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BIODEGRADATION STUDY REPORT
Revision 1
Wednesday, November 06, 2002
Biodegradation Screen Study for Telomer-Type Alcohols
PROJECT NUMBER
3M Projects ID: E01-0684 Pace Contract Analytical Projects ID: CA085
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. Bio-Analytical Services Group
1700 Elm Street, Suite 200 Minneapolis, Minnesota 55414
PROJECTDATES Project Initiation: August 9, 2000
Project Completion: September 18, 2001 AUTHOR
Cleston C. Lange, Ph.D.
Page 1 of 38
Pace Project CA085: "Biodegradation Screen Study for Teiomer Alcohols" -Revision 1 Study Director: Cleston C. Lange, Ph.D. Pace Analytical Services, Bio-Analytical Services Group Wednesday, November 06, 2002
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Table of Contents
Title Page .....................................................................................................................................................
1
Table of Contents ........................................................................................................................................
2
List of Tables & Figures ...............................................................................................................................
3
List of Appendices .......................................................................................................................................
4
Executive Summary .....................................................................................................................................
5
1.0 Project Personnel ..................................................................................................................................
6
2.0 Data Requirements and Revision Justification ......................................................................................
6
3.0 Project Objective ....................................................................................................................................
6
4.0 Test Article ..........................................................................................................................................
7-8
5.0 Reference Articles .................................................................................................................................
9
6.0 Receipt/Generation of Samples .............................................................................................................
9
7.0 Methods .................................... i ............................................................................................................
9
7.1 7S.1a.m1plCeolPlercetpioanratioofnSl.u..d..g..e................................................................................................................. 7.1.2 Culture Preparation ......................................................................................................... 7.1.3 Solid Phase Extraction (SPE) .........................................................................................
9-109 11-12 12-13
7.2 Instrumental Analysis (HPLC/MS) ................................................................................................ 7.2.1 Instrument Parameters ...................................................................................................
13 13-14
7.2.2 Qualitative Analysis of Parents and Products ................................................................ 7.2.3 Quantitative Analysis ........................................................................................................... 7.3 Data Transformations and Calculations ........................................................................................ 7.3.1 Molar Calculations ...............................................................................................................
14-15 16 16 16
7.3.2 Conversion of ng/mL to micromolar and nanomolar ........................................................... 16
7.3.3 Molar Mass Balance Calculations .......................................................................................
17
7.4 Software Versions .........................................................................................................................
17
8.0 Results .................................................................................................................................................
18
8.1 Sludge Characterization ................................................................................................................
18
8.2 Quality Control/Sample Matrix Spike Results ...............................................................................
18
8.3 Analytical Blanks Results ...............................................................................................................
18
8.4 H8.P4L.1C/QMuSaliAtantaivleysisHP.L..C.../.M...S......a..n..d....H..P...L..C../.M...S.../.M...S......R..e..s..u..l.t.s...................................................................................................................... 19-2149
8.4.2 Quantitative Analysis Results for PFOA ..................................................... ;: .................. 24-25
9.0 Conclusions .........................................................................................................................................
27
10.0 Literature Cited ..................................................................................................................................
28
11.0 Sample and Data Retention ..............................................................................................................
29
s
Page 2 of 38
Pace Project CA085: "Biodegradation Screen Study for Telomer Alcohols" -Revision 1 Study Director: Cleston C. Lange, Ph.D. Pace Analytical Services, Bio-Analytical Services Group Wednesday, November 06, 2002
List of Tables & Figures
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TABLES
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Table 1. Parent telomer alcohol expected ions......................................................................................... 14
Table 2. Expected product fluorinated fatty acid products ions ................................................................ 15
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Table 3. HPLC/MS Data Table for Parent and Product Ions observed .................................................... 23
Table 4. HPLC/MS/MS Data for !3-OxidationProduct Ions observed ....................................................... 24
:
FIGURES
Figure 1. Chemical Representationof Telomer Alcohols........................................................................... 8
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Figure 2. Chemical Structure of perfluorooctanoate (PFOA) ...................................................................... 9
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Figure 3. Plotted HPLC/MS Integrated Peak Areas for C8,C10and C12Telomer Alcohols ......................20
Figure 4. Plotted HPLC/MS Integrated Peak Areas for C6,C14and C_8Telomer Alcohols ......................20
ii
Figure 5. Plotted HPLC/MS Integrated Peak Areas for Perfluorinated Fatty Acids Detected ..................22
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Figure 6. Plotted Peak Areas for Transient Polyfluorinated Fatty Acids Detected ................................... 22
Figure 7. Plotted PFOA Concentrations Measured in Biodegradation Samples (SPE eluate 2) .............25
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Figure 8. Proposed Biodegradation Pathwayfor Telomer Alcohols
26
Page 3 of 38
PaceProjectCA085:"BiodegradationScreenStudyfor TelomerAlcohols"-Revision 1 StudyDirector:ClestonC. Lange,Ph.D. PaceAnalyticalServices,Bio-AnalyticaSl ervicesGroup Wednesday,November06, 2002
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List of Appendices
Appendix A: Approval Signatures ............................................................................................................
30
Appendix B: Final Quantitative Results for PFOA (ng/mL)
Table 1. Test Cultures Final Results ..................................................................................
31
Table 2. Abiotic Cultures Final Results ................................................................................
31
Table 3. Blank Cultures Final Results .................................................................................
31
Appendix
C: Integrated Chromatogram Peak Area Data for the Telomer Alcohols
Table 1. Telomer Alcohols Peak Areas ...............................................................................
32
Table 2. Percentage of Peak Areas For Telomer Alcohols Remaining at Time Points ........ 32
Appendix D: Table 1. Integrated LC/MS (SIR) Peak Area Data for Perfluorinated Fatty Acids ............. 33 Appendix E: Table 1. Peak Area Data for Transiently Formed Pol_uorinated Fatty Acids .................. 34 Appendix F: Table 1. Total Ion Chromatogram (TIC) for SIR Data for Biodegradation Samples .......... 35 Appendix G: Table 1. TICs for SIR Data for Day 0 and Day 16 Biodegradation Samples ..................... 36
Appendix H: Table 1. TICs for SIR Data for Day 0 and Day 16 Abiotic Control Samples ...................... 37 Appendix I: Table 1. TICs for Extracted Ion Data for Transient Polyfluorinated Fatty Acids .................. 38
Page 4 of 38
Pace Project CA085: "Biodegradation Screen Study for Telomer Alcohols" -Revision 1 Study Director: Cleston C. Lange. Ph.D. Pace Analytical Services, Rio-Analytical Services Group Wednesday, November 06, 2002
Executive Summary
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A screening study was undertaken to determine whether the fluorochemical telomer intermediate
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(telomer alcohol) biodegrades when exposed to municipal wastewater treatment sludge. The
study included the preparation of cultures for a six sample-point comparative study, and included test cultures, blank cultures, and control cultures. The microbial inoculum for cultures was sludge obtained from the Twin Cities Metro Wastewater Treatment Facility. Telomer alcohol test
substance was added to cultures at approximately 5 #M concentrations. Cultures were
incubated with shaking at 25C. Solid phase extraction of cultures for recovery of
biodegradation products and parent analytes was employed. An HPLC/MS analytical method
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was developed for analysis of the telomer alcohols and expected products. The HPLC/MS
analysis of culture extracts provided strong evidence that the telomer alcohols were
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biodegraded. The observed loss of telomer alcohols occurred concomitant with the appearance
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of several expected perfluorinated carboxylic acids. Unexpectedly, transiently formed
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polyfluorinated !3-oxidation intermediates were observed. The _-oxidation pathway was
suspected to be the major route of biodegradation resulting in the observed even-numbered
carbon chain length perfluorinated fatty acids. Minor end products, as odd-numbered carbon
chain length perfluorinated fatty acids, were also observed and were likely the products of fatty
acid co-oxidation,a minor pathway of fatty acid metabolism usually observed in branched-chain
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fatty acids. After the 16day test period, perflourinated carboxylic acids ranging from C5 to C12
were observed in the test cultures. These compounds were not in controls or blank cultures.
Pmearsflsuobraolaonctcaendoaattae (aPcFcoOuAn)t,ewd afosrthaeppornolxyimcoamteplyou6n-7d%quoaf nthtiteattoivteallytealnoamlyezreadlcaonhdo,lsbainsietidalolyn
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present in the test cultures.
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Page 5 of 38 Pace Project CA085: "Biodegradation Screen Study for Telomer AIcohols"-Revision 1 Study Director: Cleston C. Lange, Ph.D. Pace Analytical Services, Bio-Analytical Services Group Wednesday, November 06, 2002
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1.0
Project Personnel
1.1
Sponsor Company
1.2 Sponsor Representative
1.3
Contract Facility Personnel:
1.3.1 Study Director
1.3.2 Laboratory Management:
1.3.3 Sample Preparation
1.3.4 1.3.5 1.3.6
HPLC/MS Analyst Sample Custodian: Report Author
3M Dr. James K. Lundberg
Dr. Cleston C. Lange Mr. Bruce E. Warden Ms. Angela L. Schuler Dr. Cleston C. Lange Ms. Angela L. Schuler Dr. Cleston C. Lange Dr. Cleston C. Lange
2.0
Data Requirements and Revision Justification
The sponsor representative desired an aerobic biodegradability screening study to be
conducted using the te/omer alcohol mixture as test substrate. The study was initiated on August 9, 2000 as a non-GLP study.
The report issued Friday, November 16, 2001 is revised to substitute "Dupont Zonyl BA type Telomer, or Zonyl type Telomer" with Telomer. The reason for the revision is to
more accurately reflect the impact of results to the general class of telomer alcohols.
