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Biodegradation study Report
The 35-Day Aerobic Biodegradation Study of PFOS
Thursday, March 29, 2001 Project Number
3M Project ID: LIMS E01-0444 Pace Contract Analytical Project ID: CA104
Study Director Cleston C. Lange, Ph.D.
Client James K. Lundberg, Ph.D., 3M Environmental Laboratory Bldg 2-3E-09, P.O. Box 33331,
St.Paul, MN 55133-3331
Contract Laboratory Pace Analytical Services, Inc.
Contract Analytical Group 1700 Elm Street, Suite 200 Minneapolis, Minnesota 55414
Project Dates Project Initiation: September 20, 2000
Project Completion: April 1,2001
A uthor Cleston C. Lange, Ph.D.
Page 1 of 29 Pace Project CA104: "The 35-Day Aerobic Biodegradation Study for PFOS" Study Director: Cleston C. Lange, Ph.D. Pace Analytical Services, Contract Analytical Group Thursday, March 29, 2001
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Table of Contents
Title Page.............................................................................................................................................................. 1 Table of Contents................................................................................................................................................ 2 List of Tables & Figures....................................................................................................................................... 3 List of Appendices............................................................................................................................................... 3 Executive Summary.............................................................................................................................................4 1.0 Project Personnel..........................................................................................................................................5 2.0 Data Requirements...................................................................................................................................... 5 3.0 Project Objective...........................................................................................................................................5 4.0 Test A rticle....................................................................................................................................................6 5.0 Reference Articles..................................................................................................................................... 6-8 6.0 Receipt/Generation of Samples.................................................................................................................. 8 7.0 Methods
7.1 Sample Preparation............................................................................................................................... 8 7.1.1 Collection of Sludge...................................................................................................................8-9 7.1.2 Culture Preparation..................................................................................................................9-10 7.1.3 Solid Phase Extraction (SPE)....................................................................................................11
7.2 Instrumental Analysis (HPLC/MS)...................................................................................................... 12 7.3 Data Transformations and Calculations
7.3.1 Molar Calculations..................................................................................................................... 13 7.3.2 Conversion ofng/mL to micromolar and nanomolar............................................................. 13 7.3.3 Molar Mass Balance Calculations............................................................................................14 7.4 Statistics 7.4.1 Formula for Standard Deviation............................................................................................... 15 7.4.2 Formula for Relative Standard Deviation................................................................................ 15 7.5 Software Versions.................................................................................................................................15 8.0 Results 8.1 Incubator, Refrigerator, and Freezer Temperature Log D ata.......................................................... 16 8.2 Mishaps recorded during the study..................................................................................................... 17 8.3 Sludge Characterization...................................................................................................................... 17 8.4 Quality Control/Sample Matrix Spike Results....................................................................................18 8.5 HPLC/MS Calibration and Calibration Verification...................................................................... 18-19 8.6 Analytical Blanks...................................................................................................................................20 8.7 Matrix Spikes........................................................................................................................................ 20 8.8 Surrogate S pikes..................................................................................................................................20 8.9 Samples and Controls Results.......................................................................................................20-22 9.0 Conclusions........................................................................................................................................... 22-23 10.0 Sample and Data Retention....................................................................................................................24
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Pace Project CA104: "The 35-Day Aerobic Biodegradation Study for PFOS" Study Director: Cleston C. Lange, Ph.D. Pace Analytical Services, Contract Analytical Group Thursday, March 29, 2001
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List of Tables & Figures
Figure 1: Time-Plot of PFOS concentrations for Test C ultures................................................................... 22 Figure 2: Time-Plot of PFOS concentrations for Abiotic Control Cultures ................................................. 22
List of Appendices
Appendix A: Signatures of Project Personnel.................................................................................................25 Appendix B: Table 1. Biodegradation Samples Final Results..................................................................... 26
Table 2. Abiotic Controls Final Results..................................................................................... 26 Table 3. Blank Controls Final Results...................................................................................... 26 Appendix C: Table 1. Integrated LC/MS Peak Area Data...................................................................... 27-29
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Executive Summary
This screening study was undertaken to determine the aerobic biodegradation potential of perfluorooctanesulfonate (PFOS) when exposed to municipal wastewater treatment sludge. This study was conducted as a comparative six-sample point study that lasted for five weeks. Aerobic sludge from the Twin Cities Metro Wastewater Treatment Facility was used as the microbial inoculum in test cultures. The PFOS was tested at a target concentration of approximately 2.582 pg/mL (5.576 pM). Samples were prepared for quantitative analysis by solid phase extraction (SPE). Quantitative analysis of SPE extracts was conducted by LC/MS. Cultures containing municipal sludge were incubated for 35 days with no loss of PFOS over that period of time. Similarly, abiotic controls that did not contain sludge did not show a decrease in PFOS concentrations. The final comparative results showed that PFOS did not degrade during the 5 weeks of incubation. Recovery of PFOS throughout the study for all sample points was nearly 100%. Furthermore, expected biodegradation products, PFOA and PFOSulfinate, were not detected.
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1.0 Project Personnel
1.1 Sponsor Company 1.2 Sponsor Representative
3M Dr. James K. Lundberg
1.3 Contract Facility Personnel: 1.3.1 Study Director 1.3.2 Laboratory Management: 1.3.3 Analysts/Technicians
Dr. Cleston C. Lange Mr. Bruce E. Warden Ms. Angela L. Schuler Dr. Cleston C. Lange
1.3.4 1.3.5 1.3.6
HPLC/MS Analyst Sample Custodian: Report Author
Mr. Anthony E. Scales Dr. Cleston C. Lange Dr. Cleston C. Lange
2.0 Data Requirements
The sponsor representative desired an aerobic biodegradability screening study to be conducted using HPLC purified perfluorooctanesulfonate (PFOS) as the test substrate. The study was initiated on September 20, 2000 as a non-GLP study, and culture incubations proceeded through to October 25, 2000.
3.0 Project Objective
This study was conducted in order to elucidate whether or not PFOS is biodegradable under aerobic conditions using a microbial rich inoculum of municipal wastewater treatment sludge. The study design was copied from earlier studies conducted as part of Pace project CA058 and CA097 for 3M. The loss of parent material, determination of a biodegradation rate, and identification of products were critical parameters for the determination of biodegradation. The study was prepared as a six-sample point screening study, and the last cultures were harvested on the 35th day of incubation. The microbial inhibition by PFOS in aerobic sludge was not tested as part of this study, however, a previous activated sludge inhibition study for PFOS showed that PFOS is not inhibitory to microorganisms at the concentration tested here .
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Pace Project CA104: "The 35-Day Aerobic Biodegradation Study for PFOS: Study Director: Cleston C. Lange, Ph.D. Pace Analytical Services, Contract Analytical Group Thursday, March 29, 2001
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4.0 Test Article
The test article used for this study was HPLC purified PFOS (3M Std I.D. # TCR-00017046), and was provided by the sponsor. The sponsor did not provide the purity of the test article. The test article was a white crystalline solid, supplied in a clear -Chem 40 mL vial, and documentation provided with the material had an expiration date of January 1, 2010. Upon receipt at Pace, the material was given the test, control and reference (TCR) number CA-TCR02-014.
5.0 Reference Materials Reference materials were received from the 3M Environmental Laboratory on June 8, 2000 for conduct of earlier studies. Approximately 1 gram of each of the following materials was received with accompanying purity information, MSDS, and chain of custody (COC # 14681). Upon receipt at Pace, each was given a Pace test, control, & reference number: Note: M556 was received from the 3M Environmental Laboratory as a methanol solution. An MSDS, and a chain of custody were not provided for this material.
All test, control, and reference materials were stored at -85C + 15C per Pace standard operating procedures. All final data in data tables for this report were calculated using the available purity information provided for PFOS (86.4% purity, below) since it was the only HPLC/MS target analyte detected.
NC: Purity analysis was not completed at the time of the study. All purity information regarding the control & reference articles was maintained under lock and key in the laboratory in a client specific binder.
