should be further diluted and reanalyzed. 13.0 Acceptance Criteria 13.1 Chromatogram must show a peak o f a daughter ion at 369 amu from a parent o f 413 amu. The 413 amu parent corresponds to the PFOA anion, while the daughter ion (369 amu) represents the loss o fcarbon dioxide. 13.2 Method blanks must not contain PFOA at levels greater than the LOQ. If a blank contains PFOA at levels greater then 10 ng/g, then a new blank sample must be obtained and the entire set must be re-extracted. 13.3 Recoveries o f control spikes and matrix spikes must be between 70-130% of their known values. If a control spike folia outside the acceptable lim its, the entire set o f sample should be re-extracted. Any matrix spike outside 70 130% should be evaluated by the analyst to determine i f re-extraction is warranted. 13.4 Any calibration standard found to be a statistical outlier by using the Huge Error Teat, may be excluded from die calculation o f the calibration curve However, the total number o f calibration standards that could be excluded must not exceed 20% o fthe total numbero fstandards injected. 13.5 The correlation coefficient (R) for calibration curves generated must be 20.992 (RJ 20.985). I f calibration results fo il outside these lim its, then appropriate steps must be taken to adjust instrument operation, and the standards or the relevant set o f samples should be reanalyzed. 13.6 Retention times between standards and samples must not d rift more than 4% within an analytical tun. I f retention time drift exceeds this lim it within an analytics! nm then the set must be reanalyzed. 14.0 Calculations 14.1 Use the following equation to calculate the amount o f PFOA found (in ng/mL, based on peak area) using the standard curve (linear regression parameters) generated by the Mats Lynx software program: PFOA found (ng/mL) ffsgk gm - intercept) x DF x aliquot factor slope DF - factorby which tbc final volume was diluted, i f necessary. Aliquot factor- 10 Pag t o r 7 Page 57 o f 65 Exygen Research Page 101 of 111 Interim Report #12 - Analysis of Vegetation Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 Exygu fUaMtck Method Number VOOOI785 | ANALYTICAL METHOD .......... .... Method o fAnalytic for the Determination o fPeriluorooctinoic Acid (PFOA) in Small Mammal Uver by LC/MS/MS 14.2 For samples fortified with known amounts o f PFOA prior to extraction, use die following equation to calculate the percent recovery* Recovery (%) - [ totalanalyte found (ng/mL) analytefound in control (ng/rnL)] ^ analyte added(ng/mL) 14.3 Use the following equation to convert the amount o f PFOA found in ng/mL lo (PPb). PPflA fainrf fnnM - fPPnA found IW niM x fine! volume fmLll sample weight (g) Exygen Research Pate 7 o f7 Page 58 o f 65 Page 102 of 111 Interim Report #12 - Analysis of Vegetation Samples Exygen Study No.: P0001131 Exygen Protocol N um ber P0001131 ANALYTICAL METHOD Method Number VOOOl 786 Method o fA a a lyili for the Determlaatloa of PerfUiorooctanoic A dd (PFOA) in Small Mammal Serum by LC/MS/MS Analytical Testing Facility; Exygen Research 3058 Research Drive State College, PA 16801 Approved B y _L = L a _ _ _ Paul Connolly I Technical Leader, LC-MS, Exygen Research 4k. / # ) / / / JShnFlahwty ' Vice President, Operations, Exygen Research Date Date Exygen Research Total Pages: 7 Page 59 o f 65 Page 103 of 111 Interim Report #12 - Analysis of Vegetation Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 Exys Rmch Method Nuotar VOOO17*6 a n a l Vt i c a l m e t h o d Method o fAnalysis for the Determination o f Perfluorooctanoic Acid (PFOA) in Small Mammal Serum by LC/MS/MS 1.0 Scope This method is to be employed for die isolation and quantitation o f perfluorooctanoic acid by H igji Performance Liquid Chromatography coupled to a tandem Maas Spectromdric Detector(LC/MS/MS) in small mammal serum. 2.0 Safety 2.1 Always observe safe laboratory practices. 2.2 Consultthe appropriate MSDS before handling any chemical for proper safety precautions. 3.0 SampleRequirement 3.1 A t least 1mL o ftest sample for extraction. 