3.0
,Project Objective
This study was conducted in order to elucidate whether the fluoroteiomer intermediate (telomer alcohol) is biodegradable under aerobic conditions using a microbial rich inoculum of municipal wastewater treatment sludge. This study was _milar to earlier studies conducted for 3M by Pace as Pace projects CA058 1 and CA097 2 and CA104 3, CA105 4, and CA132 2. The development of an analytical method and the analysis of parent analytes and possible products were critical parameters for the determination of biodegradation.
Page 6 of 38
Pace Project CA085: "Biodegradation Screen Study for Telomer AJcohols"-Revision 1 Study Director: Cleston C. Lange, Ph.D. Pace Analytical Services, Bio-Analytical Services Group Wednesday, November 06, 2002
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4.0
Test Article
The test article used for this study was the Zonyl BA-type telomer alcohol fluorochemical surfactant intermediate. Two sources of test material were used for this study. The first source was provided by the sponsor company, 3M, as approximately 2.5 g of crystalline solid test material labeled Zonyl BA-N telomer alcohol, 3M tracability number TN-A-2186, on July 7, 2000. The sponsor did not provide an MSDS, a chain of custody, the purity information for the test article, nor an expiration date for the material. This material was given a test, control, and reference (TCR) number at Pace as CA-TCR02-009 and was stored at 4C. Material CA-TCR02-009 was used primarily for method development purposes of the study, including the frst set of cultures prepared and original LC/MS method development.
A commercial vendor, Aldrich Chemical Company, provided the second source of telomer
alcohol used. Material was purchased as Zonyl BA-L fluorotelomer intermediate (1999
Aldrich Catalog number 42,151-0), also called perfluoroalkylethanol.
The material
characteristics were boiling point 145-245C, F(CF2)nCH2CH2OH where n equals
approximately 7 to 8, and molecular weight was reported as Mn ca. 443.70 wt. An
expiration date and the purity, or percent composition, of the material were not provided.
The purchased material was received at Pace on September 22, 2000. Upon receipt at
Pace, the material was given tracability number CA-TNC00-254 and was stored at 4C,
or less. A representative chemical compilation of the Zonyl BA-type telomer alcohol is shown in Figure 1.
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Page 7 of 38 Pace Project CA085: "Biodegradation Screen Study for Telomer Alcohols" -Revision 1 Study Director: Cleston C. Lange, Ph.D. Pace Analytical Services, Bio-Analytical Services Group Wednesday, November 06, 2002
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[M + CHACO0 "]"= 323
F F FF F F
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MMolleeccuuliaarrfWrmeuiglhat ==C3s6H4,s1F04laO
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-'rllllll C,o,V,o,ec=uC,,o,,'H,o,,r_r'I F F F F F F F F
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Molecular Weight = 464.119
FF F F F FF F
[M + CHACO0 ] "= 523
IlslrJrllr F F F F F F F F F F
F F F 'F F F F F F F F _----OH 012
Molecular Weight =564.134 [M+ CHACO0 "1= 623
0
F F F FF F FF F F F F
rlrtlllrlldl F F F F F F F F F F F F F ___ OH 014
Molecular formula = C_4H5 F2_O Molecular Weight = 664.149
F F F F F F FF F F F F F F F F F F F FF F F FF F F F
[M + CH3COO "]'= 723
Molecular formula
= C18 H 5 F29 0
[M + CH3COO "]" = 823
4.
Figure 1. The telomer alcohols. Based on the HPLC/MS average peak area response observed in the six abiotic control cultures, the composition of the test material was 6.4% + 0.3% as C6 telomer alcohol, 39.3% + 1.5% as C8, 27.7% + 2.1% as C10, 17.5% + 1.2% as C12,7.2% + 1.0% as C_4, and 2.0% + 0.2% as C16.
Page 8 of 38
Pace Project CA085: "Biodegradation Screen Study for Telomer Alcohols" -Revision 1 Study Director: Cleston C. Lange, Ph.D. Pace Analytical Services, Bio-Analytical Services Group Wednesday, November 06, 2002
5.0
Reference Materials
The sponsor provided reference material. The neat material was stored at -80C.
NS: Expiration date not specified
5.1. Perfluorooctanoate Ammonium Salt (PFOA)
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Purity: 95.2 %; 3M#: TCR-99131-37; Pace #: CA-TCR02-001; Expires: NS
rlllJil, o F F F F F F F O" NH; F F FFF FF
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Figure 2. Perfluorooctanoate ammonium salt
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6.0 Receipt/Generation of Samples
Samples were not received, but were generated as an inherent part of this study. All of the experimental cultures prepared for each culture set were extracted by solid phase extraction (SPE) methodology to generate three analytical samples per culture. The analytical samples, as SPE eluates collected for each culture, were labeled as SPE eluates 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. Qualitative and semi-quantitative HPLC/MS analysis was conducted for eluate 2 only, and the resulting data was evaluated to determine whether biodegradation occurred.
7.0 Methods
7.1
Sample Preparation
7.1.1. Collection of Sludge.
The sludge for this study was obtained from the Twin Cities Municipal waste treatment facility in a manner consistent with the sludge collected for other studies listed in section 3.0. The sludge used for the preparation of the first set of cultures was collected on July 31, 2000 and
Page 9 of 38
Pace Project CA085: "Biodegradation Screen Study for Telomer Alcohols" -Revision 1 Study Director: Cleston C. Lange, Ph.D. Pace Analytical Services, Bio-Analytical Services Group Wednesday, November 06, 2002
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delivered with Pace chain of custody (COC) form 465254. The sludge for the second set of cultures was collected on September 18, 2000 and was accompanied with COC # 528791. Sludge used for the second set of cultures in this study was also used in preparation of cultures for other fluorochemical biodegradation studies (CA105 4 and CA132 s), and was shown to be active for biodegradation of other fluorochemical
compounds in those studies.
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 located in St. Paul, MN. Typically, Four liters of MLSS was collected by Pace laboratory personnel and delivered as four l-liter Nalgene polypropylene bottles containing MLSS, and were accompanied with a corresponding chain of custody with date collected. 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 least 24 hours at 4C + 3C. The settled solids "sludge" were then used to prepare MLSS plus sludge for use in preparing test cultures. 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 l-liter bottle, based on
visual observations-and was consistent with observation of earlier sludge collections.
Page 10 of 38
PaceProjectCA085:"BiodegradationScreenStudyfor TelomerAlcohols"-Revision 1 StudyDirector:Cleston C.Lange,Ph.D. PaceAnalyticalServices,Bio-AnalyticaSl ervicesGroup Wednesday,November06, 2002
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7.1.2. Culture Preparation.
The first set of cultures were prepared August 9, 2000 and incubation of those cultures continued with intermittent culture harvests until the final harvest on September 13, 2000, for a total incubation time of 35 days. The second set of cultures were prepared on September 27, 2000 and incubation of those cultures continued with intermittent culture harvests until the final harvest on October 12, 2000, for a total of 16 days incubation. The culture preparation procedure described below was used, and was documented as Pace standard operating procedure (SOP) CAG-SP-03 8
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 Na2HPO4-2H20, 0.005 g NH4CI, 0.2175 g
K2HPO4, and 0.085 KH2PO4, 0.0275 g CaCI2-anhydrous, MgSO4-7H20, and 0.00025 g FeCI3-6H20.
0.0225 g
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.
A mineral salts medium, un-sterilized and without sludge, was prepared. This mineral salts 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 regularly during dispensing in order to keep the mixture homogenous and prevent the sludge from settling out of the solution.
The test substance, either Zonyl BA-N telomer alcohol for culture set one (Stock ID CA058-SS-004 at 10,280 _.g/mL in methanol) or Zonyl BA-L
Page 11 of 38 Pace Project CA085: "Biodegradation Screen Study for Telomer Alcohols" -Revision 1 Study Director: Cleston C. Lange, Ph.D. Pace Analytical Services, Bio-Analytical Services Group Wednesday, November 06, 2002
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telomer alcohol for culture set two (Stock ID CA085-SW-001 at 7,288 j._g/mL in methanol), was added to the test cultures by transferring 5 p.L and 8pL, respectively, to the appropriate test cultures. The final test concentration in cultures was 2.056 I.Jg/mL of Zonyl BA-N telomer alcohol for set one, and 2.332 tJg/mL of Zonyl BA-L telomer alcohol for set two. Blank control cultures received 25 mL of mineral medium solution, as did the test culture, but without addition of the test substrate.
All of the day zero cultures were prepared and immediately -20C for storage until SPE preparation could be conducted.
frozen at All other
cultures were placed in temperature controlled shaking incubators that
were maintained at 25C + 3C. Cultures were removed from the incubators at designated time points. Upon removal from the incubator,
cultures were either immediately frozen, or immediately prepared for analysis by solid phase extraction.
All culture preparation information, including times, analyte additions, etc. were recorded in sample preparation worksheets and signed and dated by the preparation analyst. All original data sheets were maintained in project specific binder labeled as Project CA085.
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 7.
All 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 cartridge was packed with plug of quartz glass wool to deter plugging. 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.
Page 12 of 38
Pace Project CA085: "Biodegradation Screen Study for Telomer Alcohols" -Revision 1 Study Director: Cleston C. Lange, Ph.D. Pace Analytical Services, Bio-Analytical Services Group Wednesday, November 06, 2002
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Frozen cultures 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 I-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 I-Chem vial labeled eluate 2. Eluate 2 was expected to contain a majority of the analyte that was in the original culture.