5.1. Perfluorooctanoate Ammonium Salt (PFOA) The purity was determined at 97-99% by HPLC/MS as part of project CA058. 3M#: TCR-99131 -37 Pace #: CA-TCR02-001
F F F F F F F o' NH
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O
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Pace Project CA104: "The 35-Day Aerobic Biodegradation Study for PFOS" Study Director: Cleston C. Lange, Ph.D. Pace Analytical Services, Contract Analytical Group Thursday, March 29, 2001
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5.2. Perfluorooctanesulfinate Potassium Salt (PFOSulfinate)
Purity: NC
3M#: SD-007
Pace #: CA-TCR02-002
F F F F F FF O F F F F F FF
5.3. Perfluorooctanesulfonate Potassium Salt (PFOS)
Purity: 86.4%
3M#: SD-009
Pace #: CA-TCR02-003
FF O
ii O K* FF O
5.4. Perfluorooctanesulfonamide (FOSA)
Purity: NC
3M#: SD-029
FF F F F F F F O
i
P H' FF F F F F F F O
Pace #: CA-TCR02-004
5.5. A/-Ethyl perfluorooctanesulfonamide (N-EtFOSA)
Purity: NC
3M#: SD-012
Pace #: CA-TCR02-005
FFFF FFFFO
1
F F F F F F F F ro \
ch
5.6. [2-(Perfluorooctanesulfonamido) acetic acid] (M556)
Purity: NC
3M#: 00001-5-24
Pace#:CA-TCR02-006
FFFFFFFFO
OH
5.7. [2-(A/-Ethyl-perfluorooctanesulfonamido) acetic acid]
(A/-EtFOSAA) and was provided labeled as FC-129
Purity: NC
3M#: SE-038
Pace #: CA-TCR02-007
F FF F FF
FF FF
FF FF
FO | II
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MF i O \ ___ cchh3
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Pace Project CA104: "The 35-Day Aerobic Biodegradation Study for PFOS" Study Director: Cleston C. Lange, Ph.D. Pace Analytical Services, Contract Analytical Group Thursday, March 29, 2001
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5.8. [2-(A/-Ethyl-perfluorooctanesulfonamido) ethyl alcohol] (A/-EtFOSE alcohol)
Purity: 99.9%
3M#: SE-035
Pace #: CA-TCR02-008
6.0 Receipt/Generation of Samples
Samples were not received, but were generated as an inherent part of this study. Each of the thirty experimental cultures prepared for this study were extracted by solid phase extraction (SPE) methodology to generate ninety analytical samples. The SPE eluates collected for each sample were labeled as either SPE eluate 1, 2 or 3. Of the three eluates generated for each sample, eluate 1 was the 25 mL aqueous sample eluate and eluates 2 and 3 were 25 mL methanol eluates from the SPE cartridge. Quantitative HPLC/MS analysis was conducted for eluate 2 and eluate 3 only, and the resulting analytical data was used to determine whether biodegradation occurred.
7.0 Methods
7.1 Sample Preparation
7.1.1. Collection of Sludge.
To prepare cultures, sludge was obtained from the primary municipal waste treatment facility in the Twin Cities area. Arrangements were made for Pace personnel to retrieve fresh mixed liquor suspended solids (MLSS) from the aeration units at the Twin Cites Metro Wastewater Treatment Facility (also referred to as Pig's Eye Sewer Treatment Plant) located in St. Paul, MN. Four liters of MLSS was collected by Pace laboratory personnel on September 18, 2000 and delivered as four 1-liter Nalgene polypropylene bottles containing MLSS, and were accompanied with a corresponding chain of custody (Pace C.O.C. # 528791). Upon receipt at Pace Science Solutions, the individual bottles were labeled #1 through #4. The suspended solids in the bottles were allowed to settle at 4C from September 18th to September 20th. On September 20th, the
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settled solids "sludge" from bottle #1 was used to prepare cultures for this study. A sludge characterization analysis was not conducted as part of this screening study.
The settled sludge in each bottle constituted approximately 20% of the volume, or approximately 200 mL volume in a 1-liter bottle, based on visual observations-and was consistent with observation of earlier sludge collections.
7.1.2. Culture Preparation.
All cultures were initially prepared on September 20, 2000 and the last set of incubated cultures was harvested on October 25, 2000 (35 days total incubation). The culture preparation procedure described below was documented as Pace standard operating procedure (SOP) CAG-SP03 4.
Cultures were prepared using a mineral salts medium defined by EPA Guideline OPPTS 835.3200. The mineral salts medium pH was 7.4 and contained per liter, 0.334 g Na2HPO4-2H2O, 0.005 g NH4Cl, 0.2175 g K2HPO4, and 0.085 KH2PO4, 0.0275 g CaCl2-anhydrous, 0.0225 g MgSO4-7H2O, and 0.00025 g FeCl3-6H2O.
Two liters of a mineral salts medium containing 100 mL of settled sludge was prepared and contained 1 mL of methanol. To each test culture was added 25 mL of this mineral medium containing sludge.
Mineral Salts medium, un-sterilized and without sludge, was prepared. This mineral medium without sludge was used to prepare the 25 mL no sludge (abiotic) control cultures.
All cultures were prepared by dispensing 25 mL of appropriate mineral salts medium solution into clear sterile 125 mL Nalgene polycarbonate culture flasks containing labels with appropriate identification information. Note: The mineral salts medium that contained sludge had to be swirled
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regularly during dispensing in order to keep the mixture homogenous and prevent the sludge from settling out of the solution. PFOS was added as the test substrate to cultures by transferring 6 mL of a 10,760 mg/mL methanol solution of PFOS (stock solution CA104-SS001) to each culture flask, for a final target concentration of 2,582 ng/mL. Blank control cultures received 25 mL of equivalent mineral medium solution as test culture, but without PFOS addition. The day zero samples were prepared and immediately placed in a -20C freezer. All other cultures prepared were placed in temperature controlled shaking incubators that was maintained at 25C + 3C. Samples were pulled from the incubator at designated time points of days 2, 5, 7, 14, and 35. Upon removal from the incubator, samples were either immediately frozen, or immediately prepared for analysis by solid phase extraction.
All sample preparation information, including times, analyte additions, etc. were recorded in sample preparation worksheets and signed and dated by the sample preparation analyst. All original data sheets were maintained in project specific binders labeled CA104.
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7.1.3. Solid Phase Extraction of Cultures
The solid phase extraction procedure described below was documented as Pace standard operating procedure (SOP) CAG-SP-04 .
All Sample cultures and control cultures were prepared by solid phase extraction methodology using SEP-VAC C18 6cc SPE cartridges from Waters Corporation (Part No. WAT036905). A sample label was applied to each SPE cartridge prior to use, and each was packed with plug of quartz glass wool to deter plugging of the SPE filter. Each SPE cartridge was washed prior to use by drawing 5 mL of methanol and then 5 mL of aqueous 1% acetic acid solution through the cartridge. These wash solution eluates were discarded to waste. All of the SPE eluates for this study were collected in clear I-chem vials with labels that identified them as eluate 1, 2 or 3, as defined below.
Frozen samples were thawed at ambient room temperature before extraction. Following thawing, and prior to solid phase extraction, 0.25 mL of glacial acetic acid was added to each of the cultures yielding a final concentration of 1% acetic acid. The content of each acidified culture was swirled to mix, and then drawn by vacuum through the appropriately labeled SPE cartridge by carefully pouring the contents of the culture flask into the SPE cartridge. The aqueous eluate was collected in an -chem vial labeled eluate 1 , removed from the vacuum manifold, and capped. Then, 25 mL of methanol was added to the culture flask, the flask sealed, and vigorously shaken. The cap was then removed from the flask, and the methanol content (25 mL) drawn through the SPE cartridge, collected in an -chem vial labeled eluate 2. Eluate 2 was expected to contain a majority of the analyte that was in the original culture sample. As a precaution that some analyte may be retained in the SPE cartridge, or in the culture flask, a second 25 mL methanol step was conducted in a similar fashion to that used for eluate 2, and collected in a third I-chem vial, labeled eluate 3. Aliquots of eluates 2 and 3 were transferred to autovials, capped, and then
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quantitatively analyzed by HPLC/MS. The remaining volume of each eluate was stored at 4C.
7.1 Instrumental Analysis (HPLC/MS)
The quantitative LC/MS method described below was documented as Pace standard operating procedure (SOP) CAG-ORG-23 6.