3 2 No simple processing is needed for serum samples. However, frozen serum samples mustto allowed to completely thaw to room temperature before use. 3.3 Sample collection procedures w ill be specified in die sampling plan for this project 4.0 Reagents and Standards 4.1 W ater-HPLC grade 4.2 Methanol - HPLC grade 4.3 Acetonitrile - HPLC grade 4.4 Ammonium Acetate - A.C.S. Reagent Grade 4.5 Perfluorooctanoic Acid - Sigma*Aldrich 5.0 Instrument and Equipment 5.1 A high performance liquid chromatograph capable o f pumping up to 2 solvents equipped with a variable volume injector capable o f injecting 5-200 pL connected to atandem Mass Spectrometer(LC/MS/MS). 5.2 A device to collect raw data for peak integration and quantitation. 5.3 Analytical balance capable o freeding to 0.00001 g. 5.4 50 roL dispotable polypropylene centriflige tubes. 5.5 15 mL disposable polypropylene centriflige tubes. 5.6 Disposable micropipets (50-100uL, 100-200uL). 5.7 125-raL LDPE narrow-mouth bottles. 5.8 2 mL clear HPLC vial Ids. 5.9 Disposable pipettes. 5.10 Autopipettes (100-1000 pL and 10-100 pL), with disposable tips. 5.11 Wstera ScpPak Vac 6 cc (lg ) tC l8 SPE cartridges. 5.12 SPE vacuum manifold. 5.13 Voitexer. P tg e o P P age 60 o f 65 Exygen Research Page 104 of 111 Interim Report #12 - Analysis of Vegetation Samples Exygen Study No. : P0001131 Exygen P rotocol N um ber: P0001131 ExyaeaK cM uch Method Number V00017M I A IW Y T1C A L METHOD Method o fAnalysis for the Determination o fPerlhxxooctanoic Acid (PFOA) in Small Mammal Serum by LCSMS/MS 5.14 Wrist-action shaker. 5.15 Centrifuge capable o fpinning 15 mL polypropylene tubes at 3000 rpm. 6.0 Chromatographic System 6.1 Analytical Column: Fluophaae RP (Keystone Scientific), 2.1 mm x 50 mm. 5p (P/N: 82505-052130) 6.2 Temperature: 30*C 6.3 Mobile Phaae (A ): 2 mM Ammonium Acetate in Water 6.4 Mobile Phaae(B ): Methanol 6.5 GradientProgram: Time (mini 0.0 1.0 8.0 20.0 22.5 SLA 65 65 25 25 65 Flow Rate % B (m L/m inl 35 0.3 35 0.3 75 0.3 75 0.3 35 0.3 6.6 Injection Volume: 15 pL (can be increased to as much as 50 nL). 6.7 Quantitation: PeakArea-external standard calibration curve. 6.8 RunTime: -2 3 minutes. The above conditions are intended as a guide and may be changed in order to optimise the HPLC system. 7.0 MS/MS System 7.1 Mode: Electrospray Negative MRM mode, monitoring 413 -+ 369 m /r for PFOA. The above conditions are intended as a guide and may be changed in order to optimize the MSMS system. 8.0 Preparation o fSolutions 8.1 Mobile Phase 8.1.1 2 mM ammonium acetate in water is prepared by adding 0.1S4 g of ammonium acetate to 1000 mL o fwater. Alternate volumes may be prepared. peg3 of 7 Page 6i o f 65 Exygen Research Page 105 of 111 Interim Report #12 - Analysis of Vegetation Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 Exygee Research Method Number V000I786 I ANALYTICAL METHOD ..... Method o fAnalysis for the Determination ofPerfluorooctanoic Acid (PFOA) in Small Mammal Serum by LC/MS/MS 9.0 Standard Preparation 9.1 Standard Stock/Fortification Solution 9.1.1 Prepare a stock solution o f-100 pg/mL o f PFOA by weighing 10 mg o f analytical standard (corrected for purity) and dilute to 100 mL with methanol in a 123-raL LDPE bottle. 9.1.2 A 1.0 pg/mL fortification solution o f PFOA is prepared by bringing 1 mL o f the 100 pg/raL rolution to a final volume o f 100 with methanol in a 125 mL LDPE bottle. 9.1.3 A0.1 pg/mL fortification solution ofPFO Aia prepared by bringing 10 m Loftbe 1.0 pg/mL solution to a final volume o f 100 with methanol in a 125 mL LDPE bottle. 9.1.4 The stock and fortification solutions are to be stored in a refrigerator at approximately 4*C and are stable for a maximum period o f 6 months from the date o fpreparation. 9.2 Standard Calibration Solutions 9.2.1 9.2.2 LC/MS/MS calibration standards are prepared in methanol via dilution o fthe 0.1 jig/m L fortification solution. The following is a typical example: additional concentrations may be prepared asneeded. Concentration o fFortification Volume Diluted to Final Concentration Solution (aa/mU 100 100 (mL) 5.0 2.0 (m L) 100 100 (na/mL) 5.0 2.0 100 1.0 100 5.0 10 100 2.0 10 100 1.0 0.5 0.2 1.0 10 100 0.1 9.2.3 Store all calibration standards in 125-mL LDPE narrow-mouth bottles at 2C to 6*C, up to six months. 9.2.4 Alternate volumes and concentrations o f standards may be prepared as needed. 10.0 Bitch SetUp 10.1 Each batch o f samples extracted (typically 20 or less) must include at least one untreated control and two untreated controls fortified at known concentrations{lab control spike)to verify procedural recovery for the batch. 