Document zbbQrjvD741B0xEjEN4mVK5Xn
INTERIM REPORT # 17 - Analysis of Sediment and Water Samples
STUDY TITLE Analysis of Perfluorobutanesulfonate(PFBS), Perfluorohexanesulfonate(PFHS), and Perfluorooctanesulfonate(PFOS) in Water, Soil, Sediment, Fish, Clams, Vegetation, Small Mammal Liver and Small Mammal Serum Using LC/MS/MS for the 3M Decatur
Monitoring Program
DATA REOUIREMENTS 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 June 29,2007
PERFORMING LABORATORY Exygen Research
3058 Research Drive State College, PA 16801
Phone: 814-272-1039
STUDY SPONSOR 3M Company
3M Building 0236-0 1-B-10 St. Paul, MN 55144 Phone: 651-733-6374
PROJECT Protocol Number: POOOl 131 Exygen Study Number: POOOl 131
Total Pages: 127
Interim Report #17 - Analysis of Sediment and Water Samples Exygen Study No.: POOOl 131
GOOD LABORATORY PRACTICE COMPLIANCE STATEMENT
Exygen Study Number POOOl 131, 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.
Charles Simons Principal Investigator Exygen Research
Jaisimha Kesari P.E., DEE Study Director Weston Solutions, Inc.
Michael A. Sant#o Sponsor Representative 3M Company
Exygen Research
*@-
Date Page 2 of 127
Interim Report #17 - Analysis of Sediment and Water Samples Exygen Study No.: POOOl 131
OUALITYASSURANCE STATEMENT
Exygen Research's Quality Assurance Unit reviewed Exygen Study Number POOOl 131, 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 phases' 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 23. Raw Data and
Interim Report Review
Date Inspected
02123-24/06
Date Reported to Date Reported to
Principal
Ex ygen
Date Reported to
Investigator Management Study Director
0 3 /09/0 6
03/10/06
04/03/06
52. Final Interim Report Review
06/07/07
06112/07
06115/07
06118/07
A.
1
~SamPorter Quality Assurance Group Leader, Quality Assurance Unit
G DatellglO;L
`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
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Interim Report #I 7 - Analysis of Sediment and Water Samples Exygen Study No.: PO001 131
CERTIFICATION OF AUTHENTICITY
This interim report, for Exygen Study Number POOO1131, 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:
I
Charlgs Simons Director, Analytica Exygen Research
Exygen Research Facility Management:
Date Analytical Sciences Exygen Research
Jaisimha Kesari P.E., DEE Weston Solutions, Inc.
Sponsor Representative,3M Company:
n
sf Michael A. Sant o
Director of Regulatory Affairs
Exygen Research
?/{o*
Date
Page 4 of 127
Interim Report #17 - Analysis of Sediment and Water Samples Exygen Study No.: POOOl 131
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: EXYGEN STUDY NUMBER:
POOOl 131 POOOl 131
TYPE OF STUDY: SAMPLE MATRIX:
Residue
Sediment an1 u ater
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:
11/11/05
Interim Analytical Termination Date: 12/09/05
Interim Report Completion Date: 06/29/07
Exygen Research
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Interim Report #17 - Analysis of Sediment and Water Samples Exygen Study No.: PO001 13 1
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 Charles Simons Karen Risha Eric Edwards Christine Edwards Mark Ammerman Amy Sheehan Mindy Cressley Brittany Kravets Krista Gallant
Title
Vice President Director, Analytical Laboratory Operations
Scientist Sample Custodian
Technician Sample Custodian Associate Scientist
Technician Technician Technician
Exygen Research
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Interim Report #17 .Analysis of Sediment and Water Samples Exygen Study No .. PO001 13 1
TABLE OF CONTENTS
Paae
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 .................................................................................................... 11 1.0 SUMMARY................................................................................................................ 12 2.0 OBJECTIVE ............................................................................................................... 12
3.0 INTRODUCTION ...................................................................................................... 13
4.0 ANALYTICAL TEST SAMPLES........................................................................... 13
5.0 REFERENCE MATERIAL ........................................................................................ 14
6.0 DESCRIPTION OF ANALYTICAL METHOD ........................................................ 15 6.1 Extraction Procedure For Sediment ......................................................................... 15 6.2 Percent Solids Procedure For Sediment ................................................................... 16 6.3 Extraction Procedure for Water................................................................................ 16 6.4 Preparation of Standards and Fortification Solutions .............................................. 16 6.5 Chromatography.......................................................................................................... 17 6.6 Instrument Sensitivity............................................................................................... 17 6.7 Description of LC/MS/MS Instrument and Operating Conditions .......................... 17 6.8 Quantitation and Example Calculation .................................................................... 18
7.0 EXPERIMENTAL DESIGN ...................................................................................... 20 8.0 RESULTS ................................................................................................................... 20 9.0 CONCLUSIONS ........................................................................................................ 21 10.0 RETENTION OF DATA AND SAMPLES ............................................................. 21
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Interim Report #17 .Analysis of Sediment and Water Samples Exygen Study .No. PO001 131
Table I. Table I1.
Table I11.
Table IV. Table V .
LIST OF TABLES
Paae
Summary of PFBS. PFHS and PFOS in Sediment Samples .......................... 23
Summary of PFBS. PFHS and PFOS in Water Samples................................ 24
Matrix Spike Recovery of PFBS. PFHS and PFOS in Sediment Samples ....26
Matrix Spike Recovery of PFBS. PFHS and PFOS in Water Samples ..........28
Total Percent Solids in Sediment Samples..................................................... 34
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Interim Report # 17 .Analysis of Sediment and Water Samples Exygen Study .No. PO001131
LIST OF FIGURES
Figure 1. Typical Non-Extracted Calibration Curve for PFBS in Methanol .................36
Figure 2. Typical Extracted Calibration Curve for PFBS in Methanol ......................... 37
Figure 3. Figure 4. Figure 5 . Figure 6.
Non-Extracted Standards of PFBS in Methanol. 0.2 ng/mL
and 0.5 ng/mL. Respectively .......................................................................... 38
Extracted Standards of PFBS in Methanol, 25 ng/L
and 50 ng/L, Respectively .............................................................................. 39
PFBS in 1YOAcetic Acid Reagent Blank, 0.4 ng/mL Fortified Reagent Spk A, and 4.0 ng/mL Fortified Reagent Spk B, Respectively ........40
PFBS in Water Reagent Blank, 50 ng/L Fortified Reagent Spk A,
and 500 ng/L Fortified Reagent Spk B, Respectively .................................... 41
Figure 7. Chromatogram Representing a Sediment Sample Analyzed for PFBS (Exygen ID: C0091586, Data Set: 112105C)................................................. 42
Figure 8. Chromatogram Representing a Water Sample Analyzed for PFBS
(Exygen ID: C0091652, Data Set: 112105D)................................................. 43
Figure 9. Typical Non-Extracted Calibration Curve for PFHS in Methanol .................44 Figure 10. Typical Extracted Calibration Curve for PFHS in Methanol ......................... 45
Figure 11. Non-Extracted Standards of PFHS in Methanol, 0.2 ng/mL
and 0.5 ng/mL, Respectively .......................................................................... 46
Figure 2. Extracted Standards of PFHS in Methanol, 25 ng/L and 50 ng/L, Respectively ................................................................................................... 47
Figure 3. PFHS in 1% Acetic Acid Reagent Blank, 0.4 ng/mL Fortified Reagent Spk A, and 4.0 ng/mL Fortified Reagent Spk B, Respectively ........48
Figure 4. PFHS in Water Reagent Blank, 50 ng/L Fortified Reagent Spk A, and 500 ng/L Fortified Reagent Spk B, Respectively .................................... 49
Figure 15. Chromatogram Representing a Sediment Sample Analyzed for PFHS (Exygen ID: C0091587, Data Set: 112105C)................................................. 50
Figure 16. Chromatogram Representing a Water Sample Analyzed for PFHS (Exygen ID: C0091549, Data Set: 111405C)................................................. 51
Figure 17. Typical Non-Extracted Calibration Curve for PFOS in Methanol ................. 52
Figure 18. Typical Extracted Calibration Curve for PFOS in Methanol ......................... 53
Figure 19. Non-Extracted Standards of PFOS in Methanol, 0.2 ng/mL and 0.5 ng/mL, Respectively .......................................................................... 54
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LIST OF FIGURES (continued)
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Figure 20. Extracted Standards of PFOS in Methanol, 25 ng/L and 50 ng/L,
Respectively .................................................................................................. .55
Figure 21. PFOS in 1% Acetic Acid Reagent Blank, 0.4 ng/mL Fortified
Reagent Spk A, and 4.0 ng/mL Fortified Reagent Spk ByRespectively ........ 56
Figure 22. PFOS in Water Reagent Blank, 50 ng/L Fortified Reagent Spk A, and 500 ng/L Fortified Reagent Spk B, Respectively .................................... 57
Figure 23. Chromatogram Representing a Sediment Sample Analyzed for PFOS
(Exygen ID: C0091566, Data Set: 112105A)................................................. 58
Figure 24. Chromatogram Representing a Water Sample Analyzed for PFOS
(Exygen ID: C0091558, Data Set: 111405C)................................................. 59
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LIST OF APPENDICES
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Appendix A Study Protocol POOOl 131 (Exygen Study No. POOOl 131) with
Analytical Methods and Protocol Amendments 1 and 2 ........................... 60
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Interim Report #17 - Analysis of Sediment and Water Samples Exygen Study No.: POOOl 131
1.0 SUMMARY
Exygen Research extracted and analyzed sediment samples for the determination of perfluorobutanesulfonate (PFBS), perfluorohexanesulfonate (PFHS), and perfluorooctanesulfonate (PFOS) according to Exygen Method VOOO1782 and water samples for the determination of perfluorobutanesulfonate (PFBS), perfluorohexanesulfonate (PFHS), and perfluorooctanesulfonate (PFOS) according to Exygen Method VOOOl780 (Appendix A).
The limit of quantitation for PFBS, PFHS, and PFOS in sediment was 0.2 ng/g. The LOQ provided is assuming wet weight values, and the results for the actual samples are reported based on dry weight.
The limit of quantitation for PFBS, PFHS, and PFOS in water was 25 ng/L.
Analytical results and assessed accuracies for the analysis of PFBS, PFHS, and PFOS in sediment samples based on dry weight are summarized in Table I. Fortification recoveries for the analysis of PFBS, PFHS, and PFOS in sediment samples based on wet weight are summarized in Table 111. The overall average percent recoveries f standard deviations for PFBS, PFHS, and PFOS in sediment samples based on wet weight are 79 k 9%, 98 k 14%, and 84 f24%, respectively. Analytical results and assessed accuracies for the analysis of PFBS, PFHS, and PFOS in water samples are summarized in Table 11. Fortification recoveries for the analysis of PFBS, PFHS, and PFOS in water samples are summarized in Table IV. The overall average percent recoveries f standard deviations for PFBS, PFHS, and PFOS in water samples are 88 f 18%, 105 k 19%, and 87 f 22%, respectively.
Quantitative analytical results were obtained for all samples except for PFBS in three water samples, PFHS in one water sample, and PFOS in two sediment and two water samples that are designated as not reported (NR) due to quality control failures.
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 sediment and water according to Protocol POOOl 131 (Appendix A).
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Interim Report # 17 - Analysis of Sediment and Water Samples Exygen Study No.: POOOl 131
3.0 INTRODUCTION
This report details the results of the analysis for the determination of PFBS, PFHS, and PFOS in sediment using the analytical method entitled, "V0001782: Method of Analysis for the Determination of Perfluorooctanoic Acid (PFOA) in Sediment by LC/MS/MS" and for the determination of PFBS, PFHS, and PFOS in water using the analytical method entitled, "V0001780: Method of Analysis for the Determination of Perfluorooctanoic Acid (PFOA) in Water by LC/MS/MS."
The study was initiated on November 05, 2004, when the study director signed protocol number POOOl 131. The analytical start date for this interim report was November 11, 2005, and the analytical termination date for this interim report was December 9,2005.
4.0 ANALYTICAL TEST SAMPLES
Twelve sediment (SD) samples and one hundred and three water samples (Exygen ID COO91529 - C0091656) representing twelve sampling sites for sediment, surface water and interstitial (pore) water samples and associated trip blanks, trip blank spikes, and equipment rinseate blank samples were received on wet ice on September 2, 2005 from Tim Frinak at Weston Solutions, Inc. Please note that rinseate blank samples do not have associated field spike samples. Also note that due to the limited volume of sample available at some of the low-yielding pore water sampling locations, not all interstitial water samples include complete field spike series. The samples were logged in by Exygen personnel and placed in refrigerated storage.
Sample identification (ID) codes are of the form DNS-xx-STAOxx-x(x)-O5082x and are composed of the strings described below:
The first string defines the general sampling area where DNS = Decatur near shore.
The second string defines the matrix where SW = surface water, IW = interstitial (pore) water and SD = sediment.
The third string indicates the sampling station number (1-12).
The fourth string describes the sample aliquot where 0 = primary sample volume, DB = duplicate sample volume, LS = low spike, HS = high spike and RB = rinseate blank.
The final string is the sample collection date in YYMMDD format.
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.
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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 SP000240 1 SP0002694
Lot # 101 SE036 430 180-1
Purity (%) 96.7 98.6 101.2
Expiration Date 12/04/06 1O/ 18/06 10131107
The molecular structures of PFBS, PFHS and PFOS are given below:
PFBS
Chemical Name: Perfluorobutanesulfonate Molecular Weight: 338 supplied as the potassium salt (CdF9SO3-K')
Transitions Monitored: 299 + 99
Structure:
PFHS Chemical Name: Perfluorohexanesulfonate Molecular Weight: 438 supplied as the potassium salt (C6F13S03-K')
Transitions Monitored: 399 + 80
Structure:
F F F
\/-F
/so3
F FF F F F
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Interim Report # 17 - Analysis of Sediment and Water Samples Exygen Study No.: PO00 1131
PFOS Chemical Name: Perfluorooctanesulfonate Molecular Weight: 538 supplied as the potassium salt ( C ~ F I ~ S O ~ - K + )
Transitions Monitored: 499 -+80
Structure: FF F F
6.0 DESCRIPTION OF ANALYTICAL METHOD
The analytical methods "V0001782: Method of Analysis for the Determination of Perfluorooctanoic Acid (PFOA) in Sediment by LC/MS/MS" and "V0001780: Method of Analysis for the Determination of Perfluorooctanoic Acid (PFOA) in Water by LC/MS/MS" were used for this study.
6.1. Extraction Procedure For Sediment
Before the samples were weighed for the extraction, they were mixed thoroughly by vigorously shaking the container. A 5 gram portion of sediment was weighed into a fifty milliliter centrifuge tube for the extraction. After fortification of appropriate samples, 35 mL of 1% acetic acid in water was added to the samples. The samples were vortexed and allowed to shake on a wrist action shaker for -60 minutes. The samples were centrifuged for -20 minutes at -3000 rpm. The supernatant was then loaded onto a C18 SPE cartridge conditioned with 10 mL of methanol and 20 mL of water. The eluate was discarded. Twenty milliliters of methanol was added to the sediment samples left in the centrifuge tube. The samples were vortexed and allowed to shake on a wrist action shaker for another 30 minutes. The samples were centrifuged again for -20 minutes at -3000 rpm. The supernatant was then loaded onto the same c18 SPE cartridge. The eluate was collected into a 500 mL Nalgene Bottle. The column was washed with 4 mL of methanol. The wash was collected in the same bottle as the eluate. Approximately two hundred milliliters of water was added to the bottles. The samples were mixed by shaking and loaded onto another clg SPE cartridge conditioned with 10 mL of methanol and 20 mL of water. The eluate was discarded. Approximately five milliliters of methanol was added to the cartridge. Five milliliters of eluate was collected into a graduated 15 mL polypropylene centrifuge tube. Each sample was analyzed by LC/MS/MS electrospray.
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6.2 Percent Solids Procedure For Sediment
Percent solids were determined using the procedure indicated in Exygen method V0000427. Approximately 20 grams of sample was weighed into a pan. The weight of the sample plus the pan was recorded. The samples were then dried in an oven overnight at 104 f 2 "C. Then the samples were transferred to a dessicator and allowed to cool for -1 5 minutes. Each sample was then weighed again, including the weight of the pan. The percent solid for each sample was then calculated. See Table V for percent solids results.
6.3 Extraction Procedure for Water
A 40 mL aliquot of the water sample was used for the extraction procedure. After fortification of appropriate samples, the samples were loaded onto a c18 SPE cartridge conditioned with 10 mL of methanol and 5 mL of water. The eluate was discarded. Approximately five milliliters of methanol was added to the cartridge. Five milliliters of eluate was collected into a graduated 15 mL polypropylene centrifuge tube. Each sample was analyzed by LC/MS/MS electrospray.
6.4 Preparation of Standards and Fortification Solutions
A mixed stock standard solution of PFBS, PFHS, and PFOS was prepared at a concentration of 1000 pg/mL by dissolving 10 mg of each of the standards (corrected for purity and salt content) in methanol. From this solution, a mixed 100 pg/mL fortification standard solution was prepared by taking 10 mL of the appropriate stock solution and bringing the volume up to 100 mL with methanol. By taking 10 mL of the mixed 100 pg/mL fortification standard and bringing the volume up to 100 mL with methanol, a mixed 10 pg/mL fortification standard was prepared. By taking 10 mL of the mixed 10 pg/mL fortification standard and bringing the volume up to 100 mL with methanol, a mixed 1.O pg/mL fortification standard was prepared. By taking 10 mL of the mixed 1.O pg/mL fortification standard and bringing the volume up to 100 mL with methanol, a mixed 0.1 pg/mL fortification standard was prepared. By taking 10 mL of the mixed 0.1 pg/mL fortification standard and bringing the volume up to 100 mL with methanol, a mixed 0.01 pg/mL fortification standard was prepared.
A set of non-extracted standards containing PFBS, PFHS and PFOS was prepared in methanol as specified in Exygen method VOOOl780. The following concentrations were prepared:
Conc. of Fort
' Solution
(ng/mL) 100 100
Fort Volume
10 5 .O
Volume of Fortified Sample
(mL) 100 100
Final Conc. of Calibration Std.
(ng/mL) 10 5 .O
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100
2.0
100
2.0
10
10
100
1.o
5 .O
10
100
0.5
'
of
2.0 PFBS,
PFHS
10 and PFOS
100
0.2
The stock standard solution and all fortification and calibration standard solutions were stored in a refrigerator (4" k 2OC) when not in use. Documentation of standard preparation is located in the raw data package associated with this interim report.
6.5 Chromatography
Quantification of PFBS, PFHS and PFOS was accomplished by LC/MS/MS electrospray. The retention times of PFBS, PFHS and PFOS were -0.5 mins, -8.4 mins, and -1 1.O mins, respectively. Peaks above the LOQ were not detected in any of the reagent blank samples corresponding to the analyte retention time.
6.6 Instrument Sensitivity
The smallest standard amount injected during the chromatographic run had a concentration of 0.2 ng/mL of PFBS, PFHS and PFOS.
6.7 Description of LC/MS/MS Instrument and Operating Conditions
Instrument: Interface: Computer: Software: HPLC:
API 4000 Biomolecular Mass Analyzer
Turbo Ion Spray Liquid Introduction Interface
DELL OptiPlex GX400
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 Vol.: 15 pL Mobile Phase (A): 2 mM Ammonium Acetate in water Mobile Phase (B): Methanol
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Time (min)
%A
%B
0.0
65
35
1.o
65
35
8.0
25
75
10.0
25
75
11.0
65
35
18.0
65
35
Total run time: -1 8 min
Flow Rate: 0.3 mL/min
Ions monitored:
Analyte
PFBS PFHS PFOS
Mode
negative negative negative
Transition Monitored
299 -+99 399 + 80 499 + 80
Approximate Retention Time
(mln) -0.5 min.
-8.4 min. -1 1.O min.
6.8 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 l/x fit weighted linear regression) by Analyst software using six concentrations of standards. The concentration was determined from the following equations. Equation 1 calculated the amount of analyte found (in ng/mL, based on peak area) 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.
For samples fortified with known amounts of PFBS, PFHS and PFOS prior to extraction, Equation 2 was used to calculate the percent recovery.
Equation 2:
Recovery (YO=)
{analyte found (na/mL) - analvte in control (na/mL)) x 100%
amount added (ng/mL)
Note: For the PFBS, PFHS, and PFOS recovery calculations, the "control" is the unspiked aliquot of the primary field sample. Recovery is based on ng/mL for water and on ng/g (ppb) for sediment.
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Equation 3 was used to convert the amount of PFBS, PFHS and PFOS found in ng/mL to ng/g (PPb).
Equation 3:
Analyte found (ppb) = JAnalyte found (ndmL) x final volume ( 5 mLU sample weight ( 5 g)
Equation 4 was then used to calculate the amount of PFBS, PFHS and PFOS found in ppb based on dry weight.
Equation 4: Analyte found (ppb) dry weight = Analyte found (ppb) x [100% / total solids(%)]
An example of a calculation using an actual sample follows (calculation is for PFHS
only):
Sediment sample Exygen ID: COO91582 Spk E (Set: 112105B), fortified at 4 ng/mL
with where: peak area intercept slope dilution factor
- 547626
- 3300
- 119000
- -
1
ng/g PFHS added (fort level) = 4
amt in corresponding sample = 0.49 1
final volume <mL)
sample weight (g)
total solids (%)
From equation 1: Analyte found (ng/L)
= 1547626 - 33001 x 1 1 19000
From equation 2: % Recovery
= 4.57ng/mL
= (4.57 n d g - 0.491 ng/& x 100%
4 ng/g = 102%
From equation 3: Analyte found (ppb)
= (4.57 ng/mL x 5 mL)
5g = 4.57 ppb
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From equation 4: Analyte found (ppb) dry weight = 4.57 ppb x (100% / 56.01%) = 8.16ppb
NOTE: This value may be slightly different than that of the raw data due to rounding.
7.0 EXPERIMENTAL DESIGN
For sediment 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. For water samples designated as field matrix spikes, PFBS, PFHS, and PFOS were added at a known concentration to the sample bottles in the laboratory before being shipped to the field. The samples were filled to a 200 mL volumetric fill line in the field.
The sediment samples were extracted in four sets, one of which was a re-extraction. Each set included one reagent blank and two reagent blanks fortified at known concentrations. Two sets contained five sample sites and one set contained two sample sites. One sample was collected for each sample site. For each site, two laboratory matrix spikes and a laboratory duplicate were extracted.
The water samples were extracted in ten sets, two of which contained re-extractions. Each set included one reagent blank, two reagent blanks fortified at known concentrations. Set one contained pore water and surface water for one sample site. Set two contained pore water for two sample sites and surface water for one sample site. Set three contained pore water for one sample site and surface water for two sample sites. Sets four and five each contained pore water for two sample sites and surface water for two sample sites. Set six contained pore water for two sample sites and surface water for one sample site. Set seven contained pore water for one sample site and surface water for two sample sites. Set eight contained pore water for one sample site, surface water for one sample site, two rinseate blanks, and three trip blanks with their associated field spikes. Set nine was a re-extraction of pore water for one sample site and surface water for two sample sites. Set ten was a re-extraction of pore water for two sample sites and surface water for two sample sites. For each site and type of water, a sample, a field duplicate and two-matrix field spikes were collected. For each site and type of water, a laboratory duplicate was extracted and two laboratory matrix spikes were also extracted.
8.0 RESULTS
Analytical results and assessed accuracies for the analysis of PFBS, PFHS, and PFOS in sediment samples based on dry weight are summarized in Table I. Fortification recoveries for the analysis of PFBS, PFHS, and PFOS in sediment samples based on wet
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weight are summarized in Table 111. The overall average percent recoveries k standard deviations for PFBS, PFHS, and PFOS in sediment samples based on wet weight are 79 +_ 9%, 98 f 14%, and 84 f 24%, respectively. Analytical results and assessed accuracies for the analysis of PFBS, PFHS, and PFOS in water samples are summarized in Table 11. Fortification recoveries for the analysis of PFBS, PFHS, and PFOS in water samples are summarized in Table IV. The overall average percent recoveries k standard deviations for PFBS, PFHS, and PFOS in water samples are 88 f 18%, 105 f 19%, and 87 k 22%, respectively. Quantitative analytical results were obtained for all samples except for PFBS in three water samples, PFHS in one water sample, and PFOS in two sediment and two water samples that are designated as not reported (NR) due to quality control failures.
Accuracies were assessed for each sample by reviewing the individual QC results obtained for each sample site. For the sediment samples, there were two laboratory matrix spike recovery results available for each sample that were used to assess the accuracy. For the water samples, there were two laboratory and two field spike recovery results available for each sample site that were used to assess the accuracy. In instances of failed laboratory or field spike recoveries, the samples were not reported due to quality control failure, and recoveries associated with other spikes were used to assess sample accuracy.
9.0 CONCLUSIONS
Except as noted above, the sediment and water samples were successfully extracted and analyzed for PFBS, PFHS and PFOS according to analytical methods V0001782 and VOOOl780, respectively. There were no circumstances that would have affected the quality or integrity of the data.
