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BIODEGRAOATION STUDY REPORT Revision 1
Wednesday, November 06,2002
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Blodagradatlon Scraen Study for Talomer-Type Alcohols
PROJECT NUMBER
3M Projects ID: 601-0684 Pace Contract Analytical Projects ID: CA085
STUDY DIRECTOR
Cleston C. Lange, Ph.D.
CLIENT
James K. Lundberg, Ph.D., 3M Environmental Laboratory BIdg 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
PROJECT DATES
Project Initiation: Augusts, 2000 Project Completion: September 18,2001
AUTHOR
Cleston C. Lange, Ph.D.
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Pace Projecl CA085: "Biodegradation Screen Study for Tetomer Alcohols" -Revision 1
Study Director Cleston C. Lange, Ph.D. Pace Analytical Senrices, Bio-Anatytlcal Services Group
Wednesday, November OS, 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..................................................................................................................................... S
I.0 Project Personnel..................................................................................................................................6
2.0 Data Requirements and Revision Justification.....................................................................................Q
3.0 Project Objective.................................................................................................................................... 8
4.0 Test Article.............................................,............................................................................................7-8
5.0 Reference Articles.......--.....................--......................................................-.........--...... --...................9
8.0 Receipt/Generation of Samples................,...--........................................................................i.u.a....iS"''
7.0 Methods.................................................................................................................................................9
7.1 Sample Preparation....-..............,...............................................................--.................................9
7.1.1 Collection of Sludge....................................................................................................... ,,.9-10
7.1.2 Culture Preparation.........................................................................................................11-12
7.1.3 Solid Phase Extraction (SPE),........................................................................................ 12-13 7.2 Instrumental Analysis (HPLC/MS)................................................................................................ 13
7.2.1 Instrument Parameters ......,,........--................................................................................ 13-14 --
7.2.2 Qualitative Analysis of Parents and Products ................................................................14-15 7.2.3 Quantitative Analysis............................................................................................... 16 7.3 Data Transformations and Calculations........................................................................................ 16
7.3.1 Molar Calculations............................................................................................................raliSr.
7.3.2 Conversion of na/mL tomlcromolar and nanomolar....................................................;&.a 16, <>i ,. f, 3!..,.r. ;
7.3.3 Molar Mass Balance Calculations...............................................................................Ms.l?A7n. Cs'.ram-siitKti?.
7.4 Software Versions.................................................................................................................v-.i.w. 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 HPLC/MS Analysis........................................................................................................................ 19
8.4.1 Qualitative HPLC/MS and HPLC/M8/MS Results..-..............................-...........-....-.....19-24
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8.4.2 Quantitative Analysis Results for PFOA..............................,....,...,,.............,...................24-25
9.0 Conclusions................................................--...--.................,....................-- .....................,,........-- ....27 10.0 Literature Citad............................................................................................................................... 28
II.0 Sample and Data Retention .............................................................................................................29
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Pace Project CA085: "Biotlegradation Screen Study for Talomer Alcohols' -Revision 1 Study Director; Cleston C. Lange. Ph.D. Pace Analytical Services, Blo-Analytical Services Group Wednesday, November 05,2002
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List of Tables & Figures
TABLES
Table 1. Parent telomer alcohol expected ions........................................................................................ 14 Table 2. Expected product fluorinated fatty acid products Ions............................................................... 15 Table 3. HPLC/MS Data Table for Parent and Product Ions observed ....................................................23 Table 4. HPLC/MS/MS Data for p-Oxidatlon Product Ions observed .......................................................24
FIGURES
Figure 1. Chemical Representation of Telomer Alcohols ...........................................................................8 Figure 2. Chemical Structure of perfluorooctanoate (PFOA)..............-.....................................................9 Figure 3. Plotted HPLC/MS Integrated Peak Areas for C,, C,B and 0,2 Tetomer Alcohols......................20
Figure 4. Plotted HPLC/MS Integrated Peak Areas for Co, C, and C,a Telomer Alcohols......................20
Figure 5. Plotted HPLC/MS Integrated Peak Areas for Perfluorinated Fatty Acids Detected ..................22 Figure 6. Plotted Peak Areas for Transient Polyfluorinated Fatty Acids Detected.............,..--................22 Figure 7. Plotted PFOA Concentrations Measured in Biodegradalion Samples (SPE eluate 2) .............25 Figure 8. Proposed Btodegradation Pathway for Telomer Alcohols.........................................................26
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Pace Project CA085: Btodegradation Screen Study for Telomer Alcohols" -Revision 1 Study Director: Cleston C. Lange, Ph.D. Pace Analytical Services, Bid-Analytical Services Group Wednesday, November 06,2002
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List of Appendices
Appendix A: Approval Signatures............................................................................................................30
Appendix B: Final Quantitative Results for PFOA (ng/mL)
Table 1. Table 2. Table 3.
Test Cultures Final Results ................................................................................31 Abiotic Cultures Final Results................................................................................ 31
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 Fatly Acids .............33 Appendix E; Table 1. Peak Area Data for Transiently Formed Polyfluomated 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..................... 38
Appendix H: Table 1. TICs for SIR Data for Day 0 and Day 16 Abiotic Control Samples......................37 Appendix 1: Table 1. TICs for Extracted ton Data for Transient Polyfluorinated Fatty Acids..................38
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Pace Projecl CAQ05: ''Biodegradation Screen Study for Telomer Alcohols' -Revision 1 Study Director Cteston C. Lange, Ph.D. Pace Analytical Services, Bto-Analytical Services Group Wednesday, November 08.2002
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Executive Summary
A screening study was undertaken to determine whether the fluorochemical telomer intermediate (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 pM concentrations. Cultures were incubated with shaking at 25C. Solid phase extraction of cultures for recovery of blodegradation products and parent analytes was employed. An HPLC/MS analytical method 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 biodegraded. The observed loss of telomer alcohols occurred concomitant with the appearance of several expected perfluorinated carboxylic acids. Unexpectedly, transiently formed polyfluorinated P-oxidation intermediates were observed. The p-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 fatly acid a-oxidation, a minor pathway of fatty acid metabolism usually observed inrbranched-chain fatty acids. After the 16day test period, peiflourinatad carboxylic adds ranging from Cg to Ciz were observed in the test cultures. These compounds were not in controls or blank cultures. Perfluorooctanoate (PFOA). was the only compound quantitatively analyzed and, based on mass balance data accounted for approximately 6-7% of the total telomer alcohols initially present in the test cultures.