As a precaution that some analyte may be retained in the SPE cartridge or in the culture flask, a second 25 mL methanol eluate was collected in a similar fashion to that collected for eluate 2, and was labeled eluate 3. Aliquots of eluates 2 and 3 were transferred to autovials, capped, and then quantitatively analyzed by HPLC/MS. The remaining volume of each eluate was stored at 4C + 2C.
The final SPE extractions of the day 7, 14 and 16 day cultures from set two occurred on September 24, 2001.
7.2 InstrumentalAnalysis (LC/MS)
7.2.1 Instrument Parameters The HPLC/MS method used was a modified version of Pace method CAGORG-23 8, as described below.
Analysis of culture extracts from culture set one, prepared with Zonyl BA-N telomer alcohol, was conducted using instrument "STING" which included an HP1100 HPLC pump with Gilson 215 liquid handling system in line with a Micromass Quattro II triple-quadrapole mass spectrometer detector.
Page 13 of 38 Pace Project CA085: "Biodegradation Screen Study for Telomer Alcohols" -Revision 1 Study Director: Cleston C. Lange, Ph.D. Pace Analytical Services, Bio-Analytical Services Group Wednesday, November 06, 2002
Analysis of culture extracts from culture set two, prepared with Zonyl BA-L
telomer alcohol, were analyzed both on instrument "STING" and
instrument "10LCMS03" which consisted of a Waters 2690 HPLC system in
line with a Micromass Quattro II triple-quadrapole mass spectrometer
:",i detector. Typical conditions for analysis of telomer alcohols and
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fluorochemical acid products were as follows:
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Instrument conditions for HPLC/MS analysis.
Mass Spectrometer
HPLC
Ionizationmode: API-ES negative
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Desolvation Temperature: 200C
Time ('min).
%A %R
Source Block Temperature: 150C
0.00
97.0 3.0
RF lens: 0.2
0.50
97.0 3.0
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Extractor: 3
5.00
5.0
95.0
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Cone: 8
11.00
5.0
95.0
Capillary: 3.50 LM Resolution: 14
11.50 15.00
97,0 3.0 97.0 3.0
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HM Resolution: 14
Flow: 1 mL/min, splitter approx. 3:1
{
Ion Energy: 2.5
Solvent A= 2 mM ammonium acetate
Lens 6:2
Solvent B= Methanol
MS1 Multiplier: 650
Column Temperature: ambient
i MS./MS:Collision Energy-varied from 10 to 40 V for individual Experiments, MS/MS Collision Gas: Argon
MS2 Multiplier: 750
Typical injection volumes for samples and calibration standards were 50 t_L. The 4.6 x 150 mm Betasil C8 column used for the quantitation of
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extracts from culture set two had serial number 1101567H and the 4 x 35
mm NG1 column used had serial number 15104. A pressure-regulated
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splitter was used with an approximate split ration of 2:1 (Waste:MS)
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7.2.2 Qualitative Parent Analyte and Products Analysis.
Parent Analyte C8 telomer alcohol acetate adduct C10telomer alcohol acetate adduct C12 telomer alcohol acetate adduct C14 telomer alcohol acetate adduct
Expected ions (m/z) 423 523 623 723
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C18telomer alcohol acetate adduct
823
Table 1. Parent telomer alcohols analyzed.
Page 14 of 38
Pace Project CA085: "Biodegradation Screen Study for Telomer Alcohols" -Revision 1 Study Director: Cleston C. Lange, Ph,D. Pace Analytical Services, Bio-Analytical Services Group Wednesday, November 06, 2002
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To conduct the analysis of parent compounds and expected biodegradation products, the HPLC/MS system was set up with the same chromatographic configuration as that described in method CAG-ORG23 8. The method utilized two columns in tandem with flow to a pressure relief valve that serves as a flow through splitter to the mass spectrometer Z-spray source.
Specific MS conditions for mass analysis of the telomer alcohols and acid products were developed during this study. The mass spectrometer, Micromass triple-quadrapole mass spectrometer with Z-spray ion source, was operated with electrospray ionization in either selected ion-recording (SIR) mode or with mass-range (150-1000 m/z) scanning on MS1. Discrete chromatographic peaks with singly charged negative ions, (M + Acetate)- for parent telomer alcohols and (M - H for expected acid biodegradation products, were observed and monitored:
Expected Product
Perfluorobutyrate (C4) Perfluoropentanoate (Cs)
Expected anion structure
CF3(CF2)2CO0" CF3(CF2)3CO0"
PPeerrfflluuoorroohheepxtaannooaattee (0(C6)_) Perfluorooctanoate (C8, PFOA) Perfluorononanoate (C9) Perfluorodecanoate (Clo)
CCFF33(C(CF2F)42C)sOC0"OO" CF3(CF2)6COO CF3(CF2)TCOO CF-_(CF=)sCOO"
Perfluoroundecanoate (Cll) Perfluorododecanoate (C12) 2H, 2H-perfluorooctanoate (C8) 2H, 2H-perfluorodecanoate (C_0) 2H, 2H-perfluorododecanoate (C12)
CF3(CF2)gCOO CF3(CF2)10COO" CF3(CF2)sCH2CO0" CF3(CF2)7CH2COO CF3(CF2)gCH2COO
Expected anions (m/z)
213,169 263,219
331633,,312969 413,369 463,419 513,469
563,519 613,569
377 477 577
Table 2. Expected products, and expected anions for each.
Page 15 of 38
Pace Project CA085: "Biodegradation Screen Study for Telomer Alcohols" -Revision 1 Study Director: Cleston C. Lange, Ph.D. Pace Analytical Services, Bio-Analytical Services Group Wednesday, November 06, 2002
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..... i
7.2.3 Quantitative Analysis.
Quantitative analysis was conducted only for perfluorooctanoate (PFOA) and only on extracts of culture set two. Quantitative analysis was performed by the external standard method using SPE extracted
calibration standards (eluate 2 only) and quadratic calibration curves. The HPLC/MS analysis of culture set one extracts was used primarily for method development purposes.
Typica! injection volumes for samples and calibration standards were 50
ILL. The 4.6 x 150 mm Betasil C8 column used for the quantitation of extracts from culture set two had serial number 1101567H and the 4 x 35
mm NG1 column used had serial number 15104. The pressureregulated splitter had no identifying number to distinguish it. The part number for ordering the pressure relief valve (splitter) was AIItech catalog part number 39025.
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 when applicable. The mole conversion values for each analyte are as follows:
PFOA molecular weight, as ammonium salt, is 431.10 Zonyl BA-L telomer alcohol average molecular weight is 43_4.70.
7.3.2
Conversion of ng/rnL to micromolar (Working Examples):
(F.M) and nanomolar
(nM).
2,232 ng/mL telomer alcohol = (2,232 ng/mL)*(lnmole / 434.7 ng) =
5.135 nmole/mL = 5.135 p.mole/liter = 5.135 _.Mmassuming 100% purity.
150 ng/mL PFOA (NH4+ salt) = (150 ng/mL) * (1 nmole / 431.1 ng)
= 0,3479 nmole/mL = 0.3479 _mole/L = 0.3479 p.M = 347.9 nM
Page 16 of 38
Pace Project CA085: "Biodegradation Screen Study for Telomer Alcohols" -Revision 1 Study Director: Cleston C. Lange, Ph.D. Pace Analytical Services, Bio-Analytical Services Group Wednesday, November 06, 2002
_:':I
J
/_i
_.r"_:\I I .:, !i
I
[i__
:
7.3.3
Molar Mass Balance Calculations (Theoretical Yield): Convert all ng/mL values to their corresponding molar concentrations as
I.LM or nM (see section 7.3.2, above). Divide the sum of the analyte
concentrations by the known concentration of starting compound and
represent the final result as a percentage concentration.
of the known
starting
(Working Example): If, the starting concentration of BA-L telomer alcohol was at 5.135 pM And, after incubation, the following was determined:
If, PFOA was detected at 0.3479_M
Then, the mass balance is as follows: Mass balance = [(0.3479 _M) / 5.135 _.M] X 100% Mass balance = [0.0678] X 100% = 6.78% Or, 6.78% of the telomer BA-L alcohol was oxidized to form PFOA.
7.4
Software Versions
Microsoft TM Excel 2000 was used for data processing and producing tables. Microsoft TM Word 2000 was used for processing the analytical report text. Adobe Acrobat 4.0 was used for generation of the final electronic report. Masslynx version 3.2 was used for data collection and peak integration.
ACD Chemsketch version 4.0 was used for chemical drawings.
Page 17 of 38
Pace Project CA085: "Biodegradation Screen Study for Telomer Alcohols" -Revision 1 Study Director: Cleston C. Lange, Ph.D. Pace Analytical Services, Bio-Analytical Services Group Wednesday, November 06, 2002
-' ;i
{i
;1_:_! _.
'I
, _,_ :_" i_
_
, '" i
8.0 Results 8.1 Sludge Characterization
A chemical analysis of the mixed liquor suspended solids used for the samples prepared on August 9, 2000 was conducted, and characterization information can be found in the final report for project CA0581. The sludge for samples prepared on September 27, 2000 was not characterized, but was obtained from the Twin Cities Municipal waste treatment facility in a manner consistent with the sludge collected and used for project CA058.
8.2 Quality Control/Sample Matrix Spike Results.
The determination of the analyte recoveries from sample matrix spikes was not included as part of this screening study. Recoveries are only reflected in the ability to achieve molar mass balance based on expected parent and product yields from samples and by the use of a sludge-extracted curve for semiquantitative analysis of PFOA.
8.3 Analytical Blanks
Methanol solvent blanks were injected onto the HPLC/MS column and quantitatively analyzed to determine the instrument background analyte concentration and carry over during the analysis.