10.0 RETENTION OF DATA AND SAMPLES
When the final analytical report is complete, all original paper data generated by Exygen Research will be shipped to the 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
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Interim Report #17 - Analysis of Sediment and Water Samples Exygen Study No.: PO001 131
TABLES
Exygen Research
Page 22 of 127
Interim Report #17 - Analysis of Sediment and Water Samples Exygen Study No.: PO001131 Table I. Summary of PFBS, PFHS and PFOS in Sediment Samples
Exygen ID
Client Sample ID
Perfluorobutanesulfonate
Analyte Found Assessed
(ppb. ng/g) Dry Weight
Accuracy
(+/- 56)
Perfluorohexanesulfonate
Analyte Found Assessed
(ppb. ng/g) Dry Weight
Accuracy (+/- %)
COO915 4 7
DNS-SD-STA001-0-050824
0.979
30
1.29
30
COO91547 Rep DNS-SD-STA001-0-050824'
0.787
30
1.42
30
COO91557
DNS-SD-STA002-0-050824
0.452
30
0.435
30
COO915 5 7 Rep DNS-SD-STA002-0-050824'
0.494
30
0.427
30
COO91566
DNS-SD-STA003-0-050824
0.51 9
30
ND
30
COO91566 Rep DNS-SD-STA003-0-050824"
0.61 7
30
ND
30
COO91 5 7 9
DNS-SD-STA004-0-050825
ND
30
ND
30
COO91579 Rep DNS-SD-STA004-0-050825'
ND
30
ND
30
COO91 5 8 0
DNS-SD-STA005-0-050825
ND
30
2.08
30
COO91580 Rep DNS-SD-STA005-0-050825"
ND
30
2.03
30
COO915 8 1
DNS-SD-STA006-0-050825
ND
30
ND
30
COO91581 Rep DNS-SD-STA006-0-050825'
ND
30
ND
30
COO91582
DNS-SD-STA007-0-050825
ND
30
0.877
30
COO915 8 2 Rep DNS-SD-STA007-0-050825*
0.357
30
1.25
30
COO91583
DNS-SD-STA008-0-050825
ND
COO915 8 3 Rep DNS-SD-STA008-0-050825'
ND
30
0.421
30
30
0.428
30
COO91 584
DNS-SD-STA009-0-050825
4.13
30
6.20
50
COO915 8 4 Rep DNS-SD-STA009-0-050825'
4.72
30
6.66
50
COO915 8 5
DNS-SD-STAOI0-0-050825
0.392
30
1.30
30
COO915 8 5 Rep DNS-SD-STAOI0-0-050825*
0.430
30
1.21
30
COO91586
DNS-SD-STAOI 1-0-050825
0.582
30
ND
30
COO91586 Rep DNS-SD-STAOI 1-0-050825'
ND
30
0.393
30
COO915 8 7
DNS-SD-STAOI2-0-050825
0.822
30
0.553
30
COO915 8 7 Rep DNS-SD-STAOI2-0-050825'
0.743
30
0.905
30
C8 Sulfonate PFOS
Perfluorooctanesutfonate
Analyte Found Assessed
(ppb. n d g ) Dry Weight
Accuracy
(+/- 36)
21.1
30
21.3
30
NR NR
10.1
30
11.7
30
1.22
30
1.41
30
88.0
40
88.0
40
0.810
30
0.810
30
26.1
30
25.7
30
16.4
40
16.1
40
24.7
30
26.5
30
NR NR
4.67
40
4.53
40
15.7
30
15.6
30
'Laboratory Duplicate
ND = Not detected at or above the Limit of Quantitation (LOQ) of 0.2 ng/g (wet weight) NR = Not reported due to quality control failure.
Exygen Research
Page 23 of 127
Interim Report #17 - Analysis of Sedimentand Water Samples Exygen StudyNo.: PO001131
Table 11. Summary of PFBS, PFHS and PFOS in Water Samples
Client
C4 Sulfonate PFBS
Perlluorobutanesulfonate Assessed
Analyte Found Accuracy
COO91 529
DNS-SW-TRIPOl-0-050824
ND
30
COO91533
DNS-SW-TRIP02-0-050824
ND
30
COO91536
DNS-SW-TRIP03-0-050824
ND
30
COO91539
DNS-IW-STA001-0-050823
682
30
COO91539 Rep DNS-IW-STA0010-050823'
690
30
COO91540
D N S - I W - S T A 0 0 1- D E 4 5 0 8 2 3
666
30
coo91543
DNS-SW-STA001-0-050823
111
30
COO91543 Rep DNS-SW-STA001-0-050823'
110
30
COO91544
DNS-SW-STA001-DB-050823
101
30
COO91548
DNS-SD-STA001-RB-050824
ND
30
coo91549
DNS-IW-STA002-0050824
364
30
COO91549 Rep DNS-IW-STA002-0-050824'
368
30
COO91550
DNS-IW-STA002-DB-050824
353
30
COO91553
DNS-SW-STA002-0-050825
140
30
COO91553 Rep DNS-SW-STA002-0-050825'
130
30
coo91554
DNS-SW-STA002-DB-050825
134
30
coo91558
DNS-I W-STAOO3-0-050824
76.4
30
COO91558 Rep DNS-IW-STA003-0-050824'
79.2
30
COO91559
DNS-IW-STA003-DB-050824
77.0
30
COO91562
DNS-SW-STA003-0-050824
58.7
30
COO91562 Rep DNS-SW-STA003-0-050824'
59.5
30
COO91563
DNS-SW-STA003-DB-050824
58.1
30
COO91588
DNS-IW-STA004-0-050824
75.8
30
COO91588 Rep DNS-IW-STA004-0-050824"
77.1
30
COO91589
DNS-IW-STA004-DB-050824
61.9
30
COO91592
DNS-SW-STA004-0-050824
134
30
COO91592 Rep DNS-SW-STAOO4-0-050824'
133
30
COO91593
DNS-SW-STA004-DB-050824
131
30
COO91596
DNS-IW-STA005-0-050823
86.9
30
COO91596 Rep DNS-IW-STA005-0-050823'
90.1
30
COO91597
DNS-IW-STA005-DE-050823
86.2
30
COO91600
DNS-SW-STA005-0-050823
64.5
30
COO91600 Rep DNS-SW-STA005-0-050823'
61.4
30
COO91601
DNS-SW-STA005-DB-050823
66.9
30
COO91604
DNS-IW-STA006-0-050824
58.2
30
COO91604 Rep DNS-IW-STA006-0-050824'
56.1
30
COO91605
DNS-IW-STA006-DB-050824
59.6
30
C6 Sulfonate PFHS
Perlluorohexanesulfonate
Analyte Found
Assessed Accuracy
(ppt. ng/L)
(+/- %)
ND
30
ND
30
ND
30
790
50
832
50
793
50
24.0
30
22.4
30
18.8
30
ND
30
620
40
633
40
632
40
59.2
30
48.5
30
50.6
30
51.1
30
44.4
30
64.5
30
42.7
30
43.1
30
47.3
30
45.2
30
45.5
30
30.4
30
178
30
168
30
180
30
21.1
30
22.8
30
21.3
30
16.9
30
15.8
30
15.8
30
57.4
30
57.4
30
61 .O
30
C8 Sulfonate PFOS
Perlluorooctanesulfonate
Analyte Found
Assessed Accuracy
(ppt. nglL)
(+/- Yo)
ND
30
ND
30
ND
30
20000
50
19100
50
18500
50
164
30
148
30
128
30
ND
30
2860
40
31 70
40
3140
40
403
30
346
30
358
30
645
30
593
30
633
30
180
30
165
30
200
30
184
50
151
50
131
50
555
30
478
30
555
30
167
30
158
30
167
30
124
30
99.8
30
136
30
263
30
249
30
320
30
'Labwatory Duplicate ND = Not detected at or above the Limit of Quantitation (LOQ) of 25 na/L
Exygen Research
Page 24 of 127
Interim Report #17 - Analysis of Sedimentand Water Samples Exygen Study No.: PO001131
Table 11. Summary of PFBS, PFHS and PFOS in Water Samples Continued
COO91635
DNS-IW-STA010-0-050826
COO91635 Rep DNS-IW-STAO10-0-050826'
NR
COO91636
DNS-IW-STA010-DB-050826
NR
COO91638
DNS-SW-STA010-0-050826
73.2
40
COO91638 Rep DNS-SW-STAO10-0-050826*
73.5
40
COO91639
DNS-SW-STAOl0-DB-050826
79.2
40
COO91642
DNS-IW-STAO11-0-050826
1880
30
COO91642 Rep DNS-IW-STAO11-0-050826'
1830
30
COO91643
DNS-IW-STAOl 1-DB-050826"
1630
30
COO91644
DNS-SW-STAO11-0-050826
NR
COO91644 Rep DNS-SW-STAO11-0-050826'
NR
COO91 645
DNS-SW-STAOl1-DB-050826
NR
COO91648
DNS-I W-STA012-0-050826
8730
30
COO91648 Rep DNS-IW-STA012-0-050826'
9140
30
COO91649
DNS-IW-STAO12-DB-050826
8850
30
COO91652
DNS-SW-STA012-0-050826
53.3
30
COO91652 Rep DNS-SW-STAO12-0-050826'
56.5
30
COO91653
DNSSW-STAOl2-DB-050826
57.3
30
COO91 6 5 6
DNS-IW-STAO12-RB-050825
ND
30
818 837
53.6 52.1 57.5
844 807 750
39.4 47.0 42.4
NR NR NR
42.2 40.0 41.4
ND
40
17700
30
40
21500
30
30
139
40
30
122
40
30
177
40
30
850
30
30
756
30
30
1320
30
30
96.4
50
30
110
50
30
131
50
1330
40
1220
40
1230
40
30
142
30
30
144
30
30
187
30
30
ND
30
Exygen Research
Page 25 of 127
Interim Report #17 - Analysis of Sediment and Water Samples Exygen Study No.: PO001 131
Table 111. Matrix Spike Recovery of PFBS, PFHS and PFOS in Sediment Samples
Sample Description
DNS-SD-STA001 -0050824 (coo01547Spk C. 4 ppbSpiko) DNS-SD-STA001-0050824 (CWglMSpk 0.40ppbSpike)
-moun
w spiked
C4 Sulfonate PFBS Wet Weight
Amt Found in Sample ng/g) wet w t
Amount Recovered (ng/g) wet wt.
re cove^
(%)
C6 Sulfonate PFHS Wet Weight
Amt Found in Sample ng/g) wet wt.
Amount Recovered (ng/g) wet Wt.
Recover) (%)
C8 Sulfonate PFOS Wet Weight
Amt Found in Sample ng/g) wet w t
Amount Recovered (ng/g) wet Wt.
Recovery (%)
4
0 565
3 49
73
0 743
4 95
105
12 2
34 8
40
0 565
22 7
55
0 743
36 3
89
12 2
48 8
92
DNS-SO-STA002-0050824
(CcQ91557Spk E, 4 ppbSpike)
4
DNS-SD-STA002-0050824 (coo91557Spk F.40 ppbSpike) 40
0 236 0 236
3 05
70
0 227
27 6
68
0 227
4 03
95
NR
39 9
99
NR
NR
NR
NR
NR
DNS-SD-STA003-0050824
(coo91586Spk G. 4 ppbSpike)
4
DNS-SD-STA003-0050824 (Cwg1SBBSpk H. 40 ppbSpike) 40
0 238 0 238
3 15
73
ND
31 1
77
ND
4 16
104
4 61
38 9
97
4 61
8 31
93
28 8
60
DNS-SD-STA004-0050825
(coo81579Spk I.4 ppbSpike)
4
ND
DNS-SD-STA004-0050825
(cw91570Spk J. 40 ppbSpike) 40
ND
2 84
71
ND
28 7
72
ND
404
101
0 855
38 1
95
0 855
4 74
97
34 5
84
DNS-SD-STA005-0050825
(coo91580Spk K. 4 ppbSpike)
4
ND
DNS.SD-STA005-0050825
(cooQ1580Spk L 40 ppb Spike)
40
ND
3 33
83
123
30 5
76
123
5 49
107
52 0
37 8
91
52 0
51 1
77 5
64
DNS-SD-STA006-0050825
(coo91581Spk C. 4 ppbSpike)
4
ND
DNS-SD-STA0060050825
(cwB1581Spk D. 40 ppbSpike) 40
ND
3 63
91
ND
33 6
84
ND
4 32
108
0 584
39 4
99
0 584
5 38
120
38 7
90
DNS-SD-STA007-0050825
(cw91582Spk E. 4 ppbSpike)
4
ND
DNS.SD-STA007-0050825
(COW1582Spk F. 40 ppbSpike) 40
ND
3 47
87
0 491
4 59
102
14 6
29 8
75
0 491
38 8
96
14 6
16 1
47 4
82
DNS.SD-STA008-0050825
(CwDlM3SpkG. 4ppbSpiko)
4
ND
DNS.SD-STA008-0050825
(cw81583Spk H. 40 ppbSpike) 40
ND
3 40
85
0 251
4 43
104
9 77
30 9
77
0 251
38 4
95
9 77
11 0
31
37 4
69
DNS.SD-STA009-0050825
(coo81584Spk I.4 ppbSpike)
4
2 54
DNS-SD-STA009-0050825
(coo91584Spk J. 40 ppbSpike)
40
2 54
8 30
94
3 81
37 9
88
3 81
964
146
15 2
43 2
98
15 2
34 5
55 3
100
DNS-SD-STAO10-0050825
(COOQ1585 Spk K. 4 ppb Spike)
4
0 227
3 81
90
0 755
4 10
84
NR
- DNS-SD-STAOlO-0050825
(coo91585Spk L. 40 ppbSpike)
40
0 227
36 5
91
0 755
31 3
76
NR
*sample residue exceeds the spikinglevelSiQnificantly(3x spiking level):therefore, an accuraterewveiy value cannot be calculated ND = Not detected at or above the Limit of Ouanlitalion (LOO) of 0.2 nglg (wet webht) NR = Not reported due lo quainy control failure. Note: since this summarytable shows rounded results, recoveryvalues mayvaryslightlyfrom the values in the raw data.
NR
NR
NR
NR
Exygen Research
Page 26 of 127
Interim Report # I 7 - Analysis of Sediment and Water Samples Exygen Study No.: PO001 131
Table 111. Matrix Spike Recovery of PFBS, PFHS and PFOS in Sediment Samples Continued
Sample Description
Amoun
Spiked
&g
DNS-SD-STAO11-0050825
(cwO1586Spk C, 4 ppb Spike)
4
DNS-SD-STAO11-0050825
(cW1586 Spk D. 40 ppb Splko)
40
C4 Sulfonate PFBS Wet Weight
Amt Found
Amount
in Sample Recovered Recove?
(ng/g)
(ng/g)
(%)
0 329
3 52
80
0 329
33 8
84
DNS-SD-STAO12-0050825
(coo81587Spk E. 4 ppb Splke)
4
0 385
3 70
83
- DNS-SD-STAOl2-0050825
(COW1587Spk F. 40 ppbSplke)
40
0.385
27.5
68
Average: 79
Standard Deviation: 9
Average: 98 Standard Deviation: 14
`Sample residue exceeds the spiking levelsignificantly (3x spiking level);therefore, an accurate recoveryvalue cannot be calculated ND = Not detected at or above the Liml of Quantltation(LOQ) of 0 2 nglg (wet weight). Note: 5ince this summary table shows rounded resuns. recovery values mayvary slightly from the values in the raw data.
Average: 84 Standard Deviation: 24
Exygen Research
Page 27 of 127
Interim Report #17 - Analysis of Sediment and Water Samples Exygen Study No.: PO001 131
Table IV. Matrix Spike Recovery of PFBS, PFHS and PFOS in Water Samples
Sample Description
= houri Spikec
C4 Sulfonate PFBS
imt Found Amount
in Sample Recovered Recoveq
(nglL)
(nglL)
(x)
C6 Sulfonate PFHS
Unt Found Amount
in Sample Recovered Recover
(nglL)
(nglL)
(K)
C8 Sulfonate PFOS
bnt Found Amount
in Sample Recovered Recovery
(nglL)
(nglL)
(X)
DNS-SW-TRIPO1-LS-050824 (C0091530.1.0 ppb FieldSpike)
DNS-SW-TRIP01-HS-050824 (C0091531.10ppb FieldSpike)
1000
ND
10000
ND
913
91
8980
90
ND
970
97
ND
11100
111
DNS-SW-TRIP02-LS-050824 (CW91534.1.0 ppb Field Spike)
1000
ND
960
96
ND
1160
116
DNS-SW-TRIP02-HS-050824
ICW91535. 10ppb FieldSpike)
10000
ND
10800
108
ND
11100
111
DNS-SW-TR IPO3-LS-050824 ICOO91537.1.0 ppb FieldSpike)
DNS-SW-TRIP03-HS-050824 (COO91538.10 ppb FieldSpike)
1000
ND
10000
ND
843
84
10900
109
ND
1090
109
ND
10800
108
DNS-IW-STAOOl-0-050823
(COO91539Spk C. 1.0 ppbLabSpike)
1000
682
1710
103
790
2290
150
DNS-IW-STAOOI-0-050823 (COO91539Spk D. 10ppbLabSpike)
10000
682
10200
95
790
13000
122
DNS-IW-STA001-LS050823 (COO91541.1.0 ppb FieldSpike)
1000
682
1410
73
790
2170
138
DNS-IW-STA001-HS-050823 (CW91542, 10ppb FieldSpike)
10000
682
8520
78
790
12400
116
DNS-SW-STAOOl-0-050823
(COO91543Spk E, 10 ppbLabSpike)
10000
111
DNS-SW-STAOO1-0-050823
(C0091543SpkF. IOOppbLabSpike)
looooc
111
DNS-SW-STA001-LS-050823 (COO91545.1.0 ppb Fleld Spike)
1000
111
DNS-SW-STAOO1-HS-050823 (COO9154&10ppb FleldSpike)
10000
111
8550
84
80600
80
815
70
7970
79
24 0
9470
94
24 0
90800
91
24 0
924
90
24 0
8610
86
DNS-IW-STAO02-0-050824 (COO91549Spk C. 1.0 ppb LabSpike)
DNS-IW-STA002-0-050824 (COO91549Spk D. 10 ppbLabSpike)
DNS-IW-STA002-LS-050824 (C0091551.1.0 ppb FieldSpike)
DNS-IW-STA002-HS-050824 (CW91552.10 ppb Field Spike)
1000
364
10000
364
1000
364
10000
364
=
1120
76
8690
83
1120
76
7740
74
620
1320
70
620
9950
93
620
1240
62
620
8940
83
'Sample residue exceeds the spiking level signiticantly (3x spiking level); therefore, an accurate recovew value cannot be calculated ND = Not detected at or above the Limit of Quantitation(LOQ)of 25 ngJL. Note: Since this surnrnarytable shows rounded results. recovery values may vary slightlyfrom the values in the raw data.
ND ND
ND ND ND ND 20000 20000 20000 20000 164 164 164 164 2860 2860 2860 2860
1200
120
11700
117
1180
118
10500
105
977
98
10600
106
18700
25900
59
18400
25100
51
8870
87
85200
85
859
70
7230
71
4230
137
9740
69
3970
111
8850
60
Exygen Research
Page 28 of 127
Interim Report # 17 - Analysis of Sediment and Water Samples Exygen Study No.: PO001131
Table IV. Matrix Spike Recovery of PFBS, PFHS and PFOS in Water Samples Continued
Sample Description
_3
imounl
Spiked
(ng/L)
C4 Sulfonate PFBS
imt Found Amount
in Sample Recovered Recover!
(nglL)
(nglL)
(K)
4mt Found Amount
Amt Found Amount
In Sample Recovered Recovery In Sample Recovered Recovery
DNS-SW-STA002-0-050825
(COO91553Spk E. 1.0ppb Lab Spike)
1000
140
DN S-SW-STA002-0-050825 (COO91553Spk F. 10 ppb b b Spike)
10000
140
DNS-SW-STA002-LS-050825 (CW91555.1.0 ppb Field Spike)
1000
140
DNS-SW-STA002-HS-050825
(C0091556.10 ppb Fieid Spike)
10000
140
889
75
8820
87
989
85
7330
72
59 2
976
92
59 2
9850
98
59 2
988
93
59 2
7610
76
403
1310
91
403
9530
91
403
1670
127
403
8040
76
DNS-IW-STA003-0-050824 (COO91558Spk G.1.0ppb Lab Spike)
DNS-IW-STA003-0-050824 (COO91558Spk H.10 ppb Lab Splke)
DNS-IW-STA003-LS-050824 (COO91560.1.0ppb FieldSpike)
DN S-IW-STA003-HS-050824 (COO91561,10 ppb Fieldspike)
1000
76 4
10000
76 4
1000
76 4
10000
76 4
913
84
8130
81
849
77
8830
88
51 1
814
76
645
51 1
8820
88
645
51 1
1090
104
645
51 1
9450
94
645
1510
87
9080
84
1880
124
8350
77
DNS-SW-STA003-0-050824
(COO91562Spk C. 1.0 ppb Lab Spike)
1000
58 7
949
89
D NS-SW-STA003-0-050824
(COO91562Spk 0. 10 ppb Lab Spike)
10000
58 7
9040
90
DNS-SW-STA003-LS-050824
(COO91564.1.0 ppb Fieidspike)
1000
58 7
860
80
DNS-SW-STA003-HS-050824
(C0091565.10ppb Fieid Spike)
10000
58 7
8850
88
42 7
1140
110
42 7
11400
114
42 7
1280
124
42 7
12100
121
180
1050
87
180
8950
88
180
1380
120
180
10900
107
DN S-IW-STA004-0-050824
(COO91588Spk E. 10 ppb Lab Spike)
10000
75 8
8140
81
DNS-IW-STA004-0-050824
(COO91588Spk F. 100 ppb LsbSpike)
100000
75 8
60900
61
DNS-IW-STA004-LS-050824
(C00915W.1.0 ppb Fisid Spike)
1000
75 8
783
71
DNS-IW-STA004-HS-050824 (COO91591.10 ppb Field Spike)
10000
75 8
7010
69
45 2
9630
96
45 2
77100
77
45 2
810
76
45 2
7920
79
184
9030
88
184
73800
74
184
764
58
184
5710
55
D NS-SW-STA004-0-050824
(COO91592Spk G. 1.0 ppb Lab Spike)
1000
134
1030
90
178
1350
117
555
1540
99
DNS-SW-STA004-0-050824 (COO91582Spk H. 10 ppb Lab Spike)
10000
134
8770
86
178
11600
114
555
9240
87
D NS-SW-STA004-LS-050824 (Cw91594.1.0ppb Field Spike)
1000
134
1010
88
178
1470
129
555
1630
108
DNS-SW-STA004-HS-050824 (CW91595.10 ppb Fieid Spike)
-10000 134
8480
83
178
12500
123
555
8790
82
'Sample residue exceeds the spikinglevel significantly (3x spiking level): therefore,an accuraterecovery value cannot be calculated ND = Not detectedat or above the Limit of Quantitation (LOQ) of 25 nglL. Note: Since this summary table shows rounded results, recovery values may vary slightly from the values in the raw data.
Exygen Research
Page 29 of 127
Interim Report # I 7 - Analysis of Sediment and Water Samples Exygen Study No.: PO001 131
Table IV. Matrix Spike Recovery of PFBS, PFHS and PFOS in Water Samples Continued
Sample Description
=
C4 Sulfonate PFBS
imouni bnt Found Amount
Spiked In Sample Recovered Recove,!
(ng/L) (nglL)
(ng/L)
(46)
Imt Found Amount
Amt Found Amount
in Sample Recovered Recovery in Sample Recovered Recovery
DNS-IW-STA005-0-050823 (CW91596 Spk C. 1.0 ppb Lab Spike)
1000
86 9
945
66
21 1
1120
110
167
995
83
DNS-IW-STA005-0-050823
(C00915W Spk D. 10 ppbLabSpike)
10000
86 9
9450
94
21 1
11900
119
167
9020
89
DNS-IW-STA005-LS-050823
(C0091596.1.0ppb FieldSpike)
1000
86 9
987
90
21 1
1270
125
167
1010
84
DNS-IW-STA005-HS-050823
(CW91599. 10 ppbFieldSpike)
10000
66 9
6470
84
21 1
9370
93
167
9980
98
D NS-SW-STA005-0-050823
(C0091wO Spk E, 1.0 ppb Lab Spike)
1000
64 5
845
78
D NS-SW-STA005-0-050823
(C0091600 Spk F. 10 ppb Lab Spike)
10000
64 5
9920
99
DNS-SW-STA005-LS-050823
(CW91602.1.0ppb FieldSpike)
1000
64 5
909
84
DNS-SW-STAOO5-HS-050823
(CQQ916031.0 ppb FieidSpika)
10000
64 5
9870
98
16 9
990
97
16 9
11900
119
16 9
1200
118
16 9
13100
131
124
777
65
124
8600
87
124
863
74
124
10600
105
DNS-IW-STA006-0-050824
(C0091604Spk G. 1.0 ppb LabSpike)
1000
58 2
940
88
57 4
1150
109
263
1130
87
DNS-IW-STA006-0-050824 (CW91604 Spk H. 10 ppbLabSpike)
10000
58 2
9070
90
57 4
11000
109
263
8510
82
DNS-IW-STA006-LS-050824 (COO91606. 1.0 ppb FieldSpike)
1000
58 2
1020
96
57 4
1360
130
263
1320
106
DNS-IW-STA006-HS-050824
(CO091607. 10 ppb FieldSplke)
10000
58 2
10200
101
57 4
12800
127
263
10300
100
DNS-SW-STA006-0-050824 (C0091606 Spk I, 10 ppb Lab Spike)
10000
NR
DNS-SW-STA006-0-050824
(C0091606 Spk J, 1W ppb Lab Spike)
100000
NR
DNS-SW-STA006-LS-050824
(C0091610. 1.0 ppbFieldSpike)
1000
NR
DNS-SW-STA006-HS-050824
(CW91611. 10 ppbFieldSpike)
10000
NR
NR
NR
39 6
10700
107
NR
NR
NR
39 6
87000
87
NR
NR
NR
39 6
696
66
NR
NR
NR
39 6
9660
96
NR
NR
NR
NR
NR
NR
NR
NR
NR
DNS-IW-STA007-0-050825 (C0091612Spk C. 1.0 ppb LabSpike)
DNS-IW-STA007-0-050825 (COO91612 Spk D. 10 ppb LabSpike)
DNS-IW-STA007-LS-050825 (CW91614.1.0ppb FieldSpike)
1000
578
1M)oo 578
1000
578
=
1540
96
10100
95
1410
83
703
1720
102
16200
18100
703
11800
111
16200
26200
100
703
1670
97 I 16200 17400
'Sample residue exceedsthe spiking level significantly (3x spiking level): therefore,an accurate recoveryvalue cannot be calculated ND = Not detected at or above the Limit of Quantitation(LOQ) of 25 ng/L NR = Not reported due to quality control failure. Note: Since this summary table shows rounded results, recovery values may vary slightlyfrom the values in the raw data.