All quantitative analysis was conducted on an HP1100 high performance liquid chromatograph with mass spectrometer detector (HPLC/MSD) system. The HPLC/MSD system was set up with the same chromatography configuration as that described in method CAG-ORG-23, which utilized two columns fed into a pressure relief valve that served as a flow through splitter. The MSD was operated with electrospray ionization in negative ion mode and with selected ion
monitoring (SIM). Ions monitored were: m/z 413 (PFOA); m/z 483 (PFOSulfinate); m/z 498 (FOSA); m/z 499 (PFOS); m/z 526 (V-EtFOSA); m/z 556 (M556); m/z 584 (N-EtFOSAA); and m/z 630 (N-EtFOSE alcohol). All
analysis was quantitative, with 8 calibration standards prepared from approximately 5 to 1000 ng/mL (ppb) and containing each of the analytes being quantified. Typical injection volumes for samples and calibration standards were
50 mL, except where otherwise noted for instrumental dilutions in section 8.3 below. The 4.6 x 150 mm Betasil C8 column used for this study had serial number 0501383T, lot number P9G09 and the 4 x 35 mm NG1 column used had serial number 15123. The pressure-regulated splitter had no identifying number to distinguish it. The part number for ordering the pressure relief valve (splitter) was Alltech catalog part number 39025.
MSD Settings: Ionization mode: API-ES negative Gas Temp: 300C Drying gas: 8.0 L/minute Nebulizer Pressure: 30 psig Vcap: 3500V Fragmentor: 70V EMV Gain: 2.0 Actual Dwell for each ion: 146 SIM resolution: high
HPLC Settings:
Flow: 1 mL/min, splitter used
Time (min).
%A %B
0.00 97.0 3.0
0.50 97.0 3.0
6.00 5.0 95.0
8.50 5.0 95.0
8.51 97.0 3.0
10.50
97.0 3.0
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Pace Project CA104: "The 35-Day Aerobic Biodegradation Study for PFOS" Study Director: Cleston C. Lange, Ph.D. Pace Analytical Services, Contract Analytical Group Thursday, March 29, 2001
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7.3 Data Transformations and Calculations
7.3.1 Molar Calculations:
Because all data was collected on an ng/mL basis (part per billion, ppb), a transformation from ng/mL to molar concentrations had to be conducted to obtain mass balance information. The mole conversion values used for each of the analytes were as follows:
PFOA- NH4+ salt = 431 nanogram (ng) per nanomole (nmole) PFOSulfinate- K+ salt = 522 ng/nmole PFOS- K+ salt = 538 ng/nmole FOSA = 499 ng/nmole A/-EtFOSA = 527 ng/nmole M556 acid = 557 ng/nmole A/-EtFOSAA = 585 ng/nmole A/-EtFOSE alcohol = 571 ng/nmole
7.3.2 Conversion of ng/mL to micromolar (mM) and nanomolar (nM).
(Working Examples): 500 ng/mL PFOSulfinate = (500 ng/mL)*(1nmole/522 ng) = 0.958 nmole/mL = 0.958 mmole/liter = 0.958 mM
50 ng/mL PFOSulfinate = (50 ng/mL) * (1 nmole/522 ng) = 0.0958 nmole/mL = 0.0958 mmole/L = 0.0958 mM = 95.8 nM
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7.3.3 Molar Mass Balance Calculations: Convert all ng/mL values to their corresponding molar concentrations as mM or nM (see section 7.3.2, above). Then, sum the quantified molar values mM) for all analytes in eluate 2 and eluate 3. Divide the sum of the analyte concentrations by the known concentration of starting compound and represent the final result as a percentage of the known starting concentration. (Working Example): If, the starting concentration of N-EtFOSE alcohol was at 1800 mM And, after incubation, the following were determined: PFOA was detected at 50 mM PFOS was detected at 100 mM N-EtFOSAA was detected at 500 mM N-EtFOSE alcohol was detected at 1000 mM Then, the mass balance is as follows: Mass balance = [(50mM + 100mM + 500mM + 1000pM)/1800pMl X 100% Mass balance = (1650pM/1800pM) X 100% = 91.7%
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7.4 Statistics
7.4.1 Formula used for determination of Standard Deviation (SD):
SD =(Zx2/(n-1) )1/2 = [Z (X - X )2/(n-1)]1/2
Zc = the sum of the squared dilferences of deviations between the values of individual data points and the mean: = Z [(X - X f i
Z = The sum of all values in a population Xi = the value of an individual data point
n = the total number of samples, where one sample is represented as (i).
X = mean of all X in the sample population = (Z X) / n
7.4.2
Formula used for Relative Standard Deviation (RSD): The RSD is a ratio of the standard deviation (SD) to the arithmetic mean of the replicate analysis and expressed as a percent.
RSD = (SD / mean of Xn) * 100%
7.5 Software Versions
Microsoft TM Excel 2000 was used for data processing and producing tables. MicrosoftTM Word 2000 was used for processing the analytical report text. Adobe Acrobat 4.0 was used for generation of the final electronic report. Chemstation for the LC/MSD was used for data collection and integration.
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8.0 Results
8.1 Incubator, Refrigerator, and Freezer Temperature Log Data.
During this study, one reciprocal shaker incubator was used. The Pace ID number for the ncubator-shaker was 0269. Samples were shaken at 200 rpm, but the speed was not regularly verified.
A -20 C freezer, Norlake freezer, was used during the study to store the cultures until they could be prepared by solid phase extraction. The freezer ID was 0050 and was situated at Pace/3M Tier-2 facility in location SL-R6.
One IsoTemp ultra-low freezer -80C) was used for storage of test, control and reference materials. The freezer ID was 0241 and was situated at Pace in location SL-R4.
Two refrigeration units, a Carroll walk-in cooler and a True refrigerator, were used to store SPE-prepared samples and stock and calibration standards during the study. The coolers were identified as ID 0140 (location SL-R1) and ID 0213 (location SL-R8), respectively.
The average temperatures + the standard deviation for each incubator, refrigerator, and freezer are shown below for the dates in which they were used. The average temperatures and standard deviations are based on readings that were recorded on a daily basis, excluding weekends and holidays.
Incubator-shaker, ID 0269, from dates 9/20/00 to 10/25/00: Norlake Freezer, ID 0050, from dates 9/20/00 to 10/25/00: IsoTemp Freezer, ID 0241, from dates 9/20/00 to 10/25/00: Carroll walk-in cooler, ID 0140, from dates 9/20/00 to 10/25/00: True refrigerator, ID 0213, from dates 9/20/00 to 10/25/00:
27C + 1C -20C + 7 C -76.0C + 19C 4C + 2C 4C + 1C
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8.2 Mishaps recorded during the Study.
One sample, CA104-0920-028, was found to contain N-EtFOSE alcohol and some of its expected biodegradation intermediates. This sample was supposed to be a day-14 no sludge control (abiotic control) for PFOS. It is probable that the technician, upon transferring the sample to an auto-vial, mistakenly retrieved the wrong sample from storage and used a sample from another study. Since NEtFOSE alcohol was never used for this study, there is no other logical explanation. Furthermore, the concentration of PFOS in the sample was not at the level expected of an abiotic control for this study (expected at approximately 2.5 mg/mL, but measured at approximately 10 ng/mL). While the data for this sample is included in the final data tables in Appendix B, it is not discussed in the final conclusions and is excluded from discussions of PFOS biodegradation.
8.3 Sludge Characterization
Chemical analysis of the mixed liquor suspended solids used for this study was not conducted. However, the sludge was obtained from the Twin Cities Municipal waste treatment facility in manner consistent with sludge collected for project CA058 and was used immediately within 2 days. The sludge used for the preparation of samples in this study was also used for the preparation of samples in other biodegradation studies, and in some of those other studies, preliminary results have shown positive biological activity for degradation of fluorochemicals 7 8. Hence, it was assumed that active microorganisms were present in the sludge used for this study.
8.4 Quality Control/Sample Matrix Spike Results.
The determination of analyte recoveries from sample matrix spikes was not included as part of this screening study. However, PFOS was recovered at expected concentrations, and previous duplicated results obtained during project CA058 showed excellent recoveries from sludge matrices for all analytes pertinent to this study by the SPE method.
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8.5 HPLC/MS Calibration and Calibration Verification.
All HPLC/MS analyses were conducted in analytical sequence run D102600.s on Pace instrument "Dutch".