The no-sludge (abiotic) controls containing mineral salts medium with test analyte, and culture blanks, containing mineral salts medium with test analyte and no sludge, were prepared and analyzed. Abiotic controls and blank cultures were prepared and incubated in an identical manner to biodegradation test cultures. The results were used to determine whether the sample matrix contained any of the analytes of interest and whether biodegradation could be attributed to the bioactivity of the sludge. Fluorochemical anal_es were not detected in blanks and biodegradation did not occur in abiotic controls.
Page 18 of 38 Pace Project CA085: "Biodegradation Screen Study for Telomer Alcohols" -Revision 1 Study Director: Cleston C. Lange, Ph.D. Pace Analytical Services, Bio-Analytical Services Group Wednesday, November 06, 2002
i
:_":_ !
.:_:
'i_:i i
_J
,!
._ _[_'!_
,__
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8.4 HPLC/MS Analysis Results
Semi-quantitativeanalysiswas conductedonly for the SPE eluate 2 extractsfrom the second set of cultures, prepared September 27, 2000, and were performed on September of 2001 as sequence CA085_092401b.spl (with full scan MS data), and CA085_092701a.spl (SIR data) on instrument 10LCMS03 in the BioAnalytical services group at Pace. The qualitative HPLC/MS analyses and HPLC/MS method development for this study were conducted in September and October of 2000 as analytical sequence runs S091500.spl, S091800.spl, S092000.spl, S092100.spl, S092600.spl, Sl000200.spl, and S100400.spl on Pace instrument "STING".
8.4.2 Qualitative HPLC/MS and HPLC/MSIMS Results
The first set of test culturescontainingsludgeand BA-N telomer alcohol substrate providedevidence that biodegradation occurred. The HPLC/MS data showed near total loss of all of the telomer alcohol peaks in the HPLC/MS chromatogram, with formation of chromatographic peaks that had mass spectra consistent with perfluorinated acids. However, at that time, the analytical test method was not complete. Concerns about the integrity of the test material resulted in a second set of test cultures being prepared using commercially purchased Zonyl BA-L telomer intermediate.
Although each sample, control, and blank for the second set of cultures
was prepared and incubated in duplicate, analysiswas conducted for SPE eluate 2 extracts from one sample, control and blank per time point.
The data showed that the sample test cultures underwent rapid loss of the C8,C8, C10a. nd C12-telomeralcohols, and moderate loss of the C_4-telomer alcohol over the 16 days of incubation, as determined by decreasing peak area response for each telomer alcohol ion at the sequential sampling time points (Figures 3 and 4). The C_8-telomeralcohol was degraded very slowly and showed little loss.
Page 19 of 38 Pace ProjectCA085:"BiodegradationScreenStudyfor TelomerAlcohols"-Revision 1 Study Director:ClestonC. Lange,Ph.D. PaceAnalyticalServices,Bio-AnalyticaSl ervicesGroup Wednesday,November06, 2002
:;
:,:;::.;
.... _._,:,! _::i _'_' i :i:!
.i
'-/]
......
i
:..i !",iii
35000 . .
3000o
. ,,,
,,:,:;.,
_,. .: -.-:._........._...... ;
25000
: :'_:' ""::':_';"
] ., .:.;_,_:.,_._!]
....,.:..:...:.-.. :.:.:..:........_.::._...;._,.:,:.,..
20000 15000 10000
peak area , AC10 TA peak area
i_eC12 TA._Pea}area
5000
0
0
5
10
15
20
Figure 3. HPLC/MS integrated peak areas for the most abundant telomer alcohols (TA). Near complete degradation of all three was observed. Data plotted with the best manual fit of the data.
6OOO
5OOO
_: 4000
O
_ 3000 4[
_
n
2000
1000
C6 TA peak area
C14 TA peak area : C16 TA peak area
0
0
5
10
15
20
Incubaiton time (days)
Figure 4. HPLC/MS integrated peak areas for the less abundant telomer alcohols (TA). Near complete degradation was observed for the C6-TA. Moderate degradation of the C14-TA was observed and little degradation of the C_e-TA was observed. Data plotted with the best manual fit of the data.
Page 20 of 38
Pace Project CA085: "Biodegradation Screen Study for Telomer Alcohols" -Revision 1 Study Director: Cleston C. Lange, Ph.D. Pace Analytical Services, Bio-Analytical Services Group Wednesday, November 06, 2002
. _, ::_i, ::':::_,, "'! ,:-.
'.,' q
,_,_, i
!
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Concomitant with the loss in telomer alcohols was the formation of compounds with HPLC/MS ions and retention times consistent with perfluorinated acids (PFAs), including perfluorooctanoate (PFOA, Cs-PFA), which was confirmed as an end product. The transformation of telomer alcohols to PFAs was rapid, with a large increase in all of the PFA peak area responses at day 1, and continuing increase through day-16 (Figure 5). No degradation to form PFAs was observed in the abiotic no-sludge controls.
Although PFOA was accurately quantified, many other perfluorinated acids were qualitatively observed and their relative concentrations determined based on the observed increasing peak area response. The observed perfluorinated fatty acid end products were: perfluoropentanoic acid (Cs PFA), perfluorohexanoic acid (C6-PFA), perfluoroheptanoic acid (CT-PFA),
perfluorooctanoic acid (Cs-PFA, PFOA), perfluorononanoic acid (Cg-PFA),
perfluorodecanoic acid (C_o-PFA), perfluoroundecanoic perfluorododecanoic acid (C12-PFA).
acid (C_I-PFA)and
Transiently formed intermediate compounds were also observed at early time points following the initial exposure of the sludge to the telomer alcohol substrate (Figure 6). The transient compounds were suspected to be: 2H,
2H-perfluorooctanoate; 2H, 2H-perfluorodecanoate; 2H, 2H Perfluorododecanoate and the possible 13-oxidation pathway intermediates: 2H-perfluoro-2-octenoate, 2H-perfluoro-2-decenoate, and 2H-perfluoro-2-
dodecenoate. Table 3 shows the MS ions and retention times observed for the polyfluorinated and perfluorinated acid products and the telomer alcohol
pHaPrLeCnt/MsSu/bMsStrateDsa. ta (Table 4) support the identification of the suspected
metabolites..
""
Page 21 of 38
Pace Project CA085: "Biodegradation Screen Study for Telomer Alcohols" -Revision 1 Study Director: Cleston C. Lange, Ph.D. Pace Analytical Services, Bio-Analytical Services Group Wednesday, November 06, 2002
250000
200000
C O
._
_ 150000
100000
50000
0
0
2
4
6
8
10
12
14
16
18
,'._I
Incubation time (days)
Figure 5. Integrated peak area responses for the perfluorinated fatty acids (PFAs)
'i
observed in test culture extracts. Even numbered carbon chain length carboxylic acids
:J
were the most abundant carboxylic acid peaks and were: perfluorohexanoate (Cd-PFA),
;
perfluorooctanoate (Cs-PFA) and perfluorodecanoate (C10-PFA).
[
18000 14000 12000
i
10ooo
!/:I
a. 8000
"O
J 60oo 'Si
l
4000
2000
!
0
0
1
2
3
4
"-
5
i
IncubationTime (days)
.
Figure 6. Transient polyfluorinated fatty acid intermediates observed. The 2H-
i
_i
perfluorinated olefinic fatty acids are suspected _-oxidation intermediates that formed
rapidly upon initial exposure to telomer alcohols, and then were transformed to their
!:i!:i
corresponding perfluorinated carboxylic acids end products
Page 22 of 38
Pace Project CA085: "13iodegradation Screen Study for Telomer Alcohols" -Revision 1 Study Director: Cleston C. Lange, Ph.D. Pace Analytical Services, Bio-Analytical Services Group Wednesday, November 06, 2002
Analytes Observed
Chemical Formula
Retention Time LCIMS anions
_ii
observed
I
:
C_-telomer alcohol*
CF3(CF2)3CH2CH2OH... CHaCO0 "
7.63
323
Cs-telomer alcohol*
CF3(CF2)sCH2CH2OH.C.. HaCO0"
8.13
423
.i
Clo-telomer alcohol*
CF:_(CF2)TCH2CH2OH... CHaCO0 "
8.50
523
C12-telomeralcohol*
CF3(CF2)gCH2CH2OH.C..HaCO0 "
8.96
623
i
C_4-telomeralcohol*
CF3(CF2)I 1CH2CH2OH... CH3COO "
9.53
723
C16-telomeralcohol*
CF3(CF2)13CH2CH2OH.C..H3COO
10.25
823
2H, 2H-perfluorooctanoate*
CF_(CF2)sCH2COO
7.17
377
2H, 2H-perfluorodecanoate*
CF_(CF2)TCH2COO"
7.38
477
2HI 2H Perfluorododecanoate* CF3(CF2)_CH2COO"
7.49
577
_i',
2H-perfluoro-2-octenoate*
CF3(CFz)4CF=CHCOO"
i:__::
2H-perfluoro-2-decenoate*
CF3(CF2)sCF=CHCOO"
2H-Perfluoro-2-dodecenoate*
CF3(CF2)sCF=CHCOO"
Perfluoropentanoate
CF3(CF2)_COO"
i.J
Perfluorohexanoate
CF3(CF2)4COO"
7.14 7.35 7.52 6.62 6.88
357,293 457,393 557,493 263,219 313,269
_:_
Perfluoroheptanoate
CF3(CF2)sCOO"
]
Perfluorooctanoate
CF3(CF2)sCOO"
Perfluorononanoate
CF3(CF2)TCOO"
Perfluorodecanoate
CF3(CF2)sCOO"
Perfluoroundecanoate
CF3(CF2)gCOO
Perfluorododecanoate
CF3(CF2)_oCOO"
7.07 7.22 7.30 7.41 7.48 7.52
363, 319 413, 369 463,419 513,469 563, 519 613, 569
_il
*" Only anions corresponding to even chain length molecules were observed.