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Pace Project CA085: "BlodegradaUon Screen Study for Telower Alcohols" -Revision 1 Study Director: Claston C. Lange, Ph.D. Pace Analytical Services, Bio-Analytical Services Group Wednesday, November OS, 2002
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1.0
Prolact Personnel
1.1
Sponsor Company
1.2
Sponsor Representative
1.3
Contract Facility Personnel:
1.3.1 Study Director
1.3-2 1.3.3
Laboratory Management: Sample Preparation
1.3.4 1.3.5 1.3.6
HPLC/MS Analyst Sample Custodian: Report Author
2.0
Data Requirements and Revision Justification
3M Dr. James K. Lundberg
Or. Cleston C. Lange Mr. Bruce E. Warden Ms. Angela L. Schuler Dr. Cleston C. Lange Ms. Angela L. Schuler Or, Cleston C. Lange Dr. Cleston C. Lange
Trie sponsor representative desired an aerobic biodegradability screening study to be conducted using the telomer 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 Telorner. The reason for the revisioroisxta
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more accurately reflect the impact of results to the general class of telomer alcolaols.oi.iratiaty rafteci, thf-
3.0
Project Objective
This study was conducted in order to elucidate whether the fluorotetorner intermediate (tetomer alcohol) is biodegradable under aerobic conditions using a mfcrobial rich inoculum of municipal wastewater treatment sludge. This study was similar to eariier studies conducted for 3M by Pace as Pace projects CA05B 1 and CA097 z and CA104 3. CA105 4, and CA132 s. The development of an analytical method and the analysis of parent analyteA and possible products were critical parameters for the determination of
biodagradation.
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Pace Project CA085: "Biodegradation Screen Study for Telomer Alcohols' -Revision 1 Study Director: Cleston C. Lange, Ph.D. Pace Analytical Services, Blo-Anatytlcal Services Group Wednesday, November 06.2002
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4.0 Test Article
The lest article used far 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 first set of cultures prepared and original LC/MS method development.
A commercial vendor, Aldrlch Chemical Company, provided the second source of telomer alcohol used. Material was purchased as Zonyl BA-L fluorotelomer intermediate (1999 Atdrich Catalog number 42,151-0), also called perfluoroalkytelhanol. The material characteristics were boiling point 145-245C, FtCF^nCHaCHgOH 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-TNCOO-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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Pace Project CA08S: "Biodegradation Screen Study lor Telomer Alcohols' -Revision 1 Study Director: Cleston C. Lange, Ph.D. Pace Analytical Services, Blo-Analytical Services Group Wednesday. November 06,2002
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Molecular formula Molecular Weight
= Cg H, Fg 0 =264.089
1M+CH,C001-=323
Molecular formula '*C,HgF,iO
Molecular Weight = 364.104
(M+CH,COOT=423
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Molecular formula
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F FF F F F
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Molecular formula = C,; Hg P,, 0 Molecular Weight =564.134
[M+CH,COOT=923
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Molecular formula Molecular Weight
[M + C-..H, ,C- OO^ T,, .
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=664.149
723
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Molecular formula = C,^ H, Fg 0 Molecular weight =764.164 (M+CH3COOT=823
Figure 1. The tetomer 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 Cg telomer alcohol, 39.3% 1.5% as Ca. 27.7% 2.1% as C,i), 17.5% 1.2% as C,,. 7.2% 1.0% as 14. and 2.0% 0.2% as C,B.
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Pace Project CA085: "Biodegradation Screen Study for Tetomer Alcohols" -Revision 1 Study Director: Cleston C. Lange, Ph.D. Pace Analytical Services, Blo-Analytlcal Services Group Wednesday. November 08.2002
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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) Purity: 95.2 %; 3M#: TCR-99131-37; Pace 1k CA-TCR02-001; Expires: NS
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F F F F F F F Figure 2. Perfluorooctanoate ammonium salt
8.0 RacaiDt/Gnraon 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 eluatas collected for each culture, were labeled as SPE eluates 1, 2 or 3. Of the three efuatea generated for each sample, eluate 1 was the 25 mL aqueous sample eluate and eluates 2 and 3 were 25 mL mathanol eluatas.(ro(n: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 biodegradatlon
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
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Pace Project CA085: 'Biodegradation Screen Study forTelomer Alcohols* -Revision 1 Study Director Cleston C. Lange. Ph.D. Pacs 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 S 528791. Sludge used for the second set of cultures In this study was also used In preparation of cultures for other fluorochemical biodegradation studies (CAWS 4 and CA132 '), 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 1-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 MLS5 plus sludge for use in preparing test cultures. A sludge characterization analysis was not conducted as part of this screening
study.
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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-ahd was consistent with observation of earlier sludge collections.
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Pace Project CA085: "Biodegradadon Screen Study for Telomer Alcohols"-Revision 1 Study Director Claston C. Lange, Ph.D. Pace Analytical Services. Bio-Analytical Services Group Wednesday, November 06.2002
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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 Ume 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 6.
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. Na;,HP04-2HzO, 0.005 g Nh^CI. 0.2175 g KzHP04, and 0.085 KH2PO, 0.0275 g CaCh-anhydrous, 0.0225 g MgSO-7H20, and 0.00025 g FeCls-SlW.
Two liters of a mineral salts medium containing100 mL of settled sludge was prepared and contained 1 mL of methanol. To each test culture was added 25 niL 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.
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All cultures were prepared by dispensing 25 mL of appropriate mineral salts medium soluUon 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 (ig/mL in methanol) or Zonyl BA-L
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Pace Project CA085: "Biodegradation Screen Study for Telomer Alcohols" -Revision 1
Study Director. Cleslon 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 l.ig/mL In methanol), was added to the test cultures by transferring 5 iL and 8(iL. respectively, to the appropriate test cultures. The final test concentration In cultures was 2.056 ug/mL of Zonyl BA-N telomer alcohol for set one, and 2.332 pg/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 frozen at -2CTC for storage until SPE preparation could be conducted. All other cultures were placed in temperature controlled shaking incubators that were maintained at 25C t 3'C. 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-04T.