Although this study targeted the quantitative analysis of PFOA, PFOS and PFOSulfinate, the HPLC/MS multi-component calibration standards used in this study contained PFOA, PFOSulfinate, PFOS, FOSA, N-EtFOSA, M556, NEtFOSAA and N-EtFOSE alcohol. Eight calibration standards were prepared and contained each of the analytes at nominal concentrations of 5, 10, 20, 40, 100, 200, 500 and 1000 ng/mL. The calibration standards used were labeled from low concentration to high concentration as CA105-MW-001 to CA105-MW008, respectively. The calibration standards were prepared for use in projects CA105 and CA132 at Pace and preparation sheets can be cross-referenced to those projects. For final quantitation of unknowns, the exact concentration of the standards were used in calibration curves. Calibration standards were prepared in methanol in 10 mL quantities and were stored in clear -chem vials at 4C in refrigerator ID 0213. Aliquots were transferred to autovials and capped for use in HPLC/MS runs. Continuing calibration verification standards (CCVs) were used in sequence runs, and included both CA105-MW-004 (approximately 20 ng/mL) and CA105-MW-006 (approximately 200 ng/mL). CCVs were used for verifying the integrity of the calibration curves for quantitation throughout the sequence run.
Instrument calibration was performed for each analyte by use of either the low 5 calibration points (5 to 100 ng/mL) or the high 5 calibration points (40 to 1000 ng/mL). Analyte concentrations below 100 ng/mL were reported based on the value given for ol w calibration curve data and residuals defining the quantitation limits were 100% + 25% of the actual value. Values reported between 100 and 1000 ng/mL were determined from high calibration curve data, and residuals defining the quantitation limits were 100% + 25% of the actual value.
For samples with concentrations determined to be above 1000 ng/mL, the samples were re-injected as 10 mL injections and quantified off of calibration curves in which 50 mL of calibrant was injected, creating an instrumental dilution
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factor of 5. Final concentrations of dilution injections were corrected for the dilution factor automatically by the instrument, and final reported values were used. Final quantitative data for dilutions was obtained using the high five standards (40-1000 ng/mL), residual values in the range of 100% + 15%. The minimum limit of quantitation (LOQ) for the targeted analytes for analysis conducted were: PFOA (11.0 ng/mL), PFOS (4.4 ng/mL-adjusted for purity 86.4%), PFOSulfinate (5.5 ng/mL), FOSA (5.3 ng/mL), N-EtFOSA (5.9 ng/mL), M556 (5.0 ng/mL), N-EtFOSE acid (5.3 ng/mL), and N-EtFOSE alcohol (6.0 ng/mL-adjusted for purity 99.9%). Calibration check and verification standards (CCVs) were used after 15 sample injections and included both calibration standards for verification of low and high range curves. CCVs were flanked by methanol blanks. All quantitation was conducted using averaged calibration curves flanking no more than 30 samples, one set of CCVs, and the accompanying solvent blanks. All calibration curves were slightly quadratic and had R-values of at least 0.990. All data was collected as external standard calibration, without the use of an internal standard. Instrumental sequence runs were setup as described in method CAG-ORG-23.
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8.6 Analytical Blanks
Methanol blanks were injected onto the HPLC/MS column and quantitatively analyzed to determine the background analyte concentration in the methanol used for sample preparation.
Sample blanks, consisting of either mineral salts medium or mineral salts medium containing sludge, and no analyte addition. Sample blanks were prepared and incubated in an identical manner to true samples. The sample blank results were used to determine whether the sample matrix contained any of the analytes of interest. No fluorochemical analytes were detected in the sample blanks.
8.7 Matrix Spikes
Sample matrix spikes were not employed for this study.
8.8 Surrogate Spikes
Surrogate spikes were not employed during this study.
8.9 Sample and Control Sample Results
8.9.1 Result of Incubations with PFOS.
The final reported data were calculated using the sponsor-provided purity information from the Centre Analytical Laboratories, Inc. certificate of analysis # 023-018B for PFOS lot 171 (3M I.D. SD-009). The PFOS lot 171 material was used as reference material for preparation of calibration standards (PFOS reference material at 86.4% purity). In the test material PFOS (HPLC purified, 3M Std I.D. # TCR-00017-046), no significant impurities were detected in samples for this study. From the graphs in Figure 1 and Figure 2, it is obvious that degradation of PFOS did not occur during the 35-day incubation experiment. Furthermore, PFOA and
Page 20 of 29 Pace Project CA104: "The 35-Day Aerobic Biodegradation Study for PFOS" Study Director: Cleston C. Lange, Ph.D. Pace Analytical Services, Contract Analytical Group Thursday, March 29, 2001
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PFOSulfinate, expected products from biodegradation of PFOS, were not detected (Table 1, Appendix B).
The average concentration of PFOS for cultures containing sludge throughout the study was 2,553 ng/mL + 102 ng/mL, for an RSD of 4.0% during 35 days of incubation. The average concentration of PFOS for abiotic control cultures was 2,654 ng/mL + 83 ng/mL, for an RSD of 3.1%. The average PFOS concentration determined for all test and control cultures was 2,601 ng/mL + 105 ng/mL, with an RSD of 4.0%. The data presented in Appendix B and Figure 1 and Figure 2, below, demonstrate that PFOS was not degraded in any fashion during the course of this screening study.
y = 1.4771x + 2537.5
0 10 20 30 40 Incubation (days)
Figure 1. The data plot for the 35-day incubation of PFOS with aerobic sludge showing that no significant biodegradation occurred. The data is fit with a linear equation and error bars representing one standard deviation from the mean. The slope of the fit line is slightly positive in nature, while degradation would produce a negative slope (due to loss of PFOS).
Page 21 of 29 Pace Project CA104: "The 35-Day Aerobic Biodegradation Study for PFOS" Study Director: Cleston C. Lange, Ph.D. Pace Analytical Services, Contract Analytical Group Thursday, March 29, 2001
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a
3 4000
f 3000
C
I 2000
t ta
i
y = 1.6892x + 2647.3 4
1000
c o 0
0
T
10 20 30 Incubation (days)
40
9.0 Conclusions
Figure 2. The data plot for the 35-day incubation of PFOS in abiotic controls, showing that no significant degradation occurred. The data is fit with a linear equation and error bars representing one standard deviation from the mean. The slope of the fit line is slightly positive, while degradation would produce a negative slope (due to loss of PFOS).
This study determined that PFOS is not biodegraded by the inherent microbial populations of municipal waste treatment facilities under these defined test conditions and at the tested concentration of 2.582pg/mL (5.576 pM) of PFOS. The PFOS concentration in all test cultures and control cultures remained unchanged throughout the 35 days of incubation. The measured concentration of PFOS in all cultures was always within 100 + 7% of the expected concentration of 2.582 pg/mL after adjusting for reference material purity.
The results of this study support the postulation that PFOS may be an end-point metabolite in the biodegradation of fluorochemical polymers and fluorochemical monomers (see final reports for project CA058 and CA097 ). The results of this study corroborate those of project CA097, which showed no biodegradation of PFOS, but did show biodegradation of other perfluorooctanesulfonyl-based chemistries .
Page 22 of 29
Pace Project CA104: "The 35-Day Aerobic Biodegradation Study for PFOS" Study Director: Cleston C. Lange, Ph.D. Pace Analytical Services, Contract Analytical Group Thursday, March 29, 2001
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Although toxicity was not addressed as part of this study, the microbial respiration inhibition by PFOS has been tested previously, and PFOS was found to not be a significant inhibitor of microbial respiration processes at (13% inhibition at 2.7 mg/mL) .
10.0 References
1. Final Reports for Pace Project CA058, "2-Week N-EtFOSE Alcohol Biodegradation Screen Study Report" and "Aerobic Biodegradation of N-EtFOSE alcohol Study Report". 3M LIMS Project Number: E00 -2252. Author: Cleston C. Lange, Ph.D.
2. Final report for Pace Project CA097, "The 18-Day Aerobic Biodegradation Study of Perfluorooctanesulfonyl-based Chemistries." 3M LIMS Project Number: not available. Author: Cleston C. Lange, Ph.D.
3. Activated Sludge Respiration Inhibition Test for PFOS. Robust Summary for 3M Environmental Laboratory Lab Request number U2723. Test Conducted under GLP requirements for 3M by Wildlife International, Ltd. Easton, MD.
4. Pace method CAG-SP-03. "Culture Preparation for Assessment of Aerobic Biodegradability of Fluorochemicals Using Municipal or Industrial Sludge as Microbial Inoculum."