Table 3. Parent and products anions observed in by HPLC/MS analysis of SPE eluate 2 extracts from test cultures containing sludge and telomer alcohol substrate. The parent anion signals decreased in
test cultures over time concomitant with increases in product signals. Many of the telomer alcohol
..,
signals decreased below detection limits in test cultures.
s,.
!
Page 23 of 38
Pace Project CA085: "Biodegradation Screen Study for Telomer Alcohols" -Revision 1 Study Director: Cleston C. Lange, Ph.D. Pace Analytical Services, Bio-Analytical Services Group Wednesday, November 06, 2002
i
,i `!
'i ,_
'_:' -_
E
_?ii:i
_:,_'_.:_ .I ....
Product Analytes Observed
2H,2H-perfiuo rooctanoate* 2H,2H-perfluorodecanoate* 2H,2H Perfluorododecanoate* 2H-perfluoro-2-octenoate* 2 H-periluoro-2-decenoate* 2H-Perfluoro-2-dodecenoate*
Chemical Formula
CF3(CF2)sCH2COO" CF3(CF2)7CH2COO CF3(CF2)gCH2COO" CF3(CF2)4CF=CHCOO CF3(CF2)6CF=CHCOO CF3(CF2)eCF=CHCOO"
Retention Time
7.17 7.38 7.49 7.14 7.35 7.52
LC/MS/MS Parent anion
377 477 577 357 457 557
LCIMS/MS daughter anions observed
333,313,293 413,393 -
313,293,243,143,119 413,393,343 493
Table 4. HPLC/MS/MS data for suspected -oxidation products. Parent and daughter anions from the HPLC/MS/MS analysis of the SPE eluate 2 extract from the 1-day biodegradation sample (CA0850927-SA-033 E2). The parent anions were observed as transiently formed anions in early sample point biodegradation samples only, with the most intense signals for the ions observed at day one. The MS/MS data collected for each product was consistent with the predicted chemical formula. Common mass loss from the parent anion, attributed to loss of CO2 (mass 44) and HF (mass 20), were observed.
8.4.2 Quantitative Analysis of PFOA
The HPLC/MS multi-component calibration standards used in this study for product quantitation contained the target analytes: PFOA, PFOSulfinate, PFOS, FOSA, N-MeFOSA, M556, M570, and N-MeFOSE alcohol. Five calibration standards were prepared by addition of a known amount of_. perfluorinated analytes to a culture medium with sludge, and then extraction by solid phase extraction in manner identical to the treatment of samples. The PFOA was the only target analyte of that mixture that was used for this study. The exact concentrations of the standards were used in calibration curves for quantitative analysis.
Page 24 of 38
Pace Project CA085: "Biodegradation Screen Study for Telomer Alcohols" -Revision 1 Study Director: Cleston C. Lange, Ph.D. Pace Analytical Services, Bio-Anaiytical Services Group Wednesday, November 06, 2002
i_ ."i
,. !
_!
i?'
i :_:,_
! i ._ i
iL_'i.!
All calibration standards and sample eluates were stored at 4C in refrigerator ID 0213 until analysis. Aliquots were transferred to autovials and capped for use in HPLC/MS runs. The Instrument calibrations were performed for PFOA by use of a five point calibration with quadratic fit of the data. The LLOQ was 11.0 ng/mL PFOA, and the coefficient of determination (r) for PFOA was 0.965. Quantitative sequence runs contained calibration standards at the beginning and end of the run.
The biodegradation measured levels
of the telomer alcohols resulted in an increase in the of PFOA during the 16-day study (Figure 7).
180.0
160.0 140.0
_' 120.0 100.0
< 80.0 o _. 60.0
40.0
20.0
0.0
0
2
4
6
8
10
12
14
16
18
Inciibat]on
tJrNe
(days)
Figure 7. The measured perfluorooctanoate (PFOA) in cultures containing sludge and
fslluuodrgoete. loDmaetra aplloctotheodl.witThhtehePbFeOsAt mwaansuanloftitmoef athsuerdaabtlea ainndcuclotunrceesnttrhaatitodnisd snhootwcnowntearien not adjusted for reference material purity of 95.2%.
o.
i.!
Page 25 of 38 Pace Project CA085: "Biodegradation Screen Study for Telomer Alcohols" -Revision 1 Study Director: Cleston C. Lange, Ph.D. Pace Analytical Services, 8io-Analytical Services Group Wednesday, November 06, 2002
' "i
i
''
_
:_i
" i
:
_
" _
_'., .:_",'; ._i,,J
_:ii!
!
.i !_:_
FFFFFF FF
FFFFFFFF
C,o Telomer Alcohol
I d_dr_enase
FFFFF FFF
IIFI fl_ II
F
FFFFF FFF
0
I'F det_y_'ogenase
,,, , i TTTTTTT F F F F F F F F O
Oxidative decarboxylation
e
(_-oxidation)
F F FF F F F F
OH
,,,.1 " .II_I_I.I.I.I__-o
oddase
Ilia2r
F F F FF F FF
O"
iF F F IF III1_ F F F F 0
Perfluorononanoate
+
C02
ase
13-Oxidation
0" F F F F F F F ,_
,,,bl ' II}IIII F F F F F F F F
hydtatase
TiTT T " ,,,b2' F F F F F F F F o>O" I--
_ detly_rogenase
IIIb3
Jv_ J3-ketoacyfmiolase
FFF_FF F
"
- _.
Perfluorooctanoate
H3C_. 0 _-
O"
Acetate
Figure 8. Proposed biodegradation pathway for telomer alcohols, shown for C10-telomeralcohol as example. The 13-oxidation pathway branch (lllb) involves primary formation of a fatty acyI-CoA thioester that is not shown, to clearly depict the products observed by HPLC/MS. The fatty acyI-CoA thiol bond is readily hydrolyzed to yield the corresponding fatty acids observed.
Page 26 of 38
Pace Project CA085: "Biodegradation Screen Study for Telomer Alcohols" -Revision 1 Study Director: Cleston C. Lange, Ph.D. Pace Analytical Services, Bio-Analytical Services Group Wednesday, November 06, 2002
9.0 Conclusions
This studyhas demonstratedthatfluorochemicaltelomer alcoholsare biodegradableand
are transformed to perfluorinatedacid end products. Althoughthe telomer alcoholsare
I
supplied only as even numbered carbon chain compounds, the end product
perfluorinated acids consist of both even number and odd number carbon chains, with
even numbered carbon chain length acids predominating as the major end products.
This observation suggests that, following the initial oxidation of the hydroxyl-carbon to form the primary carboxylic acid (telomer carboxylate), two oxidation mechanisms exist as shown in Figure 8,.111.The first, less utilized route (Figure 8, Ilia), involves oxidation
of the c{-carbon concomitant with decarboxylation (oxidative decarboxylation) to form an
i...!
odd chain length perfluorinated carboxylic acid, which does not undergo further
biotransformation. The second observed route of oxidation (Figure 8, IIIb) is the more
;
common route of fatty acid p-oxidation, as evidenced by the formation of detectable
transient 13-oxidationpolyfluorinated fatty acid intermediates and the more abundant even
i! [ number carbon chain length carboxylic acids
;
Although toxicity of the telomer alcohols was not addressed as part of this study, the
microbial degradation observed here over the entirety of the 16-day study suggests that
microorganisms present in the sludge were not significantly inhibited by the tested
_
concentration of the test material.
Page 27 of 38
PaceProjectCA085:"BiodegradationScreenStudyfor TelomerAlcohols"-Revision 1 StudyDirector:ClestonC. Lange,Ph.D. PaceAnalyticalServices,Bio-AnalyticaSl ervicesGroup Wednesday,November06, 2002
-i
._ :.'.4 ;_:] :_
, -! =I
'_::.,._:i
" : _i
i_!i_i
_
2._.i
_,i .
-.i
8.0 Literature Cited
1.Final Reports for Pace Project CA058 (3M # E00-2252). "2-Week N-EtFOSE Alcohol Biodegradation Screen Study Report" and "Aerobic Biodegradation of N-EtFOSE alcohol Study Report". Author: Cleston C. Lange, Ph.D.
2. Final report for Pace Project CA097 (3M # E01-0415), "The 18-Day Aerobic Biodegradation Study of Perfluorooctanesulfonyl-based Chemistries." Author: Cleston C. Lange, Ph.D.
3. Final report for Pace Project CA104 (3M # E01-0444), "The 35-Day Aerobic Biodegradation Study of Perfluorooctanesulfonate (PFOS)." Author: Cleston C. Lange, Ph.D.
4.Final Report for Pace Project CA105 (3M # E01-0683), " Fluorochemical Adipate (L15468) Biodegradation Screen Study." Final report issued July 10, 2001. Study Director: Cleston C. Lange, Ph.D.
5.Final Report for Pace Project CA132 (3M # E01-0682), "The Aerobic Biodegradation Study of the Fluorochemical FC807-Diester". Final report issued May 29, 2001. Study Director: Cleston C. Lange, Ph.D.
6.PaBcieodmegertahdoadbilCitAyGo-Sf FPl-u0o3ro. c"hCemulitcuarles PrUespinagratMiounnfiocripAalssoer sIsnmduesntrtiaol fSAluedrogbeicas Microbial Inoculum."
7.Pace method CAG-SP-04. "C18 Solid Phase Extraction Procedure for Fluorochemicals Recover,/from Aqueous/Sludge Matrices."