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 l-Chern 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: Cieston C. Lange, Ph.D. Pace Analytical Services. Bio-Analytical Services Group
Wednesday. November 06.2002
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Frozen cultures were thawed al 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 swifted to mix, and then drawn by vacuum through the appropriately labeled 5PE cartridge by carefully pouring the contents of the culture flask into the SPE cartridge. The aqueous eluate was collected in an l-Chem vial labeled eluate 1, removed from the vacuum manifold, and capped. Then, 25 mL of melhanol was added to the culture flask, the flask sealed, and vigorously shaken. The cap was then removed from the flask, and tha methanol content (25 mL) drawn through the SPE cartridge, collected in an l-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 eluatss 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 + 2'C.
The final SPE extractions of the day 7, 14 and 16 day cultures from set two occurred on September 24,2001.
7.2 Instrumental Analysis (LC/MS)
7.2.1 Instrument Parameters The HPLC/MS method used was a modified version of Pace method CAGORG-238, 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 HPl 100 HPLC pump with Gllson 215 liquid handling system in line with a Micromass Quattro H triple-quadrapole mass spectrometer detector.
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Pace Project CAOS5: "Biodegradation Screen Study for Telomer Alcohols* -Revision 1 Study Director; Cleston C. Lange, Ph.D. Pace Analytical Services, Bio-Analytlcal Services Group
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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 11 tripte-quadrapole mass spectrometer detector. Typical conditions for analysis of telomer alcohols and fluorochemica] acid products were as follows:
Instrument conditions for HPLC/MS analysis.
Mass Spectrometer
HPLC
lonization mode: API-ES negative
Desolvatlon Temperature: 200'C Source Block Temperature: 150'C RF lens: 0.2
Time (mm). 0.00 0.50
3^ ^B
97.0 3.0 97.0 3.0
Extractors
5.00
5.0
S5.0
Cone:8
11.00
5.0
95.0
Capillary: 3.50
11.50
97.0 3.0
LM Resolution: 14
15.00
97.0 3.0
HM Resolution: 14
Flow: 1 mL/min, splitter approx. 3:1
Ion Energy: 2.5
Solvent A5 2 rnM ammonium acetate
Lens 6:2
Solvent 8= Methanol
MS1 Multlpliar: 650
Column Temperature: ambient
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 ut. The 4.6 x 150 mm Betasil C8 column used for the quantitatlon of 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 splitter was used with an approximate split ration of 2:1 (Waste:MS)
7.2.2 Qualitative Parent Analyte and Products Analysis.
Parent Analyta Cg telomer alconol acetate adduct Cio telomer alcohol acetate adduct Ctz telomer alcohol acetate adduct CK telomer alcohol acetate adduct Cm telomer alcohol acetate adduct
Expected tons (m/z) 423 523 623 723 823
Table 1. Parent telomer alcohols analyzed.
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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 B. 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 2-spray source.
Specific MS conditions for mass analysis of the telomer alcohols and acid products were developed during this study. The mass spectrometer, Micromasa triple-quadrapole mass spectrometer with Z-spray ion source, was operated with electcospcay ionization in either selected ton-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 tetomer alcohols and (M - fT)" for expected acid biodegradation products, were observed and monitored:
Expected Product
Perfluorobutyrate (64) PerBuoropentanoate (Cs) Perfluorohexanoate (Ce) Pertluoroheptanoate (7) Perfluorooctanoate (Ci, PFOA) Perfluorononanoate (C) Perfluorodecanoate (C,o) Perfluoroundecanoate (Cn) Perfluorododecanoate (C,z) 2H, 2H-perfluorooctanoate (Ca) 2H. 2H-perfluorodecanoate (C,o) 2H. ZH-perfluorododecanoate (Cu)
'
Expected anion structure
CF3(CF2hCOO" CF3(CF2)3COO CF3(CF2)<COO CF(CF;)5COOCF3(CF2)eCOO CFalCFzbCOOCF3(CF2).COOCF3(CF2hCOOCF3<CF2)ioCOO CF3(CF2)sCH;COO" CF,(CF2)7CHaCOO CF3(CFz)sCH;COO-
Expactad anions (m/r)
'
i
213,169
283,29Qw'v;'wWt:'. 313;t26a8fohaKatS6?
363,a^!9t^>^?<^wn^1
413,369 463, 419 513,469 563,519 613.569
377 477 577
Table 2. Expected products, and expected anions for each.
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Pace Project CA085: "Biodegradation Screen Study for Telomer Alcohols" -Revision 1 Study Director: Cleaton C. Lange, Ph.D. Pace Analytical Services, Bio-Analytical Services Group Wednesday, November 06,2002
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7.2.3 Quantitative Analysis.
Quantitative analysis was conducted only for perfluorooctanoate (PFOA) and only onextracts-of-culture set two; - Quantitative analysis was performed by the external standard method using SPE extracted calibration standards (etuate 2 only) and quadratic calibration curves. The HPLC/MS analysis of culture set one extracts was used primarily for method development purposes.
Typical injection volumes for samples and calibration standards were 50 pL. The 4.6 x 150 mm Betasil CB column used for the quantftallon 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 Alltech catalog part number 39025.
7.3 Data Transformations and Calculations
'
7.3.1 Molar Calculations:
''* -;alcuias:ti
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. Ie 431.10 Zonyl BA-L telomer alcohol average molecular weight is 434.70.
7.3.2
Conversion of ng/mL to micromolar (pM) and nanomolar (nM). (Working Examples}: 2,232 ng/mL telomer alcohol = (2,232 ng/mL)'(1nmole / 434.7 ng) = 5.135 nmole/mL = 5.135 timole/liter == 5.135 (iM--assuming 100% purity.
150 ng/mL PFOA (NH4+ salt) a (150 ng/mL)" (1 nmole /431.1 ng)
= 0.3479 nmole/mL = 0.3479 (imole/L = 0.3479 pM = 347.9 nM Page 16 of'38
Pace Project CA085: "Biodegradation Screen Study for Tetomer Alcohols" -Revision 1 Study Director: Claston C. Lange, Ph.D. Pace Analytical Services, Bio-Analytieal Services Group
Wednesday, November 06.2002
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7.3.3
Molar Mass Balance Cafcuiations (Theoretical Yigtd): Convert all ng/mL values to their corresponding molar concentrations as |iM 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 of the known starting concentration.