Exygen Research
Page 30 of 127
Interim Report # 17 - Analysis of Sediment and Water Samples Exygen Study No.: PO00 1131
Table IV. Matrix Spike Recovery of PFBS, PFHS and PFOS in Water Samples Continued
Sample Description
=
C4 Sulfonate PFBS
m o u n 4mt Found Amount
Splked In Sample Recovered Recover
(ng/L) (nglL)
(nglL)
(X)
C6 Sulfonate PFHS
unt Found Amount
In Sample Recovered RecoveQ
(nglL)
(ng/L)
(K)
C8 Sulfonate PFOS
imt Found Amount
in Sample Recovered Recovery
(ng/L)
(ng/L)
(X)
DNS-SW-STA007-0-050825
(COO91615Spk E. 1.0 ppb Lab Spike)
1000
132
979
85
56 9
1100
104
388
986
60
DNS-SW-STA007-0-050825 (COO91615Spk F. 10 ppb LabSpike)
10000
132
9300
92
56 9
9920
99
388
9060
87
DNS-SW-STA007-LS-050825
(CW91617.1.0 ppb Field Spike)
1000
132
995
86
56 9
1280
122
388
1290
90
DNS-SW-STA007-HS-050825
(CW91618.10 ppbField Spike)
10000
132
8960
88
56 9
11000
109
388
7570
72
DNS-IW-STA008-0-050825 (Cw91619Spk 0.1.0 ppb Lab Spike)
1000
6020
6810
5190
6930
90 1
1610
91
DNS-IW-STA008-0-050825
(COO91619Spk H. 10 ppb LabSpike)
10000
6020
15400
94
5190
17100
119
90 1
8370
75
DNS-IW-STA006-LS-050825 (Co091621.1.0 ppb Fieldspika)
1000
6020
6180
5190
7310
90 1
1900
100
DN S-IW-STA008-HS-050825
(C0091622.10ppbField Spike)
10000
6020
14500
85
5190
16300
111
901
6950
60
DNS-SW-STAO08-0-050825 (COO91623Spk I. 1.0 ppb Lab Spike)
DNS-SW-STA006-0-050625 IC0091623 Spk J. 10 ppb Lab Spike)
DNS-SW-STA008-LS-050825 (C0091625. 1.0ppb Field Spike)
DNS-SW-STA006-HS-050625 (C0091626.10ppb Field Spike)
1000
217
10000
217
1000
217
10000
217
1160
94
9440
92
934
72
8200
80
158
1320
116
NR
158
11900
117
NR
158
1090
93
NR
156
10600
104
NR
NR
NR
NR
NR
NR
NR
NR
NR
DNS-IW-STA009-0-050826 (COO91627Spk C. 1.0 ppb Lab Spike)
DNS-IW-STA009-0-050826 (COO91627Spk 0. 10 ppb LabSpike)
DNS-IW-STA009-LS-050826 (C0091629.1.0 ppb Fieldspike)
DNS-IW-STA009-HS-050826 (C0091630.10 ppb FleldSplke)
1000 10000 1000 10000
8560 8580 8580 6580
11100
22400
138
11700
19500
109
11600
12800
11800
27600
158
11800
13500
11800
24700
129
3260 3260 3260 3260
4920
12400
91
5500
14500
112
DNS-SW-STA009-0-050826
(COO91631Spk E,1.0 ppb LabSpike)
1000
53 4
909
86
40 7
1130
109
134
932
80
DNS-SW-STA009-0-050826
(COO91631Spk F. 10 ppb Lab Spike)
10000
53 4
18700
186
40 7
11500
115
134
8850
87
DNS-SW-STA009-LS-050626
(C00915633,1.0 ppb Field Spike)
1000
53 4
958
90
40 7
1290
125
134
1080
95
DNS-SW-STAOO9-HS-050826
(CW91634.10ppb Fleid Spike)
10000
53 4
9100
90
-
40 7
12600
126
134
8500
84
'Sample residue exceeds the spiking level significantly (3x spiking level):therefore, an accurate recovetyvalue cannot be calculated ND = Not detected at or above the Limit of Quantitation(LOQ) of 25 ng/L NR = Not reported due to quality control failure. Note: Since this summary table shows rounded results, recovery values may vary slightlyfrom the values In the raw data.
Exygen Research
Page 31 of 127
Interim Report # 17 - Analysis of Sediment and Water Samples Exygen Study No.: PO00 1131
Table IV. Matrix Spike Recovery of PFBS, PFHS and PFOS in Water Samples Continued
Sample Descrlption
DNS-IW-STAOI0-0-050826 (COO9163SSpk 0.1.0 ppb LabSpike)
DNS-IW-STAO10-0-050826 (C0091635Spk H. 10 ppb Labspike)
DNS-IW-STAOI0-LS-050826" (COO91637,1.0 ppb FieldSpoke)
=
C4 Sulfonate PFBS
imount \mt Found Amount
Splked In Sample Recovered Recoveq
- (ng/L)
(ng/L)
(%)
tint Found Amount
Amt Found Amount
In Sample Recovered Recovery in Sample Recovered Recovery
(nglL)
(ng/L)
(%)
(nglL)
(ng/L)
(%)
1000
NR
10000
NR
NR
NR
846
2130
128
19200
19200
NR
NR
846
12500
117
19200
28800
96
10528
NR
NR
NR
846
7900
67
19200
27500
DNS-SW-STAO10-0-050826 (COO91638Spk C. 10ppbLabSpike)
DNS-SW-STA010-0-050826 (COO91638Spk 0.100 ppbLabSpike)
DNS-SW-STAO10-LS-050826 (C0091640. 1.0 ppb Field spike)
DNS-SW-STA010-HS-050826 (C0091641.10ppb Fieldspike)
10000 100000
1000 10000
73.2 73.2 73.2 73.2
16800
167
63600
64
758
68
6990
69
53.6
12800
127
53 6
86500
86
53.6
1080
103
53 6
10200
101
139
18400
183
139
66500
66
139
832
69
139
6580
64
DNS-IW-STAO11-0-050826
(COO91642Spk E, 1.0 ppbLabSpike)
1000
1880
2840
96
DNS-IW-STAOI1-0-050826
(COO91642Spk F. 10ppbLabSpike)
10000
1880
10700
86
844
2020
118
650
1730
88
844
11900
111
850
7860
70
DNS-SW-STAO11-0-050826
(COO91644Spk G. 10ppb LabSpike)
10000
NR
DNS-SW-STAOI1-0-050826
(COO91644Spk H,100 ppb Labspike)
100000
NR
DNS-SW-STAO11-LS-050826 (COO91645.1.0ppb FieldSpike)
1000
NR
DNS-SW-STAOI1-HS-050826 (COO91647.10 ppb FieldSpike)
10000
NR
NR
NR
39.4
9310
93
96.4
7110
70
NR
NR
39.4
97500
97
96.4
78500
78
NR
NR
39.4
746
71
96.4
607
51
NR
NR
39.4
6640
66
96.4
4700
46
DNS-IW-STAO12-0-050826
(COO91648Spk C. 1.0 ppb LabSpike)
1000
8730
9060
NR
DNS-IW-STAOI2-0-050826
(COO91648Spk D. 10ppbLabSpike)
10000
8730
19300
106
NR
DNS-IW-STAO12-LS-050826
(COO916M. 1.0 ppb Field Spike)
1000
8730
8840
NR
DNS-IW-STAOI2-HS-050826
(COO91651.10ppb FieldSpike)
10000
8730
16100
74
NR
NR
NR
NR
NR
NR
NR
NR
NR
7
'Sample residue exceedsthe spiking level significantly (3x spiking level); therefore.an accurate recovew value cannot be calculated Asamplebottle was received mth only 19 mL of water ND = Not detected at or above the Limit of 9uantitation (Log)of 25 nglL NR = Not reported due to quality control failure. Note: Since this summary table shows rounded results. recovery values may vary slightlyfrom the values In the raw data.
1330
2090
76
1330
10800
95
1330
2030
70
1330
7650
63
Exygen Research
Page 32 of 127
Interim Report #17 - Analysis of Sediment and Water Samples Exygen Study No.: PO001 131
Table IV. Matrix Spike Recovery of PFBS, PFHS and PFOS in Water Samples Continued
Sample Description
DNS-SW-STA012-0-050826 (COO01652 Spk E, 1.0 ppbLabspike)
DNS-SW-STA012-0-050826 (COO91652Spk F. 10ppbLabSpike)
DNS-SW-STA012-LS-050626 (C0091654. 1.0 ppb FieldSplke)
DNS-SW-STA012-HS-050626 (CwB1655.10 ppb FieldSpike)
- imounl
- Spiked
&gLJ
C4 Sulfonate PFES
imt Found Amount
in Sample Recovered Recovey
(nglL)
(ng/L)
(K)
4mt Found Amount
Amt Found Amount
in Sample Recovered Recovery In Sample Recovered Recovery
(ng/L)
(nglL)
(46)
(nglL)
(ng/L)
(X)
1000
53.3
666
a3
42 2
1160
112
142
1000
66
10000
533
9030
90
422
10500
105
142
7660
77
1000
53.3
670
62
42 2
I100
106
142
97 1
63
10000
53.3
7620
76
-
Average: 88 Standard Deviation: 18
422
10300
103
Average: 105 Standard Deviation: 19
142
7500
74
Avenge: 87 Standard Deviation: 22
`Sample residue exceeds the spiking level significantly (3x spiking level): therefore, an accurate rewvery value cannot be calculated ND = Not detected at or above the Limit of Quantitation(LOCI) of 25 ng/L Note: Since this summary table shows rounded results, recovery values may vary slightly from the values in the raw data.
Exygen Research
Page 33 of 127
Interim Report #17 - Analysis of Sediment and Water Samples Exygen Study No.: PO001131
Table V. Total Percent Solids in Sediment Samples
Exygen ID
coo91547 COO91557 COO91566 COO91579 COO91580 COO91581 COO91582 COO91583 COO91584 COO91585 COO91586 COO91587
Client Sample ID
DNS-SD-STA001-0-050824 DNS-SD-STA002-0-050824 DNS-SD-STA003-0-050824 DNS-SD-STA004-0-050825 DNS-SD-STA005-0-050825 DNS-SD-STA006-0-050825 DNS-SD-STA007-0-050825 DNS-SD-STA008-0-050825 DNS-SD-STA009-0-050825 DNS-SD-STAO10-0-050825 DNS-SD-STAOI1-0-050825 DNS-SD-STAO12-0-050825
Total Percent Solids (%)
57.70 52.23 45.84 70.28 59.06 72.13 56.01 59.64 61.43 57.91 56.54 46.84
Exygen Research
Page 34 of 127
Interim Report # 17 - Analysis of Sediment and Water Samples Exygen Study No.: PO001 13 1
FIGURES
Exygen Research
Page 35 of 127
Interim Report ##17 - Analysis of Sediment and Water Samples Exygen Study No.: PO001 131
Figure 1. Typical Non-Extracted Calibration Curve for PFBS in Methanol
112105A Sediment.rdb (PFBS): "Lineal' Regression c1 / Y weighting): y = 7 . 6 6 e + 0 0 4 x + 4.33e+003 ( r = O.Qg88)
7.915 7.5e5 7.0e5 6.5e5 6.0e5 5.5e5 5.0e5
-(D 4.5e5
c
3 0 u 4.0e5
m-
<E 3.5e5
3.0e5 2.5e5 2.0e5 I. 5 e 5 1.Oe5 5.0e4
Concentration. nglrnL
Exygen Research
Page 36 of 127
Interim Report # I 7 - Analysis of Sediment and Water Samples Exygen Study No.: PO001131
Figure 2. Typical Extracted Calibration Curve for PFBS in Methanol
111405C Pore Water.rdb (PFBS): "Linear' Regression ("1 /*'weighting): y = 157 x + 0.000389( r = 0.9987)
m
c
c 3
0 u
Concentration. ng/L
Exygen Research
Page 37 of 127
Interim Report # 17 - Analysis of Sediment and Water Samples Exygen Study No.: PO001 131
Figure 3. Non-Extracted Standards of PFBS in Methanol, 0.2 ng/mL and 0.5 ng/mL, Respectively
I - SS0070869 PFBS (5tanrdard)2 9 1 W 1 0 amu -sampk 7 of 24froo~1772705CwiR Area: 76048 counrtr M i g h t : 7.37e+003 cpz RT: a532 minr
I-1 Time,min SSOOO!374h). PFBS (Standard)299.0#9.0 a m u . sample 2 o f 2 4 f r o m 112105C.wiff Area: 4 0 6 2 3 counts Height: 3.29e+003 cps R T : 0.523 rnin
2000
u)
a u
1000 500 0
_r
1
2
3
4
5
6
7
8
D
10 11 1 2 13 1 4 1 5 16 17
Time, min
Exygen Research
Page 38 of 127
Interim Report #17 - Analysis of Sediment and Water Samples Exygen Study No.: PO001 131
Figure 4. Extracted Standards of PFBS in Methanol, 25 ng/L and 50 ng/L, Respectively
IXC112305-1-PFBS partlard) 298WLO am# -sample 2 oF44from 112705awiff Area: 4871counrtr #Mgh?: 397e+002c p RT: a607 mi# 0.61
w Q u
g c
a,
c -c
1.54
Time, rnin XC112305-2. PFBS (Standard)299.OR9.0 arnu. sample 3 of 4 4 f r o r n 112105D.wiff
I Area: 8948 counts Height: 7.52e+002 cps RT: 0.600 rnin
::k700
w
Q 0
p
c
(11
300
-c 4-.
200
100
0
- T
1
2
3
4
5
6
7
-- _--
IO
11
12
13
14
15
18
17
Time, rnin
Exygen Research
Page 39 of 127
Interim Report #17 - Analysis of Sediment and Water Samples Exygen Study No.: PO001 131
Figure 5.
PFBS in 1% Acetic Acid Reagent Blank, 0.4 ng/mL Fortified Reagent Spk A, and 4.0 ng/mL Fortified Reagent Spk B, Respectively
I - Reagent Control PFBS @ f k r o w n ] 29Ra/9ROamu -sample 8 0 f 24from 1127OSCwifl @eak not found)
i i 3 4 j: 6 i d i I b 1; 1; I B 12 l k Ib 1;
I Time. min ReagentSpkA. PFBS(QC)2880rWO amu-sample8 of24from 112105Cwiff
Area 32007 counts Height 2 57e+003 cps R T 0 525 min
0.52
Eu) 2000-
&E 1000-c(I)
c
0
1.30
1
2
3
4
5
6
7
8
Q
10 11 12 13 14 15 16 17
I Time, min Reagent Spk B . PFBS (OC)2QQ.ORQ.O amu. sample 10 of 2 4 f r o m 112105C.wiff Area: 302171 counts Height: 2.48e+004 cps RT: 0.524 min
0.52
Time, min
Exygen Research
Page 40 of 127
Interim Report # 17 - Analysis of Sediment and Water Samples Exygen Study No.: PO00 1131
Figure 6. PFBS in Water Reagent Blank, 50 ng/L Fortified Reagent Spk A, and 500 ng/L Fortified Reagent Spk B, Respectively
- Reagent Control PFBS (I,4tknownr] 29L(VsLOamar -sample 9 o f 44fronrr 112105awitT @eak not foarnd)
12 18
Lo
15
gY Q 10
ca,
5
c -c
0
1
2
3
4
5
6
7
8
9
10 11 12 13
14 15 16 17
Time. min
Reagent S p k A - PFBS(QC)298.0iQ8.0 amu-sample 10 of44from 112105D.wiff Area: 8575 counts Height: 6.79e+002 cps RT: 0.611 min
0.61
I . Time, min Reagent Spk B . PFBS (QC)299.0199.0 amu sample 11 of 4 4 f r o r n 112105D.t~iff Area: 93444 counts Height: 8.15e+003 cps RT: 0.825 min
06 2
Lo
2 6000-
$j 4000-
c
d-c 2000-
' 0
I 2
3
4
5
6
7
8
9
10 11 12
13
14 15
16 17
Time, min
Exygen Research
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Interim Report # 17 - Analysis of Sediment and Water Samples Exygen Study No.: PO001131
Figure 7.
Chromatogram Representing a Sediment Sample Analyzed for PFBS (Exygen ID: C0091586, Data Set: 112105C)
I - COO97586 PFBS (Wknowrr) 299.WAO aftfly -sa:arrrpk15 of 24froftf 772105Gwiff Area: 27754courrb Height: 1.68e+003cpo RT: a528min
3 1600 1500 1400
1300
1200
1100
1000
m
gau 900
c
800
a,
c -c 700
600
500
400
300 200 100
0
1
2
3
4
5
6
7
8
g
10 11 12 13 14 15 16 17
Time, min
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Interim Report # 17 - Analysis of Sediment and Water Samples Exygen Study No.: PO00 1131
Figure 8.
Chromatogram Representing a Water Sample Analyzed for PFBS (Exygen ID: C0091652, Data Set: 112105D)
I - COO91652 PFBS (lbtRnrowrr) 29Ldr99.0 amu -sample 23of 44frorrr 772105awiff Area: 9132courrts Height: 631e+002cps R7: a642mirr I 600 550 500 -
460 ~
400.
"Lo
Q
350 -
ELcalo 300 -
c -c
250 -
200 -
150 -
1.72
100 -
50 ~
0-J
--
--- - .
1
2
3
4
5
6
7
8
Q
10 11 12 13 14 15 16 17
Time, min
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Interim Report #17 - Analysis of Sediment and Water Samples Exygen Study No.: PO001 131
Figure 9. Typical Non-Extracted Calibration Curve for PFHS in Methanol
1 1 2 1 0 5 A Sedimenkrdb (PFHS): "Linea?' Regression r1/ Y weighting): y = 1.24e+005 x + 777 ( I = O.QQQ0)
1.30e6
1.20e6
l.lOe6
1.00e6
Q.00e5
8.00e5
5+u- ) 7.00e5
0 Y
me 6 . 0 0 e 5
=I 5.00e5
4.00e5
3.00e5
2.00e5
1 .OO e5
1
2
3
4
5
6
7
8
Q
10
Concentration. nglmL
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Interim Report # 17 - Analysis of Sediment and Water Samples Exygen Study No.: PO001 I3 1
Figure 10. Typical Extracted Calibration Curve for PFHS in Methanol
111405C Pore Water.rdb (PFHS): "Lineat` Regression c1 /*`weighting): y = 376 x + 839 (r= 0.W63)
1.-I-
0
100
200
300
400
500
600
700
800
900
1000
Conoentration. nglL
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Interim Report #17 - Analysis of Sediment and Water Samples Exygen Study No.: PO001 131
Figure 11. Non-Extracted Standards of PFHS in Methanol, 0.2 ng/mL and 0.5 ng/mL, Respectively
- SS0070869 PF#S (starrdardj39Lo18aOa m .sample 7 of 24fmtt 172705Cwit7
Area: 24963couttis might: 1.OQ+003 cps RT: a32 mitt
8.32
I SSOOO974 - PFHS (Standard)399.0180.0
Time, min amu. sample 2 of24from 112105C.wiff
Area: 58135 counts Height: 2 . 4 3 e + 0 0 3 cps R T : 8 . 2 9 min
8.29
Time. min
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Figure 12. Extracted Standards of PFHS in Methanol, 25 ng/L and 50 ng/L, Respectively
XC112305-7. PFM (standard) 39%0/8&0 amu -ramp!@2 of Wfrom 11210mwitT Area: 7724COUMtt Might: 165e+002 cps R E I43miu
350 -:
300 j
200 ~ 150 100 : 50
6.97. 1
Time, rnin XC112305.2. PFHS (Standard)399.0180.0 arnu. sample 3 of 44frorn 112105D.wiff Area: 1 4 8 7 2 counts Height: 5.90e+002 cps RT: 8.44rnin
500
400
300
200
1 7.59
lo0 o 1
2
3
4
5
6
7
8 1, . , ,
,
g
1 0 11 12 1 3 1 4 1 5 1 6 17
Time, rnin
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Interim Report # 17 - Analysis of Sediment and Water Samples Exygen Study No.: PO00 131
Figure 13. PFHS in 1 % Acetic Acid Reagent Blank, 0.4 ng/mL Fortified Reagent Spk A, and 4.0 ng/mL Fortified Reagent Spk B, Respectively
- Reagent Cortrol PFH5 (7btkrowr) 39%.0/8O.O amu -sample 8 of Mfrom 772705Cwifl geak r o t found)
- 0.47
15 P
mn u 10
8.70
- c
a,
5-
c c
1.79
n
1
2
3
4
5
6
7
8
9
10 11 12 13 1 4 15 16 17
I Time, min Reagent Spk A - PFHS (C!C)399.0#0.0 amu. sample 9 of 24from 112105C.wiff Area: 48045 counts Height: 2.16e+003 cps RT: 8.38 rnin
IReagent Spk B -
PFHS (RC)3Q!3.0#0.0
amu.
Time, min sample 10 of 24from 112105C.wiff
Area: 4Q7983 counts Height: 2.06e+004 cpr RT: 8.35 min
2.0e4 -
0)
u Q
$$ 1.0e4-
c
-a,
1 c
Time. min
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Interim Report #17 - Analysis of Sediment and Water Samples Exygen Study No.: PO001 131
Figure 14. PFHS in Water Reagent Blank, 50 ng/L Fortified Reagent Spk A, and 500 ng/L Fortified Reagent Spk B, Respectively
- Reagent &n?ro/ PFlls &&known) 39~maoamu -sample 9 of&from ? ? 2 ? 0 m w i t ~ $eak not found)
0.50
ln
15-
- Q
u 10
- c
a,
- c
2.35. 2.87 A3.68 p.18
I Time, min Reagent Spk A - PFHS (OC)300.0#0.0 arnu. sample 10 o f 4 4 f r o m 112105D.wiff Area: 15580 counts Height 7.58e+002 cps RT: 8.55 min
15.05
.gln
u Q
500-
400-
c
2
200-
- c
I Time, rnin Reagent Spk B - PFHS (QC)3Oa.O#O.O a m u . sample 11 of 4 4 f r o r n 112105D.wiff Area: 143728 counts Height: 6.78e+003 cps R T : 8.58 min
ln 5000-
Q
u
4000 -
-g-c
a, 2 0 0 0 -
LII 0.84
7.72 Time, rnin
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Interim Report # 17 - Analysis of Sediment and Water Samples Exygen Study No.: PO001 131
Figure 15. Chromatogram Representing a Sediment Sample Analyzed for PFHS (Exygen ID: C0091587, Data Set: 112105C)
I - COO92587 PFHS w k ~ o w r3]9~0/8aoamu -sample 22of 24from 2 1 2 1 0 ~ ~ w i f f Area: 32362 courts Might: 22&+003 c p RT:845 mi# e 2200
2000 1800
1600
1400
u)
g Q
0 1200
c
22 1000 - c
800
600
400
0...5.0
200 - I
7.88,
2 g
10 I 1 12 13 14 15 16 17
Time, min
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Interim Report #17 - Analysis of Sediment and Water Samples Exygen Study No.: PO001 131
Figure 16. Chromatogram Representing a Water Sample Analyzed for PFHS (Exygen ID: C0091549, Data Set: 111405C)
I - COO97549 PFHS m&nrownr] 391Q/8&0amu -sample 76 O f 3 8 from 777405Cwift Area: 234368 counts Might: 7.8Se+004cps RT: ll2minr
I.8e4 -
1.7e4-
1.6e4-
1.5e4 I.4e4-
1.3e4-
1.2e4-
l.le4-
(D
n. u 1.0e4-
$ 9000.0:
e
0
- 8000.07000.0 - 6000.0
5000.0 4000.0 -
1 I
Time, rnin
1'1
12
13
14
15
16
17
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Figure 17. Typical Non-Extracted Calibration Curve for PFOS in Methanol
1 1 2 1 0 5 A Sediment.rdb (PF0S):"Lineat' Regression C'1 Id'weighting): y = 1.15e+005 x + 5 . 6 e + 0 0 3 ( r = 0 . g Q g l )
1.15e8 I.lOe6 1.05e8 1.00e8 9.50e5 9.00e5 8.50e5 8.00e5 7.50e5 7.00e5
8UI
E 0.50e5
3 0.OOe5
m-
5.50e5
c 5.00e5 4.50e5 4.00e5 3.50e5 3.00e5 2.50e5 2.00e5 1.50e5 1.00e5 5.00e4
1
2
3
4
5
6
7
8
g
10
Concentration. nglmL
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Interim Report #17 - Analysis of Sediment and Water Samples Exygen Study No.: PO001 131 Figure 18. Typical Extracted Calibration Curve for PFOS in Methanol
111405C Pore Water.rdb(PFOS):"Lineat'Regressionrl I k " w e i g h t i n g ) : y = 3 0 Q x + 5 6 7 ( r = O . g ~ 8 0 )
ul
c c
0 u3
me -
Q
Concentration. nglL
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Interim Report #17 - Analysis of Sediment and Water Samples Exygen Study No.: PO001 131
Figure 19. Non-Extracted Standards of PFOS in Methanol, 0.2 ng/mL and 0.5 ng/mL, Respectively
I - SS0070869 PFOS Wanrdard)49Lo18aOamu -sample 7 of 24from 772705Cwiff Area: 24587 counrts M i g h t : 7.56e+O03 cps RT: 77.0 mi# 11.02
I . Time. rnin SSOOOQ748 - PFOS (Standard)WQ 01800 amu sample 2 of 24from 112105C wiff Area 59399 counts Height 3 84e+003cps RT 11 0 rnin
3500
3000
g(cDz 2500
u
2000 c c 1500
- e
1000
500
0
Time. rnin
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Figure 20. Extracted Standards of PFOS in Methanol, 25 ng/L and 50 ng/L, Respectively
- X C I I U O S - I PFOS (Standard) 49LWaO autu -sample 2 o f M f r o u t 11210Wwiff
Area: 8283 counts M i g h t : 535e+002 cpo R R la9 mitt
1 500
400
300
200 10.4,
I0
1
2
3o 4
5
6
7o 8
Q
10
Time, min XC112305.2. PFOS (Standard)49.0#0.0 a m u . sample 3 o f 4 4 f r o m 112105D.wiff
Area: 1 5 2 9 1 counts Height: Q.73e+002 cps RT: 1 1 . 0 min
I s1 12 13 14 15 16 17
10.95
800 -
u)
a u
600 -
Time, rnin
v 12
13
14
15
16
17
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Figure 21. PFOS in 1% Acetic Acid Reagent Blank, 0.4 ng/mL Fortified Reagent Spk A, and 4.0 ng/mL Fortified Reagent Spk B, Respectively
- Reagent Control PFOS (lhknown) 491w8aOamdl -sample 8 o f 24from 712705Gwiff
Area: 935counts might: P1&+001cpsRT: Il.Omin
grn
30-
E
20 -
Time, min
Reagent S p k A . PFOS (RC)4QQ.OBO.O arnu. sample Q of 2 4 f r o m 112105C.wiff
Area: 47406 counts Height: 3.13e+003 cps RT: 11.1 rnin
- 3000
VI
gau 2000 ~
-cm 1000 -
c c
VI Q
2.0e4-
25 1.0e4-
c -c
0.0 ~ . I
Time, rnin
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Interim Report #17 - Analysis of Sediment and Water Samples Exygen Study No.: PO00 131
Figure 22. PFOS in Water Reagent Blank, 50 ng/L Fortified Reagent Spk A, and 500 ng/L Fortified Reagent Spk B, Respectively
V)
6.0- 0.63
gY Q
- c
+ a,
2.0-
c
1.76 2.32
4.42.5.28.