8. Pace method CAG-ORG-23 "Quantitative Analysis of Fluorochemicals by High Performance Liquid Chromatography with Mass Spectrometric Detection."
Page 28 of 38
PaceProjectCA085:"BiodegradationScreenStudyfor TelomerAlcohols"-Revision 1 StudyDirector:Cleston C. Lange,Ph.D. PaceAnalyticalServices,Bio-AnalyticaSl ervicesGroup Wednesday,November06, 2002
,_: :! :_i
!:_:"1 :,_ I
! _ i 'i
.._..!i
_ i. J
:
9.0 Sample and Data Retention
At a minimum, one copy of all pertinent raw data and one copy of the signed final report will be retained in the Pace Analytical-Tier 2 data archives for a minimum period of 2 years after completion of the project. The remaining sample extracts will be retained at the Pace Analytical facility for a period of 2 years after completion of the project at 4C in the Carroll walk-in cooler (Pace ID 0140) located in the Pace Analytical-Tier 2 facility.
The following will be provided to the sponsor (3M):
The original signed analytical report and one copy of the signed original. The final scanned report (read only) and pertinent electronic data on a CD. All original Data, correspondence, chromatograms, sample & standards prep sheets, etc. Upon request before 2 years, the stored samples may be sent to the sponsor.
All electronic copies of the instrumental raw data will be archived onto CD disks and one copy provided to 3M and one copy retained at Pace.
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
Page 29 of 38
PaceProjectCA085:"BiodegradationScreenStudyfor TelomerAlcohols"-Revision 1 StudyDirector:ClestonC. Lange,Ph.D. PaceAnalyticalServices,Bio-AnalyticaSl ervicesGroup Wednesday,November06, 2002
APPENDIX A
i'i
i
APPROVAL SIGNATURES
,i, i I
Project Title:
Biodegradation Screen Study for Telomer Alcohols
_,7
Client Project ID:
E01-0684
t
Contract Analytical Project Number: CA085
L
ii
Mourad Rahi
.!
t
I
Bruce E Warden
"-::';
Laboratory Management:
/
Signatur v ,- -
.... _'_-" Signat'ure__---"_"
Date/i/_7--O
_ _SJ'
i'
! _.J
Page 30 of 38
Pace Project CA085: "Biodegradation Screen Study for Telomer Alcohols" -Revision 1 Study Director: Cleston C. Lange, Ph.D. Pace Analytical Services, Bio-Analytical Services Group Wednesday, November 06, 2002
'?i 't i
_:_
i r_ ;
_:_
Ii " -J
Appendix B Final Quantitative Results for PFOA in samples, controls and blanks
Table 1. The measured PFOA concentrations in test cultures during the 16-day screening study for telomer alcohol aerobic biodegradability.
Sample _ay 0 blode_radatlonsample. CA085-0927-SA-031, E2 _ay 1 b=ode_ta0at6osnample. CA085-0927-SA-033_E2 Day 2 blode_rud_ion sample CA085*0927-SA-035 E2 Day 5 I_(xJL_t_lgtlonsample, CA085-0927-SA-037, E2 D,ay 7 l_oOe_r_ation sample,CA085-0927-SA.03g. E2 Day 14blode_radationsample,CA0_5-0927-SA-041. E2 Oay 18 i_e_rgd3tion sample.CA085-0927-SA-043. E2
Concentralon (n_/mL_ [Etuate 2] PFOA 00 252 65 0 1114 13g8 158.8 160.0
Table 2. The measured PFOA concentrations in the abiotic control cultures during the 16-day screening study for telomer alcohol aerobic biodegradability.
Sample _ay 0 no s_u_l_ecor_tml,CA.0_0927-SA-053, E2
3ay 1 no slud_lecontrol.CA_27-SA-055,
E2
_ay 2 no sludgeco,ntmLCA085-0927-SA.056, E2
bay 5 no slUC_lecontrol,CA085-O927-SA-(_g, E2 _)a),7 no sludge COntrolC, A085-0927.SA.060. E2
_a}t 14no sludgecon/ml_CA08-5-0927.SA-062E2 )ay 16no sludgecontrol,CA085-092?.SA.064, E2
Concentration (n_/ml.) [Eluate 2
-- , ,
PFOA 00
00 00
00 0.0
00 00
Table 3. The final data results for sludge blanks during the 16-day screening study for telomer alcohol aerobic biodegradability. PFOA was not detected in any of the sludge blanks.
Sample Day 0 slu(Igeb|ank, CA085-0927.SA-045. E2 Day 1 s_ud_le_,ank. CAO85-Og27-SA-046E. 2
Dlaayy 25 sslluuddggee bbllaannkkt CCAA0O8S5,-_0-9O29T2.7S-AS-A0-4074,8EE,Z2 3ay 7 sludge blank. CA085-0927-SA-049. E2 la}_14 sludge blank,CAO85-0927-SA._50. E2 3ay 16 slud_leblank. CA085-0927-SA-051, E2
Concentration
(n_lmL_ lE;uate 2] PFOA
00 O0 .
,
0O00
00
00
00
._
. Page 31 of 38
Pace Project CA085: "Biodegradation Screen Study for Telomer Alcohols" -Revision 1 Study Director: Cleston C. Lange, Ph.D. Pace Analytical Services, Bio-Analytical Services Group Wednesday, November 06, 2002
i '.',,.'-I
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' ..._. '_"_ _,_,'i 'i
! i_ .I
! I
Appendix C Integrated Chromatogram Peak Area Data for telomer alcohols
Table 1. Integrated LC/MS peak area raw data for the parent telomer alcohols substrates. acquired from sequence run CA085_092701a.spl as SIR data.
Data
Sample ,
"FllelD ,,
,0a.y0 b_Ode__:lat_osnampleCAO85.0927.SA-031. E2 , C,A085 092701a 008
D.,a,.Iy bloclejrada|,onsamole, CA,08._-0927.SA.033.E2 Day 2 bK_e_taOabon sample CA085-0927.SA.035, E2
CA085 CA085
092701a 092701a
009 01,.0
Day $ b,odegr_at=on sample, CA085-0927.SA-637. E2
CA085 092701a 011
Oa_ 7 b=oOe_lraOatlosnample, CA085-0927.SA.039, E2
CA085 092701a .012
...0. c,.. 2.7..3E2 Da.v14blode_radalionsample. CA085.0927-SA-041. E2 /
CAO85 092701a. ,013
II I
3aV 0 no sludgecontrol.CA0&5-0927.SA-053, E2
CA085, ,092701a.016
3ay 1 no sJud_econtrol.CA085-0927-SA-055. E2
CA085.092701a
017
)ay 2 no sJud_econtrol, CAO85-O927-SA.OS&E2 3a.y5 no sludge con.troIC, A085-0927- SA-05g. E2
CAO85092701a
O18
CA085,..092701 a019
IDa_7 no sludcjecon|rol,CA085-0927-SA-060, E2 Oay 14no s&Kl_lecontrol, CA_,-Og27-SA-O_2 E2
3a;* 16no sluClgecontrot,CA085-0927-SA-064, E2
"" ...... Da,f 9 slud(_eblank. CA085-0927-SA-045 E2 Day I slud_leblank.CA085.0927-SA.045. E2
Oa,y2 st,L,_geblank.,CA085-0927-SA-047, E2
Da'/5 slud, _e blank. CA085-0927-SA;048, E2
Day 7 sludge blank. CAD85.0927-SA..049.E2
Oay 14 slud(p blank, CA085-O927-SA-050. E2
,.
Day 16sludge blanl_,CA085-0927-SA.051. E2
CA085, 092701a.020 CAO85,=O92701aO21
CA085 I092701a I022
CA085.092701a
044
CA085 092701a =045
CA085,092701a_046
0A085 092701a 047
CA085,.092701a,.048
CA085=092701a .049 CA085 ,,,092701 a_050
C6 3503 NO ND
12 NO
.oNO
5072 4891 5198 4383
3515 33_9
4602
NO ND NO NO 37 NU 17
C8 21704
91 , 268 690 631
.3594
30841 31305 3J882 27486
21460 21284
27329
47 ND NO NO 162 211 68
Telome Alcohol C10 28940 1309 262 103 ND
N1O ,.
InteQrated Peak Neas
C12
C14
18446
5558
8833
4766
6877
4483
3070
3701
1263
3248
670
,275 1324
C16
SUM
1328
1326
1340
1340
1213
1213
1140
1140
1343
.. 1343
1,37 1093 ,,37 1093
225{}4 20373
24414 22470
13734 11273
13888 12856
5014 4712
5379 4941
1492 1477
1462 1393
1492 1477
f462 1393
13621 14366
10429 10228
4252 4992
1095 1265
1095 1265
17948
12215
5230
1478
1478
NO
NO
ND
NO
NO
NO
NO
N0
ND
NO
ND
ND
NO
269
NO
NO
NO
NO
NO
NO
NO
NO
NO
NO
NO
NO
ND
ND
NO
ND
NO
NO
NO
NO
NO
,',:"I I,:,L_:I
!_,!, _i';,_
:: .;
; : ...._ ,
"i
Table 2. Percentage of integrated peak area remaining at different time points with respect to the
peak area at time zero for their respective culture types. Near complete degradation of several telomer alcohols (TA) was observed in test cultures only. Near 90% remained in control cultures for all except C10TA, which was 79.4%.