(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 0.3479uM
Then, the mass balance Is as follows: Mass balance = ({0.3479 pM) / 5.135 pM] X 100% Mass balance = [0.0678] X 100% = 6.7B% Or, 6.78% of the telomer BA-L alcohol was oxidized to form PFOA.
7.4 Software Versions
MicrosoftTM 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. 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
Paca Project CA085: Biodegradation Screen Study for Telomer Alcohols" -Revision 1 Study Director Cleston C. Large, Ph.D. Pace Analytical Services, Bio-Analytlcal Services Group Wednesday. November 06,2002
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8.0 Results 8.1 Sludge Characterization
A chemical analysis of the mixed liquor suspended solids used for the samples " "" prepared on August"^; 2000 was conducted, and" characterization information
can be found in the final report for project CA058'. 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 analyta 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-siudge (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 analytes were not detected in blanks and biodegradation did not occur in abiotic controls.
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Pace Project CA085: "Biodegradalion 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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8.4 HPLC/MS Analysis Results
Semi-quantitative analysis was conducted only for the SPE aluate 2 extracts from the'secQnff'set of cultures, prepared September 27. 2000. and were performed on September of 2001 as sequence CA085J)92401b.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.sp], S092000.spl, S092100.spl, S092800.spl, S1000200.spt, and S100400.sp( on Pace instrument "STING".
8.4.2 Qualitative HPLC/MS and HPLC/MS/MS Results
The first set of test cultures containing sludge and BA-N telomer alcohol substrate provided evidence 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 dhromatographic 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.
"w i'-.; i:'
Although each sample, control, and blank for the second set of cultures was prepared and incubated in duplicate, analysis was conducted for SPE eluate 2 extracts from one sample, conlrot and blank per time point.
The data showed that the sample test cultures underwent rapid loss of the Ce. Ca, do.and Cij-telomer alcohols, and moderate loss of the Ci4-telorner alcohol over the 16 days of incubation, as determined by decreasing peak area response for each tetomer alcohol Ion at the sequential sampling time points (Figures 3 and 4). The Gia-telomer alcohol was degraded very slowly and showed little loss.
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Pace Project CA085: "Biodegradation Screan 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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C8 TA peak area
A C10 TA peak area
C12TA peak area
Figure 3. HPLC/MS integrated peak areas for the most abundant talomer alcohols (TA). Near complete degradation of all three was observed. Data plotted with the best manual fit of the data.
-'3( '' c
AC8TA peak area C14 TA peak araa C16TA peak area
5
10
15
Incubaiton tima (days)
Figure 4. HPLC/MS Integrated peak areas for the less abundant telomer alcohols (TA). Near complete degradation was observed for th e C(-TA. Moderate degradation of the Ci4-TA was observed and litHe degradation of (ha Cis-TA was observed. Data plotted with the best manual fit of the data.
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Pace Project CA085: "Biodegradation Screen Study for Telorner 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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Concomitant with the loss in telomer alcohols was the formation of compounds with HPLC/MS ions and retention times consistent with periluorinated acids (PFAs). including perfluorooctanoate (PFOA, Ca-PFA), which was confirmed as an end product. The transformation of telomer alcohols to PFAs was rapid, with a large increase in alt 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 add (C; PFA), Derfluorohexanolc acid (CyPFA). perfluoroheptanoic acid (CT-PFA), perfluorooctanoic acid (Cg-PFA, PFOA), perfluorononanoic acid (Cg-PFA), peifluorodecanoic acid (Cic-PFA), pernuoroundecanoic acid (Cu-PFA) and perfluorododecanoic acid (C(Z-PFA).
Transienlly 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-perfluorooclanoata|2H, ZB-Riarfluorodecanoate; 2H, 2H
.
Pertluorododecanoatelliad the-possible-p-oxidation pathway intermediates:
2H-perfluoro-2-octenoate. 2H-perfluoro-2-decenoate. and 2H-perfIuoro-2dodecenoate. Table 3 shows the MS ions and retention times observed for the polyfluoi-inated and perfluorinated acid products and the telomer alcohol
parent substrates. HPLC/MS/MS Data (Table 4) support the identification of the suspected
metabolites..
<ii?i'.:, ,'r::. ,:;,w'n'>',-
' - .!staaat: SH W -,. ?',',' ' :-!.'tee^^mwB,tl WA tia- isoartbte f'
--
,
-4i; :mw< '}>.
Page 21 of 36
Pace Project CA085: "Blodegradation Screen Study for Telomer Alcohols" -Revision 1 Study Director: Cleston C. Lange, Ph.D. Pace Analytical Services. Bio-Analylical Services Group Wednesday. November 06.2G02
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Figure 5. Integrated peak area responses for the pcrfluorinatod fatty acids (PFAs) observed in test culture extracts. Even numbered carbon chain length carboxylic adds were the most abundant carboxylic acid peaks and were: pertluorohexanoata (Cs-PFA), pei-fluorooctanoate (Ca-PFA) and perfluorodecanoate (Cm-PFA).
Figuro 6. Transient polyfluorinated fatly acid intermediates observed. The 2Hperfluorinatetj olefinic fatty acids are suspected (l-oxidation intermediates that formed rapidly upon initial exposure to telomer alcohols, and then were transformed to their corresponding psrfluorinated carboxylic acids end products
Page 22 of 36
Pace Project CA085: "Biodegradation Screen Study for Telomer Alcohols" -Revision 1 Study Director. Cleston C. Lange, Ph.D. Pace Analytical Services, Bio-Analytfcal Services Group Wednesday, November OS, Z002
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Analytas Observed
Chemical Formula
Ce-tetomer alcohol*
Ca-telomer alcohol'
Cto-telomer alcohol*
Cirtelomer alcohol*
CM-telomer alcohol*
Cwtetomer alcohol*
2H, 2H-perfhiorooctanoate*
2H, 2H-pertluorodecanoate" 2H, 2H Perfluorododacanoate*
2H-pe(fluoro-2-oct9noata*
2H.perfiuoro-2-dec6fioate*
2H.Perfluoro-2-dod8cenoat8"
Perfluoropentanoate
Perfluorohexanoate
Pertluofoheptanoate
Perfluorooclanoate
Pertluorononanoate
Se(o(0(ie6afioata
r^f.:-^^-^.;,^^^^"-:.^:^
^ .