5.86 7.14
1.1 'nn
10.63,
I!
8.74
I
12.91
16.20
A.
1
2
3
4
5
6
7
8
9
10 11 12 13 1 4 15 16 17
I Time, min Reagent Spk A . PFOS (QC)499.0#0.0 amu. sample 10 o f 4 4 f r o m 112105D.wiff Area: 16685 counts Height: l.O6e+003 cps RT: 11.0 min
1000
V)
Q u
500
c
a,
c -c
0
I . Time, min Reagent Spk B . P F O S (QC)499.0#0.0 amu sample 11 of 44from 112105D.wiff Area: 166657 counts Height: 9.71e+003 cps RT: 11.0 rnin
11.03
(0
Q u
g 5000:
c
-c11)
c
0-
Time. rnin
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Interim Report # 17 - Analysis of Sediment and Water Samples Exygen Study No.: PO001 131
Figure 23. Chromatogram Representing a Sediment Sample Analyzed for PFOS (Exygen ID: C0091566, Data Set: 112105A)
- COO91566 PFOS (Irkknrownr]4980$8a0am# -sample 24of 37from 11210W.wiff
Area: 537869co#nrtO M i g h t : 892@+004cpoRT: 11.f mi#
3.8e4 3.Be4 3.4e4 3.2 e4 3.0e4 2.8e4 2.Be4 2.4e4
au) 2 . 2 e 4 u
3 2.0e4
(0
c ar 1.8e4
c -c
1.Be4 1.4e4 1.2e4 1.0e4 8000 0 6000.0 4000.0 2000.0
0 .o
1 i1
1 0 .B(
1
2
3
4
5
B
7
8
Q 10
Time, min
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Interim Report # 17 - Analysis of Sediment and Water Samples Exygen Study No.: PO001 13 1
Figure 24, Chromatogram Representing a Water Sample Analyzed for PFOS (Exygen ID: C0091558, Data Set: 111405C)
- COO91558 PFOS (7b#known4)YLW110amu -sample 32 of 38 from 111405Cwiff
Area: 199544courtr might: 1.28e+004cpo RT: 123 min
1.2084
1 !8
1.10~4
1.OO e4
0000.00
8000.00
7000.00
gc 8000.00
-c0)
I
5000.00
11.84.
4000.00
3000.00
2000.00
1000.00
0 .oo
Time, min
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APPENDIX A
Study Protocol PO001131
(Exygen Study No. P0001131) with Analytical Methods and
Protocol Amendments 1 and 2
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Exygen Protocol Number: PO00 1 13 1
STUDY PROTOCOL
Study Title: Analysis of Perfluorobutanesulfonate(PFBS),
Perfluorohexanesulfonate(PFHS), and Perfluorooctanesulfonate (PFOS) in Water, Soil, Sediment, Fish, Clams, Vegetation, Small Mammal Liver and Small
Mammal Serum Using LC/MS/MS for the 3M Decatur Monitoring Program
Exygen Protocol Number: POOOl 131
Performing Laboratorv: Exygen Research 3058 Research Drive State College, PA 16801 Phone: (814) 272-1039
SDonsor R m r e s e n t a t i v e : Michael A. Santoro Director of Regulatory Affairs 3M Building 0236-01-B- 10 St. Paul, MN 55 144 Phone: (651) 733-6374
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Interim Report #17 - Analysis of Sediment and Water Samples Exygen Study No.: PO001 13 1
Exygen Protocol Number: PO001 13 1
+
DISTRIBUTION:
1) JaisimhaKesari, Study Director,Weston Solutions 2) John M. Flaherty, Principal Investigator,Exygen Research 3) Michael A. Santoro, SponsorRepresentative,3M Company 4) Exygen Research Quality Assurance Unit
e-
+ Exygen Research
Page 2 of 65
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Interim Report # 17 - Analysis of Sediment and Water Samples Exygen Study No.: POOOl 13 1
Exygen Protocol Number: PO00I I3 1
n
PROTOCOL APPROVAL
Study Title: Analysis of 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 MonitoringProgram
Exygen Protocol Number: POOOl 131
APPROVALS
Weston Solutions
w 4 A S .Michael A. Safitoro.. Sp.onsorRepresentative 3M Coinpdy
John M. Flaherty, hncipal Investigator
Date
/ Exygen Research
Date
Exygen Research
-.
Pugr 3 uJ 6.5
.... ..
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Exygen Protocol Number: PO001 13 1
TABLE OF CONTENTS
TITLE PAGE ..................
............................................................... 1
DISTRIBUTION.
............................................................... 2
P R O T O C O L A P P R O V A L ................................................................................................................................ 3
TABLE O F CONTENTS ...................
.................................................................. 4
INTRODUCTION.. ........................................................................................................................................... 5
TEST MATERIALS ......................................................................................................................................... 5
OBJECTIVE
............................. 6
TESTING FACILITY .............................................................................................................................. 6
STUDY DIRECTOR ........................................................................................................................................ ?
S P O N S O R REPRESENTATIVE .........
..................................
.......................... ..?
PRINCIPAL INVESTIGATOR ........................................................................................................................ 7
PROPOSED EXPERIMENTAL START AND TERMINATION DATES
................................. 7
IDENTIFICATION A N D JUSTIFICATION OF T H E TEST SYSTEM .................................................
SAMPLE PROCUREMENT, RECEIPT AND RETENTION ......................................................................... 8
F SAMPLE IDENTIFICATION .............
............9
ANALYTICAL PROCEDURE SUMMARY ................................................................................................... 9
VERIFICATION OF A N A L Y T I C A L P R O C E D U R E
........................................................ 9
METHOD FOR CONTROL OF BIAS .................................................................................
...........I I
STATISTICAL METHODS .......................................................................................................................... I I
GLP STATEMENT .............
.............
II
REPORT ........................................................................................................................................................... 1 1
SAFETY AND HEALTH .............................................................................................................................. I Z
AMENDMENTS TO PROTOCOL
DATA RECORD KEEPING .................................................... QUALITY ASSURANCE ..............
..................................... 13
RETENTION OF DATA AND ARCHIVING
........................ 14
APPENDIX I. ANALYTICAL METHODS ................................................................................................... I S
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Exygen Protocol Number: PO00I 13 I
P--
INTRODUCTION
The purpose of this study is to perform analysis for perfluorobutanesulfonate (PFBS), perfluorohexanesulfonate (PFHS) and perfluorooctanesulfonate (PFOS) in water, soil, sediment, fish, clams, vegetation, small mammal livers and small mammal serum using L C M S M S 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 of 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 (CdFpSO3.K') Lot Number: 101 Purity: 96.7%
Transitions Monitored: 299 + 99
Structure:
PFHS Chemical Name: Perfluorohexanesulfonate Molecular Weight: 438 supplied as the potassium salt (C,jF,,SO,-K') Lot Number: SE036 Purity: 98.6%
Transitions Monitored: 399 + 80
Structure:
FFF
F
FFF
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Exygen Protocol Number: PO00IIZ I
PFOS Chemical Name: Perfluorooctanesulfonate Molecular Weight: 538 supplied as the potassium salt (CgFl7SO3-K') Lot Number: 217 Punty: 86.9%
Transitions Monitored: 499 + 99
Structure:
OBJEXTIVE
The purpose of this study is to perform analysis for perfluorobutanesulfonate (PFBS), perfluorohexanesulfonate (PFHS) and perfluorooctanesulfonate (PFOS) in water, soil, sediment, fish, clams, vegetation, small mammal livers and small mammal serum for the 3M Decatur Monitoring Program using the current versions of the following Exygen analytical methods:
VOOOl780: V0001781: VOOOl782: V0001783: V0001784: V0001785: VOOOl786:
"Method of Analysis for the Determination of Perfluorooctanoic Acid (PFOA) in Water by LC/MS/MS" "Method of Analysis for the Determination of Perfluorooctanoic Acid (PFOA) in Soil by LC/MS/MS' "Method of Analysis for the Determination of Perfluorooctanoic Acid (PFOA) in Sediment by LC/MS/MS" "Method of Analysis for the Determination of Perfluorooctanoic Acid (PFOA) in Fish and Clams by LC/MSIMS" "Method of Analysis for the Determination of Perfluorooctanoic
Acid (PFOA) in Vegetation by LCIMSIMS" "Method of Analysis for the Determination of Perfluorooctanoic Acid (PFOA) in Small Mammal Liver by LC/MS/MS" "Method of Analysis for the Determination of Perfluorooctanoic Acid (PFOA) in Small Mammal Serum by LC/MS/MS"
TESTING FACILITY
Exygen Research 3058 Research Drive State College, PA 16801 Phone: (814) 272-1039
Page 6 of65
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-
STUDY DIRECTOR
Jaisimha Kesari P.E., DEE Weston Solutions, Inc. 1400 Weston Way
West Chester, PA 19380
Phone: (610) 701-3761 F a : (610) 701-7401 j.kesari@westonsolutions.com
Exygen Protocol Number: PO001 13 I
SPONSOR REPRESENTATIVE
Michael A. Santoro 3M Company Director of Regulatory Affairs 3M Building 0236-01-B-10 St. Paul, MN 55144 Phone: (651) 733-6374
c1
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 of 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.
Puge 7 o/ 65
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Exygen Protocol Number: PO001 13I
c.
IDENTIFICATION AND JUSTIFICATION OF THE TEST SYSTEM
The following are the test systems for this study: 0 Water (groundwater and surface water) 0 Soil 0 Sediment Fish Clams 0 Vegetation 0 Small Mammal Liver 0 Small Mammal Serum
The samples will be collected by Weston Solutions. The control samples will be purchased and prepared by the testing facility. Purchase and processing details for the control samples will be included in the final report associated with this study.
The test systems were chosen to access the environmental impact of PFBS. PFHS and PFOS in the Decatur, Alabama area.
r
SAMPLE PROCUREMENT, RECEIPT AND RETENTION
Water, soil, sediment, fish, clam, vegetation, small mammal liver and small mammal serum samples will be received at Exygen directly from Weston Solutions. The details of sample procurement for this study are outlined in the 3M work plan entitled "Phase 2 Work Plan for Sampling Environmental Media." The number and types of samples collected will vary depending availability in the field. The total number of 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 2OC-8'C. 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 5 -10C. Small mammal whole blood samples will be centrihged in the field at the time of collection and the serum fraction will be used for the study. Small mammal serum will be stored frozen at 5-10C.
The receipt and processing of the samples will be documented in the final report and raw data associated with the study.
___
__ -
Page 8 of 65
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-
SAMPLE IDENTIFICATION
Exygen Protocol Number: PO00113I
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: VOOOl780: VOOOl781: VOOOl782: V0001783: VOOOl784: VOOOl785: VOOOl786:
"Method of Analysis for the Determination of Perfluorooctanoic Acid (PFOA) in Water by LCMSIMS' "Method of Analysis for the Determination of Perfluorooctanoic Acid (PFOA) in Soil by LCIMSIMS" "Method of Analysis for the Determination of Perfluorooctanoic Acid (PFOA) in Sediment by LC/MS/MS" "Method of Analysis for the Determination of Perfluorooctanoic Acid (PFOA) in Fish and Clams by LCIMS/MS" "Method of Analysis for the Determination of Perfluorooctanoic Acid (PFOA) in Vegetation by LC/MS/MS' "Method of Analysis for the Determination of Perfluorooctanoic Acid (PFOA) in Small Mammal Liver by LCIMSIMS" "Method of Analysis for the Determination of Perfluorooctanoic Acid (PFOA) in Small Mammal Serum by LCIMS/MS"
The above methods use analytical conditions capable of separating the isomers of PFBS, PFHS and PFOS. The final report will include the isomers summed into total PFBS, total PFHS, and total PFOS found.
VERIFICATIONOF ANALYTICAL PROCEDURE
A laboratory control sample will be used for the preparation of 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
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Exygen Protocol Number POOO 1 I3 I
collection at the testing facility and have been shown to be free o f PFBS. PFHS and PFOS. Samples will be added to each container to a volumetric fill line at 200 mL. A field duplicate, a low field spike and a high field spike of each sample will be collected. The low and high field spike bottles will contain PFBS, PFHS and PFOS as well as perfluorooctanoic acid (PFOA) and 1.2-13C perfluorooctanoic acid ("C PFOA). PFOA and I3C PFOA are included in the solutions used to spike the samples. The results for PFOA and I3C PFOA will not be reported in this study. Exygen will supply one field blank (control water) and two field blank spikes (control water fortified with PFBS, PFHS and PFOS at a low and high level) for every twenty samples collected. At the testing facility, each water sample (excluding field duplicates and field spikes) will be extracted in duplicate and will also be fortified at a low and high concentration with PFBS, PFHS and PFOS and processed through the described procedure to determine method accuracy and to check for bias.
For soil, sediment, clams, and vegetation, Exygen will supply one 500 mL precleaned Sci/Spec Premier wide mouth HDPE bottle per sample collected or a zip-seal bag. All containershags used for sample collection will be shipped to the sample location. Samples will be added to each container or bag in the field. At the testing facility, each sample will be extracted in duplicate and will also be fortified at a known concentration with PFBS, PFHS and PFOS at both a low and high level and processed through the described procedure to determine method accuracy and to check for bias.
For small mammal liver, Exygen will supply a 50 mL polypropylene centrifuge tube. For small mammal serum, Exygen will supply a collection kit for each sample containing s e m 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.
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ExygenProtocol Number: PO001 13 1
Recoveries are anticipated to be between 70% and 130% of the fortified levels; however, the exact precision and accuracy will be determined by the analysis of the quality control samples described above. A statement of accuracy will be included in the final report.
METHOD FOR CONTROL OF BIAS
Control of bias will be addressed by taking representative sub-samples from a homogeneous mixture of each matrix from untreated control samples, and by analyzing at least two levels of fortifications.
STATISTICAL METHODS
Statistics will be limited to those specified in the subject methods and to the calculation of average recoveries, as applicable.
GLP STATEMENT
/-
All aspects of 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 of the study. The report will include, but will not be limited to, the following:
The name and address of the Study Director, Sponsor Representative, and of the testing facility.
A statement of GLP compliance (any related documentation, such as chain-of-custody records, must be in the study records).
P-
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Exygen Protocol Number: POOOl 131
The signed and dated statement by the Exygen Research Quality Assurance Unit regarding dates of study inspections and dates findings were reported to the Study Director and Management. A description of the exact analytical conditions employed in the study. If the subject method was followed exactly, it is necessary to include only a copy of the analytical method. Any modifications to this method will be incorporated into the report. If the method is photo-reduced, the project number and page number must be included on each page.
Description of the instrumentation used and operating conditions.
All results from all sets analyzed. Control and fortified samples will be
identified and the data table will include sample number and fortification level.
Representative chromatograms for each analyte in each matrix, including chromatograms of 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 of 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 of an amendment signed by the Study Director. The amendment shall clearly identify that part of 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 of study results as per 40 CFR 792.185.
SAFETYANDHEALTH
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 OJ 65
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Exygen Protocol Number:POOO I I3 I
AMENDMENTS TO PROTOCOL
All significant changes to the analytical protocol outlined here will be expressed in writing, signed and dated by the Study Director and Sponsor Representative. Amendments usually will be issued prior to initiation of study plan change. However, when a change is required without sufficient time for the issue of a written amendment, that change may be effected verbally with supporting documentation signed and dated by the Study Director and followed with a written amendment as soon as possible. In this case, the effective date of the written amendment will be the date of the documented change. Copies of 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 promplly to the Sponsor Representative.
DATA RECORD KEEPING
Records to be maintained include the following (as appropriate):
Sample tracking sheet(s) 0 Sample receipt records, storage history, and chains of custody
History and preparation of 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 0 Sample extraction and analysis dates
A complete listing of study personnel, signatures and initials e Chronological presentation of all study correspondence
Any other documentation necessary for the reconstruction of the study
Chromatograms-All chromatograms will contain the following:
Sample identification, injection date, arrow or other indication of the area of interest, and injection number corresponding to the run.
Additionally, fortifications will include the amount of analyte added and the sample number of the sample that was fortified.
rrr
Analytical standard chromatograms will additionally include the
concentration (e.g., pg/mL).
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Exygen Protocol Number: PO001 13 1
As part of the documentation the following sheets will be included in each analytical set: a run sheet listing the samples to be run in the set, and an instrument conditions sheet describing the instrument type and operating conditions.
QUALITY ASSURANCE
The QA Unit of Exygen Research will inspect the study at intervals adequate to assure compliance with GLP's, and will report the findings of audits to the Study Director, Exygen Management, and the Sponsor Representative.
RETENTION OF DATA AND ARCHIVING
All hard copy raw data, including, but not limited to, the original chromatograms, worksheets, correspondence, and results shall be included with the data package submitted to the Study Director. These will be archived with the original study plan, amendments, final report, and all pertinent information from the Sponsor. The testing facility shall keep all electronic raw data and any instrument, equipment, and storage logs for the period of time specified in 40 CFR 792.195. An exact copy of 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.
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Exygen Protocol Number: POOOl 13 1
APPENDIX I
ANALYTICAL METHODS
V0001780: VOOOl781: VOOOl782: V0001783: VOOOl784: V0001785: VOOOl786:
"Method of Analysis for the Determination of Perfluorooctanoic Acid (PFOA) in Water by LC/MS/MS" "Method of Analysis for the Determination of Perfluorooctanoic Acid (PFOA) in Soil by LC/MS/MS" "Method of Analysis for the Determination of Perfluorooctanoic Acid (PFOA) in Sediment by LC/MS/MS" "Method of Analysis for the Determination of Perfluorooctanoic Acid (PFOA) in Fish and Clams by LC/MS/MS" "Method of Analysis for the Determination of Perfluorooctanoic Acid (PFOA) in Vegetation by LC/MS/MS" "Method of Analysis for the Determination of Perfluorooctanoic Acid (PFOA) in Small Mammal Liver by LCMSIMS' "Method of Analysis for the Determination of Perfluorooctanoic Acid (PFOA) in Small Mammal Serum by LC/MS/MS"
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Exygen Protocol Number: PO00I 131
ANALYTICAL METHOD
McchodNumber: VOW1780
Mctbod of Analyaiafor tbc Determlnntlon of Pcrfluoroocta~olrA d d (PFOA) io Water by LC/MSiMS
Analytical Testing Facility:
Exygm RuKsrch 3058 ResearchDnvs
State Collage,PA 16801
Appmvcd By:
Paul Connolly
I
TechnicalLeader. LC-MS. @xygcn Resoarch
f/dh-- 71/77
JohnFlahay / Vice President, Operations,Exygm Rescvch
A Daw
Exygen Research
Total Pages: 7
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Exygen Protocol Number: POOOl 13 1
ErmaR w m h
McUlod Numkr V(xMI780
I 1 AYALYTICAL METIfOD Method of Analysis for the Dewmination of Pertluoroocmoic Acid (PFOA) in Water by
LCIMSIMS
1.0 scope
Thir method is to be employed for the isalalion and quamitation of perfluomctanoic acid by High Porformnnoc Liquid Chromalograpby couplcd to a tandem Mass Spccmmehic Dctcctor( L U M S I M S ) in water.
2 0 Safety
2.I Alwsp observe safe laborplory pmlicco. 2.2 Conaull the approprialeMSDS bofore handling any chemical for proper safely
prccwtiona.
3.0 Sample Requirement
3.I A1 lust 40 mL of test sample for cxlraction. 3.2 No sample pmcnring is needed for w n m samples. 3.3 Samples stored nliigeraled should be allowed lo qutlibratc IO room
trmpashw. 3.4 All sample6 mwt k thoroughly mixed before being sampled for extraction 3.5 Any m p l a containing pnnicles shculd be centrifuged at -3000 rpm for - 5
minuter Mdthe rupcmatantused for the cxmtion. 3.6 Sample collcction procedures will be spccificd in the sampling plan for [his
pmjecr
4.0 Reagents and Sturdudl
4.1 Water- HPLC grade
4.2 Methanol - HPLC grade 4.3 Ammonium Acetate - A.C.S. Reagent Grade 4.4 Peduomoclanoic Acid - Sigma-Aldrich
5.0 htlumenl and Equipmrnt
5.1 A high p e r f o m c liquid chromalograph capable of pumping up lo 2 solvents quipped with a variable volume injcclor capable of injecting 5-?W pL COMS~C~ OI a tudm Mass Spocmmncr( L W S I M S ) .
5.2 A device to eollecIraw d m for p a k integration and quantitation. 5 3 Analytical balancecapableo f d i n g lo 0.0MMI g. 5.4 50mL dirporablcpolypmpylenc centrifugetubes. S S IS mL d*porsble plypmpylme centrifugetub-. 5.6 Dirpossble micropipeu(50-1OOuL. loO-ZoouL). 5.7 125-mLLDPE nmw-mouth b o n k 5.8 2 mL clur HPLC vial kit. 5.9 Dirpose,bls pipottcr. 5.10 Autopipcttes(100-1000)ILmd 10-100pL),withdisposabletips 5.1 1 Watcn Sop Ppk Vac 6 EC (Ig) IC18 SPE cartridges.
Page17of6j
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Exygen Protocol Number: POOO I 13 1
Exysm Rnorrsh
Mabad humbn VWOIIUU
I 1 ABALYTICAL.METHOD Mahod of Anal@ for the LMcdnrtion ofPcrtluomoclanoic Acid (PFOA) in Water bv
LUMS/MS
5.12 SPE vpcuum manifold. 5.13 Centrifigocapable ofspinning 50 mL polypropylenetubes at 3000rpm.
6.0 Chmmatopphic System
6. I Analytical Column:Fluophnrc RP (Keystone Scientific). 2.I nun x 50 mm. Sbi (PM:82505-052130)
6.2 Tempanturr: 3 W C 6.3 Mobile Phase(A) : 2 mM Ammonium Acetote in Water 6.4 Mobile P h w (B) ! Mahurol 6.5 Gradient Rugrun:
Tlmc(Mn)a
0.0
65
I .o
65
8.0
25
20.0
25
22.5
65
Flow Rate
%BbllLexd
35
0.3
15
01
75
0.3
75
0.3
35
0.3
6.6 6.1 6.8
- - hjcction Volume: 15 pL (Can be incrcaacd to pli much as 50 pL).
Q u ~ t i t a t i ~P:eak Area external ataadard calibration curve. R U ~~i m e : 23 min~tcs.