Sample Description
Day
% TA Remaining versus Day 0
Day 0 biodegradation sample, CAOBS-0927-SA-031, E2
C8 TA
C8 TA C10 TA C12 TA C14 TA C16 TA
0
100.0% 100.0% 100.0% 100.0% 100.0% 100.0%
Day 1 biode_radation sample, CA085-0927-SA-033, E2
1
0.0%
0.4%
4.5%
46.8%
85.8% 101.1%
Day 2 biode_radation sample, CA085-0927-SA-035, E2
2
0.0%
1.2%
0.9%
36.2%
80.7%
91,5%
DaY 5 biodegradation sample, CA085-0927-SA-037, E2
5
0.3%
3.2%
0.4%
16.6% 66.6% 86.0%
)ay 7 biodegradation sample, CA085-0927-SA-039, E2
7
0.0%
2.9%
0.0%
6.8%
58.4% 101.3%
Day 14 biode_radation sample, CA085-0927-SA-041, E2 14
0.0%
2.7%
0.0%
3.6% "23.8% 82.4%
I :)ay 16 biodegradation sample, CA085-0927-SA-043, E2 16 "0.0%
3.0%
0.4%
3.7%
23.0% 85.7%
3ay 0 no sludge control, CA085-0927-SA-053, E2
0
100.0% 100.0% 100.0% 100.0% 100.0% 100.0%
3ay 1 no sludge control, CA085-0927-SA-055, E2 _)ay2 no sludge control, CA085-0927-SA-056, E2
,Day 5 no sludge control, CA085-0927-SA-059, E2
1
96.4% 101.5% 90.2% 82.1%
94.0%
99.0%
2
102.5% 103,3% 108.1% 101.0% 107.3% 98.0%
5
86.4%
89.1%
99.5% 93.6%
98.5%
93.4%
;Day,7 no sludge control, CA085-0927-SA-060, E2 Day 14 ,no sludge control, CA085-0927-SA-062 E2 [Day 16 no sludge control, CA085-09'27-SA-064, E2
7
69.3%
69.6%
60.3% 75.9%
84.8%
73.4%
14
65.4%
69.0% 63.6% 74.5% 99.6% 84.8%
16
90.7%
88.6% 79.4% 88.9% 104.3% 99.1%
Page 32 of 38
Pace Project CA085: "Biodegradation Screen Study for Telomer Alcohols" -Revision 1 Study Director: Cleston C. Lange, Ph.D. Pace Analytical Services, Bio-Analytical Services Group Wednesday, November 06, 2002
i
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_i h
ii !
;j
Appendix D Integrated Peak Area Data for Perfluorinated Acid End Products
Table 1. Integrated LC/MS peak area raw data for the end products formed. Data acquired from sequence run CA085_092701a.spl as ,SIR data.
Sample Da;/0 b_odecJradatlo_n,ampleCA085-OO27-SA.031 E2 Day.I b_odecjradat_osnample_CA085-0927.SA-033. E2 Da,v2 mo,J_.<jtaclatJsoanmple. CA085-0927-SA-035, E2 Day 5 oloclecJradatbsoanmple. CA085-0927-SA.037, E2 Day 7 O_ooegrudattosnample CA085-0927.SA-039 E2 _a'( 14 b,o0ec_radatiosnample. CA085-O927.SA-041, E2
/ / / _aIv ;6_od e_raclatmnsarn_e. CA085-0927-SA-043. E2
_ay.0 no slud_lecontrol.CA085.0927.SA-053. E2
:)a_ I no sludge control,CAO85-Og27.SA-O55E, 2 :)ay2 no slu_e control CA_5-O927-SA.058, E2 )a_ 5 no sludge conm_, CA085-O927-SA.059, E2 _a_,7 no sI.JOcJceontrol.CA085-0927-SA-060. E2 _ay.14no sludgecontrol, CA085-0927-SA.062 E2
/ I Day 16no sluclaecontrol, CA085.0927.SA-064, E2
Day 9 sluclcjeblank CA085-0927-SA-045, E2 Da_ t sluclc_betank CA085-0927.SA-048, E2 DaZ 2 sJu_e blank, CAO85-0927-SA-047. E2
Day 5 sludc_eblankCA085*0927-SA.048, E2 Day 7 sludgeblank, CA085.0927.SA-049. E2 Da Z 14sludge bJank.CA085-0927-SA-050. E2 Da_,16sludc_eblank. CA085-0927-SA-C_31E, 2
'
liD'
:
CA085..0927016 008 CA085 0927016 009
CA085 0927016 010
CA085 0927016 011 CA085 0927016 012
CA085 0927016 013
I CA0851019127016 014 I CA085 0927016 016
CA085 0927016 017 CA085 0927016 018 CA085 0927016 019
CA085 0927016 020 CA085 0927016 021
I I CA085 0927016 022
CA085 0927016 044
CA085 0927016 045 CA085 0927016 046
CA085 0927016 047 CA085 0927016 048 CA085 0927016 049
CA085q0927016.050
Porfluoopentanoate
276 13796 46384 68648 72870 78976 79828
193
288 351 475 457 581 " 611
Perfluorohexanoate
3576 46183 127207 204221 210569 221543 215870
861
1722 1437 1744 1843 1731 1851
618
525
683
152
685
510
--
:
320
248
Rz_
J
237
elt4
j
285
Perfluoroo heptarmate
12127 15464 25641 35747 43227 47751 47315
9244
16885 17394 17948 15212 16978 17910
Integrated Peak Areas
Perfluorooctanoata
2715 41821 88508 140409 170923 190903
192075
Perfluoroo nonaanoat6
6818 6386 8267 9375 13751 15382
15669
I Porfluoro decanoate 253 6428 17566 44419 75377 92168
96834
1057
4382
184
1695
5516
233
1649
5958
164
1683
7420
278
1583
6328
173
1680
7129
214
1837
7203
233
31
117
18
0
76
166
262
0
51
228
722
0
66
223
392
0
62
581
247
0
86 81
I
731 645
314 397
0 0
Perfluoroundecanoat(
1569 1281 1376 1641 2644 3071 3276
1283
1692 1178 1928 1015 1675 1342
0 0 0 0 24 24 21
Perfluorododecanoat_
315 395 ]192 2865 {]047 11078
11603 re
49
51 21 61
25 66 34
,,.,t 0
0 48
0
42
I
29 0
Page 33 of 38
Pace Project CA085: "Biodegradation Screen Study for Telomer Alcohols" -Revision 1 Study Director: Cleston C. Lange, Ph.D. Pace Analytical Services, Bio-Analytical Services Group Wednesday, November 06, 2002
_:' :'"_'_'r
. -_
_Ii, .t
J _!
i
Appendix E
Integrated Peak Area Data for Transiently formed Polyfluorinated Acid Products
Table 1. integrated LC/MS peak area data for transient products. Data from sequence CA085 092401b'spl as full scan MS data from m/z 100 to m/z 1000.
i:
' :_ '
Sample. .
_',,,
)ay 0 I;,()de_raOat=osnampJe, CA085-0927-SA-031, E2
_ay 1b:odegraaationsample, CA085-O927.SA.033, E2
)a'f 2 b_0degraOaUosnample. CA08.5-0927.SA.035, E2
)ay 5 bio0e_ra0ath0nsample. CA085-OQ27.SA-O37,E2 Day T bu0de_radadonsample, CAO85-Og27.SA-039.E2 Day 14 Oiode_radationsample, CA065-0Q27.SA-041. E2
ODa=yy 0Innoo.ss_itxuld_geeccoonnltrroo.l..JCC,AA00&855--00992277--SSAA--005653.. EE22 Da)_2 no sJtJd_econ_.l. CA085-O927-SA-068. E2 Day 5 no s_udgecontrol CA085-0927-SA-0_9 E2 Day 7 no sludge control,CA085_927-SA-O60, E2 Day 14 no sl_e control, CA085-O927.SA.062E2
1 Day 10 no sludgecontrol, CA085-0927-SA-064, E2
Day0 s_udgeblank. CA085-0927-SA-045, E2 Day 1 sJud(jeblank, CA085.0927.SA-046, E2
Day2 sludgeblank. CA085-0927-SA-047, E2
33aayy 57 sslluudcgl_eebbllaannkk,. CCAA008855--0O992277-.SSAA--00448g., EE22 _ay 14s/u_e .blank, CAO85.0927.SA.050, E2 :3ay16sludgeblank, CA085-0927-SA*051, E2
"
:.
:
::, :_i;:.i;_ '_
FilelD
"
' CA085 0924016=008
CA085 0924016 009
CA085 092401b 010
,, CA085 0924016 011 ,_ CAO850924016 0f2 CA085 092401b 013
CCAA008855 D09g224400116b810718 CA065 092401b 018 CA085 0924016 019 CA085 0924018 020 CA085 0924018 021
I I CA085 0924016 022
CA085 092401b 030 CA085.092401b 031
CA085 0924816 032
CCAA0088.55 009922440011b6 003334 , CAO850924016 =035 CA085_092401b038
2Hperfluoro-2-
octenoate 1040 3429 223 NO NO NO
"
NNOO N ND NO NO NO
NO NO NO
NNOO N0 NO
2Hperfluoro-2-
decenoate ND 780 NO NO NO NO
7
NNO ND NO NO NO NO
NO ND ND
NNOO NU NO
Inte_lrated
2Hperfluoro-2,
do.decenoate NO 249 NO NO NO NO
"
NNDO ND NO NO NO NO
Peak Areas
1H,1Hperfluoro*
octanoate 30_5 15825 8020 236 157 68
'"
NNOO ND N NO NO NO
NO
NO
NO
NO
NO
NO
,, NNOO
NNOO
NO
NO
NO
NO
1H,1Hperfluoro,
decanoate 211 7055 1802 NO NO NO
"
tN_D) , NO ND ND NO ND
NO NO NO
NNOO NO NO
1H,1Hperfluoro-
dodecanoate ND 958 263 NO NO ND
"
NNDD , NO NO NO NO.... NO
NO NO NO
NNOD NO NO
J
6,.