5. Pace method CAG-SP-04. "C18 Solid Phase Extraction Procedure for Fluorochemicals Recovery from Aqueous/Sludge Matrices."
6. Pace method CAG-ORG-23 "Quantitative Analysis of Fluorochemicals by High Performance Liquid Chromatography with Mass Spectrometric Detection."
7. Project CA105, "35-Day Fluorochemical Adipate Biodegradation Screen Study". Initiated September 2001--in progress, Study Director: Cleston C. Lange, Ph.D.
8. Project CA132, "35-Day FC807-Diester Biodegradation Screen Study ". Initiated September 2001--in progress, Study Director: Cleston C. Lange, Ph.D.
Page 23 of 29 Pace Project CA104: "The 35-Day Aerobic Biodegradation Study for PFOS" Study Director: Cleston C. Lange, Ph.D. Pace Analytical Services, Contract Analytical Group Thursday, March 29, 2001
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9.0 Sample and Data Retention
Original facility records and original data and a copy of the signed final report will be retained in the Pace Analytical-Tier 2 data archives for a period of 2 years after completion of the project. At the end of 2 years, or upon request before that time, the data will be sent to the 3M Environmental Laboratory for ultimate long-term archiving.
The following will be provided to 3M personnel:
The original signed analytical report and one copy of the signed original All original Data, correspondence, chromatograms, sample & standards prep sheets, etc., upon request.
Upon request of original data and records, copies of all original raw data in the form of chromatograms, reduced data, and written records concerning this project will be prepared and retained in the Pace Analytical-Tier 2 data archives.. All electronic copies of the instrumental raw data will be archived onto CD disks (read-only) and a copy provided to 3M upon request, or after 2 years, which ever is first.
Facility data will be retained for a period of 10 years. Facility data is available for inspection and includes the following records:
Training records Controlled storage temperature logs Standard preparation logs Calibration and maintenance logs Chemical and solvent traceability logs Standard Operating Procedures Methods pertaining to the conduct of this project
The remaining sample extracts will be retained at the Pace Analytical facility for a period of 1 year after completion of the project at 4C in the Carroll walk-in cooler (Pace ID 0140) located in the Pace Analytical-Tier 2 facility.
Page 24 of 29 Pace Project CA104: "The 35-Day Aerobic Biodegradation Study for PFOS" Study Director: Cleston C. Lange, Ph.D. Pace Analytical Services, Contract Analytical Group Thursday, March 29, 2001
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Appendix A Signatures of Project Personnel
Project Title: 5-Week A/-EtFOSE Alcohol Aerobic Biodegradation Study
Client Project ID:
LIMS E01-044
Contract Analytical Project Number: CA104
The following individuals participated in the conduct of this project:
Study Director: Cleston C. Lange
_________________________ Signature
Laboratory Management: Bruce E Warden
Report Author: Cleston C. Lange
Signature Signature
Sample Preparation Analyst: Angela L. Schuler
____________________ Signature
HPLC/MS Analysis Conducted by: Anthony E. Scales
____________________ Signature
Angela L. Schuler
_________________________ Signature
Date
Date Date
Date
Date ________
Date
Page 25 of 29
Pace Project CA104: "The 35-Day Aerobic Biodegradation Study for PFOS" Study Director: Cleston C. Lange, Ph.D. Pace Analytical Services, Contract Analytical Group Thursday, March 29, 2001
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Appendix B Final Concentration Results: PFOS Adjusted for Purity
Table 1. Final data results for sample cultures during this 35-day screening study. PFOS was
the only target analyte detected in sample cultures, and the mean concentration during the entire
study for sample cultures, plus/minus the standard deviation, was 2,553 ng/mL + 102 ng/mL, and
with a relative standard deviation of 4.0%. The final concentration values shown are adjusted for
the PFOS reference material purity reported at 86.4%. The PFOS measured versus the expected
concentration (2,582 ng/mL) is presented as a percentage in the last column
PFOS
Sample ID
Sample Information
ng/mL
CA104-0920-SA-001 Day 0 PFOS biodegradation sample
2620
CA104-0920- SA-002 Day 0 PFOS biodegradation duplicate
2710
CA104-0920- SA-003 Day 2 PFOS biodegradation sample
2374
CA104-0920- SA-004 Day 2 PFOS biodegradation duplicate
2425
CA104-0920- SA-005 Day 5 PFOS biodegradation sample
2522
CA104-0920- SA-006 Day 5 PFOS biodegradation duplicate
2558
CA104-0920-SA-007 Day 7 PFOS biodegradation sample
2485
CA104-0920- SA-008 Day 7 PFOS biodegradation duplicate
2695
CA104-0920- SA-009 Day 14 PFOS biodegradation sample
2477
CA104-0920- SA-010 Day 14 PFOS biodegradation duplicate
2559
CA104-0920-SA-011 Day 35 PFOS biodegradation sample CA104-0920- SA-012 Day 35 PFOS biodegradation duplicate
2585 2626
LOQ for PFOS 4.4 ng/mL
LOQ for PFOA 11.0 ng/mL
LOQ for PFOSulfinate 5.5 ng/mL
N D: peak not detected
PFOA ng/mL
ND ND ND ND ND ND ND ND ND ND ND ND
PFOS Sulfinate ng/mL ND ND ND ND ND ND ND ND ND ND ND ND
m/z 556 ng/mL
ND ND ND ND ND ND ND ND ND ND ND ND
W-EtFOSE acid ng/mL ND ND ND ND ND ND ND ND ND ND ND ND
FOSA ng/mL
ND ND ND ND ND ND ND ND ND ND ND ND
W-EtFOSE Alcohol ng/mL ND ND ND ND ND ND ND ND ND ND ND ND
W-EtFOSA
ng/mL ND ND ND ND ND ND ND ND ND ND ND ND
PFOS Recovery
% 101.5% 105.0% 91.9% 93.9% 97.7% 99.1% 96.3% 104.4% 95.9% 99.1% 100.1% 101.7%
Table 2. Final data results for the abiotic control cultures during this 35-day screening study. PFOS was the only analyte detected, and the mean concentration plus/minus the standard deviation determined for all abiotic controls through the study was 2,653 ng/mL + 83 ng/mL, with a relative standard deviation of 3.1%. Note: Sample CA104-0920-028 results were invalid because of a sample preparation error--this sample is likely a positive control culture with NEtFOSE alcohol as a test substrate, which was prepared for another study. The final concentration values are adjusted for PFOS reference material purity at 86.4% pure._____________
PFOS
Sample ID
Sample Information
ng/mL
CA104- 0920-SA- 019 Day 0 PFOS abiotic control
2522
CA104- 0920-SA- 020 Day 0 PFOS abiotic duplicate
2530
CA104- 0920-SA- 021 Day 2 PFOS abiotic control
2704
CA104- 0920-SA- 022 Day 2 PFOS abiotic duplicate
2744
CA104- 0920-SA- 023 Day 5 PFOS abiotic control
2744
CA104- 0920-SA- 024 Day 5 PFOS abiotic duplicate
2751
CA104- 0920-SA- 025 Day 7 PFOS abiotic control
2610
CA104- 0920-SA- 026 Day 7 PFOS abiotic duplicate
2635
CA104- 0920-SA- 027 Day 14 PFOS abiotic control
2681
CA104- 0920-SA- 028 Invalid Sample
4.3
CA104- 0920-SA- 029 Day 35 PFOS abiotic control
2588
CA104- 0920-SA- 030 Day 35 PFOS abiotic duplicate
2683
LOQ for PFOS 4.4 ng/mL
LOQ for PFOA 11.0 ng/mL
LOQ for PFOSulfinate 5.5 ng/mL
ND: peak not detected
PFOA ng/mL
ND ND ND ND ND ND ND ND ND ND ND ND
PFOS Sulfinate ng/mL ND ND ND ND ND ND ND ND ND ND ND ND
m/z 556 ng/mL