The above ccnditionaam intandcd pli a guide and may be changed in order to optimize the HF'LC system.
7.0 M S M S Systm
7.1 Mode: Elatrorpny Negative MRM mode. monitoring 41 3 -+ 369 mm
'Ihe above conditionr arc i n t d c d aa a guidc and m a y be changed in order IO optimize the MSMS rystcm.
8.0 Prspprorionof Solutiona 8.1 Mobilc Phase
8.1.1 2 mM m o n i u m 1ce1.1~in water is p r e p 4 by adding 0.154 g 01' ammonium acetate to 1000 mL of water.
A l m a l e volumes may be prcpnrcd.
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Exygen ProtocolNumber: PO001131
LCiMSiMS
..
9.0 Sundard Repemtion
9.1 S t a m i d StocWFottification Solution 9.1.1 Pnpue s Itak aolution of -100 p#mL of PFOA by weighing IO ntg ofMJytiul s u n b r d (corrected for purity) end dilute to 100 mL willt mcthawl in a 125-mL LDPE bode. 9.1.2 A IO p&nL fortificetionsolutionof PFOA is prepared by bringing It1 mL ofthe 100 p g h L sohlion to a final volume of 100 wilh methsnnl in a 125 mL LDPE bonk 9.1.3 A 1.0 p#mL fortification solution ofPFOA is prepared by bnnging 111 mL of the IO p#mL aolution lo a final volume of 100 with methanol III a 125 mL LDPE botlle. 9.I .4 A 0.1 p#mL fortification solution of PFOA is prepared by bnnging II1
mL of the 1.0 pe/mL solutionto a fml volumeof 1W with meihanolin a 125 mL LOPE bonle. 9.1.5 A 0.01 p%mLfortification solution of PFOA is prepared by bringing IO mL of the 0.1 p#mL solution to a final volumc of 100 wilh meth.nol in a I25 mL LDPE bonk. 9.1.6 The stock nnd fottification solutions arc lo be stored in a rekigeralor al sppmxlmtdy 4OC and arc stable for a maximum penod of 6 niomlis
from the &IC of preparation.
9.2 StandardCalibration Solution8
9.2.1 9.2.2
LUTvlShB calibration nanddr arc p r e p d in HPLC water. The calibration stand=& .ro pmcssisd through the extraction procedure. identical to ~ m p l c ~ .
The following ia a typical cxample: rdditional cowenbations may bc propared w needed.
Final
Concentration Fortlficatiw V o h of Concmtrauon of Calibration
of Fomfsilion Volume Fomficd Cwfml Calibration
Standard ID
Solution@pb) (PL) Sanmle (mL) Slanhrd ( ~ 1 ) ' (CXMPIC)
0
0
40
0
XCmmddn 0
10
100
40
21,
XCmmddyy I
IO
100
40
50
XCmmddyy 2
10
400
40
IW
XCmmddyy.1
100
IW
40
250
XCmmddyv4
100
200
40
JW
XCmmddyy.5
-* 100
400
40
1000
XCmmddyy6
The Qmcled COnECnmttonof thc calibration standard i s equal Io Bx its initial
wncmmtion, due lo tho concmmtton of UK alandard dunng the extraction (WE)
XC extmccod calibration standard
.
Exygen Research
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Exygen Protocol Number: POOOl 131
ErypcnIhnouoh
Method Number VWOllBO
I ANALYTICAL METHOD Method of Analysis for the Determination ofPcduorowtanoic Acid (PFOA) in Water by
LCNSNS
9.2.3 A ZQD slnndard solution (reagent blank) wt be D I C U w~ ith each set of S t M W cxtmcted.
9.2.4 Stom all cxtmctcd calibration stMdard8 in 15-mL polypropylene tubes at ZC to 6'C. up to IWO we&.
9.2.5 Allmratc volume$ and conconeationsof standards may be prepared as needed.
10.0 Batch Set Up
10.1 Each batch of u m p l s ~ ~ a ~(tyt pcicadlly 20 or Ius) must include at least one reagent conbol (mahad blank using HPLC water) and two reagent controls fortified at know concentrations (lab control spike1 IO verili pmcedural rsoovery for the batch.
10.2 Requirements for field and laboratory duplicatcs and spikes will be specified in the quality assurance plan for this project.
1I .O Sample Extiaction
11.1 MCMUC40 mL of sample or a portion of sample diluted to 40 mL with n'ater into 50 mL polypmpylcne CenKifupC Nbes (forhfy as needed,replace lid and mix well).
11.2 Condition the CIS SPE carbidges ( I g. 6 mL) by passing IO mL methano. followedby 5 mL of HPLC water (- 2 drop/scc). Do not let column run dry
11.3 Losd rynplc on conditioned CI, SPB cartridge. Discard eluate. 11.4 Elute with -5 mL 100% methanol. Collect 5 mL of eluate into graduatsd
I5 mL polypropylene centrifugetubed (final volume = 5 mL). 11.5 Analyrc s q l u wing elcc.~~ospmyLCIMSIMS.
12.0 Chmmtopaphy
12.1 Inject the same mount of each .tandnrd, sample and fortified sample into the Lc/Ms/MS system. A calibration standud must pmedc and follow JII uulyzed ssmplw.
12.2 Sunduds of PFOA conupondq to at least five or man ~ncennarionlevels must ba included in an nnalylical set.
12.3 An entiro set of exliacted calibration r t d a r d r must bo included ai in? beginning and at thc end of a sample ret. Extracted standards must hb interspersed betwoon wmy 5-10 samples. As an alternative. an entire set 01 extracted calibration stMdarQ may bo in]ected at the beginning of a sci followed by exaa~tedcalibration standards iII1KSpCISCd every 5-10 samples (to account for a asond rst of exKacted standards). In either case. extracted calibration s t d n r d r murtbe the fimf and Inst tnjection in a sample K I
12.4 Use linear rt&d curved for quanlilation. Lineu slandsrd curves are gonmtcd for the uulyte by linear rcpcssion using I l x weighting of peak area
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Exygen Protocol Number:PO00I I3 1
Exypen Rorarcb
MemodNumbn VoOo1?80
I ANALYTICAL METHOD Mahod ofAnalysu forthe DctnmiolltionofPorflwmlanoic Acid (PFOA) in Water by
LCIMSIMS
v m calibntionMandard concentrationusing MassLynx 3.3 (or equivalent) aoflwue system. 12.5 Sample ~ p o n r should not ncsed standPrd responses. Any samples that c x c a d s t d a r d ruponrcr should be further diluted and reanalyzed.
13.0 Acceptance Critoria
13.1 Chromatogram mum show a peak of a daughter ion at 369 m u from a pareni of 413 MU. The 413 amu parent w m p o n d r lo the PFOA anion, while ilic daughtaion (369 MU) represents Le loss ofcarbon dioxide.
13.2 Mcthod bllnlrs must ~t contain PFOA a1 levels pcater than the LOO. Il'd blank contains PFOA at lcvels greater than 50 n g l l thcn a ncw blank sampie must be obtained rad the entire set must be re-extracted.
13.3 Recoveries of wnml ipikes and matrix spikes must be between 70-130% of their born values. If a wntml spike falls outside thc ncccptablc limits. thc entiru set of m p l a should be re-extracted. Any matnx rpikc outside 70. 130% should be evaluUcd by the analyst to determine if rc-cxuaction is wM.nfcd.
13.4 Any calibmion smdnrd found to be a statiatical outlier by using the Huge Emr Test, may be n c l u d d fmm the calculation of the calibration curve Howcvcr, the Wfal number of exoactvd calibration smdards that could be excluded must not a c c d 20% of the WtDl numbs of extracted standards injected.
13.5 The correlation cwfficient (R) for calibration curves generated must be M.992(R220.985). If calibration mulls fall outside thew limits. then appmpribte stqr must be lakcn to adjust insbumenl operation, and the standda or the relevant of samples should be reanalyzed.
13.6 Retention times baoucen standards and samples must not drifl more thm f 4 K within an annlytkal run. If retention time drifl exceeds this limit wirhui an analytical run then the set must be reanalyzed.
14.0 Calculations
14.1 Usc the followmp quation W calculate the amount of PFOA found (In ndL. based on peak area) using the standard c w e (lurcar regression parametrnt generated by the Mslu Lynx soAwars program:
- PFOA found ( n a ) UW arc^ .interncot). X DF slope
-DF factor by which the f w l volume was diluted, if necessary.
Exygen Research
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Exygcn Protocol Number: PO001 13 1
Exypra Rerarch
I M e h d of Andy!&
McrhodNvmkr VWO I780
ANALYTICAL M E M O D for tho Delenninntionof PcrfluomoctlnoicAcid (PFOA) in Water by
LCJMSiMS
142 For urmplol fonificd with known amounts of PFOA prior to extraction. use
- the followingq
Recovery ('A)
w
i
o
n
IO
calculptc
t
k
psrcwt
recovery.
1 (na) , totalMllytefound
.lnalytc found in control (ng/L)] oo
lnalytc eddcd (ndL)
Exygen Research
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Exygen Protocol Number: POOOl 131
ANALYTICAL METHOD
MnhodNumbcr. V0001781
Methodof Analylb for tbe Detsrmlmatlomof Periluorooct.no1c Acid (PFOA) In Sol1 by LCrmSrmS
Analytical TcdtinpFacility
Exygen Rcssanh 3058 Research Drive SWe College.PA 16801
Appmved By:
Paul Comlly Technical Leader, LC-MS. Exygcn Rcacarch
Exygen Research
TomlPngea: 7 Page 23 of65
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Exygen Protocol Number: Po001 I3 I
Exypn Rravch
MnbDdNumbcr VwO1781
1 I m.urrica rnmnoD Mahcd of Analylu for the DctenninsriM ofPefflu0rooctanoic Acid (PFOA) in Soil by
LCIMs/MS
1.0 scope
This method is to be omplopd for lhe isolation and quantitntion ofperfluorooctancic acid by High Paformunc Liquid Chromatography coupled to a LYldcrn Mass SwlromCeic Detector (LCMS/MS)in soil.
2.0 safety
2 I Always observe ssfe laboratorypractices 2 2 Consult the appropnite MSDS before handling any chemical for pwper rslel)
precautions.
3.0 Sample Requitwnent
3.1 At least I5 g o f t a t sample for cxlraclion. 3.2 No sunple procaiing isneeded for soil samples. 3.3 Sampler stored rcliigeratcd should be rllowcd to cquilibmc to room
tempmnrrc. 3.4 All ramplcc must be lhoroughlymixed before be& sampled for extraction. 3 5 Sample collktim pmccdmes will be rpccilid in the sampling plan for this
project
4.0 Reagent8 and Standards
4.1 Watcr-HPLC l p d e 4.2 Melhnnol- WPLC @de
4.3 AmmoniumAcclate- ACS. Reagent Grade 4.4 Pcrfluorooctanoic Acid - Sigma-Aldrich
5.0 Inslrurncntand Q u i p m m
5.1 A hi& performulcs liquid chmmatopph capable of pumping up to 2 solvcnviequipped with a variable volumo injector capable of injecting 5.200 pLconnwtedto a tandom M s u S p c c m e t n (LCMSIMS).
5.2 A device to wllcctrw data for peak integration and quanlitation. 5.3 Annlylical b a l m capable of reading to 0.00001g. 5.4 50 mL disposablepolypropyleneccntrihge tuber. 5.5 15 mL disposablepolypropylenecentrifuge tuber. 5.6 Disposablemicropipeti (50-10OuL. 100-2OOuL). 5.7 125-mL LDPE narrow-mouthbottles. 5.8 2 mL clear W L C vial kit. 5.9 Disposablepipettes. 5.10 Autopipettes(lOO-looOpL and 10-100 pL), with disposable tips. 5.11 WatcnScpPakVu:6cc(lg)tC18SPEcanridger. 5.I 2 SPE vwum manifold. 5.13 UlepaoNc bath.
Page 24 of 65
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Exygen Protocol Number: POOOl 13 1
Exyson R.uusb
MsfiwdNumbcrVW1781
I 1 ANALYTICALMETHOD Mabod of AnaIyk for the Ddmmirutionof Pafluomoctmoic Acid (PFOA) in Soil by
Lc/MsIMs
5.14 Wrist-action 5.15 Centrifugecapable of spinning50 mL plypmpylcnc tubes a1 5000 rpm
6.0 ChmmatogrlphicS y a m
6.I Andylical Column:FluophareRP (KeyrtoneScientific). 2.I mm x 50 mm. 51 (PM: 82505452130)
6.2 T c m p n a ~ n3: 0 T 6.3 Mobile Phue (A) : 2 mM Ammonium Acctafe in Watcr 6.4 Mobile P h w (E) : Methanol 6.5 Gradient Progrm:
Timc(min)u
0.0
65
1 .o
65
8.0
25
20.0
25
22.5
65
Flow Rate
Yi.Bl!dAw!l
35
0.3
35
0.3
15
0.3
75
0.3
35
0.3
6.6 6.7 6.8
- Injection Volume: 15 pL (canbe i n c m c d to u much &r 50 pL).
Qumtilation: Peek h-cxtanrl irmdudcalibration c w c . Run T i c : 23 minutea.
mu: P
The aboveconditions are intcdcdu I fide and msy be changedin order ts
optimizc the
cyrtm.
7 0 MSNSSystcm
7.1 Mode: ElcctrorprayNegative MRM mode,monilonng 413 + 369 m k for
PFOA.
The above conditionsare intendedu a guide and may be changod in order IO optimizethe MSMS ryalom.
8.0 Preparationof Solutions 8.1 Mobile P h w
8. I .I 2 mM ammonium acstple in wafer is prepared by adding 0.I54 g or ammonium pccIafe IO 1000mL of water.
AltaMte volumes maybe pcpprcd.
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Interim Report # 17 - Analysis of Sediment and Water Samples Exygen Study No.: PO00 1131
Exygen Protocol Number: PO001 13 1
@-
Exypen Research
Mstbad Number VWOl181
I I ANALYTICAL METHOD Method of h d f l i r for the Dstorminationof PsrIluomoctMoic Acid (PFOA) in Soil by
LC/MS/MS
9.0 StandardP r e p d o n
9.1 StandardStockEonification Solution 9.1.1 Prqwc Irtock lolution of -100 fie/mL of PFOA by wcighing IO rng of analytical s t M W (corrsctcd for purity) and dilute to 100 mL with melhmwl in a I25.mL LDPE bottle. 9.1.2 A IO )Ig/mL foltificntionsolution of PFOA is prepared by bnnying Iil
mL of he 100 pg/mL solution to a final volume of IO0 with m r l h m l
in a 125 mL LDPE botlle. 9.1.3 A 1.0 pe/mL fortificationsolution of PFOA is prepared by bnnging I O
mL of chc IO fig/mLsolution to a final volume of IO0 with methanol 111 a 125 mL LDPE bottle. 9.1.4 A 0.1 pe/mL fonificntion solution of PFOA is prepared by bnnging 10 mLofw I.Opg/mLsoldontoafinal volumcof 100 withmsthsnol in a 125 mL LDPE bottle. 9.1.5 A 0.01 pe/mL foltificalion lolutiou of PFOA is prepared by bnnging IO mL of thc 0.1 pe/mL solution lo a final volumc of 100 with mectnrol in 8 125 rnL LDPE bottle. 9.1.6 Tb. stock urd fortificationsolutions .are to be stored in a refrigerator at lpproximntely4*C mi are stable for a maximum period of 6 months from the date of preparation.
9.2 StandardCalibrationSoluliont
9.2.1 LC'MS/MS calibration sltariduds M prepared in HPLC water The calibration standard8 u e procarscd through the extraction proccdure. identical to samplw.
9.2.2 The following is a lypicnl example: additional wncentralions may bc prepared u nceded.
Find
Conccntrsaon Fmrificarion Volume of Concentration 01 Callbralion
of Fodifhtion Volume FortifiedCanml C~libration Standard ID
Solunon (ppb) (rL)
Sample (mL) Standard (ppt)'
(exarnplcl-
0
0
40
0
XCmmddyy-0
IO
100
40
25
XCmmddyy-l
IO
200
40
50
XCmmddyy-2
IO
400
40
IO0
XCrnmddyy.1
100
100
40
250
XCrnmddyy-4
100
200
40
500
XCmmddn-5
LOO
400
40
1000
XCmrnddn.6
-Ihc cmsctod 0onccnhDtionof the calibration -dad i: q u a l lo 8r its initial
concanIration,duo to the Mnsmeationof the atMdard during the extraction (SPE) XC urmctcdcalibration 8tmdard.
Page 26 of 65
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Page 86 of 127
Interim Report # I 7 - Analysis of Sediment and Water Samples Exygen Study No.: POOOl 131
Exygen Protocol Number: POOOl 13 I
Exygm Rncucb
M E WNvmbv VwO1781
I J ANALYTICALMETHOD Metbod ofAndysL for Ibs DaenninltionofPcrtluorooctMoic Acid (PFOA) in Soil by
LC/MS/MS
9.2.3 A zao standard Mllulion (reagent blank) wi be D r e D m wtih each
rst of atanduds extracted. 9.2.4 Store 111utncted calibration standards in 15-mL polypropylene tuber
n t 2 T to 6'C, up to two wccks. 9.2.5 Allemate volumes and concentrations of standards may be prepared as
noodcd.
10.0 Batch Set Up
10.1 Each batch of uunples extracted (typically 20 or less) must include ai ledst one reagent contml (method blank using 5 mL of methanol) and iwo reagent conlmll fortified ai known wnccnlrations (lab control spikr) to verity proccdunl rtcovay for the batch.
10.2 Requimncntr for field and laboratory duplicates and spikes will bc rpcciilcd IUthe quality wsuranco plan for Ibis project.
11.0 Sample Extraction
11.1 Weigb 5 g of ample into 50 mL polypropylene centrifuge tuba (fortify as n d d , replacc lid 4mix well).
11.2 Add 5 mL ofmahmol lad &&e on a Wrist action shaker for -I 5 minutes 11.3 Transferthc tuba to an ulepsonic bitb and eonicatc for -1 5 minuies. 11.4 S h g the volume up to 40 mL with watu in the 50 mL polypropylene
ccntrifugc Nbc. 11.5 Cmntrifugc for -10 minutes at -3OOO ipm.
11.6 Condition lho CI, SPE cartridges (1 g, 6 mL) by psssing IO mL methanol followed by 5 mL of HF'LC watm (- 2 dmp/sec). Do nor let column run dry
11.7 Load (decant) the sample on the conditioned CIS SPE ciulridpe Discard
- elunte.
11.8 Elute with -5 mL lOOK methanol. Colla1 5 mL 01"eluate inlo gradualell 15 mL polypropylcncccnVifUpctube8 (final volume 5 mL).
11.9 A n a l p samples using eleetrospray LCIMSIMS.
12.0 Chrmnatogmphy
12.1 Inject lho same u ~ u nolf each standard. sample and fortified sample into the LoMSlMS system. A calibration standard musi precede and follow all nndyrod samples.
12.2 Standards ofPFOA comcpondmg to ai Iwt five or more concentration levels must be ineluded in an .rul)4'c.l set.
12.3 An mtim $e4 of sxesCted calibration standards must be included ai Ihr: beginning and at the cnd of a sample Bet. Extnctcd standards must be intmpauod batwwacvwy 5-10 samples. As an alternative. M entire sst of
PI^ 5 017
F-
Poge 2 7 oJ 65
. ._
Exygen Research
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Interim Report #17 - Analysis of Sediment and Water Samples Exygen Study No.: PO001 13 1
Exygen Protocol Number: PO00 1 13I
Exygrn.Rouuch
Mehod Nun&, VWO1781
I I ANALYTICALMETHOD M&od of Analysis for theDctdnPlion of Pcrfluomtanoic Acid (PFOA) in Soil by
LCIMWMS
nlnclcd calibration rtandPrds may be injected SI the beginning of a set followed by ubaclcd cnlihtion strmdsrb intorsporwd cvory 5-10 samples
(to Lccount for a second 6el of oxtrscled standards). In either cwe. extracted calibration reuuiardr muat be the f a t and l u t injection in a ample sei 12.4 Use linw standud CWCI for quantitatioo. Linear standard curves art. gcncrslcd for the anal9 by lincarr e p s i o n using I/x weighting of peak arcs vmus calibralion itandnrd concentration usrng MassLynx 3.3 (or quivallcn~~ soffwplo sylum. 12.5 Sample rupousc should Mt cxcecd standard rcsponscs. Any samples that e x c d s l u d v d ruponsea should be further diluted and reanalyred
13.0 AcceptMce Criteria
13.1 Chromtopnmmust show 8 pork of 8 daughter ion at 369 a m u from a panni
of 413 mu. The 413 m u parent corresponds to the PFOA anion, while the
daughler ion (369 mu) rqrrrrentsthe loss of carbondioxide.
13.2 Method bla& must wI contain PFOA 81 levels p e a t n than the LOQ. I f d
blank contuns PFOA at Iwcla greater UW 50 ngL. lhen a new blank sample
muit be obtained and the entire le1must be re-extracted.
13.3 RWVU~CIof conkol spiker Md mabix spikes must be buween 70-130% of
their known valua. If a conlrol spike fslls outride the acceptable limits. the
entire set of l~lplnshould be re-cxtnelcd. Any mntrix spike outside 70.
130% should be FvlluDtcd by the analyst to determine if re-extraction is
,-
warranted
13.4 Any calibration ltandard found to be a atatistical ourtier by using the Huse
Enur Teat. may bo cxcludcd fmm tho calculation of the calibration curve
However, thc tot.1 number of cxesctod calibrarion standards that could bc
excluded must not cxcccd 20% of the total number of cxuacied stdndards
injatcd.
13.5 The cornlalion coefficient (R) for calibration curves generated must hu
20.992 (R' rn.985). If calibr8uon results fall outside these lim~is.tlicli
appropriate slop6 mu81 be Wen to adjust instrument operation. and llie
swdsrds or the relcvanl act ofsunplea ahould be rcMalyzed.
13.6 Retention times bMwm ruaduds and samples must not drift moic than
i 4 K within an d y t i c d fun. If relention time drifl exceeds this limit within
an analytical run then the set must be rearulyd.
Exygen Research
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Page 88 of 127
Interim Report # 17 - Analysis of Sediment and Water Samples Exygen Study No.: POOOl 131
Exygen Protocol Number: POOOl 131
e X y p Ruearch
MclhodNumber VOW1781
1 AXUUYTICAL METHOD
I
Method of ANlyxir for the Dctmnination of PcrtluomoctPaoicAcid (PFOA) in Soil by
LC/MSiMS
14.0 Cslculaliom
14.1 Use the following cgu.tion la calculate the mount of PFOA found (in ndL.
bucd on peak area) using the standard curve (lincar repcssion parameters)
generatedby the M.u L y u aothvarep m g r m :
- PFOA found (n@)
Y DF rlopc
-DF factor by which the find volume w a d~iluted,if necessary.
14.2 For sampkr fmificd wich h o w amounts of PFOA pnor to extraction. use
- the following
Rccovcry (%)
quation
to
caloulatc
the
pacent
recovery.
14.3 Use the following equation to convcr( the amount of PFOA found in ng/L lo
n%s@PW.
PFOA found @pb)
-
sampleweight (5 8)
14.4 Use the following equation to calculste the amount of PFOA found in pph
- bascd on dry weight.
PFOA found @pb) dry weight PFOA
found (ppb) x
[ 100%
/
total
solids(%)]
Exygen Research
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Interim Report # 17 - Analysis of Sediment and Water Samples Exygen Study No.: PO00 1131
Exygen Protocol Number: Po001 131
ANALYTICAL METHOD MclbodNumber: VOOO1782
Method of Andydr for the Determlnrtlonof Perfluoroodanoic Acid (PFOA) in Sedlrnent by LUMSIMS
hnalytical Testing Facility:
Exygen Research 3058 R m h Drive State College, PA 16801
Approval By:
Paul Connolly Techniul Leader, LC-MS, ExygmRosoush
10IZbld Date
L Date
a+-
?
Exygen Research
TotalPagos: 7
Page 30 of 65
..
Page 90 of 127
Interim Report #17 - Analysis of Sediment and Water Samples Exygen Study No.: POOOl 131
Exygen Protocol Number: POOOl 131
1.0 scopc
This mahod is to be employed for the isolation and qwntitation of perfiuoroocmolc acid by High Pcrformlncc Liquid Chmmatopphy wupled IO a tandem Mass Specoomohic Detector(LC/Ms/MS) in rcdlnmt.
2.0 safely 2.1 Away, obcave d e labommypractices. 2.2 Consult the appmpriuc MSDS before handling any chemical for proper safety prreautiw.
3.0 Sample Requinmcnt 3.I AI lea81 30g of lest sample for exlrsclion. 3.2 No samplc processing is needed for rcbunenr samples. 3.3 Smplcr stored rofrigecrstcd should be allowed to equilibrate io Toon: tmpanturc. 3.4 All rpmpla must be thoroughly mixed before being smpled for exmction 3.5 Sample collection prowdurn will be specified in the sampling plan for 1111s projd
4.0 RagcntsuulStandards 4.1 Water- HF'LC grndc
- 4.2 Mothano1 HPLC grade - 4.3 Acctic Acid Reagent g ~ d e - 4.4 Ammonium Acetate A.C.S. Reagent Gradc
4.5 PWnwoocwOic Acid -Sigma-Aldrich 5.0 lnstnuncnl and Equipment
5.1 A high performrmcc liquid chromatograph capable of pumping up to 2 solvmtr equippedwith I variable volumo injwtor capable of injecting 5.200 pL connected to a tandm Ma6r Spcclrometcr(Lc/Ms/MS).