10.2 Requirements for field and laboratory duplicates and spikes w ill be specified in the quality assurance plan for this project. Pig 4 of 7 Page 62 o f 65 Exygen Research Page 106 of 111 Interim Report #12 - Analysis of Vegetation Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 ExygnJCaiMich Method Number VQOO1716 1 a n a ly tica l m e t h o d Method o fAnalysis to the Determination o fPerfluorooctanoic Acid (7FOA) in Small Mammal Seram by LC/MS/MS 11.0 Sample Extraction 11.1 Measure l mL o f sample into a SOmL polypropylene centrifuge tubes (fortify as needed, replace lid and mix well). Note that alternate volumes o f serum maybe measured depending on the sample size available for use. 11.2 Add water to the sample to final volume o f20 mL. Cap tightly 11.3 Vortex for -1 minute. 11.4 Transto 1 mL o f (he sample using a disposable pipette into a 15 mL disposable centrifage tube. ! 1.5 Add 5 m L ofacetonitrile and shake for -20 minutes on s wrist-action shaker. 11.6 Centrifuge the tubes at -3000 ipm for -5 minutes. 11.7 Decant the supernatant into a 50 mL disposable centrifuge tube and add 35 m L o fwater. 11.8 Condition the Cis SPE cartridges (1 g, 6 mL) by passing 10 mL methanol followed by 5 mL o fHPLC water (-2 drop/sec). Do not let column run dry 11.9 Load the sample on conditioned Cis SPE cartridge. Discard eluate, 11.10 Elute with -2 mL o f methanol. Collect 2 mL o f eluate into a graduated 15 mL polypropylene centrifoge tube (final volume 2 mL). 11.11 Analyze simples using electrospray LC/MS/MS. 12.0 Chromatography 12.1 Inject the same amount o feach standard, sample and fortified sample into the LC/MS/MS system. A calibration standard must precede and follow all analyzed samples. 12.2 Standards ofPFOA corresponding to at least five or more concentration levels must be included in an analytical set. 12.3 An entire set o fcalibration standards must be included at the beginning and at the end o f a sample sec. Standards must be interspersed between every 5-1o simples. As an alternative, an entire set o f calibration standards may be injected at the beginning o f a set followed by calibration standards interspersed every 5-10 samples (to account for a second set o f standards). In either case, calibration standards must be the first and last injection in a sample set 12.4 Use linear standard curves for quantitation. Linear standard curves are generated for the analyte by linear regression using 1/x weighting o fpeak area versus calibration standard concentration using MassLynx 3.3 (or equivalent) software system. 12.5 Sample response should not exceed standard responses. Any samples that exceed standard responses should be farther diluted and reanalyzed. Page 5 of 7 Page 63 o f 65 Exygen Research Page 107 of 111 Interim Report #12 - Analysis of Vegetation Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 Exygen Rcacatcb | 1ANALYT CALM KTHOD 1M ethod Num ber VOOO 7K6 ~ Method o f Analysis for the Detenninstion o fPerftuorooctanoic Acid (PFOA) in Small Mammal Serum by LC/MS/MS 13.0 Acceptance Criteria 13.1 Chromatogram must show a peak o f a daughter ion at 369 amu from a parent o f 413 amu. The 413 amu parent corresponds to the PFOA anion, while the daughter ton (369 amu) represents rite loss o fcarbon dioxide. 13.2 Method blanks must not contain PFOA at levels greater than the LOQ. If a blank contains PFOA at levels greater than 10 ng/mL, then a new blank sample mustbe obtained and the entire set must be re-extracted. 13.3 Recoveries o f control spikes and matrix spikes must be between 70*130% of their known values. I f control spike falls outside the acceptable lim its, the entire set o f samples should be re stricte d . Any matrix spike outside 70 130% should be evaluated by the analyst to determine if re-extraction is warranted. 13.4 Any calibration standard found to be a statistical outlier by using the Huge Error Test, may be excluded from the calculation o f the calibration curve However, the total number o f calibration standards that could be excluded must not exceed 20% o fthe total numbero fstandards injected. 13.5 The correlation coefficient (R) for calibration curves generated must be 20.992 (Ra 20.985). I f calibration results foil outside these lim its, then appropriate stepe muat be taken to adjust instrument operation, and the standards or the relevant set o f samples should be reanalyzed. 