,
^ .
Periluoroundecanoate
;
J|y|$lBg^^canoa^..^
CF3(CF!)3CH2CHzOH...CH3COO" CF3(CF2,sCM2CH20H..,CH,COO' CFa(CF2)7CH2CH20H...CHCOO' CF3<CF2)9CH2CHaOH...CH3COO"
CFa(CF2)CH2CH20H...CH3COO
CF3(CF2)iCH2CH20H...CH3COO CF^Cy^iCHaCOO CF3(CF2)rCH2COO CF(CF2)sCH2COO" CF3<CF2)4CF=CHCOO' CF3(CF2)BCF=CHCOOCF3<CF2)eCF=CHCOO' CFs(CF2)3COO CF,(CF2),COOCF3(CFt)5COO' CFa(CF2lsCOOCFatCFzWOO CF}(CF2)aCOOCFa(CF2)COO CF3<CFs)io00
Retention Time LCIUS anions
obasrvad
7.63
323
8;13
423
8.50
523
8.96
623
9.53
723
10.25
823
7.17
377
7.38
477
7.49
577
7.14
357,293
7.35
457.393
7.52
557.493
6.62
263.219
6.88
313.269
7.07
363,319
7.22
413,369
7.30
463.419
j (^tS^wA-'a1 o-M-.513.469
; PSi'SASF^
563,519
; L "^
;, 8.13,589
Table 3. Parent and products anions observed in by HPLC/MS analysis of 5PE eluate 2 extracts from i 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.
Pace 23 of 38
Pace Project CA085; "Blodegradation Screen Study for Telomer Alcohols" -Revision 1 Study Director Clasion C. t-ange, Ph.D. Pace Analytical Services, Bio-Analytical Services Group Wednesday. November 06,2002
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Product Analytoc Observed
2H.2H-perfluorooctanoate* 2H.2H-petfluorodecanoate* 2H.2H Perfiuorododecanoate' 2H-perfluoro"2"octenoate* 2H-p9riIuoro-2-decenoate* 2H.Perfluoo-2-dodecenoatB*
Chemical Formula
CF3(CF2)5CHaCOO" CFaiCFiWteCOO CFi(CFz)iCH2COO CF3<CFz)tCF=CHCOOCF3(CF2)tCF=CHCOOCFa(CF2)CF=CHCOO-
Retention Tlm 7.17 7.38 7.49 7.14 7.35 7.52
LC/M5/MS Parent anion
377 477 577 357 457 557
LC/MS/MS daughter
anions otufvad
333.313.293 413.393
--
313.293,243.143.119 413,393.343 493
Tabia 4. HPLWMS/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 (CA08&0927-SA-033 62). The parent antons were observed as transienlly formed anions in early sample point biodesradation 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 (onTnjla;;i3o(nnion :.
| mass loss from the parent, anion, attributed to loss ofCOz (mass 44) and HP (mass 20),;werfl<bbsee(ed/!ai-n' >.' ;
8.4.2 Quantitative Analysis of PFOA
The HPLC/MS multi-component calibration standards used in this study for product quantttation contained the target analytes: PFOA, PFOSulfinate, PFOS, FOSA, A/'MeFOSA. M556, M570. and A/-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 CA065: "BtodegradaUon Screen Study far Telomer Alcohols" -Revision 1 Study Director Cleston C. Lange, Ph.D. Pace Analytical Services, Blo-Analytfcal Services Group Wednesday. November 06.2002
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All calibration standards and sample eluates were stored at 4C in refrigerator ID 0213 unti 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 PFOAwas 0:965. Quantitative sequence runs contained calibration standards at the beginning and end of the run.
The biodegradation of the telomer alcohols resulted in an increase in the measured levels of PFOA during the 16-day study (Figure 7).
Figure 7. The measured perfluofooctanoate <PFOA) In cultures containing sludge and tluorotalomer alcohol. The PFOA was not measurable in cultures that did not contain sludge. Data plotted with the best manual fit of the data and concentrations shown were not adjusted for reference material purity of 95.2%.
Page 25 of 38
Pace Project CA085: "Biodegradation Screen Sludy for Tetomer Alcohols" -Revision 1 Study Director: CIsston C. Lange, Ph.D. Pace Analytical Sen/Ices, Blo-Analytical Sen/Ices Group Wednesday, November 06,2002
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Ff F f Pf F
TTV-oH
C in Telomer Alcohol
de)))dTOBflnaM
^
III
Oxidativ decarboxylatlon
Ira-oxidation)
llla1
'"
FFF F
jaoibu
:
amyavieMS
Hlb1
P-Oxidation
HillifK "te^ l!la2
r r f r r f -f^^''-,' o'
.Ulb2 1'"
--|-4444-H4-(
a
Parfluorononanoate
laflitrwwt
i-
I^JaiacyllhUau
CO,
lilb3
t 1. ^ F F f
'
Perfluorooctanoale
^^0.
0'
Acetate
Figure 8. Proposed biodegradaUon pathway for telomer alcohols, shown for Cio-te)omer alcohol as example. The p-oxidation pathway branch (IHb) involves primary formaSon of a fatty acyl-CoA thioesler that is not shown, to clearly depict Hie products observed by HPLC/MS. The fatty acyl-CoA thtol bond is readily hydrolyzed to yield the corresponding fatty acids obseived.