5.2 A device to wllccl raw d a h for pcak inlegration and quamitarton. 5.3 ANlyrioal b l l u r e capableof d i n g to 0.00001 g. 5.4 SO mL diapotable polypropylene ccnaiffip tubes. 5.5 I5 mL dinporablo pol~mpylonccnlrifugelubes. 5.6 Disposablemicropipets (SO-IOOuL,100-200uL). 5.7 125-mLLDPE narrowmouth bottler. 5,s ZmLclearHPLCvillkit. 5.9 Dispopolablepipsttcs. 5.10 Autopipltes (100.1OOO pL and 10-100pL). with disposable tips. S.11 WatonSopPakV~c6cc(lp)rC18SPEcartridgcr. 5.12 SPE vacuum nunifold.
F
Page 31 of65
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Exygen Research
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Interim Report #17 - Analysis of Sediment and Water Samples Exygen Study No.: PO001 131
c
E x y g e n Protocol Number: PO00 I I3 1
Exma Rclcvsh
Method Nvmbcr VW01782
5.13 Vortcxcr. 5.14 Wrist-mion .hJrsr. 5.15 CenPifugecapable of spinning50 m l p o l p m p y l u w N b a at3000 rpm.
6.0 Chromatographic System
6 I Analyticll Column: Fluophasc RP (Keyslone Scientific). 2. I m m x 50 mrn. 5 ~ 4 (PM: 82505-052l10)
6.2 Tcmpcrm: 300C 6.3 Mobile Pbuc (A) : 2 mM AmmoniumAcorate in Water 6.4 Mobile P b (3): M e h o l 6.5 W c n t h g r s m :
Tichin)
0.0
65
1.0
65
8.0
25
20.0
25
22.5
65
Flow Rate
%!Jh!&!nJ
35
0.3
35
0.3
75
0.3
75
0.3
35
0.3
6.6 6.7 6.8
- - Iqjcction Volume: IS pL(can be inclrsred to as much 81 50 pL).
Quntitation: Peak Area external rtMdardcalibrationcurve. Run Time: 23 minutu.
The above conditionsarc inlcrdcd 81 a guide and may be changed in order ID
optimizethe HPLC syetem.
7.0 MSMS Syrtem
7.1 Mode:ElcctmlprayNegUive MRM mode, monitoring413 -+ 369 d z for
PFOA.
The above conditionsarc intended 81 8 guide and may be changed in order to optimizethe MSMS ryltem.
8.0 Prepmion of Sohtionr 8.1 MobilePhvc
8.1.1 2 mM ammonium aceWe in water is picparod by adding 0.154 y o f ammonium poctate to loo0 mL of water.
Exygen Research
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Page 92 of 127
Interim Report #17 - Analysis of Sediment and Water Samples Exygen Study No.: PO001 131
Exygcn Protocol Number PO001 13 1
F
E x y m Rc-b
McrhodNumber VooO1782
L J ANALYTICAL METHOD
Method ofArulysis for the Dstaminafion of Pcrflwmoclanoic Acid (PFOA) in Sediment by
LciMSlMS
8.2 Extraction Solutim
8.2.1 I% Pcnic acid in wafer is p m p d by adding IO mL of acetic acid ID IWO mL of water.
Allemalevolumes maybe p'oparcd
9.0 S~andudPrcparation
9.1 Swdrrd SUrWorrificotion Solution 9.1.1 Prepare 8 atock wlution of -100 &mL of PFOA by weighing 10 iiiy of ~ r l y r i ~ l l s U n d r (rcdorrected for purity) and dilute to 100 mL w d i methanolin a I25-mLLDPE bottle. 9.1.2 A IO j~&& forIification solution of PFOA is prepared by bringing I I)
mL of lhe 100 &mL solution to a fmal volume of 100 with mthnol in a 125 mL LDPE bonk. 9.1.3 A 1.0 pg/mL fonifiution solution of PFOA is prcpared by bnnginy IO mL of the IO p h L wiulion to a final volume of 100 with methanol in a IZS mL LDPE bottle. 9.1.4 A 0.1 j~ghLforlificationsolution of PFOA is prepared by bnnpng IO mLofthe 1.0p~~solutionloafinalvO~um1e0o0fwithrnclhanolin a 125 mL LDPE bottle.
9.1.5 A 0.01 pg/mL forlification wlution of PFOA i s prepared by bringing 10 mL of the 0.1 PdmL wlution to a final volume of I00 wiih moUmolina125mLLDPEbonlc.
9.1.6 The m k ud fomficationsolutions are to be stored in a refrjgerafor at
appmximauly 4`C and ITC stable for a maximum period of 6 monlhs h m cho doto of preparation.
9.2 Standard c.libration Solutions
9.2.1 Lc/Ms/MS ulibntion rlandard8 arc prcpared in methanol via dilulion
of the 0.1 &nL fatificuion solution.
9.2.2 `Ihe following is a lypical example: additional concentrationsmay be
VrcPKal aY d e d .
Conantntim
Final
of Fonidution Vohrmo
Lhlutcd 10
COnCenmlion
Solution (@I&) (mL)
(mL)
(ng/mL)
1.0.0.
10
1.W.
10.0
100
$
100
50
too
2
100
20
10
10
100
IO
5
IO
100
05
Exygen Research
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Page 93 of 127
Interim Report #17 - Analysis of Sediment and Water Samples Exygen Study No.: POOOl 131
..--
Exygen Protocol Number: POOOl 13 I
Ex.m_mRororrch
M N k d Nvmbrr VOOOl782
1 1 ANALYTICAL METHOD
Method of h l p i r for the Determinotionof Perfluomoctlnoic Acid (PFOA) in Sediment by
Lc/MS/MS
9.2.3 S t m all cslibruion rkndnrd6 in 125-mL LDPE nlumw-mouth bottles .tZ'C to 6C.up to six monthr.
9.2.4 Allemate volumcn wl wnccntrations of slMdards may be prepared as needed.
10.0 Bnlch Set Up
10.1 Each batch of rpmplw exIxaclcd (typically 20 or las) mun include a1 least one unlratcd mIrul and hvo unlnalod controls fortified at known CornartraIionr(lob mtml spike) to V d f yprocedural recovNy for the batch
10.2 Rquimsntr for field 4lrbontory duplicates and spikes will be soattied
in the quality LUYMEC plvl for this pmjat.
1I .O Sample Extraction
11.1 11.2 11.3 11.4 11.5 11.6
11.7 11.8 11.9 11.10 11.11 11.12 11.13
Weigh 5 g of iample into 50 mL plypropylene m t n f u g e tubes (forllfy ds
nccdcd. nplsce lid mimix well).
Add 35 mL of 1% acetic acid. cap. vortex mi shake on a wnrt action shaker
for -60 minuta. Cennifves UK tubed u -3000 rpm for -20 minuter.
Condition the CII SPE cutrid~er( I g 6 mL) by passing IO mL methanol
followedby 20 mL ofHPL water (- 2 dmp/scc). Do no! let column NII dry
Load (-1)
the u ~ p okn the conditioned C M SPE cartridge. Discard
eluate.
Add 20 mL of m c h o l to the a d i e n t IcA in the bottom of the SO rnL
ccnbifugc tube. Cap, vortex md shako on a Wrist action shaker for -30
minuta.
Ccnnifugo tho tuba nt -3000 QUI for -20 minuter.
DwMt the methanol onto the 8am SPE cutridge. Collw1 the eluate
Wash the wlumn with 4 mL of mcthpnol. Collect rhe eluate and add it to lhc
elur(s Eollatcd in ntep 1I .8.
Condition a m o d Cta SPE cartridge ( I g. 6 mL) by passing 10 mL methanol
followed by 20 mL of HPLC water (- 2 drop/crcc). Do not let ~ l u m rnun dry
Add the mslhnol to -200 mL of wrtcr m d load on the saond conditiotied
- SPEd x e .
Elute with 5: mL LOO% m c h l . CoUoct 5 mL of eluate into graduated 15 mLpolypropylcne centrifuge lubes (final volume 5 mL). Anll~eplauringolccborpr~yLCIMSIMS.
Exygen Research
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Page 94 of 127
Interim Report #17 - Analysis of Sediment and Water Samples Exygen Study No.: POOOl 131
Exygen Protocol Number: POOOl 13 I
1 I ANALYTICALMETHOD
Methodof Andyns for the Dstamiwion ofPorflwmoetanoic Acid (PFOA) in Sediment bv LC/MS/MS
12.0 Chmmatography 12.1 Inject the same amount of each standard, sample and fortified sample into the LCIMSIMS ryltcm. A calibration standard must precede and follow ail uulpcd rampla. 12.2 Standard6 ofPPOA corraponding to at least five or more concentralion ievels muat be included inan wlyiical set. 12.3 An entire set of extracted calibration standuds mu1 be included al rhv beginning and at the end of a sample set. Standards must be inrerspened bmucon o v q 5-10 m p l a . h an .Iternalive, M enlire set of calibration rtand.rdr m a y be injected at the beginning of a set followed by calibratiun standuds inknpencd every 5-10 ampler (to account for a second set of aunduds). B eitha c w , calibration 6Iandards must be the first and ldsi injectionin Isamploset 12.4 Use limu standmtd c w c s for gumtitation. Linear standard curves arc gcnaplodfor the d y t c by linear regression using l/x weighting of peak are.! vmus calibration standard concentration using MarrLynx 3.3 (or equivalent) WoAwSro syrm. 12.5 Sample rwponrc should not excad rmdard rcsponsu. Any samples lhat exceed atnn.ndardrcsponaes a b u M bo m e r dilutcd and rmalyred.
13.0 Acccptanw Critcria 13.1 ChromatogrunmyI( h o w a peak ofa daughter ion at 369 m u from d' parent of 413 MU. Thc 413 MU parent cormponds to the PFOA anion. whiie the daughter ion (369 m u ) r c p m b the loas of &on dioxide. 13.2 Method blanks must MI contdn PPOA at levels greater than the LOQ. I f a b l d conuins PFOA at lcvela greater than 0.2 ng/mL. then a new blank sample muat be oblnined and the entiro sd must be re-extracted. 13.3 Reeovericr of conml #pika and matrix &ea must be between 70-:30% of their known vrlua. If a control #pike hUs outside tho acceptable limits. the entire act of samples t b u l d be re-extracted. Any matrix spike oulstde 701300h lhould be evrluatod by the analyst to determine if re-extraclion i s wartanted 13.4 Any calibration rtandard found to be a statistical outlier by using the HUBE Enor Test,m y be excluded from the calculation of the calibration CUM However. the totll number of cxtnctcd calibration standards that could be excluded must not excad 20% of the total number of extracted standards injstai. 13.5 The conehtion oooficicnt (R) for calibration curves generated must he 20.992 (R' 20.985). If calibralion rclults fall outside these limits. tliun Bppmpriak ncpa must be taken to adjust instnunen1 operation. and Ihe standard,or the relevant set of sampler should be reanalyzed.
Exygen Research
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Page 95 of 127
Interim Report # 17 - Analysis of Sediment and Water Samples Exygen Study No.: PO00 1 131
Exygen Protocol Number: PO001 I31
Exygen Rowrcb
MclbodNumbn V W 1 7 8 2
t 1 ANALVTKX.L METEOD
Methodof AndyQ forthe Dncrminrdion ofPduorooclanoicAcid (PFOA) in Scdimcni by
Lc/MsIMS
__
13.6 Retention time8 bawm rtandards and ramplm must no1 dnR more lhdn f 4 % within m snrlyticalrun. If retcntlon timedriI? excads thls l m t within u1 lnnlyticd NO then the set must be rcanalyzod.
14.0 Cdculations
14.1 Use rho following equation to cllculptethe mount of PFOA found (In n u m i . b w d on p u k UM) wing the standard C U N ~ (linear ngmsion paramclcn) generated by Ibc Mw Lyw (ofhYp10program:
- - PFOA found (ng/mL) PcJr area i n t c n a x DF slops
-DF factor by which UK finalvolume was diluted, if necessary
14.2 For rsmplm fortified with known amounts of PFOA prior to extraction. use
- the following
Rccovsry (%)
equl(i0n
to
cdculate
the
patent
ncovny.
14.3 Uac the following equation to convcn the amount ofPFOA round in nghL in
- n%n @iW. PFOA found @pb)
PFOA found W m L ) x final volume ( 5 mLU sample weight (5 8 )
14.4 Usc the following equation (if necwuy) to calculate the amount of PFOA
- found in ppb based on dry woight.
PFOA found (ppb) dry weight PFOA found (ppb) I [ 100%/ total sol~ds(%ll
F-
Pnge 36 .f 65
Exygen Research
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Interim Report #17 - Analysis of Sediment and Water Samples Exygen Study No.: POOOl 131
Exygen Protocol Number: POOOl 13 1
ANALYTICAL METHOD Method Number: V0001783
Method of Andyalr for tbe Detcrml.afion of Perfluorooctanolc Acid (PFOA) in Fish and C l a m by LClMSlMS
Analytical Twtinp Facility:
Exygen R w m h 3058 Rsscuch Drive State College, PA 16801
Approved By:
Date
Exygen Research
Page 37 016j
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Interim Report #17 - Analysis of Sediment and Water Samples Exygen Study No.: PO001 131
Exygen Protocol Number: PO001 13 1
Exmen R e s w h
M c h d Nuder VW01783
I I ~ A L Y ~ I C AMLETHOD Mcthod of Anslylirfor lhc DetorminrtionofPcrfluomoctanoic Acid (PFOA) in Fish and
C h r by LcIMS/MS
1.0 scope
Tbir method is to bc employed for the isolation and quantitation of perfluoroosianoic acid by High Performance Liquid Chromatography coupled to a iandeni Masr SpcclrometricDckdor (LC/MS/MS) in h h and clams.
2.0 safely
2.1 Alwayx observe d e labmatory practices. 2.2 Consul1 the appropnale MSDS before handling any chemical for proper rafeij
procautions
3,O Sample Reguiremcnl
3. I AI I& 20 g of test m p l c for nrmctian. 3.2 Samples should be proccased bsforc cxesction. Plncc the frozen sample in a
food pmscssur and homogcnix with dry ice. Place the samples in containers snd leavo open in frozen sloragc ovanight lo allow for carbon dioxide rublimdon. Seal ~d place the u m p l a in fiozcn storage unhl time of anslyria. 3.3 Sample mllection pmcedwa will be specified in the sampling plan for this project.
4.0 RwcntsandStPndYb
4.1 water-HPLC grade
4.2 AectoNUile - HPLC @e 4.3 Carbon (120-400 mesh) - RklgCnl grade
- 4.4 Mclhanol WW grade
- 4.5 Silica gel (60-200mesh) Reagent grade
- 4.6 Ploriril(aO-l00 mesh) Reagont grade
- 4.7 ~upCl'ClCMLC-NHz Rspgsnl grade
4.8 1-&1MOI -
@C
- 4.9 L-Ascorbic acid Reagent grade
- 4.10 Dimethyldichbro~ilm Reagent @e
- 4. I 1 Tolucnc Reagent pndc
4.12 Ammonium Acocstc - A.C.S. Reagent Grade
4.13 P ~ u o m o c t a n o i cAcid- Sigma-Aldrich
5.0 Inshummuand Equipment
5.1 A high pcrfomuncc liquid chmmatograph capable of pumpin8 up to 2 solvents equipped with L variable volume injoctor capable of injtciing 5.200 pL connected lo a tandem MMSSpcclromcter(LCIMSIMS).
S.2 A device to collccl raw d.(. for peak integration and quantitation. 5.3 Analytical balance capable of d i n g to 0.WWI g.
Exygen Research
Page 98 of 127
Interim Report # I 7 - Analysis of Sediment and Water Samples Exygen Study No.: POOOl 131
Exygen ProtocolNumber: POOOl 131
Exyya W
M&cdNwnbcrVW0178)
I 1 ANALITICALMETHOD Method of M y a i r for the Datormination of Pcrfluorowtanoic Acid (PFOA) In Flsh and
Clamr by LC/MSIMs
5.4 R o w svsporalor. S.5 Tirfllmizcr. 5.6 125 mL pea-shaped flpEkl. 5.7 50 mL diapoiible polypropylenecentrifuge tubs.
5.8 I5 mL dimoslble ~olv~rcmvlmcecntrifunc tubes. 5.9 Disposlbk micro&;; (~6100uL.100-~00uL). 5.10 IZS-mL LDPE mow-mouth bonlcs. 5.1 I 2 mL clcu HPLC vial kit. 5.12 Disposablepipdtu.
5.13 AulopipOnu (100-1000 pL and 10-100 pL), with disposable tips. 5.14 SPE tuber (2%) (Supclcocat. no. NO57177).
5.15 Wrist sction shakos. 5.16 Centrifugecapable of apinning 50 mL polypropylene tubes at 2000 rpni.
6.0 ChromatographicSymm
6.1 Analytical Column:Fluopbaoe RP (Keystone Scientific), 2. I m m x 50 mrn. j g t ( P N 82505.052130)
6.2 Tcmpcmnur: 30T 6.3 Mobile Phuc (A) : 2 mM Ammonium A ~ ~ t aitneWater 6.4 Mobile Phuc (E) : Methanol 6.5 GradientRogram:
Trmc(minlu
0.0
65
1 .o
65
8.0
25
20.0
25
22.5
65
Flow Rate
mluum
35
0.3
35
0.3
75
0.3
75
0.3
35
0.3
6.6 6.7 6.8
- - InjectionVolume: I5 pL (CMbe increased to as much as 50 pL). titation: Peak Arw external r t d a r d calibratron curve. 2 T h e : 23 minutes.
The above condition4PIC intended as a guide d may be changed in order to optimize the HPLC system.
Exygen Research
Puge 39 o/65
.
Page 99 of 127
Interim Report #17 - Analysis of Sediment and Water Samples Exygen Study No.: PO001 131
Exygen Protocol Number: PO00I I3 I
1 I ANALYTICAL METHOD Method of Anelyalr f a Ihs Dstarmnlclooof Pafluomoclanoic Acid (PFOA) in Fish and Cluns by LCMSIMS
7 0 MSNSSptcm
7 I Mode ElccDospny Negative MRU mods. moniionng 413 --t 169 .ti I ' 0 1 PFOA
The above conditions are intended M a guide and may be chuiged in oraer IO opnmize the MSMS system
8.0 Preparation of Solutionr
8.1 Mobile Phase
8.1.1 2 mM munonium acctate in wator is: prepand by adding 0.154 g o f munoniwnacetate to loo0mL of water
8.2 Exeaction S O ~ U ~ ~ O M
8.2.1 2% ascorbic acid in methanol is prepared by dissolving 2 g of ascorbic acid in 100 mL of methanol.
8.2.2 30% Dimethyldicb!-am6ilanein toluene is prepared by bnnging 3 rnl of dimcthyldichlororilane to a Final volumc of 10 mL with toluene
Alternate volumes may be pnparod.
9.0 Standard Prcpmtion
9.1 Standard StocWortifioation Solution
9.1.1 Prepare a mock solution of-100 &nL of PFOA by weighing IO m g of lnalyticnl standard ( c o r n l e d for purity) and dilute to 100 rnL with methanolin a 125-mL LDPE bottle.
9.1.2 A 1.0 p#mL fortification solution of PFOA is prepared by bringing I mL of the I 0 0 p@L iolution to a final volume of 100 with methanol in P 125 mL LDPE bottle.
9.1.3 A 0.1 po/mt fortification solution of PFOA is prepared by bringing I O mLofthe l.O)~#mL~clutionlo~fi~lvolwn1c0o0fwith mrihunul iii a 125 mL LDPE bottle.
9.1.4 A 0.01 p#mL fortificationsolution of PFOA is prepared by bnnging IO mL of the 0.1 p#mL solution to a final volume of 100 w:h mcfhuuJIn a 125 mL LDPE bottle.
9.1.5 The it& and fortification solutions arc to be stored in a refrigerator a i approximately 4 O C and arc rtnblc for a maximum perid or 6 monihs hom the date ofpreparation.
. ...
...
Page 4 n i 8
Page 40 of 65
Exygen Research
Page 100 of 127
Interim Report #17 - Analysis of Sediment and Water Samples Exygen Study No.: PO001 131
Exygcn Protocol Number: PO001 I3 I
Exygen Rcnvch
Me1bodNumkrYW01781
1 I ANAJ.tTiCM METHOD Method of Analycii fm tbc Dclaminuion of Pcrtluomcctanoic Acid (PFOA) in Fish and
C l m by L c l M S M S
9.2 Stpndprd Calibration S O ~ U ~ ~ O M
9.2.1 LC/MS/MS calibrationstandards am propard in methanol via dilution of thc I .O&mL fortificationrolunon.
9.2.2 7he following is a typical wamplc: additional wncmtr81~oMmdy be prepared 81 needed.
ConoCntr.tirm
Final
of Foltirulion Solution (11BhnL)
1 .o I .o I .o
Volvms I d)
5.0
2.5
I .o
Dtluted to Id) 100 IO0
100
Concentration (re'mL)
0.0s
0.025 0.01
0.0s
IO
100
0.005
0.025
to
100
0.0025
0.1
10
100
0.001
0.005
IO
I00
0.0005
9.2.3 Store all calibration ttPndrrdr in 125-mL LDPE nmw-mouth bottles
at 2-C to 6OC. up Io six rnonlhr.
9.2.4 Alternate volumes and conccnIrationsof standards may bc prepared as
needed.
10.0 Batch Scl Up
10.1 Epcb batch of umpb cxtrpcted (typically 20 or Ius)mu81 include al least one unt?catsd contml and two unmated controls foniflcd at know w w s n t n t i o ~(la~b controlspike)to veri@ pmcedunlrecovery for the batch
10.2 Rquiransnlr for field and labomtory duplicates and spikes will be specified in the quality o 9 8 u ~ p~lacn for this project.
1 I .O Sample Exlraction
11.1 Weigh 5 g of frozen sample into 50 mL polypropylcnc centnfuge tuDes (foniw Ine&. replace lid and mix well).
11.2 Add 30 mL ofacelonilrile Mdshake on a wrist Bclion shaker for -1 5 minutcs 11.3 Place the tuber in a freslcr for -I hour. 11.4 Pack and wndition thc SPE and silnnizc the psar-shaped flasks. 11.5 Pack the 20 mL SPE t u b a in sequwcc with 2 g florisil. 2 g silica gel, 2 g
carbon, and I g LC-NHz.Condition lh wlumns with 20 mL of methanol.
thcm 20 mL of acetoNbilc. Discard all wlshca. Do not allow the column to
dry. 11.6 S i l h the 125 mL popr-sh.pcd flasks by rinsing wilh the 30%
dimcthyldishlomdlanc in tolume solution. Rim the flask with toluene once.
followed by mcthvlol (three times). Dry the flanks completely bsfore use. cilhaby airdyingor with a mwm of Ntrogsn.
Page 41 of 65
Exygen Research
Page 101 of 127
Interim Report # 17 - Analysis of Sediment and Water Samples Exygen Study No.: PO00 113 1
Exygen Protocol Number:PWOI 13 I
erypsnR o m h
MethodNumber VooOtl8)
I I ANALiTICAL METHOD Mcihod of Analnis for the Dctminltion of Perfluomtanoic Acid (PFOA) in Fish and
Clams by LCIMSMS
11.7 11.8
11.9
11.10 11.1 I 11.12 11.13
11.14 11.15 11.16 11.17 11.18
Cmtrihrge b e 50 mL polypropylene tubes containing sample at -2000 rpm
for -10 minutes.
Dccant the extract on to a conditioned SPE column fittcd inside the mouth of the pear-she@ flask. Collect the eluate in the 125 mL silanized pcar-shape
flak. Add 10 mL of acetonitrile 10 the sample in the 50 mL centrifuge tube
Homogenize the frozen fat phaae using a tissumizor for -30 seconds and nnse the i i i z o rwith -10 mL of acctoNeile into the tube.
- Sh&c the sampleagain for -10 minuter on a wrist-aftion shaker.
Place the tubes in a k n for I hour more. Cartrifige thc 50 mL polypmpylcnc tubes containing sample at -2000 rpm for -10 minutea.
D a m the exmot onto the same SPE calm. Collect the eluate into the same pear-shaped fld and combine with the eluent from the inmal
CXtrWtiOCL
Pass 20 mL of Icatoniailethough thc SPE column and combine the eluate in the same pw-shaped fluk. Add 3-4 dmpr of I-octanol to Ihe extract in the pear-shaped flask and cvaponts at r e d d prcsaurc wing a rotaryevaporator(at < 4OOC). Make the Garl volume, by adding 2 mL of 2% ascorbic acid in methanol io thcpw-shaped flask mdswirl to mix/diwlvc. Transferthe extncta to HPLC vials wing disposable pipets Analyze SPmplU uing elcctmprsy LC/MS/MS.
12.0 Chromatography
12.1 Inject thc u m c amount of each stwlard. sample and fonified sample into the LC/MS/MS syatcm A ulibmtion standard muit precede and fallow all d y r d IMplU.
12.2 Standard8 ofPFOA oonwponding to at lcast live or more concentralion l e ~ r l s mua be included in an analyticalsot.