ND ND ND ND ND ND ND ND ND 9.9 ND ND
W-EtFOSE acid ng/mL ND ND ND ND ND ND ND ND ND 86.6 ND ND
FOSA ng/mL
ND ND ND ND ND ND ND ND ND 7.6 ND ND
W-EtFOSE Alcohol ng/mL ND ND ND ND ND ND ND ND ND 150 ND ND
PFOS W-EtFOSA Recovery
ng/mL
%
ND 97.7%
ND 98.0%
ND 104.7%
ND 106.3%
ND 106.3%
ND 106.5% ND 101.1%
ND 102.1%
ND 103.8%
ND Not applicable
ND 100.2%
ND 103.9%
Table 3. The final data results for sludge blanks during the 35-day study. Target analytes were not detected in any of the sludge blanks.______________________________________________
Sample ID 10260017.D 10260022.D 10260051.D 10260056.D 10260085.D 10260090.D ND: peak not detected
Sample Information Day 0 PFOS sludge blank Day 2 PFOS sludge blank Day 5 PFOS sludge blank Dav 7 PFOS sludqe blank Day 14 PFOS sludge blank Day 35 PFOS sludge blank
PFOS ng/mL
ND ND ND ND ND ND
PFOA ng/mL
ND ND ND ND ND ND
PFOS Sulfinate ng/mL ND ND ND ND ND ND
m/z 556 ng/mL
ND ND ND ND ND ND
W-EtFOSE acid ng/mL ND ND ND ND ND ND
FOSA ng/mL
ND ND ND ND ND ND
W-EtFOSE Alcohol ng/mL ND ND ND ND ND ND
W-EtFOSA
ng/mL ND ND ND ND ND ND
Page 26 of 29
Pace Project CA104: "The 35-Day Aerobic Biodegradation Study for PFOS" Study Director: Cleston C. Lange, Ph.D. Pace Analytical Services, Contract Analytical Group Thursday, March 29, 2001
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Appendix C Integrated Chromatogram Peak Area Data
Table 1. Actual integrated LC/MS peak area data for calibration standards, samples, controls, blanks, and CCVs. Data obtained from LC/MS batch reports for sequence D102600.S run on
LC/MS instrument "Dutch". Peaks that were not detected resulted in a reported peak area of 0.00. The results for SPE eluate 2 are designated by E2 and results for SPE eluate 3 are designated by E3._______________________________________________________________
Sample ID Methanol Blank CA105-MW-001
CA105-MW-002 CA105-MW-003 CA105-MW-004 CA105-MW-005 CA105-MW-006
CA105-MW-007 CA105-MW-008 Methanol Blank CA104-SA-001 E2 CA104-SA-002 E2
CA104-SA-013 E2 CA104-SA-019 E2 CA104-SA-020 E2 CA104-SA-003 E2 CA104-SA-004 E2
CA104-SA-014 E2 CA104-SA-021 E2 CA104-SA-022 E2 Methanol Blank CA105-MW-004
CA105-MW-006 Methanol Blank CA104-SA-001 E3 CA104-SA-002 E3 CA104-SA-013 E3
CA104-SA-019 E3 CA104-SA-020 E3 CA104-SA-003 E3 CA104-SA-004 E3 CA104-SA-014 E3
CA104-SA-021 E3 CA104-SA-022 E3 Methanol Blank CA105-MW-001 CA105-MW-002
CA105-MW-003 CA105-MW-004 CA105-MW-005 CA105-MW-006 CA105-MW-007
CA105-MW-008 Methanol Blank CA104-SA-005 E2 CA104-SA-006 E2 CA104-SA-015 E2
CA104-SA-023 E2 CA104-SA-024 E2 CA104-SA-007 E2 CA104-SA-008 E2 CA104-SA-016 E2
CA104-SA-025 E2 CA104-SA-026 E2 Methanol Blank CA105-MW-004 CA105-MW-006
Methanol Blank CA104-SA-005 E3 CA104-SA-006 E3
File ID 10260005.D 10260006.D
10260007.D 10260008.D 10260009.D 10260010.D 10260011.D
10260012.D 10260013.D 10260014.D 10260015.D 10260016.D
10260017.D 10260018.D 10260019.D 10260020.D 10260021.D
10260022.D 10260023.D 10260024.D 10260025.D 10260026.D
10260027.D 10260028.D 10260029.D 10260030.D 10260031.D
10260032.D 10260033.D 10260034.D 10260035.D 10260036.D
10260037.D 10260038.D 10260039.D 10260040.D 10260041.D
10260042.D 10260043.D 10260044.D 10260045.D 10260046.D
10260047.D 10260048.D 10260049.D 10260050.D 10260051.D
10260052.D 10260053.D 10260054.D 10260055.D 10260056.D
10260057.D 10260058.D 10260059.D 10260060.D 10260061.D
10260062.D 10260063.D 10260064.D
PFOS AREA 0.00 14935.93
30181.21 60707.00 136328.78 305656.84 648404.88
1436899.38 2749046.50
0.00 6957170.00 6305939.00
0.00 6291996.00 6393484.00 5646777.00 6028145.50
0.00 6741408.50 6487929.50
0.00 119812.42
613307.56 0.00
396809.47 824812.50
0.00
1115791.25 900586.75 1364811.50 1301216.88
0.00
811296.81 1358693.38
0.00 13926.68 30499.03
57042.59 115962.44 306848.22 624188.44 1394744.88
2612717.25 0.00
6786659.00 6006282.50
0.00
6788747.50 6410242.00 6488784.50 6665772.00
0.00
6924641.50 6109320.50
0.00 119866.02 598445.38
0.00 845802.25 1369521.63
PFOA AREA 0.00 17005.95
18006.10 39103.28 78302.48 192285.58 383602.31
810410.25 1568670.38
0.00 0.00 0.00
0.00 0.00 0.00 0.00 0.00
0.00 0.00 0.00 0.00 74300.85
374440.19 0.00 0.00 0.00 0.00
0.00 0.00 0.00 0.00 0.00
0.00 0.00 0.00 16257.32 19643.56
35963.19 72333.11 184666.11 362352.81 812106.06
1473658.50 0.00 0.00 0.00 0.00
0.00 0.00 0.00 0.00 0.00
0.00 0.00 0.00 70442.73 366501.22
0.00 0.00 0.00
PFOSulfinate AREA 0.00 11417.06
21815.46 43160.50 91770.55 212681.84 442822.63
926250.81 1687577.50
0.00 0.00 0.00
0.00 0.00 0.00 0.00 0.00
0.00 0.00 0.00 0.00 82843.96
395532.88 0.00 0.00 0.00 0.00
0.00 0.00 0.00 0.00 0.00
0.00 0.00 0.00 9813.04 20608.71
43763.26 84334.57 211118.88 423281.03 907407.63
1696326.63 0.00 0.00 0.00 0.00
0.00 0.00 0.00 0.00 0.00
0.00 0.00 0.00 85432.81 407539.69
0.00 0.00 0.00
FOSA AREA 0.00 32261.59
51792.79 107790.25 240400.95 589225.94 1215682.00
2680574.00 5255131.00
0.00 0.00 0.00
0.00 0.00 0.00 0.00 0.00
0.00 0.00 0.00 0.00 210264.64
1170395.00 0.00 0.00 0.00 0.00
0.00 0.00 0.00 0.00 0.00
0.00 0.00 0.00 26289.82 56804.00
109354.09 219859.73 584366.00 1196495.38 2672052.00
5336807.00 0.00 0.00 0.00 0.00
0.00 0.00 0.00 0.00 0.00
0.00 0.00 0.00 222467.50 1179220.63
0.00 0.00 0.00
M556 AREA 0.00 12498.49
27038.68 51526.13 105243.81 244543.95 494728.31
949330.00 1658535.88
0.00 0.00 0.00
0.00 0.00 0.00 0.00 0.00
0.00 0.00 0.00 0.00 98867.90
438835.00 0.00 0.00 0.00 0.00
0.00 0.00 0.00 0.00 0.00
0.00 0.00 0.00 12067.14 25511.25
49171.96 100589.99 245054.34 460255.28 959887.38
1724157.38 0.00 0.00 0.00 0.00
0.00 0.00 0.00 0.00 0.00
0.00 0.00 0.00 97611.95 501073.41
0.00 0.00 0.00
N-EtFOSE acid AREA 0.00
6516.98
13797.29 25951.37 58180.79 150302.11 296591.75
650015.88 1103419.25
0.00 0.00 0.00
0.00 0.00 0.00 0.00 0.00
0.00 0.00 0.00 0.00 63633.68
290054.69 0.00 0.00 0.00 0.00
0.00 0.00 0.00 0.00 0.00
0.00 0.00 0.00 8051.56 15130.46
31349.53 62632.75 155416.72 334414.94 682568.50
1200336.75 0.00 0.00 0.00 0.00
0.00 0.00 0.00 0.00 0.00
0.00 0.00 0.00 59434.46 325603.88
0.00 0.00 0.00
N-EtFOSE Alcohol AREA 0.00 21793.25
34541.48 68317.75 148115.28 394311.94 745335.31
1667151.00 3026545.75
0.00 0.00 0.00
0.00 0.00 0.00 0.00 0.00
0.00 0.00 0.00 0.00 144413.31
787338.31 0.00 0.00 0.00 0.00
0.00 0.00 0.00 0.00 0.00
0.00 0.00 0.00 22119.35 37607.71
66553.86 146629.98 360967.19 780334.31 1736950.75
3244554.00 0.00 0.00 0.00 0.00
0.00 0.00 0.00 0.00 0.00
0.00 0.00 0.00 150306.63 786183.63
0.00 0.00 0.00
N-EtFOSA AREA 0.00
34484.85
71779.78 145654.98 309278.94 829537.06 1610657.00
3691885.25 6929020.50
0.00 0.00 0.00
0.00 0.00 0.00 0.00 0.00
0.00 0.00 0.00 0.00 308571.63
1637672.38 0.00 0.00 0.00 0.00
0.00 0.00 0.00 0.00 0.00
0.00 0.00 0.00 36496.52 72426.15
138753.38 303927.13 762607.75 1613388.25 3800280.25
7224467.50 0.00 0.00 0.00 0.00
0.00 0.00 0.00 0.00 0.00
0.00 0.00 0.00 306529.22 1615856.13
0.00 0.00 0.00
Page 27 of 29
Pace Project CA104: "The 35-Day Aerobic Biodegradation Study for PFOS" Study Director: Cleston C. Lange, Ph.D. Pace Analytical Services, Contract Analytical Group Thursday, March 29, 2001
BACK TO MAIN
Table 1, continued.