Page 34 of 38
Pace Project CA085: "Biodegradation Screen Study for Telomer Alcohols" -Revision 1 Study Director: Cleston C. Lange, Ph.D. Pace Analytical Services, Bio-Analytical Services Group Wednesday, November 06, 2002
i = ' "_; !
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i ' "
_
_I
Appendix F
Typical Total Ion Chromatograms for SIR Data Showing Telomer Alcohols and Polyfluorinated Fatty Acid Products from Day-0 to Day-16 for HPLC/MS Analysis of
Biodegradation Culture SPE eluate 2.
SIR of 17 anions: m/z 263, 313, 363, 413, 463, 513, 563, and 613 for perfluorinated acids; m/z 377, 477 and 577 for polyfluorinated acids; and m/z 323, 423, 523, 623, 723,and 823 for
telomer alcohols.
CAG85 Or_TOla 014
lo0;,
-
,
%!
Day-16 o:
.:ADPS_g_2?OIa 013
100_
'_"-
Day-14 0; ........... 1_...................
CA085_Oc_jT01a_012
Day-7
"0: '
CAO85_C92701a_011
100:
1e9
Day-5
_i
CAO85_092701a_010 1_O
'
Day-2 _0---!.
"----'_1".J)0
.
Perfluorinated Fatty Acid Peaks
8 6.8
I 721 _ 740
8 8}'7 22 '_!1 r_ 740
6Js,_j' L:r-rJi!,=( _
691722
0.62
=---
9.82
87_8),' 722 _I _"
, ;p_._.7.,..4...,........... 0- _....;_0_
10.17
747
SIR of 1i ChanneLs ESTIC
1,6_e6
8JR of "_7 CtI_ncLs E._ T._,
I 6,)e5
SIR of 1; Cha/_ne_s ESTIC
_ 52e6
SIR OI 17 Ctta_'lneLs E STIC
153e6
SIR of 17 ChanneLs EST)C
I 02e6
100 :
_
4.23
_
" 9 09 i_ _'0 _ _
.....
T_C
100
200
300
4 (30
5.00
600
7.00
800
_00_
10.00
11.00
12.00
1300
14.00
Telomer Alcohol Peaks
i I
Page 35 of 38 Pace Project CA085: "Biodegradation Screen Study for Telomer Alcohols" -Revision 1 Study Director: Cleston C. Lange, Ph.D. Pace Analytical Services, Bio-Analytical Services Group Wednesday, November 06, 2002
.: ;;,'ii i _"I
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{ ';
!
i'
:-.i
L'_;
_
.__J :i
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i::
Appendix G
SIR data Showing Ions for Telomer Alcohols at Day-0 and Day-16 from Analysis of the Biodegradation Culture SPE eluate 2.
CA085-0927-SA-031 E2, day 0 biodeg sample
C1A0085 _.)2101d 008
%
0 CA085J_JC70t,;_.)OS 100
CA085_092701a_008 100
i
%:
CA085 092701a_008 100 :
CA085_092701a_008 100
%1
CAO.85'CJg_701aO08
100
! %;
CA08:_ _701a_008
100_
,%-
0 L..... 1.0(3
,.CO - "_--_'_- -
2.00
3.00
53 O._D
A
a,3
783 813
O50
,,,
7"14748 I
,,,:_" -.';"
,,
, 89_
4.00
5.00
6.00
7.00
800
9.00
I0.00
SiR of 17.C_2h3a.nCtv_at iCsEOSD- a
SIROf 17,_,rle_/E1S1."._s 72.3COt CODa 50de5
SiR of 17Channels ES623,00 1,000a
s.co_
SIR of t7 Channe_ ES523.COt.O00a 5.C_16
SIR of 17CharlnetsES.t23 COf (X)Oa
SIR of 17 CZlanrlel._%S32300 1 _'_Oa
s0oes
,,., - ..................
I t.O0
12.00
1300
SIR of 17ChannelsESTIC
5 (X)e5
1400
Time
CA085..0927-SA-043 E2, day 16 blodeg sample CA_t35 _-'_2.70t3 0t4
SIR of 17 C_3nrlel.=E.S-
%i
CA085_b_J2701a_014
100 o:--
CA085 092701a_0t4 f00 :
%i
o CAOBSO9270_a_O_4 100
% (}:.......................................... CA085_092701a 014 )00 %
,_A_hS__2?')la 9_4
.... ,- ,. r
SIR oi 17Channel_E.$.5 0C_,
723 0_ 1 _Oa
SIR Of 17crlar_els ES623.00 t .(X_a
S_RO117 Chant,.el_ES-
_
523.00 15,.00000ea5
S_R0f 17 ChannelsES4_.3CO15.000_e')5D_3
SiR 5f 17ChannelsES-
%
CAU85.C'0270_1 0t4 100
%0 i ..........
100
'"
=............
2.00
3,00
430
4.00
5.00
eS2_ L
_741
17 j
.....
s.J%- ............
6.00
7.00
8(30
9.CO
10.00
1100
"_IRc_f17 Channel_E_ 5 _eT.4_C
12.CO
1300
f4 O0
Page 36 of 38
Pace Project CA085: "Biodegradation Screen Study for Telomer Alcohols" -Revision 1 Study Director: Cleston C. Lange, Ph,D. Pace Analytical Services, Bio-Analytical Services Group Wednesday, November 06, 2002
::,_'h_
:
;:i
14 i ' : _i L"" I'
i
_i _ _;.'_
.... .
iJ ,.
I' ,,; _
Appendix H
SIR data Showing
Ions for Perfluorinated Fatty Acids at Day-0 and Day-16 from Analysis of the Biodegradation Culture SPE eluate 2.
CA085-0927-SA-031 CA085_0_27015_008 t00
%
E2, clay 0 blodeg sample
CA005_09270 la_00_ 100 %;
CAQ05 0927015 008 100:
%:
SIR of 17C_anneL_ES51300 i 0coa 20056
SIR of 17 Cr_nnels ES5_3 00 1000;I 20Oed
SIR Qf 17Cr_nneis ES51300 t2COCO,D_a
CAQ05 092701a 0C8 tOO %1
CA085_0927015_008 1OO
%i
CA085_0927015.008 1OO
%!
CA0__0_i27015.008 100 : %
CAS_JS._Og_.T7_0 C_38
1OO %,
o' CA_,P,5..O9270ta _
16o
85O
7 57 7.52
SiR _f 17CManne..LE_S, 4"_3L,O_ _OCa 20C_5
SIR of 17 Clla,,1,--,eElsS41300 I O00a
2oo_
SIR Of 17Channel=ES363.OO1.00Oa 2.OO56
SIR orl I Y Cflanne_ ES313 00 1.00Da 20056
SIR Of f7 Cf1_r_'h_iE_S253 O0 2 (O_Oes_31
SiR of 17C.'lan_,_ E S2_ T_C
..... ,.oo 2.oo 3.oo ,,.o0 5.0o_- _.00' 7.00'.............. e.oo...._.00' i0'.0O ,i0o ' _ ...... ;2.OO_"i_OO" _,.OO Time
CA08S-0927-SA-043
CA0_5_OO2701a_014 100
E2, day 18 blodeg
sample
% 0: .................... CA085 0927015_014 IOO
752 _", ,
SIR of 17 C_nnels ES81300 100_
SIR of 17Cllanne_sES5630o _ OOOa
O: CA,'3__092701a_Q14 100.
%i 0 !CAQ05 0927015 014 100
,i
CA085_092701a 014 1OO.
%
O: ................................... CA085_0927015_014 IOO
%:
CA_85. 0927015_014
100
r
741 '_'_ ............
_ .......
7.30
722
_
-*-'%
7,07 6 88
_IR 01 17C,_,_i_l__e- ES51300 _ OOga 2 (X_e_
_[R of 17 Cnanne_ ES463.00 I 00C;I
zooes
*_ SIR of 17Cbannets ES413.00 1000a
2OOe5
SIR of 17ChanneL_ES* 3_3 O012000_056
SIR of 17C,'_lnrezsE$3_3 00 ? _Da 2 3Ce-
0
............................................................".=.".....................................................................
C._._5_L,L19*P70(1};1t4 l O0.
SIR Qf i 7 _l_t_n_.i5 _-S263 :-"";_: ;,3JCLe__DJ
%
862
CAr)_5.0927013.014
_OO
%
0
1 00
1 99 200
3.OO
4.OO
5.00
6.OO
5_7_ 7_
_ _ _ "" 700
B.00
8 62 9,OO
10.00
11.00
SIR O__7 C,'l.'_nr_ $-
2 _Te6_C
12.OO 1300
14 OO
Page 37 of 38
Pace Project CA085: "Biodegradation Screen Study for Telomer AJcohols" -Revision 1 Study Director: Cleston C. Lange, Ph.D. Pace Analytical Services, Bio-Anal_tical Services Group Wednesday, November 06, 2002
Appendix I
Chromatograms of Extracted ions m/z 377, 477, and 577 and Mass Spectra for Each Peak
.i
for Polyfluorinated Acid Intermediates Formed Transiently in Biodegradation Cultures at
Day-0, Day-l, Day-2 and Day-5. Suspected 13-Oxidation olefinic acid intermediates.
t,
i
'
q
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Page 38 of 38
Pace Project CA085: "BiodegradationScreen Study for Telomer Alcohols"-Revision 1 Study Director:Cleston C. Lange, Ph.D. Pace Analytical Services, Bio-AnalyticalServices Group Wednesday, November06, 2002