13.6 Retention times between standards and samples must not d rift more than 4 % within in analytical run. I f retention time d rift exceeds this lim it within an analytical run then the setmust be reanalyzed. 14.0 Calculations 14.1 Use the following equation to calculate the amount o fPFOA found (in ng/mL. based on peak area) using tile standard curve (linear regression parameters) generated by the M an Lynx software program: PFOA found (ng/mL)" (Peak area- intercept) x DF x aliquot factor slope DF foctorby which the final volume wasdiluted, i f necessary. Aliquot factor 20 14.2 For samples fortified w ith known amounts o f PFOA prior to extraction, use the following equation to calculate the percent recovery. Recovery (%) [total analytefound(ng/mL) - analyte found in control(ng/mL)] analyte added(ng/mL) Page 6 of7 Page 64 o f 6S Exygen Research Page 108 of 111 Interim Report #12 - Analysis of Vegetation Samples Exygen Study No.: P0001131 Exygen Protocol Number: P0001131 ExysmlUttttch Mttbod Number VOO1736 I A N A irn C A L METHOD Method o fAnalysis for the Determination o fPerfluorooctanoic Acid (PFOA) in Small Mammal Snum by LC/MS/MS 14.3 Uae the following equation to convert the amount o f PFOA found in ng/mL to ppb. PFOA found (ppb) fPFQA found fiiy/m U x final volume f m ll ample volume (mL) Exygen Research Page 7 P Page 65 o f65 Page 109 of 111 Interim Report #12 - Analysis of Vegetation Samples Exygen Study No. : P0001131 3058 Research Drive Phone: 814-272-1039 S tate College, PA 16801 Fax: 814-231-1580 PROTOCOL AMENDMENT Amendment Number 1 Effective Date: 0 1/19/05 Exygen Study Number P0001131 Client Study N um ber Page 1 of 1 D ESC R IPTIO N O F AM ENDED S E C fi N 1) Analytical Procedure Sum m ary V0001780:Section 9.1 2 ) Verification o f Analytical Procedure None AMENDED TO 1) Add to Section 9.1: Section 9 .1 .6 , A lternate weights of standards m ay be used to prepare alternate concentrations o f stock solutions as necessary. Atterriate levels of fortification solutions m ay also be prepared. 2 ) Low and high spiking levels o f the analytes for each m atrix m ay be altered depending on sam ple size available for extraction and/or to cover analyte concentrations expected in the sam ples. BfiTIPNALE 1 ) Higher concentrations o f standards need to be prepared in order to spike the sam ple bottles at higher levels. 2 ) The sam ple size avalab le fo r sm all mammal liver and serum w as sm aller than expected. Spiking at the pre-determ ined levels in the protocol puts the spiked concentration low er than the detection lim it Also, the analyte levels In the ground w ater sam ples a re expected to greatly exceed the pre-determ ined spiking levels listed in the protocol. W hen the levels In the sam ples greatly exceed the spiking levels, an accurate recovery value cannot be calculated for the Q C sam ple. Higher spiking levels in the bottles w ill cover the analyte concentrations expected in the w ater sam ples. IM PACT O N S TU D Y T h e LO Q is 100 ng/g for a 0.1 g sam ple o f small mammal liver and Is 1000 ng/m L for a 0.01 mL sam ple o f sm all mammal serum . Higher levels of spiking fo r the w ater sam ples will ensure that more Q C recovery data can be used. LIBRARY ID: W 0001226-61 . . ' ADMINISTRATIVE FORM Exygen Research Page 110 of 111 Interim Report #12 - Analysis of Vegetation Samples Exygen Study No.: P0001131 3058 Research D rive Phone: 814-272-1039 S tate College, PA 16801 Fax: 814-231-1580 Amendment Number Effective Date: Exygen Study Number PROTOCOL AMENDMENT 2 03/07/05 P0001131 Client Study Num ber Page 1 of 1 None D ESC R IPTIO N O F AM ENDED SE C TIO N 1) Report, page 11 o f 65 2 ) Test M aterials, page 6 o f 65: PFO S transition monitored 499 -> 99. AMENDEDTO 1) Instead o f one final report, Interim reports will be Issued. 2 ) P FO S transition monitored m ay also be 4 9 9 -> 80. RATIO NALE 1) Due to the excessive sizes o f the data sets, interim reports will be issued to allow the client to receive data in a tim elier-m ane* -W 2 ) The A P I 4 0 0 0 LC /M S/M S systems detect the 4 9 9 -> 80 PFO S transition with greater sensitivity than the 4 9 9 -> 99 transition. IM PACT O N S TU D Y 1) T h e client will be able to receive and review the data m ore quickly. 2 ) The 4 9 9 -> 80 transition can be detected with greater sensitivity; therefore, giving better chromatography. LIBRARY IO: V 0001228-S '- Exygen Research ADMINISTRATIVE FORM Page 111 of 111