Appendix C Integrated Chromatogram Peak Area Data
Sample ID
CA104-SA-015 E3 CA104-SA-023 E3 CA104-SA-024 E3 CA104-SA-007 E3 CA104-SA-008 E3
CA104-SA-016 E3 CA104-SA-025 E3 CA104-SA-026 E3 Methanol Blank CA105-MW-001
CA105-MW-002 CA105-MW-003 CA105-MW-004 CA105-MW-005 CA105-MW-006
CA105-MW-007 CA105-MW-008 Methanol Blank CA104-SA-009 E2 CA104-SA-010 E2
CA104-SA-017 E2 CA104-SA-027 E2 CA104-SA-028 E2 CA104-SA-011 E2 CA104-SA-012 E2
CA104-SA-018 E2 CA104-SA-029 E2 CA104-SA-030 E2 Methanol Blank CA105-MW-004
CA105-MW-006 Methanol Blank CA104-SA-009 E3 CA104-SA-010 E3 CA104-SA-017 E3
CA104-SA-027 E3 CA104-SA-028 E3 CA104-SA-011 E3 CA104-SA-012 E3 CA104-SA-018 E3
CA104-SA-029 E3 CA104-SA-030 E3 Methanol Blank CA105-MW-001 CA105-MW-002
CA105-MW-003 CA105-MW-004 CA105-MW-005 CA105-MW-006 CA105-MW-007
CA105-MW-008 Methanol Blank CA104-SA-001 E2 CA104-SA-002 E2 CA104-SA-013 E2
CA104-SA-019 E2 CA104-SA-020 E2 CA104-SA-003 E2 CA104-SA-004 E2
File ID
10260065.D 10260066.D 10260067.D 10260068.D 10260069.D
10260070.D 10260071.D 10260072.D 10260073.D 10260074.D
10260075.D 10260076.D 10260077.D 10260078.D 10260079.D
10260080.D 10260081.D 10260082.D 10260083.D 10260084.D
10260085.D 10260086.D 10260087.D 10260088.D 10260089.D
10260090.D 10260091.D 10260092.D 10260093.D 10260094.D
10260095.D 10260096.D 10260097.D 10260098.D 10260099.D
10260100.D 10260101.D 10260102.D 10260103.D 10260104.D
10260105.D 10260106.D 10260107.D 10260108.D 10260109.D
10260110.D 10260111.D 10260112.D 10260113.D 10260114.D
10260115.D 10260116.D 10260117.D 10260118.D 10260119.D
10260120.D 10260121.D 10260122.D 10260123.D
PFOS AREA
0.00 498743.13 1086776.88 768729.00 447635.56
0.00 801625.56 1471700.13
0.00 13982.04
29987.14 57815.73 120780.75 281840.94 582970.25
1360512.75 2716866.75
0.00 6546132.50 6927869.00
0.00 6125243.50
12923.94 7176159.50 6938688.50
0.00 6555500.50 6113930.50
0.00 114039.99
590903.75 0.00
523538.56 278158.69
0.00
1810332.13 0.00
244567.91 251377.70
0.00
245937.13 997470.25
0.00 13783.06 29314.46
50766.63 116922.48 290955.97 610388.56 1420859.75
2440706.50 0.00
1567058.63 1543427.88
0.00
1382376.50 1426916.38 1248381.63 1290877.75
PFOA AREA
0.00 0.00 0.00 0.00 0.00
0.00 0.00 0.00 0.00 16699.33
19143.72 36066.05 73131.43 175630.80 344841.03
778203.38 1538752.63
0.00 0.00 0.00
0.00 0.00 0.00 0.00 0.00
0.00 0.00 0.00 0.00 72349.25
347965.78 0.00 0.00 0.00 0.00
0.00 0.00 0.00 0.00 0.00
0.00 0.00 0.00 16971.07 16183.18
35860.38 76600.59 173415.73 368883.84 811055.88
1431169.13 0.00 0.00 0.00 0.00
0.00 0.00 0.00 0.00
PFOSulfinate AREA
0.00 0.00 0.00 0.00 0.00
0.00 0.00 0.00 0.00 9979.43
21326.87 41587.84 85434.55 201832.98 394586.31
905614.13 1670244.88
0.00 0.00 0.00
0.00 0.00 0.00 0.00 0.00
0.00 0.00 0.00 0.00 83337.26
380184.41 0.00 0.00 0.00 0.00
0.00 0.00 0.00 0.00 0.00
0.00 0.00 0.00 10400.72 19628.93
37017.05 77199.96 197817.11 381316.00 880515.69
1573230.13 0.00 0.00 0.00 0.00
0.00 0.00 0.00 0.00
FOSA AREA
0.00 0.00 0.00 0.00 0.00
0.00 0.00 0.00 0.00 25697.01
55001.61 111607.10 225726.55 538672.31 1127954.38
2775788.75 5418179.50
0.00 0.00 0.00
0.00 0.00 35557.76 0.00 0.00
0.00 0.00 0.00 0.00 228457.66
1120460.75 0.00 0.00 0.00 0.00
0.00 0.00 0.00 0.00 0.00
0.00 0.00 0.00 23816.95 51018.12
106005.91 231666.81 511399.22 1124824.63 2824799.25
5090895.00 0.00 0.00 0.00 0.00
0.00 0.00 0.00 0.00
M556 AREA
0.00 0.00 0.00 0.00 0.00
0.00 0.00 0.00 0.00 12133.76
25088.65 50632.96 97380.48 234026.91 451452.41
966946.69 1755200.00
0.00 0.00 0.00
0.00 0.00 24310.33 0.00 0.00
0.00 0.00 0.00 0.00 102876.83
443270.28 0.00 0.00 0.00 0.00
0.00 0.00 0.00 0.00 0.00
0.00 0.00 0.00 10909.54 24116.90
49735.05 95368.06 236238.25 449826.97 972162.31
1639724.38 0.00 0.00 0.00 0.00
0.00 0.00 0.00 0.00
N-EtFOSE acid AREA
0.00 0.00 0.00 0.00 0.00
0.00 0.00 0.00 0.00 7161.94
16386.03 32367.63 61900.77 156350.09 302950.56
694380.88 1224527.13
0.00 0.00 0.00
0.00 0.00 103052.61 0.00 0.00
0.00 0.00 0.00 0.00 68451.39
304462.94 0.00 0.00 0.00 0.00
0.00 27767.98
0.00 0.00 0.00
0.00 0.00 0.00 6845.91 15959.31
31492.54 66819.37 158163.89 322770.94 658313.31
1193133.25 0.00 0.00 0.00 0.00
0.00 0.00 0.00 0.00
N-EtFOSE Alcohol AREA
0.00 0.00 0.00 0.00 0.00
0.00 0.00 0.00 0.00 22058.21
35591.88 72765.41 146651.70 345086.19 753909.56
1755616.00 3052786.50
0.00 0.00 0.00
0.00 0.00 394497.41 0.00 0.00
0.00 0.00 0.00 0.00 149993.77
769036.19 0.00 0.00 0.00 0.00
0.00 60579.23
0.00 0.00 0.00
0.00 0.00 0.00 19189.26 33522.69
69082.69 136360.42 364269.91 722861.75 1587305.25
3142575.75 0.00 0.00 0.00 0.00
0.00 0.00 0.00 0.00
N-EtFOSA AREA
0.00 0.00 0.00 0.00 0.00
0.00 0.00 0.00 0.00 35894.19
75564.77 146495.67 302634.66 753119.63 1621473.00
3769292.50 7069280.00
0.00 0.00 0.00
0.00 0.00 0.00 0.00 0.00
0.00 0.00 0.00 0.00 314206.38
1652037.25 0.00 0.00 0.00 0.00
0.00 0.00 0.00 0.00 0.00
0.00 0.00 0.00 31411.06 73106.06
149169.80 305432.00 790519.06 1547735.88 3652949.75
7039522.00 0.00 0.00 0.00 0.00
0.00 0.00 0.00 0.00
Page 28 of 29
Pace Project CA104: "The 35-Day Aerobic Biodegradation Study for PFOS" Study Director: Cleston C. Lange, Ph.D. Pace Analytical Services, Contract Analytical Group Thursday, March 29, 2001
BACK TO MAIN
Table 1, continued.