Page 26 of 38
Pace Project CA085; "Biodegradatfan Screen Study for Telorner 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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9.0 Conclusions
This study has demonstrated that fluorochemical telomer alcohols are biodegradable and are transformed to perfluorinated acid end. products. Although the telomer alcohols are supplied only as even numbered carbon chain compounds, the end product perfluorinated adds consist of both even number and odd number carbon chains, with even numbered carbon chain length adds predominating as the major end products. This observation suggests that. following the initial oxidation of the hydroxyl-carbon to form the primary carboxylic add (telomer carboxylale). two oxidation mechanisms exist as shown in Figure 8, III. The first, less utilized route (Figure 8, Ilia), involves oxidation of the a-carbon concomitant with decarboxylation (oxidative decarboxylation) to form an odd chain length perfluorinated carboxylic acid, which does not undergo further biotransformation. The second observed route of oxidation (Figura 8, Illb) Is the more common route of fatty add p-oxidation, as evidenced by the formation of detectable transient p-oxidation polyfluorinated fatty acid Intermediates and the more abundant even number carbon chain length carboxylic adds
Although toxicity of the telomer alcohols was not addressed as part of thisostudy, 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
Pace Project CA085: "Biodegradation Screen Study for Telomer Alcohols' -Revision 1 Study Director Cleston C. Langa, Ph.D. Pace Analytical Services. Bio-Analytlcai Services Group Wednesday. November 06,2002
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8.0 Literature Cited
1.Final Reports for Pace Project CAOS8 (3M EOO-2252), "2-Week M-EtFOSE Alcohol Biodegradation Screen Study Report" and "Aerobic Biodegradation of N-EIFOSE alcohol Study Report". Author Cteston C. Lange, Ph.D.
2.Final report for Pace Project CA097 (3M # E01-0415). "The 18-Day Aerobic Biodegradation Study of Periiuorooctanesulfonyl-based Chemistries." Author. Cleston C. Lange, Ph.D.
S.Final report for Pace Project CA104 (3M # E01-0444). "The 35-Day Aerobic Biodegradation Study of Perfluocooctanesulfonate (PFOS)." Author: Cleston C. Lange, Ph.D.
4. Final Report for Pace Project CA105 (3M # E01-0683)," Fluorochemical Adipate (LI 5468) 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
S.fl.-sat HB{.<?-
Biodegradation Study ot the Fluorochemical FC807-Diester". Final report 'vsac'
Issued May 29,2001. Study Director: Cleston C. Langs, Ph.D.
6. Pace method CAG-SP-03. "Culture Preparation for Assessment of Aerobic Blodegradabllity of Fluorochomicals Using Municipal or Industrial Sludge as Microbial Inoculum."
T.Pace method CAG-SP-04. "CIS Solid Phase Extraction Procedure for Ftuorochemicals Recovery from Aqueous/Sludge Matrices.*
8. Pace method CAG-ORG-23 "Quantitative Analysis of Fluorochemicals by High Performance Liquid Chromatography with Mass Spectrometric Detection."
Page 26 of 38
Pace Project CA085: "Biodegradalion Screen Study for Telomer Alcohols' -revision 1 Study Director: Cleston C. Lange. Ph.D. Pace Analytical Services, Bio-Analytical Services Group Wednesday.November06.20Q2
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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 extractswill be retained at the Pace Analytical facility for a period of 2 years after completion of the project at 4C in the Can-oil walk-in cooler (Pace ID 0140) located in the Pace Analytical-Tier 2 faciHty.
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, chromalograms, 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 b retained for a period of 10 years. Facility data is availabtesfofciti be .-.WMW
Inspection and Includes the following records:
"< "s-, am; fn'auisw ry W"M)"
Training records Controlled storage temperature logs Standard preparation logs Calibration and maintenance logs Chemical and solvent Iraceability logs Standard Operating Procedures Methods pertaining to the conduct of this project
i
Page 29 of ^8
Pace Project CA085: "Biodegradation Screen Study for Telonw Alcohols" -Revision 1 Study Director Cleston C. Lange, Ph.D. Pace Analytical Services, Bio-Analyflcal Services Group Wednesday. November 06.2002
0^ 0.0--0<.31.
EID839576
APPENDIX A APPROVAL SIGNATURES
Project Title:
Biodegradatlon Screen Study for Telomer Alcohols
Client Project ID:
E01-0684
Contract Analytical Project Number: CA085
Report Revised by: Mourad Rahl
Laboratory Management: Bruce E Warden
^/ ^ ^ y Signature' ^ / o _DAate T-^
W-L^
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Pace Project CA085: "Biodegradatton Screen Study for Telonw Alcohol*" -Revision 1 Study Director: Cleston C. Lange, Ph.D. Pace Analytical Services, Bin-Analytical Services Group Wednesday. November 06,2002
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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.
eaiMirl< Kwnil.1 BliMft 11
D 1 bIMndM. Mi. aUIB7.tX. B
HO
Mr < naMmw unM. cr.aKor. E;
lll.t
B,uaMswH.cuusa.wai.a
11
314>lDllMkn>Mfk,CMU?-fMM1.f;
W^'f^&W^-t.Wf -.w.AajfcsBAsi
^sBiaga-i-isA.
Table 2. The measured PFOA concentrations in the abiotic control cultures during ?0 16-day
screening study for telomer alcohol aerobic biodegradability,
--ite
ConcitrXtoilwlmUlthiM.ll W>
:y ifwdujMBBittai.CAMft^wf-aA^as.ea
0.4
Sa^B.A-B.-afl.tMKnnE.SMB.B
a
IHS^fuSWW.CMIWim.SM.ti--------------------------
My 7 no ^MOq*UMMI.CAn64flar-SA48a.C2
0.0
, 1< m ibdM MW, WSlUWHI-auSt a
0.0
a,iia,mwa.ciswarw,.a
9.0
Table 3. The final data results far sludge blanks during the 16-day screening study for telomer alcohol aerobic biodegradability. PFOA was not detected in any of the sludge blanks.
*. 5 1 mg. UM.CMISWU-WW. tt
31 .HXlo.l----.WMBr.SMM.El ;W< UoteNMc. CWMKff-MW. B! BW 5 >bd UMI. CMtMW-SMW, (1
3y 10 lludM <*. CAQQUT-3A41. E2
sM""!MJiaiWt,lS!asa wo*
11.0
ao HI
tt
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Pace Project CA085: "Blodegradation Screen Study for Telomer Alcohols" -Revision 1
Study Director. Cleston c. Lange. Ph.D.
Pace Analytical Services, Bid-Analytical Services Group Wednesday. November 06.2002
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Appendix C Integrated Chromatogram Peak Area Data for talomer alcohols
Table 1. Integrated LC/MS peak area raw data for the parent telomer alcohols substrates. Data acquired from sequence run CA085_0927Q1a,spl.as SIR data.