12.3 An entire s(1 ofcalibruion standardsmust be included at the beginning and 31 the ond of a l~mpleset. Standard8 must be interspersed bctwcm every 5-1 0 sunplcs. Ar an alternative. an entire w1 of calibration standards may bc injected at the beginning of I ret followed by calibration slandards intaspmcd every 5-10 samples (to m u n t for L second set of standards) In either w e . ulibration slmdan% mum be the first and lwt in~cctionin il
sample set. 12.4 Use linw standard curves for quantitation. Linear standard curves are
gcnctatedfor the analylc by l i w ngrarion wing I l x weighting ofpeak area v m u l cdibntion aIandd concentration wing MauLynx 3.3 (or equivalent) ioRwarc syrtom.
... .
P i p 6 01 I
Page 42 of65
Exygen Research
Page 102 of 127
Interim Report #17 - Analysis of Sediment and Water Samples Exygen Study No.: PO001 131
Exygen Protocol Number: PO001 13 1
exygca RcKUch
M c d d N v m k r VwO1781
t 1 ANALYTICAL METHOD Method ofAnrlyiir for the Dotermination ofPorfIuomoctanoic Acid fPFOA) in Fish and
Clams by LUMS/MS
12.5 Sample respme should not excad standard responses. Any samples thri exwed standardr s r p o ~ eshould be W e r diluted and reanalyzed
13.0 Accsptance Criteria
13.1 Chmmatogrnm must show a puk of a daughter ion at 369 a m u from a pareni of 413 m u . The 413 m u p m l wrresponds to tho PFOA anion, while rhe daugbterion (369MU) rcpmcntithe loss of carbon dioxide.
13.2 Metbod blanks must not contlin PFOA at levels greater than the LOQ. If a blank contain# PFOA at levels greater lhan 0.S ppb. then a new blank sample mug be obtained ad the atire wt mustbe re-extrseted.
13.3 Rcwvsria of m l m l spikor and matrix lpikcs must be bclwecn 70.130% o f the knownv h . If a oollaol spike falls outside the acceptable limits. Ihe antire wt of camp108 should be re-extracfed.
13.4 Any ulibntion standard found to be a statistical outlier by using the Huge Enor Test, may be excludd from ulc calculation of the calibration CUNC Howeva, thc total nwnbcr of calibration m d u d r that could be excluded must not a c e d 20% of the total numbuof standards injected.
13.5 The cormlation coeltlciont (R) for cPlibrstion curves generated must be 20.992 (R' 20.985). If calibration multr fall outside these limits. then sppropr*to ncps mufl be taka Io adjust instnrmmt operation. and the P w d d or the mlevant wt of samples should be reanal@.
13.6 Retention times bctwcoo r t m d s r b and m p l e r musi not dnR more lhan f 4 Yo within an laplytical run. If releation time drill exceeds this l m i wiiluri an anrlytiul run then the ret must be reanalyzed.
14.0 Calculations
14.1 Uae the following equation to ulculats the amount of PFOA found (in ng/mL. b a d on peak arcs) wing the standard curve (linear regressionparameters) gareraledby the Muo Lynx soflware program:
PPOA found (ng/mL) -(Peak area. i n t a slope
14.2 Uw the following equation to w n v m the amount of PFOA found in ne/& to
no/8 @Pb).
- PFOA found @pb)
7 x fin-sI v
sample weight ( 9 )
-DF falor by which the final volume was diluted, if necessary
Puge 43 oj6S
Exygen Research
Page 103 of 127
Interim Report # 17 - Analysis of Sediment and Water Samples Exygen Study No.: POOOl 13 1
Exygen Protocol Number: POOOl 13 1
I I
Method of hoalyais for the Dstcrminationof PcrflwmoctMoic Acid (PFOA) in Fish and Clmr bv LCiMs/MS
14.3 For samples fortified with known mounts of PFOA prior 10 cxtraclion. ure the followingequation to calculate the pmcsnl recovery.
Recovery (%) =
1toid radytcfo~nd .ana~ytofoundin convol (ng/g)L ,oo
d y t c nddd(ng/8)
P-
Exygen Research
Page 44 o/ 65
Page 104 of 127
Interim Report # 17 - Analysis of Sediment and Water Samples Exygen Study No.: PO00 1131
ExygenProtocol Number:PO001 131
ANALYTICAL METHOD
Method Number: VWO1784
Method of Andy& lor the Dtterminnllon of Perfluorwclanoic Acid (PFOA) in Veprlation by LCIMSIMS
Analyaoal T&ng Facility:
Exygcn Rssoprch 3058 Rcsurch Drive Slate Colloge, PA 16801
Appmvcd By:
-I,
Paul Connolly Tscbcd Lcadcr, E - M S . Fxygcn Rcacuch
hnRphelly Vice Pnridcnl. Opcrationr, Exygcn Research
.
..
Exygen Research
TolalPagsr: 7
.__
Page 45 oj6S
Page 105 of 127
Interim Report # 17 - Analysis of Sediment and Water Samples Exygen Study No.: PO001131
Exygen Protocol Number: PO00 I 13 I
Exygcn R u r u s h
M c W Number VOllUliliJ
I I
AXALb 1ICAL METHOD
I
Method of Anslyria for the Detaminarionof Pe~uorooctmoicAcid (PFOA) in Vepeiai!on
by Lc/MS/MS
1.0 scop
This mclhod is Io be cmployod for the isolation m d quantitationofperfluorowtunoic r i d by High Paformurcc Liquid Chromatography coupled to a tandem Mass SpectrometricDctector (LC/MS/MS) in vegetation.
2.0 Safety
. . 2.1 Alwaya obKrvc d e laboratorypractices. 2.2 Consult thc a.m.rop.riatc MSDS before handlin-a any.chemical for DroDer sal`ctv
pmcautiolu.
3.0 SampleRequirement
3.1 At I m20 g of tert sample for extraction. 3.2 Samplw should be pmcused before exmtion. Place the frozen sample in a
food prucusor and homogenizewith dry ice. Place the samples in containers and leave open in frozen storage ovamight lo allow for carbon dioxide sublmstian. SUI urd place the ramplea in frozen storage until time o! analysis. 3.3 Sample collection proocduns will be specified in the sampling plan for :his project.
4.0 Reagents and Standuds
4.1 water - HPLC g n d o 4.2 Acetonitrile- HPLC grade 4.3 Carbon (120-400 mesh) - RmgenIprulc 4.4 M e h O l -HPLC grde 4.5 Silica gel (60-200 meah) - Reagent grade
- 4.6 Florisil(60-100 mesh) Reagent grade - 4.7 Supmlean LC-NHz Reagent grad0
4.8 I-Oct.nol- HF'LC @e
- 4.9 L-Aswrbic sfid Rugcnt grade - 4.IO D m c t h y l d i c h l o r u m ~ Rcngcnt p i e
4.1 I Toluene - Reagent @e
- 4.12 Ammonium Acetate AC.S. Reagent Gndc - 4.13 PafluomocUmic Acid Sigma-Aldrich
5.0 lnsmrmcnt and Equipment
5.1 A high pcrfomuacc liquid chromatograph capable of pumping up to 2 mlventa quipped with I variable volume injector capable of injecting 5-200 )ILconnsned IO m h Mrsr Spectrometer ( L C M W S ) .
5.2 A device to coUect nw data for peak integration and quantitation. 5.3 Analytical balancecapable of reading to O.oooO1 g.
Payc 2 "1 7
Page 46 of 65
Exygen Research
Page 106 of 127
Interim Report #17 - Analysis of Sediment and Water Samples Exygen Study No.: PO001 131
Exygen Protocol Number: PO001 13 1
E x y i m llrreush
Mclhod Number V W I 784
1 1 .WALYTICAI.METHOD
M e b d of hnalysh for tho Detminacion of Poduomoctanoic Acid (PFOA) in VcgeLauon
by LIMs/MS
5.4 Rotvy cvspontor. 5.5 125 mLpear-Ehapcd tlaah. 5.6 50 mL dispoublc polypmpyhs ~ S n t n f u tgu~ba. 5.7 I5 mL dirporrbl0polypropylalc calltrihrgelubes. 5.8 D i s p a ~ b l cmimpipeta (SO-lWL, loCr2OOuL).
5.9 12s-mL LDPE numw-mouth bottles. 5.10 2mLclcsrHPLCvialkit. 5.1 1 Disposable pipettes.
5.12 Autopipctta(IoCrl0WpLMd IO-IOOpL).withdisposabletips. 5.13 SPEtubsa (2OmL) (Supclcoc~tn.o.N057177). 5.14 Wrist action shaker. 5.I5 CentrifugeCQnblc of spinning50 mL polypropylenetuber at 2000 rpm
6.0 Clwmalogrsphic Swan
6.1 Analytical Column: FIuophrscRP(Keystom Scientific), 2.1 mm x 50 mm.511 ( P N 82505-052130)
6.2 Tempalhuc: 3 0 T 6.1 Mobile Phase (A) : 2 mM llmmoniwn A ~ ~ t aint sWater
6.4 Mobile Phase(B): Mclhanol
6.5 GndiontRogram:
- ,
Timcou
u FlowwRate
0.0
65
15
0.1
I .0
65
15
0.3
8.0
25
75
0.3
20.0
25
75
0.3
22.5
65
35
0.3
6.6 6.7 6.8
- - lajcaion Volumo: I5 pL (can be increased to SJ much BS 50 pL).
Quanticstion: Peak Area external standard calibrationcurve. Run Time: 23 rninutu.
The above conditionsMintadcd u a guide Mdmay be changod in order IO optimii the HPLC ryrtom.
7.0 MS/MSSyltem
7.1 Mode:ElectronprayNegative MRM made. monitoring411 + 169 mir for
PFOA.
P.pe 3 "1 7
Page 47of6S
Exygen Research
Page 107 of 127
Interim Report # 17 - Analysis of Sediment and Water Samples Exygen Study No.: PO001 131
Exygen Protocol Number: PO001131
Gxypon Rnursh
Mcrhcd Nvmbcr VaW I784
I 1 Ati.uYrica mrnon
Method of Analpis for tho Dctcrminationof Porfluomcranoic Acid (PFOA) in Veyetalion
bv LCMSMS
The above condition8arc intended so a p d e and may bo changed in order to optimize the MSMS system.
8.0 WUafhlOf SOlUtiOM
8.1 Mobile Phase
8.1.1 2 mh4 ammonium scctate in water i6 prepared by adding 0.154 g of ammonium lcetatc to 1000 mL of water.
8.2 Extraction Solutim
8.2.1 2% u c o h i c acid in methanolis prepared by dissolving 2 g of ascorbic wid in 100mLof mahmol.
8.22 30% Dimethyldkhlomihe in lolucae is p n p d by bringing 3 mL of dknahyldichlomlilure to a final volume of IO mL with toluene
Alternate volumcr may be preplrod
9.0 Standard Rcparation
9.1 Standard StocWonification Solution
9.1.1 Prepare Istwk solution of-100 rB/mLof PFOA by weighins I O my of uulfical dandard (comted for punty) and dilute to 100 mL wuh methanol in a l 2 5 m L LDPE bottle.
9.1.2 A 1.0 FdmL fonification solution of PFOA is prepared by bnnping I mL of the 100 p g / d solution Io a h l volume of 100 with methanol in a I25 mL LDPE bottle.
9.1.3 A 0.1 pg4nL fortificationlolutionof PFOA is prepared by bringing IO mLof the I .O p@mLlolution to a find volume of 100with mcthanol m I125 mL LDPE bonk.
9.1.4 A 0.01 pvmL fortificationrolution of PFOA is prepared by bnnginp IO mL of the 0.1 pg/mL solution to a final volume of 100 with mcthnol in a I25 mL LDPE bottle.
9.1.5 The stock and fortifcation solutions arc to be stored in a refrigeratorai appmximstcly4'C md arc stable for a maximum period of 6 months fmm the date of prepantion.
9.2 Standard Calibration Solution8
9.2.1 LCIMSIMS calibration MndarJi PR prepared in methanol via dilution ofthe 1.0p g h L fortificationsolution.
Page 48 0165
Exygen Research
Page 108 of 127
Interim Report # 17 - Analysis of Sediment and Water Samples Exygen Study No.: POOO 1131
Exygen Prorocol Number: POOO I I3 I
1 I A h A L Y I ICAI. Ib1L rHOD
Method of A d y P i a for the Delmmination of PnlhorooctMoic Acid (PFOA) In Vcgelai,arl by LCMs/MS
9.2.2 The following is a typical exmple: additional concentrations may be p r c p d as n W .
..
..
I.o
1.5
100
0.025
1.0
1.0
100
0.01
0.05
IO
100
0,005
0.025
IO
100
0.0025
0. I
10
I00
0.001
0.005
IO
100
0.WOJ
9.2.3 Slon dl c s l i h d o n alandvds in 125-mL LDPE narrow-mouth bottles
.t2OC to P C , up to six months.
9.2.4 Allemotevolumes and conccneations of standards may be prepared ai
needed.
10.0 BatchSctUp
10.1 Each batch of urnplea crarctod (typically 20 or less) must include ai least one unlrcaled control and two untrsatcd controls fortified a! known conccntrationa (lab control spike) lo vcriY) procedural recovery for the baich
10.2 Requiremeno for field and liborslory duplicates and spikes will be specilied inthe qualityulurance plan for this project.
11.0 Sample Exmction
11.1 Weigh 5 g of frozen sample into 50 mL polypropylene centrifuge iubes (fortiw a needed, rcplscc lid and mix well).
11.2 Add 30 mL of acctOniailcand shake on a wrirt action shaker for -I J minuter 11.3 Centrifuge the 50 mL polypropylene tuba containing sample ai -2000 pi11
for-IO minuta. 11.4 Pack and condition the SPE tube# Md rilnnizc the pcu-shaped flasks. 11.5 Pack the 20 mL SPE rubm in requencc with 2 g llonril. 2 g silica gel. 2 p
carbon, and 1 g LC-NHI. Condition the columns with 20 mL of methanol. thm 20 mL of acetonibilc. Diacard all washm. Do no1 allow the column io
11.6 Silanize the 125 mL peu.shaped flasks by rinsing with the 30%
dimcthyldichlomilanc in toluene solution. Rinse the flask with toluene oncc. followed by motbawl (Uuoo times). Dry the flasks completely before use. either by &-drying m with a atnm ofnitrogen. 11.7 Decant the exlmclon to conditioned SPE column fitted inside the mouth o i the pur-&sped flak. Collect the eluate in the 125 mL silanizd pear-rhapr flask.
Exygen Research
Page 49 of 65
- -.
Page 109 of 127
Interim Report #17 - Analysis of Sediment and Water Samples Exygen Study No.: POOOl 131
Exygen Protocol Number: POOOl 13I
Exypcn Rrrsuch
MelhodNumbcr \IwOI:14
- 1 A ~ A L Y T I C A L%IWHOD Method of Analyrir for the Detamination of Perflwmocranoic Acid (PFOA) in Vegetation
by LC/Ms/MS
-
11.8 11.9 11.10
11.11
11.12 11.13
11.14
11.1 5 11.16
Add 20 mLof ScaOnitrilcto thc sample in the 50 mL centrifuge tube. Shakethe $ampleagain for -10 minute8 on a wrist-action shaker. C~ntritUgt~hc 50 mL polypmpylene tub= containing sample at -2000 tpm for -5 minutes. DcoMt thc extnct onto the s m c SPE column. Collect the eluate into rhe m e pur-rh.pod fhsk and combine with the eluent horn the initial
extraction. Rcpcitsccp~11.81hrough11.11again.
Add 3-4 drops of I-oclpnol to thc extnct in the pear-shaped flask and evaporite rl d u c e d p ~ u r wcing a m*ry cvlpontor (at 40QC). Make Ihc fwl volume, by adding 2 mL of 2% ascorbic acid in methanol lo the pur-shrpcd flask Mdd r l to mix/diualvc.
Transfer thscxwctl to HPLC vials wing disposable pipets. Arulylel~~plasvsingclcctroapnLyC I M S I M S .
12.0 Chromatography
12.1 Inject the SMC amount of a c h smdard. sample and forltficd sample into Iht:
LClMSlMS ayatmn. A ulibntion standard must precede and follow JII
analyzcdralnpln.
12.2 Standuds ofPFOA conesponding to at loart five or more concentration levels
must be included in an analyical set.
12.3 An entire set of extracted calibration standards must be included at the
F-
b e e n g and at Ihe cad of a sample set. Extracted standards must be
i n t c r s p d between e v a y 5-10 samples. As an alternative. an entire set of
exerctsd calibration srenddn may be iqiatcd U the beginning of a set
followed by extracted ulibrption atpndards intcrrpcrsed every 5-10 samples
(to account for a m n d set of extracted 8tandards)). In either case. extracted
c a l i b h n standardsmu1 be the first and Inst injection in a sample set.
12.4 Use linear standad cwu for qumtitation. Linear slandard cuwcs arc
gnraatsed for the mlytc by linear regrossion using Ih: weighting of peak area
venus calibration atandud concentration using MassLynx 3 3 (or cquivalcnt~
aofhvmsptm.
12.5 S m p k I S s p o M c sbould MH exced standard reqonses. A n y samples Ihdl
exceed rundud rcrponstsshould be lirtiwdiluted and reanalyzed
13.0 Acceptance Criteria
13.1 Chmmato&?unmust chow a peak of I daughter ion at 369 m u from a parent of 413 mu. ?he 413 m u p m t conaponds to the PFOA anion. while the daughter ion (369mu) reprsscntsthe loss of carbon dioxide.
Page SO of 65
Exygen Research
Page 110 of 127
Interim Report #17 - Analysis of Sediment and Water Samples Exygen Study No.: PO001 131
Exygen Protocol Number: PO001 13 I
E i y ph u r c h
> -
ASALYT~CALhlETHOD
Mechod Numbcr VWO1784
`Metbod of Annlyaia for the I k I m o n of Pufluomocunoic Acid (PFOA) in Vegeration by LCIMs/Ms
13.2 Method blank# muat not conlain PFOA at levels p a t n than the LOO. I f il blank Ccnuins PFOA 91 levels matcr than 0.5 ppb. then a ncw blank sample m w be obuined and the enlire wt mutt be re-exfracted.
13.3 Rocovnies of conlml rpikcr md d x spikes must be bctwecn 70.130% or their known valua. If a conhul spike fallr outside Ihc acceptable limits. the entire set of samplesnhould bc rc-exxtRctod.
13.4 Any cplibntion atmdud found to be a statistical outlier by using the Huge
Error TwS may ba excluded from the calculPdon of the calibration curve Howwer, Ihc totd numba of calibrstion standards that could be excluded must not exceed 20% of the told number of standards injsled. 13,s The conelstion cocficicnt (R) for calibration curves generated must hc
20.992 (R' 20.985). If ulibntion rerulu fall ouUide lhcsc limits. rhen appropriate step8 mutt be laken to adjust inshumcnt operation. a d Ihc standmb or the nlcvmt wt of tampla ihould bc rcanalyzcd. 13.6 Retention limcr betwan slMdnrdn and samplcs must not drift more lhari
f 4 K within an .naly(icd run. If retention timc drift cxcceds this limii within Manalytical runthen the sct must bc reanalyzed.
14.0 Calculation8
14.1 Usc thc following equation to cdculatc the mount of PFOA found (in npiniL. bprcd on pak MI) wing tbs nandard curve (linear regression parameters)
- genmtedby the Mpu Lynx wfiwln pmgam: PFOA found (nglmL) peak area - imcrCc0t) slops
14.2 Ute tha following equation to conv@tilhe amount of PFOA found in ng/mL to nglg @rW.
PFOA found @pb) =
DF
-
factor
by
which
the
final
ramplc weight (p) volume ww diluted, if
necessary
14.3 For samples fortified with known amounts of PFOA prior to cxtract~on.use tho following q u a i o n to calculatetha pcrccntrecovery.
slwvrry (St=
. ~totalanaIylsfound(ngl/p)wlytcfoundincontrol(np/g)~xlOO
~ a l y t ~ a d d(cndug)
Exygen Research
Page SI of 65
.--
Page 111 of 127
Interim Report #17 - Analysis of Sediment and Water Samples Exygen Study No.: PO001 131
Exygen Protocol Number: PO00 I I3 I
ANALYTICAL METHOD
Method Number: V0001785
Metbod of Andylb for the Daermln8tion ofPefluorooct8nolc A d d (PFOA) In Small Munmd Liver by LCMS/MS
Analytical Testing Fncility:
Exygon R-b
3058 R-h
Drive
Slate College. PA 16801
Approved By:
Paul Connollv
I
Technicrl b&r. LC-MS, Exygm Relurch
f-
Exygen Research
Total Pipes: 7 Page 52 of 65
Page 112 of 127
Interim Report # 17 - Analysis of Sediment and Water Samples Exygen Study No.: PO00 1131
Exygcn Protocol Number:POW 1 13 1
Eiyprn Rclurch
MclhdN&r Vu001781
I 1 M U M I C A L kWrHOD Method of M y r i c for Ute Delmiartion of Pertluorwctvloic Acid (PFOA) in Small
MDmmal Livn by Lc/MS/MS
1.0 scope
Thir mcthod ia to be anployod for the isolation and qumtilption ofpcrfluoroocranotc acid by High Pafomunsc Liquid C h m m b g n p h y coupled to a tandem Mass SpccoomctricDaector (LCIMSIMS) in small manmid liver.
2.0 Safety
2 I Alwsya & m e safe laboratorypncbcca 2 2 Consult the appropnam MSDS before bandling any chemical far prupcr spirt%
prcuuaoni
3.0 Sample Rcquimcnl
3.1 AI least 5 g of terl m p l c for exesction. 3.2 Sampla rhould be pmccrlod bcfore waactioo. Place the frozen sample in a
food prrxwor md homogenize with dry ice. Plaffi the ~amplssin containers and luvc opm in bum Mnge overnight to allow for carban dioxide sublimation Sen1 md place tho campier in frozen storage u n ~ i ltime n( analyois. Altaantely. if there ir M insufficient amount of sample (-less thsn 5 g). ulcn no pmccssing is nccsssaryand the sample can be used as supplied 3.3 Sample wllcction proccdurcs will be specified in the sampling plan for this project,
4.0 Reagentsand S l u d n n l
- ,
4.1 Water- HPLC grade
4.2 Mahanol-HPLCgmdc
- 4.3 Acelonitrile HPLC grade - 4.4 Ammonium Acct.(c A.C.S. Reagent Grade - 4.5 Pafiuorooctawic Acid SipwAldrich
5.0 Insuumcnl and Equipment
5.1 A high performwtcc liquid chromatograph capable of pumping up to 2 IolVQltrcquippod with a variable volume injector capable of injecting 5.200 )rL connectedto atandem M u r Spcctmmetn (LC/MS/MS).
5.2 A dcvicc 10 collect nw d a b for pesk integralion and quantitation 1.3 Analyticalbalance capabk of reading to 0.MXK)I g. 5.4 50 mL dirpouble polypropylenecentrifuge w k . 5.5 15 mL disposablepolypropylenecenuifuge rubes. 5.6 Disporablc micropipar (5O-lM)uL. 100-2WuL). 5.7 125-mL LDPE wmw-mouth bottled. 5.8 2 mL clear HPLC vial kir
Exygen Research
Page 53 of 65
Page 113 of 127
Interim Report #17 - Analysis of Sediment and Water Samples Exygen Study No.: PO001 131
Exygen Protocol Number:PO00I I3 I
1 1 MALYTICAL hlE7HOD M W o f h d n i t for Ihe Daonnhrhon of PSmuoroalnnoic Acid (PFOA) in Small Munmd Liver by L U M S M S
5.9 Dirpoaablc pipma.
5.10 Aul~pipo(ta(lO~IOOpOL m d IO-IM) pL). with disposable lips. 5.11 WalnrSepP&kVu:6cc(lg) tCl8SPEcuuidgsr. 5.12 SPE vacuum manifold 5.13 Tirwemizor.
5.14 Wrisl-action hkm. 5.15 Centrifuge capableof spinning I5 mLpolypropylene tubes at 3000 rpm
6.0 C h r o ~ g r a p h i cSyltcm
6.1 Analytical Column: plwpbpccRP (Keystone Scientific). 2.1 m m x 50 rnm. 511 (PN:82505-052130)
6.2 Tempmlure: 30T 6.3 Mobile Pharc (A) : 2 mM Ammonium Acetate in Water 6.4 Mobile P b w (8) : Methanol 6.5 Gradient P r o m :
YI .o
8.0
20.0 22.5
Y Flow Rate
35
0.3
65
35
0.3
25
75
0.5
25
75
0.3
65
35
0.3
- 6.6 Injection Volume: I5 pL (can be increased M as much as 50 pL).
6.7 Quantitalion: Peak h- e x t c d slnndard calibration c w e . 6.8 Run Time: 23 minuter.
The above MnditioM uc intended u a guide and may be changed in order 10 optimize the HPLC syakm.
7.0 MSlMSSystom
7.1 Mode: Elecmeprny Negative MRM mode. monitoring 413 + 369 mk foi
PFOA.
The above canditiona YC intcnded as a guide and may bo changed in order lo optimizc the MSMS syatan.
P-
Page 54 o/ 6j
Exygen Research
Page 114 of 127
Interim Report #17 - Analysis of Sediment and Water Samples Exygen Study No.: PO001 131
Exygen Protocol Number: PO001131
Exyyn Rurvsb
M o W Nwnbrr VMMIl85
1 ANk!.\TlCAL%ETHOD
I
Method of Andnis for the Determination of Per!luoroaelanoic Acid (PFOAI in Small
Mammal Livor by LUMSMS
8.0 Prspmtion ofSolutiona 8.1 Mobile Phslc
8.1.1 2 m M ammonium ncctUe in water is prcpamd by adding 0 154 g 01 ammonium acelatc~o1M)OrnLofwatcr.