Appendix C Integrated Chromatogram Peak Area Data
Sample ID CA104-SA-014 E2 CA104-SA-021 E2
CA104-SA-022 E2 Methanol Blank CA105-MW-004 CA105-MW-006 Methanol Blank
CA104-SA-005 E2 CA104-SA-006 E2 CA104-SA-015 E2 CA104-SA-023 E2 CA104-SA-024 E2
CA104-SA-007 E2 CA104-SA-008 E2 CA104-SA-016 E2 CA104-SA-025 E2 CA104-SA-026 E2
Methanol Blank CA105-MW-004 CA105-MW-006 Methanol Blank CA104-SA-009 E2
CA104-SA-010 E2 CA104-SA-017 E2 CA104-SA-027 E2 CA104-SA-028 E2 CA104-SA-011 E2
CA104-SA-012 E2 CA104-SA-018 E2 CA104-SA-029 E2 CA104-SA-030 E2 Methanol Blank
CA105-MW-001 CA105-MW-002 CA105-MW-003 CA105-MW-004 CA105-MW-005
CA105-MW-006 CA105-MW-007 CA105-MW-008 Methanol Blank
File ID 10260124.D 10260125.D
10260126.D 10260127.D 10260128.D 10260129.D 10260130.D
10260131.D 10260132.D 10260133.D 10260134.D 10260135.D
10260136.D 10260137.D 10260138.D 10260139.D 10260140.D
10260141.D 10260142.D 10260143.D 10260144.D 10260145.D
10260146.D 10260147.D 10260148.D 10260149.D 10260150.D
10260151.D 10260152.D 10260153.D 10260154.D 10260155.D
10260156.D 10260157.D 10260158.D 10260159.D 10260160.D
10260161.D 10260162.D 10260163.D 10260164.D
PFOS AREA 0.00 1542179.63
1465341.63 0.00
115161.10 569541.31
0.00
1425852.63 1347554.38
0.00 1615440.38 1512463.88
1419311.50 1596666.38
0.00 1484539.25 1372794.75
0.00 116771.37 570884.63
0.00 1459985.63
1548178.13 0.00
1330363.75 0.00
1568615.88
1591005.88 0.00
1570424.50 1492012.63
0.00
13339.08 25805.36 51243.50 114950.23 285749.47
612202.88 1336394.25 2668366.75
0.00
PFOA AREA 0.00 0.00
0.00 0.00 72027.19 345411.16 0.00
0.00 0.00 0.00 0.00 0.00
0.00 0.00 0.00 0.00 0.00
0.00 70822.80 340879.81
0.00 0.00
0.00 0.00 0.00 0.00 0.00
0.00 0.00 0.00 0.00 0.00
17440.71 17887.09 34595.46 74509.45 173712.80
377687.50 816368.25 1505736.25
0.00
PFOSulfinate AREA 0.00 0.00
0.00 0.00 77033.35 401612.88 0.00
0.00 0.00 0.00 0.00 0.00
0.00 0.00 0.00 0.00 0.00
0.00 78991.95 393441.50
0.00 0.00
0.00 0.00 0.00 0.00 0.00
0.00 0.00 0.00 0.00 0.00
9950.26 18693.97 39270.14 78962.37 205597.61
419857.09 883441.13 1665229.50
0.00
FOSA AREA 0.00 0.00
0.00 0.00 224267.55 1078812.50 0.00
0.00 0.00 0.00 0.00 0.00
0.00 0.00 0.00 0.00 0.00
0.00 222036.23 1135857.38
0.00 0.00
0.00 0.00 0.00 0.00 0.00
0.00 0.00 0.00 0.00 0.00
25193.90 50273.33 105613.48 230707.58 569895.75
1123617.13 2664414.75 5137724.00
0.00
M556 AREA 0.00 0.00
0.00 0.00 99030.34 474330.34 0.00
0.00 0.00 0.00 0.00 0.00
0.00 0.00 0.00 0.00 0.00
0.00 98022.34 452548.31
0.00 0.00
0.00 0.00 0.00 0.00 0.00
0.00 0.00 0.00 0.00 0.00
11523.59 23402.56 44237.55 100317.29 243329.03
484254.34 940157.13 1700229.00
0.00
N-EtFOSE acid AREA 0.00 0.00
0.00 0.00 65506.07 322461.78 0.00
0.00 0.00 0.00 0.00 0.00
0.00 0.00 0.00 0.00 0.00
0.00 64854.18 312251.16
0.00 0.00
0.00 0.00 0.00 19335.43 0.00
0.00 0.00 0.00 0.00 0.00
7477.15 14347.88 30264.75 70063.81 154189.81
340038.13 686594.19 1182218.63
0.00
N-EtFOSE Alcohol AREA 0.00 0.00
0.00 0.00 134408.08 730636.06 0.00
0.00 0.00 0.00 0.00 0.00
0.00 0.00 0.00 0.00 0.00
0.00 143022.80 758448.81
0.00 0.00
0.00 0.00 0.00 72679.98 0.00
0.00 0.00 0.00 0.00 0.00
0.00 32397.41 61003.66 149303.59 339701.94
652429.19 1667519.50 2862431.75
0.00
N-EtFOSA AREA 0.00 0.00
0.00 0.00 289903.72 1518741.38 0.00
0.00 0.00 0.00 0.00 0.00
0.00 0.00 0.00 0.00 0.00
0.00 294664.34 1639711.25
0.00 0.00
0.00 0.00 0.00 0.00 0.00
0.00 0.00 0.00 0.00 0.00
0.00 65195.19 135146.89 310741.59 760203.31
1479341.63 3684853.75 6919559.00
0.00
Page 29 of 29
Pace Project CA104: "The 35-Day Aerobic Biodegradation Study for PFOS" Study Director: Cleston C. Lange, Ph.D. Pace Analytical Services, Contract Analytical Group Thursday, March 29, 2001
A ppendix A Signatures of Project Personnel
BACK TO MAIN
Project Title: 5-Week A/-EtFOSE Alcohol Aerobic Biodegradation Study
Client Project ID:
LIMS E01-044
Contract Analytical Project Number: CA104
The following individuals participated in the conduct of this project:
Study Director: Cleston C. Lange
Laboratory Management: Bruce E Warden
Report Author: Cleston C. Lange
Date
Sample Preparation Analyst: Angela L. Schuler
HPLC/MS Analysis Conducted by: Anthony E. Scales
Angela L. Schuler
iature
Page 25 o f 29
Pace Project CA104: "The 35-Day Aerobic Biodegradation Study for PFOS" Study Director: Cleston C. Lange, Ph.D. Pace Analytical Services, Contract Analytical Group Thursday, March 29, 2001
Original