Table 2. Percentage of integrated peak area remaining at different time points with respect tatne 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 Cm TA. which was 79.4%.
[Sampit Detcriptlon
|Pay0 blodearadatlon sample. CA085-0927-SA-031. E2
Pay 1 Biodegradation aacnple, CA065-0927-SA-033, E2 Day 2 bkxiegradalion sample. CA065-0927^A-Q35.'EZ'
pay 5 biodearadation sample. CA085-0927-SA-037. E2
Day 7 biodegfadation sample, CAOa5-0927.SA.039. E2
H3ay14 biodegradalion sample. CAOB5-0927-SA-041. 62
ilr- --.
-
j1*6~ b- i n-^d^'"""^T".-. -.
.*.--_'"'lia" ,
C*~Ai^ 0n8n5if-0f^9m2S7^-S'*AM -0n 4j 3n ,
' .
t.Z
pay 0 no sludae oonliol. CA08'-092T.SA-Q53, E2 pay 1 no sludge control. CA08S-0927-SA-055. ET
Pay 2 no sludge control. CAOa5.0927-SA-056, E2 pay 5 no sludge control. CA085-0927-SA-059. E2
p3W 7 no sludge control. CA08S.0927-SA-Q60. E2
pay 14 no sludge control. CA085-0927-SA-062 62
pay 16 no sludge control, CA085-0927-SA-064, E2
VEsauEgsssaaEssx -mar.i^Kmr^Krmr^KiCTri^TO.x^
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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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EID839579
Appendix D Integrated Peak Area Data for Perfluorinatad 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.
-
--.-....-.-._-
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Pace Project CA085: "Biodegradation Screen Study for Telomer Alcohols" -Revision 1 Study Director Claston C. Lange, Ph.D. Pace Analytical Services, Bid-Analytical Services Group Wednesday. November 06,2002
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Appendix E
Integrated Peak Area Data for Transiently formad Polyfluorinatad 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 1001o m/z 1000.
--
SM lay 0 BlxNtfatfloB mnstf. CUM&WXTSI^aa
^^a, 2H.': *. priluoro-2'a;':'^ OCt--Mt* aO_Ot;MII OM
CM WM11, 00.
gntW^i^--^c*auM^^B --
In 1t.a.ial.te .mfc CXUMMTJMM. Cl
A9Mmr.5M
OCMj M_1ro~I"D.''1"1 exm.iUMiiiniia
.CCTCaaEKiaB-
-.!^_-
_ifi_
!&saKB!sa>S!fssazfi&fi__
6.ylnart^w.CAaT-Sfc<M.
exm-iMmi. M; BiHijm
-SS-
IQO>Tna e. Mimi. oalMKr.y.-oM. a
uwuvr-SMKta eviMi-w-u-im.n
-EHtsssaa&aL
^-
par I J B. CMIMgr.M.WII. tZ
pay Z rtnipt uws. CMM-aw-SA-WIT. Ba |0..iumo.blfcCAOM-<l<gr-5A^g.
CMOUM.OIb COO
o>oa_oi>i6 oil ciKM.ngMn .on
-e-
pr T ftuawmi*. e/kWHaT-SA-iM. f
Pit ll . C*0M!7-iA.mi. 81
BlLMj!!SttflHLES25>a2asa!-E2-
CUM IMWU 038 iMgMm IM
-!S-
2Hparfluoro-2-
lnlmftd PMfc <r
W-
1H.1H-
PflUO>D4> pwfluonr
dsgwnCTitt
'"
-iE-
ecMBMl* ".
^-
_S-a_
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-ia-
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Page 34 of 38
Pace Project CA085: "Biodegradation Screen Study for Telomer Alcohols' -Revision 1 Study Director: Cleston C. Lange, Ph.D. Pace Analytical Sen/ices, Bid-Analytical Services Group Wednesday, November 06.2002
000036
EID839581
Appendix F
Typical Total Ion Chrornatograms for SIR Data Showing Telomar Alcohols and Polyfluorinated Fatty Add Products from Day-0 to Day-16 for HPLCTMS Analysis of
Biodegndation Culture SPE eluato 2.
SIRof17anibns: m/z263,313.363, 413,463, 313, 563;antf613forperf!uorinated acids; m/z 377.477 and 577 for poiyfluorinated acids; and mix 323,423,523,623,723,and 823 for
telomar alcohols.
Perfluorinatad Fatty Add Peaks
^
cjuttijnw 014
!.
Day-ie't
,,,
'
cjuicwQu,fi:!
1M-
Day-14^1;
CAown?a.i.^o.i3.....u,...........
TOO-
Day-7 ^
CAOUOBS70U011
100-.
/
^
E'Aiff-l.. II ^tV 5 ;^ ^yp A 1J-'" '" .L-' ^--S w , TJ"
SiRorirCtuMfEB. Tie
i.w*a
SIRofirCMRfMUES?c
1M!
I
C.
3U(ofrCtn>HMkE&.
TC
i&a8
ftRofawu^BESTC
1.SM
Days 7;
CMB.OnWJtIl
100.
Day-2 ^
CiU?.lWT3-B.&*< 100
A-...... .
n
^"...--^.-...." .. .. r.?
ftRaf'trawiwcstC
*02rt
flAafVCtwmref!. TC
TS3i^
^ jy^v Day.1
.^
.A.-t.C1,-?3-.,;-
100
Day-0 <;
-'^ w.. .y. .'.'.-'--'...."?....
tilft *4' <rwnt F.f; TIC ilflrt
^x.t^^4. ..,,..., 100
2.00
300
4 DO
&M
aoQ
T O
06
i\. 1000 1100 12.M
1&09
14.00
Tetomer Alcohol Peaks
Page 35 of 38
Pace Project CA085: "Biodegradation Screen Study for Telomer Alcohols" -Revision 1 Study Director: Cleston C. Lenge, Pti.D. Pace Analytical Services, Blo.Analytical Services Group Wednesday, November 06,2002
000037
EID839582
Appendix G
SIR data Showing Ions for Telomer Alcohols at Day-0 and Day-16 from Analysis of tha Biodegradatlon Culture SPE eluate 2.