Altunnle volvmcs may be prepard.
9.0 StandardPnparation
9.1 Standard StocWortifidon Solution 9.1.1 Prepare a stock solution of -100 @O/mLof PFOA by weighing IO iny of analyticals h d a r d (comted for punty) and dilute to 100 mL with methanol in a 125-mLLDPE bonk. 9.1.2 A 1.0 &IUL forti6c.tion solution of PFOA is prepared by bnnging I mL of thc IO0 &mL solution to a final volume o f 100 with methanol in a 125 mL LDPE b01Ilc. 9.1.3 A 0.1 pghnL fortiAcationsolution of PFOA is prepared by bringing 10 mL ofthc 1.0 pdmL wlution IO a final volume of 100 with methanol in a I25 mL LDPE bottle. 9.1.4 The stock and forrification solutions arc lo bc slored in a refrigerator at approximUcly 4-C 4arc 6lable far a muimum period of 6 months fmm the dale ofprspuuion.
9.2 Stand.rdCalibration S O ~ U ~ ~ O N
9.2.1 LC/Ms/MS calibrltion swdards yc prepared in methanol vra dilution of Ihe0.1 &mL forti6cationsolution.
9.2.2 The following is a typical example: additional wnccn~rationrmay bc prepared u needed.
cOmsnmtiOT,
Final
of Fortifiution Soluhon (nUmL)
100
Volumc (mL) 5.0
Diluted to (nu) 100
Concentrnhon (nUmL)
I .o
too
2.0
100
20
IW
I .o
IW
IO
5.0
IO
IW
0.5
2.0
10
IO0
0.2
I .O
10
IO0
0.1
9.2.3 Store all calibration il&& in 125-mL LDPE narrow-mouthboules
U 2'C to 6*C, up to 6iX months.
9.2.4 Alternate volume8 and concmtralions of standards may be prepared ds
needed.
Page 55 of 65
Exygen Research
Page 115 of 127
Interim Report # I 7 - Analysis of Sediment and Water Samples Exygen Study No.: PO001 131
Exygen Protocol Number: PO00113 1
L A.I.U'YTICAL METHOD
J
Method of M y * c for rhc Ducrmiwion of Perlluomoctwic Acid (PFOAI ir Small
M m a l Liver by LCiMyMS
10.0 Batch Set Up
10.1 Each batch of tunpla cxuactcd (typically 20 or less) must include a1 lrasi ona unlmakd contml and two UMUled controls fortified at known commlrUionr (lab contml spike) lo vaiSy procedural rccovcry for rhe batch
10.2 Rcquiromsnta for field lad lpboratoryduplicated and spikes will be spccificd in the quality assurancep l fo~r this project,
11.0 Sample Fxxtrpction
11.1
11.2 11.3 11.4
11.5 11.6 11.7
11.8
11.9 11.10
11.11
Weigh 1 g of umplc into a 50 mL polypropylene centrifuge tubes (fonify ,IS
needed. r c p l w lid lad mix well). Note that alternate weights ollircr may br mcanucd depending on the umplc size available for use. Add water to thc lsmplc for n final volume of IO mL.
Homo~mizclsmple winga tisruemizcrfor -1 minute. Transfer I mL of the sample wing a diiporable pipette inro a IS mL di8popalablc ccmtlifugc lube. Add 5 mL of a c h i t r i l c ud shakc for -20 minuter on a mil-action shaker Centrifuge thc tuba nt -3000 'pm for -5 minutes.
b m t the supomallat mto a 50 mL dispoaablc cmtnfugc tube and add 35 mL of water.
Condition thc Cis SPE d d g m ( I g, 6 mL) by passinp IO mL methanol followedby 5 mL of HPLC watcr (- 2 dmp/scc). Do not let column run dry
- Load thc umplc on conditioned Ctg SPE cuhidge. Discard eluate.
Elute with -2 mL of methanol. Collect 2 mL of cluatc into a graduated 15 mL polypmpyla~centrifuge tub+ (final volumc 2 mL).
Andp aunploa wingclccuospray m s / M s .
12.0 Chronulography
12.1 Injixt thc s m e y110uotof a c h atandud, a m p l e ud fortified ~ m p l icnto ihe LclMSlMS systcm. A calibration atandard muel precede and follow all andywjample#.
12.2 Stmdub of PPOA conrsponding to a1 Isrut live or mors wnccnmation levels mY.1be includedin urdyticd act.
12.3 An cntim MIof calibration uandards must be included at the beginning and ill the md of II umplc rd. S t u d u d s must be intcrspened between every S - l t l m p l s r . AS an dtanstivc, la mtirc set of calibration standards ma) hc injected U the bcgmning of a act followed by calibration standard, IIItUspmcd ~ a 5-1y0 samplcs (to account for a scwnd set of slandards) In either c w . cnlibration cundardr must be h e first and last injection in A umple IC(.
12.4 Use linur atandard c w c s for quanlitation. L i n w standard curves arc gsnonted for thc d y 1 c by line81 r c m o n wing I/x weighting of peak area
Page 5G of 6j
Exygen Research
Page 116 of 127
Interim Report # 17 - Analysis of Sediment and Water Samples Exygen Study No.: POOOl 131
ExygenProtocol Number: POOOl 131
ExypollRupucb
McmodNumbaVOW1781
I ANALI'NCAL METHOD
Mcthod of Analvlis for the Daminatinnof Per!luomocrPnoic Acid (PFOA) in Small M m a l Liver by LCIMSIMS
vcnus cllibration standard ooncmmtion usingMaasLynx 3.3 (or equivnlenl) softwm syabm. 12.5 Sample responce should not exceed standard responses. Any samples lhdi exceed standard nrponses should be hurhcr diluted and reanalyzed.
13.0 Acccptanw Cntcria
13.1 Chmmatopm must show a pcak ora daughter ion at 369 m u from a parcrii of413 m u . Tbc 413 m u pmnt ooncspoadr to the PFOA anion. while [he dpuehtaion (369m u ) rcprcaent8the loss of carbon dioxide.
13.2 Method blsnLr mwt not contain PFOA 81 levels greater than the LOQ. if a bluJc contains PFOA at levels p a t a than IO ng/g. then a ncw blank sample must be obtained d the a h rct m u t be re-extracted.
13.3 R a o v d a of c~ntmslpikes and m t r i x spikes must be between 70-130% of their known V~UCS. If a cantrol spike falls outside the scccptablc limits. the enth scl of sunplu rbould be re-extrsoted. Any matrix spike outside 70130% should be evaluated by the analyst lo determine if reextraction is
Wprrpstcd
13.4 Any calibntion rtvldnrd o fundto be a atatistical outlier by using the H U ~ K Error Test. m y be excluded from the calculation of the calibration CUNK However, the total number of calibration smdards that could be excluded must not e x d 20% of tho total number of standuds injected.
13.5 Tho comlr(ion cacflicicnt (R) for calibration curvrs generated must be 20.992 (R' 20.985). If calibration remlt8 fall outside thnc limits. then appropriale nspr mun be taken to adjust instrument operation. and the standards or the ~ k v l nK l oIf urnplea should be reanslyzed.
13.6 Rotention limn behvcsn standanla and samples must not dnR more than f 4 K within an W l y t i c a l m . If retention time driA cxceeds this limit w h i n an annlyriul W I I then lhc sct must be rrmnlyrad.
14.0 Calculationc
14.1 Us0 the foUowingequation to calculate the mount of PFOA found (in ngmL. bucd on puk uu)using tho standard e w e (linear repssion parameters) generatedby tho M u s Lynx w f h m pmgnm:
PFOA found (ng/mL) =
x DF x aliquot factor
- - slope
DF futor by which the find volume waa diluted. if newwary Aliquot factor IO
Exygen Research
Page 5 7 of 65
Page 117 of 127
Interim Report #17 - Analysis of Sediment and Water Samples Exygen Study No.: POOOl 131
Exygen Protocol Number: POOOl 131
ExyarnRneuch
MolhodNvmkr VOOO1765
I I ANA1.Y I ICAL METHOD Method of AnrlyDir for tbe Determination ofPcrklwrooclmoic Acid (PFOA) in Small
MammalLiver bv L c / M s / M S
14.2 For rpmples fortified with known mounts of PFOA prior 10 exrracuon. use the following aquaionto calculatethe pwonirocovcry.
R a o v q (W)-
14.3 Use thc following cquauon 0 wnvcn the mount o f PFOA found in nUmL IU W g @pW
PFOA found @pb) =
x fine1- v rmplc wcrghl(g)
Exygen Research
Page 58 of 65
Page 118 of 127
Interim Report # I 7 - Analysis of Sediment and Water Samples Exygen Study No.: POOOl 13 1
Exygen Protocol Number: POOOl 13 1
ANALYTICAL METHOD Method Number: VoOOl786
Method of Anllyrlc for tbe DetermLaUon of Pcfiuorooctaoolc Add (PFOA) in Small Mammal Serum by L C l M S l M S
Analytical Testing Facility:
Exygen Research 3058 Research Dnve Stale College, PA 16801
Approved By:
Paul Connolly
I
Tcshnio~Ll rdcr,LC-MS,Exy#co Rcrarsh
Exygen Research
Total Pager: 7
Poge 59 of65
Page 119 of 127
Interim Report #17 - Analysis of Sediment and Water Samples Exygen Study No.: PO001 131
Exygcn Protocol Number: POOOl13 I
1 ' ExygmR-cb
MclhodNumbrr VOW1786
ANAL'ilICAL JhlETtlOD Mcthcd of Antllylia for thc Dctenninationof Porfluomocranoic Acid (F'FOA) in Smull
Mammal S a m by LC/MS/MS -
I .o QOPc
This method is to bc omploy4 for (he iwlation and quantitation of perfluorooctanoic acid by High Performance Liquid Chromatogrsphy coupled to a tandem Mars SpatromStric Detector (Lc/MsIMS) in mall m m e l swum
2.0 Safety
2.1 Always observe ride laborslorypncticcs. 2.2 Consult (he appmgristc MSDS before handling any chemical for DroDer ralilv
precautions.
3.0 Sample Rcquirmcnt
3.1 At least I mL of ICrt m p l c Lr extraction. 3.2 No ample pmcoUing is needed for auum s ~ l p l c s .However, frozen serum
sampled must to allowedto complaoly thaw to mom tamperahue before use 3.3 Sample collection proccduru will bc specified in the sampling plan for this
project.
4.0 Reagentsw d Standards
- 4.1 W.ter HPLC &e
4.2 Methanol - HPLC gadc
- 4.3 AcctOnit& WLC @WJC
,+-
4.4 AmmoniumAKi.tc -A.C.S. Reagent Grade
- 4.5 Pcrtluoroocmoic Acid Sigma-Aldrich
5.0 hawnsot and Equipment
5.1 A high pcrform~ccliquid chromatograph capable of pumping up lo 2 solvents equipped with a variable volume injector capable of injecting S.2(JI) pL comccted lo LI tandem Mas8 Spectmmsln (LCIMSMS)
5 2 A device to collect RW d m for pet& integration and quantitaiion 5.3 Analyticalbalancecapableofreadii 10 0.00001g. S.4 SO mL disposablepolypropylenecentrifuge rubes 5.5 15 mL dirpouble polypropylenecentrifuge tubes. S.6 Dirpasrblc micmpipar (50-looUL. 100-2WuL).
5.7 125-mL LDPE narmw-mouth bottles.
5.8 2 mL clou HPLC vial kit. 5.9 DispM.blc pipcac+. 5.10 A u t o p i p ~ ( l O O - l O W ~ L a nIdO-IOOpL), withdisposabletips 5.1 I Weten Scp Pak V.c 6 cc (le)C I S SPE cartridges. 5.12 SPEv.cuummmifold. 5.13 Vomxn.
Page 60oj 65
Exygen Research
Page 120 of 127
Interim Report #17 - Analysis of Sediment and Water Samples Exygen Study No.: POOOl 131
Exygen Protocol Number: POOOl 131
1 ' Exma! R-ch
Mclhod Number VW017Ub
At`&rLk"llCAL METHOD
Muhod ofAnalysis for the Dclorminstionof Pcrfluomocwoic Acid (PFOA)in Small Mammal S c m by LC/MSMS
5.14 Wrist-actionshaker. 5.15 Centrifuge capableofspinning 15 mL polypropylene tubes at 3000rpm
6.0 Chromatogaphic System
6.1 AnalyticalColumn: Flwphasc RP (Keystone Scientific). 2.1 mm x 50 mm. S p (PM:82505-052130)
6.2 TwaprrshUs: 30-C 6.3 Mobile PhuK (A) : 2 mM Ammonium Acetate in W a t n 6.4 MobilePhuc(B): Mahvlol 6.5 Gradient Program:
Timc(minlu
0.0
65
I .o
65
8.0
25
20.0
25
22.5
65
Flow Rate
u(mVmin)
35
0.3
35
0.3
75
0.3
75
0.3
35
0.3
- 6.6 InjectionVolumc: IS pL (can be increasedto u much as 50 pL).
6.7 Quantitation: Peak Area - external standard calibmtion curve.
6.8 Run Time: 23 minute&
The above condition8we intended as a guide and may be changed in order to optimze the HPLC iystcrn.
7.0 MSIMSSyrtun
7.1 Mode: ElecmspmyNegative MRM mode, monitoring 4 I3 3 369 rniz for PFOA.
The above wnditions PIS intended u a guide and may be changed in order to optimize the MSMS syatcm.
8.0 Preparationof Solutionr 8.1 Mobile Phue
8.1.1 2 mM ammonium acet~tein water IS preppared by adding 0 154 y o f ammo01um acetate to loo0mL of watcr.
Altcmate volumes maybe p n p d
Pnge 61 1$6J
Exygen Research
Page 121 of 127
Interim Report # 17 - Analysis of Sediment and Water Samples Exygen Study No.: PO001 131
Exygen Protocol Number: PO00I 131
Exypon Rcrouth
McUlodNumkr VOoO178b
1 ANALYTICAL IWTHOD Method ofAnaly.is for the DotonninaIion ofPsMuomoctMoic Acid {PFOA) in Small
Msmmal Scrum by LCfluIs/MS
9.0 Standard P r e p d o n
9.1 Standard StocWFomfication Solution 9.1.1 Prspprs a stock solution of-100 & n L of PFOA by weighinn 10 m g of analylicll standard (corrected for purity) md dilute to I00 mL u ti11 methanol in a 125-mL LDPE bottlc. 9.1.2 A 1.0pg/mL fmtificationsolution of PFOA is preparcd by bringins I mL of the 100 pdmL solution lo a find volume of 100 with methanol in a 125 mL. LDPE bonle. 9.1.3 A 0.1 pg/mL foriifiution solutionof PFOA IS prcpared by b r i n p s IU mL of the 1.0 &nL mlution to a final volumc of 100with methanol in a I25 mL LDPE boHlc.
9.1.4 The stock urd fortification mlutimu are to bc stored in a refrigerator at approximately 4 T and an stublc for n maximum period of 6 months from the date of prepmilion.
9.2 Standard Chlibration Solutiona
9.2.1 LC/MsIMscalibralion itandards an prepared in methanol via dilurion ofthc 0.1 p g h L fortificationsolution.
9.2.2 T k following ir a typical cxunple: additional concenuations may be prepared BS needed.
Concmmllon
Final
ofFatih8llon Volvmc
BIutcd to
Concentration
Solvoo" (np/mL) 1..0.0
(mL) 5.0
(Id)
IO0
(nn/mL)
so
100
2.0
IO0
2.0
100
1.0
too
I .o
5.0
IO
100
os
2.0
10
100
0.2
1.0
10
1M1
01
9.2.3 Storc dl ulibmtion atandudr in 125-mL LDPE narrow-mouth bolllcs
a1Z'C to 6C up Io 8ix months.
9.2.4 Altcmslc volumes and conccntrptions of standards may be prcpared as
ncedcd.
10.0 Batch Set Up
10.1 Each batch of umplcr cxtrsctod (tupically 20 or Icss) muat include at least one unbuted control and two untreatsd controls fortified a1 known WIIC~I~O~~OIU(lab conbul spike)to verify pmcedwd recovery for the batch
10.2 Requiranenw for field and l h t o r y duplicates and spikcs will be specifieil in the quality BSSUMOC plan for this project.
.
Exygen Research
Page 62 of 65
Page 122 of 127
Interim Report #17 - Analysis of Sediment and Water Samples Exygen Study No.: POOOl 131
Exygen Protocol Number: POOOl131
Exypen Rewmch
Mclbod Number VOW1 78b
I AFinLYTlCAL. METHOD [ Mcthod of Anrlytis for tho Delenuhation of Pwfluomoctanoic Acid (PFOA) in Small
Mammal Serum by LCIMS/MS
1 I .O Sample Extraction
11.1
11.2 11.3 11.4
I I .5 11.6 11.7
11.8
11.9 11.10
11. I I
Mc~iurs1 mL ofsample into 8 50 mL polypropylene centrifuge tubes (fonify u d c d . rcpllcc lid and mix well). Note that alternate volumes of seruni may be mwuIcd dependingon the m p l e lire available for use.
Add water lo thc rpmple for a l i ~VOl lUmC of 20 mL. Cap tightly Vortex for-I minute. Tranrfcr 1 mL of the sample wing a dispowblc pipette into a I 5 mL disposable ccn!ihge tube. Add 5 mL of acetonitrile and ahake for -20 minutes on a wrist-action shaker Ccnaihrge the t u b a at -3000 rpm for -5 minutes.
Decant the supcmatant into a $0 mL disposable centrifuge tube and add 35
mL of witor.
Condition tbc CI, SPE carlridgcs (1 & 6 mL) by passing 10 mL rnethanul followed by 5 mL of HPLC water (- 2 draplscc). Do not let column run dry Load the sample on conditioned CIISPE cartridge. Discard eluate
Elute with -2 mL of methanol. Collcct 2 mL of eluate into a grddualcd IS mLpolypmpylene cmtrifbge tube (finalvolume = 2 mL). Analyze rampla wing clcchospny LUMSMS.
12.0 Chmmatography
12.1 lnjecl the auric m u n t ofcoch s(snd.rd,s m p k and fonified sample into Ihc LCIMSIMS ryrtcm. A calibration standard must precede and follow all Malylcd rampla.
12.2 S w W ofPFOA corresponding to at loast fivo or more concentration levels mwt be included in M analyrical set.
12.3 An entire wt ofcalibration standards must be included at the kginning and ai the end of a r m p l c ld. Slandardc must k interspersed between every 5- I II ~amplcr. As M altanativo, an entire wt of calibration standards may bu injcctal at the beginning of a set followed by calibration standards inrcrspcncd every 5-10 ample8 (to pf~ounftor a second set of siandnrds). In oitha~cam. ulibrstion Etand.rds must be the first and last injection in ii cMIpIc let.
12.4 Use linear Mndprd c w c a for quantitation. Linear standard CUNFE arc generatedfor the U y t c by l i n w regression win$ I l x weighting ofpcak area
vmw calibration standard concentration usmp MassLynx 3.3 (or equivalenll
soewsrs syrtsm. 12.5 Sample raponre should not cxsced standard rcoponscs. Any samples ihal
exceed iluuiprd ruponrca should be furtherdiluted and reanalyzed.
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Interim Report # 17 - Analysis of Sediment and Water Samples Exygen Study No.: PO001 131
h
c
Exygen Protocol Number: PO001 131
I 1 ANALYTICAL METHOD Method of Analysisfor the Delamination of PulluomoctMoic Acid (F'FOA) in Small MammalSerum by LOMSMS
13.0 Accepuvlce Cdtoria
13.1 Chromatognm must show a peak of a daughter ion at 369 m u from a parent of 413 mu. The 413 MU parent corresponds to the PFOA anion. while tliC daughta ion (369 mu) rcprrscnu the loa8 of carbon dioxide.
13.2 Method blank# mw not conlain PFOA at levels greater than the LOQ. If d blnnk conlaina PFOA e4 levels p e a t u than IO ng/mL. then a new blanh
s ~ l p l mc ust be obtained and thc enlire set must be re-extracted. 13.3 R%ovaicr of contml spikes and matrix spikn must be between 70-130% of
their known vslua. If D coni101 spike falls ouuide the acceptable limits. Ihe entire set of umpla should be re-extracted. Any matrix spike outside 70130% should bc cvdultcd by the d y s t 10 detorminc if re-extraction is WVnntCd. 13.4 Any calibntion 6tPndPrd found to bc a ~latisliclol utliu by using the Huge Enor Tu!,may be excluded b r n the ulculation of the calibration curve However. the told number of calibntion atandards that could be cxcludcd must not excad 20% of the totd number of standards injectcd. 13.5 The correlation cocfficicnt (R) for calibration curyes gmented musl bc 20.992 (R' 20.985). If calibration results fall outside thcse limits. ihrn qpropriale etspl murt bo tnkcn to adjust itutrumcnt operation. and i i i c standardsor the relevant sot of smplcs should be reanalyzed 13.6 Relenuon times beween standards and samples must not driti rnvre thdu
f 4 Y. within M Mslyticd run. If retention time drift exceeds this limii witliiii an analytical N n thDn thC Set muIt be remdyted.
14.0 Calculations
14.1 Use the fouowing quation tu cllculate the amount of PFOA found (in ngtnL. b a d on pcnk area) using the itandard curve (linear regression pardmetrrrl generated by the MPsn Lyu software program:
PFOA found (nplmL) =
x DF x aliquot factor slope
- DF = factor by which
Aliawl factor 20
the
final
volume
wan
diluted.
if
necessary
14.2 For ~ p m p flo~nihcd with known munta of PFOA pnor to cxtrac1ion. UK' the followingquation to calculate the percent recovery
Recovery (%) =
.
. ..
Puge 64 of65
Exygen Research
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Interim Report #17 - Analysis of Sediment and Water Samples Exygen Study No.: POOOl 131
Exygen Protocol Number: POOOl 13 1
Exyson Rrrsvcb
~ ~ t NU&h' ~ vdwo11a6
I - - a
ANALY7ICAL METHOD
I
Methodof M y i s for the DcIorminatioo of PnfluorwclanoicAcid (PFOA) In Small
MunmalSerum by LC/MS/MS
14.3 Use the following quation la convert tho amount of PFOA found In nUmL IO
- Ppb. PFOA found @pb)
IPFOA found C&&l x Tml vplumc hL)J aampk volume (mL)
Exygen Research
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Interim Report # 17 - Analysis of Sediment and Water Samples
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Exygen Study No.: POOOl 131
RESEARCH
3058 Research Drive Phone: 814-272-1039 State College, PA 16801 Fax: 814-231-1580
PROTOCOLAMENDMENT
Amendment N u m b e c l
Effedive Date: 01/19/05
ExygenStudy Number: POOOl 131 Client Study Number: None
..
Page 1 of 1
DESCRIPTION OF AMENDED SECTION
1) Analytical Procedure Summary V0001780:Sedion 9.1 2) Verification of Analytical Procedure
AMENDED TO 1) Add to Section 9.1: Section 9.1.6,Alternate weights of standards may be used to
preparealternate mncentmtions of stock so~utionsasnecessary. mernate levels of fortification solutions may also be prepared.
2) Low and high spiking levels of the analytes for each matrix may be altered depending on sample size available for extraction a n d h to cover analyte concentrations expected in the samples.
RATIONALE 1) Higher concentrations of standards need to be prepared in order to spike the sample
bottles at higher levels.
2) The sample size available for small mammal llver and serum was smaller than
expected. Spiking at the predeterminedlevels in the protocol puts the spiked
concentration lower than the detection limit. Also, the analyte levels in the ground water
samples are expected to greatly exceed the predeterminedspiking levels listed in the protocol. When the levels in the samples greatly exceed the spiking levels, an accurate
recovery value cannot be calculated for the QC sample. Higher spiking levels In the
bottles will cover the analyte concentrations expected in the water samples.
IMPACT ON STUDY The LOCI is 100 ng/g for a 0.1 g sample of small mammal liver and is 1000 ng/mLfor a 0.01 mL sample of small mammal serum.
Higher levels ofspiking for the water sampleswill ensure that more QC recoverydata
can be used.
1
LIBRARY ID: WOOO12256i .-
Exygen QAU Review
~
ADMINISTMTNE FORM
Exygen Research
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Interim Report ## 17 - Analysis of Sediment and Water Samples Exygen Study No.: POOOl 131
tqqy@ R E S E A R C H
3058 Research Drive Phone: 814-272-1039
State College, PA 16801 F a : 814-231-1580
Amendment Number: Effective Date: Exygen Study Number
~~
PROTOCOL AMENDMENT 2
03/07/05 POOOl 131 Client Study Number:
Page 1 of 1
None . .
DESCRIPTIONOF AMENDED SECTION 1) Report, page 11 of 65
2) Test Materials, page6 of 6 5 PFOS transition monitored499 -> 99.
AMFNDEO TO 1) Instead of one final report,interimreportswill be issued. 2) PFOS transition monitored may also be 499 -> 80.
RATIONALE
.- 1) Due to the excessive sizes of the data sets,interim reports will be issued to allow the
client to receive data in a timelier
msnncr' oSh/ar /kc*
2) The API 4000 LClMSlMS systems detect the 499 -> 80 PFOSt r a n s i t i with greater
sensitivity than the 499 -> 99 transition.
IMPACT ON STUDY
1) The client will be able to receive and review the data more quickly.
2) The 499 -> 80 transition can be detected with greater sensitivity; therefore, giving better chromatography.
LIBRARY 1Dwx)o122%E . L .
Exygen Research
ADMINISTRAW FORM
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