CM-MiT-3A-M1 Ci. dw HUMa WISH wisssam'u <m
- Wvt'rSwwIrES- vsmiwiDa
SAJpE
CMDS.I.O^Ou.'Mfl
C*0.Ce270H_ffi IOC
CM)..U2lI..Oal K
: CM.O..o.-n.,,n.--uj.i.H....
.___ , .
C.>OK._>B;70H
_OO_B_
.
!
.
_
_
_
log-
.."a.
2jxi
3jia
i.oa
sja
CAIfr4*2T-SAt3 El. dw 11 bIlxKj uiiwte
C-l3AS.OU7niJ Q14
<aa a( 17 Ciwwi ES. ^IDD lOOOii i00f5
-...IS..,
X
- 1y J S K . ,
SiRolnCnatulies. B3J010001
lOOeS
SM<lfl70lMlrilESR3.BD 1.0001 aaus
sno**7CnwES230QIOODB SOWS
J^L.
siaonTewiitties. KHX) iooo
MM
TM *13
56
'^^..XAXjg^^.- -..."a....- ^-i=?S-----,----..----,----------i----,.---------.^^,.------,,..
.
-^--,
a.m
y-oo
&aa
aoo
iff.oo
itfio
IZJM
1100
SWBll'Cn.nxtESTC
ti.WtS
l4.t
SIR oil? Ciunn.m'5. XIi.CO;9UOa aoix.l
CA-MS.OiffTOHJ**-*
siRoflPCnaWbES-
7S3LS01A)0 501M
CAOe6J182701i_QI
sB()CBii-ies52^001000 SOOri
CAt5.02701_'< 10ft
SRgrirciunn--es-
523.001JXIO*
3AO_OB2yoia_01<
SIR o(17 ChMrxA ei:)M1000a
SOQJS
Vl"t.mtTlH.3U
3iR bf 17 Chwwiaii es.W50t<a.i sabiA
ai0.aifl'')' 3 "
..'-2'...
"^"-i ;"'
:. 1.0.0..-..2.^M ^^l.o.o....-<^....s..o.a.....I.^M ...J7.0.1-^.^i.o;o ^-.l..o-a.^.u-o:o.:.^11..0.0 -...a.n-....a.o.o
SIR117ChOTCi>ES-
rc
SOOf
Page 36 of 38
Pace Project CA08S: "Siodegradalion Screen Study for Tetomer Alcohols' -Revision 1 Study Director: Cleston C. Lange, Ph.D. Pace Analytical Services. Ho-Analytlcal Services Group
Wednesday, November 06,2002
ftOOO^iR
EID839583
Appendix H
SIR data Showing Ions for Perfluorlnated Fatty Adds at Day-0 and Day-16 from Analysis of th Biodegradation Culture SPE eluate 2.
CJUMS4*ar.SA411 E2, Or a blodsg Mrnpl*
CAI.CXH7IlH.a
SIBrfUOonWtlS-
613.001.DOD*
' 2.00BB ~
i:4Cfi&_:JK!T01,fa
SiRofiZQriMwesM3.00100DH ?OM
C*OB_1)1C'OH_
>
SIR 0117 OWIIWE& snooiaci 3tl0c9
C*C.t270f(*_Ofl8
1
auxi.winiig.iiw
100
-
si<i'eu6!i. *^UOH01 tiioea
SiRllCnmiBE!r
19.GO 1 (XOB
i.ma
CAtK.CtCTOH.OOt
c*i,rotTOn_aB
IHO.
CAOA^ COiTOI.l.ffift
CAm& oBBroiu.ron
jaL
~!S-
_..
SiBclUCtunn--ttMa-OOI^MDJ 2.0M
siKi7CniMeaai?.mi.ioi
zooefl
SIRof(7Cn3mMlGS2I(X)10(10a
iM
SIR<l?Owt5.
TiC ZCCtfS
-asir-a- .'"ys-y!
.,it.M-.
4J
6.00
ftOO
7.00
&OQ
0.00
10.00
11X10
12.00
13.00
1400
CAOI&0r-!l&<US EZ dly It UMfg UII1(lk
CMM.lBaon.OH C*M5_lTOron.tU
-.!&-
>, .SRelircMnoltESHHMIOtOl 2.0M
"'SIRBlHOwtl--ESM3C41000X
ZOOM
c*fljja2raia..oi*
180.
*1
x
CAJX.nazrote.oi4
iw
s<iiirc.-urmi.e&
&1J1. t iXICa
SUM
SMdnCWMtSStU.MllOBEl
2BOeB
CAOC6_u2roM_ai
T
cju>n.]N2;ou_or
rOT C*0,<lB370ta.OM
CM):..1>21'OU 11M
tp
CAOUI Mzrtila.ou
i"fr.4,
..!---.
..^';^'-i,-^-.i.S..
100
2.00
XOO
00
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&00
7.00
I.M
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10.00
It.O
13.00
Page 37 of 38
Paca Project CA085: "Biodegradation Scraen Study far Tatomer Alcohols" -Revision 1 Study Director: Cleston C. Lange, Ph.D. Pace Analytical Services. Blo-Analytical Services Group
Wednesday, November 08,2002
SRafi7aiwes<H.K]IOMH 2.00M
smcincmnWtES.
33 00 t-OOOll 2.0D
SfR6f)7CnjmwlKES. 3t3<10l)0
iCtei
SRfif 17C!WTa6S.
figwiuxi Z'JCftI
SIR'jl ITCamtt^ES. Til;
SOM
1300
1400
000039
EID839584
Appendix I
Chromatograms of Extracted ions m/z 377,477, and 577 and Mass Spectra for Each' Peak for Polyfluorinated Acid Intermediates Formed Transientiy In Biodegradation Cultures at
Day-0. Day-1, Day-2 and Day-5. Suspected p-Oxidation oleflnic acid intermediates.
Dsy-Q
6elEftSiWB-WHISCt
ISM
IW i. IX > US t 700 M l ll nge l;w if.ia ILK
Page 38 of 38
Pace Project CAOflS: "Biodagradation Screen Study for Telomer Alcohols" -Revision 1 Study Director: Cleston C. Lange. Ph.D. Pace Analytical Services. Bio-Analytical Services Group Wednesday. November 06.2Q02
0000^0
EID839585
