Document em24gY3z04zmxYXGvKprz1K79
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
Project Dates
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 Telomer Alcohols" -Revision 1 Study Director: Cleston C. Lange, Ph.D. Pace Analytical Services, Bio-Analytical Services Group Wednesday, November 06, 2002
Table of Contents
Title P age.......................................................................................................................................................................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....................................... '........................................................................................................................ 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 (S P E )................................................................................................... 12-13
7.2 Instrumental Analysis (H PLC /M S)........................................................................................................... 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............................................................................................................................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 HPLC/MS Analysis......................................................................................................................................19
8.4.1 Qualitative HPLC/MS and HPLC/MS/MS Results................................................................. 19-24
8.4.2 Quantitative Analysis Results for PFO A......................................................... v.....................24-25
9.0 Conclusions........................................................................................................................................................27
10.0 Literature C ited................................................................................................................................................. 28
11.0 Sample and Data Retention...........................................................................................................................29
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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
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 (3-Oxidation 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 C8, C 10 and
C 12Telomer Alcohols.............. 20
Figure 4. Plotted HPLC/MS Integrated Peak Areas for C6, C 14 and
C 16Telomer 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 Biodegradation Samples (SPE eluate 2 ) ............. 25
Figure 8. Proposed Biodegradation Pathway for Telomer Alcohols...............................................................26
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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
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 Polyfluorinated Fatty A cids................... 34 Appendix F: Table 1. Total Ion Chromatogram (TIC) for SIR Data for Biodegradation Sam ples.......... 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, Bio-Analytical Services Group Wednesday, November 06, 2002
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 biodegradation 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 fatty acid a-oxidation, a minor pathway of fatty acid metabolism usually observed in branched-chain fatty acids. After the 16day test period, perflourinated carboxylic acids ranging from C5 to C 12 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.
Page 5 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
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 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 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 fluorotelomer intermediate (telomer alcohol) is biodegradable under aerobic conditions using a microbial rich inoculum of municipal wastewater treatment sludge. This study was sTmilar to earlier studies conducted for 3M by Pace as Pace projects CA058 1 and CA097 2 and CA104 3, CA105 4, and CA132 5. The development of an analytical method and the analysis of parent analytes and possible products were critical parameters for the determination of biodegradation.
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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
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 first 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)nCH2CH20H 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.
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
FFFF
FFFF F F
FFFFFFFF
F-------------------------------------
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Molecular formula = C6 H5F9O Molecular Weight = 264.089 [M + C H 3C O O '] ` = 323
Molecular formula = C8 H5 F ,3 0 Molecular Weight = 364.104 [M + C H 3C O O ' ] ' = 423
Molecular formula = C 10Hs F 17O Molecular Weight = 464.119 [M + CH3C O O T = 523
FFFFFFFFFF
F-----------------------------------------------
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Molecular formula = C 12 Hs F21 O Molecular Weight = 564.134 [M + C H 3C O O T = 623
FFFFFFFFFFFF F--------------------------------------------------------------------------------------
FFFFFFFFFFFF
'1 4 OH
Molecular formula = C,,, H5 F25 O Molecular W eight =664.149 [M + C H 3C O O T = 723
FFFFFFFFFFFFFF FFFFFFFFFFFFFF
Molecular formula = C 16 H5 F29O Molecular Weight = 764.164 [M + C H 3C O O T = 823
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 C 10, 17.5% + 1.2% as C12, 7.2% 1.0% as C 14, and 2.0% + 0.2% as C 16.
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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
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 #: CA-TCR02-001; Expires: NS
NH4
i
Figure 2. Perfluorooctanoate ammonium salt
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
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 5), 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 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 1-liter bottle, based on visual observations-and was consistent with observation of earlier sludge collections.
Page 10 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
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 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 Na2HP04-2 H20 , 0.005 g NH4CI, 0.2175 g K2H P 0 4i and 0.085 KH2P 0 4, 0.0275 g CaCI2-anhydrous, 0.0225 g M g S 04-7H20 , and 0.00025 g FeCI3-6H20 .
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 pg/mL in methanol) or Zonyl BA-L
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telomer alcohol for culture set two (Stock ID CA085-SW-001 at 7,288 ng/mL in methanol), was added to the test cultures by transferring 5 p.L and 8|iL, respectively, to the appropriate test cultures. The final test concentration in cultures was 2.056 pg/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 -20C for storage until SPE preparation could be conducted. 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 l-Chem vials with labels that identified them as eluate 1, 2 or 3, as defined below.
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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
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 l-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 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 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 4 aC + 2C.
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-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.
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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
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 detector. Typical conditions for analysis of telomer alcohols and fluorochemical acid products were as follows:
Instrument conditions for HPLC/MS analysis.
Mass Spectrometer
HPLC
Ionization mode: API-ES negative
Desolvation Temperature: 200C
Time (min).
%A %B
Source Block Temperature: 150C
0.00
97.0 3.0
RF lens: 0.2
0.50
97.0 3.0
Extractor: 3
5.00
5.0 95.0
Cone: 8
11.00
5.0 95.0
Capillary: 3.50
11.50
97.0 3.0
LM Resolution: 14 HM Resolution: 14
15.00 '
97.0 3.0
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
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 pL. 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. 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 Analyte C8 telomer alcohol acetate adduct C 10telomer alcohol acetate adduct C 12 telomer alcohol acetate adduct C 14 telomer alcohol acetate adduct C 16 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
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
Expected anion
structure
Perfluorobutyrate (C4)
CF3(CF2)2COO '
Perfluoropentanoate (C5)
CF3(CF2)3COO
Perfluorohexanoate (C6)
CF3(CF2)4COO
Perfluoroheptanoate (C7)
CF3(CF2)sCOO
Perfluorooctanoate (C8, PFOA)
CF3(CF2)6COO '
Perfluorononanoate (C9)
CF3(CF2)7COO '
Perfluorodecanoate (C10)
CF3(CF2)8COO
Perfluoroundecanoate (Cn)
CF3(CF2)9COO
Perfluorododecanoate (C12)
CF3(CF2)ioCOO
2H, 2H-perfluorooctanoate (C8)
CF3(CF2)sCH2COO
2H, 2H-perfluorodecanoate (C10)
CF3(CF2)7CH2COO '
2H, 2H-perfluorododecanoate (C 12) CF3(CF2)9CH2COO '
Expected anions (m/z)
213,169 263,219 313, 269 363,319 413,369 463,419 513,469 563,519 613,569
377 477 577
|
i
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: Cleston C. Lange, Ph.D. Pace Analytical Services, Bio-Analytical Services Group Wednesday, November 06, 2002
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.
Typical injection volumes for samples and calibration standards were 50 pL. 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 Alltech 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 m ole 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 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/m LnInm ole / 434.7 ng) = 5.135 nmole/mL = 5.135 pmole/liter = 5.135 pM-- assuming 100% purity.
150 ng/mL PFO A (N H 4+ salt) = (150 ng/mL) *(1 n m o le /431.1 ng)
= 0.3479 nmole/mL = 0.3479 pmole/L = 0.3479 pM = 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
7.3.3
Molar Mass Balance Calculations (Theoretical Yield): Convert all ng/mL values to their corresponding molar concentrations as pM 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 0.3479pM
Then, the mass balance is as follows: Mass balance = [(0.3479 pM) / 5.135 pM] 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
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.
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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
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 semi quantitative 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 analytes 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
8.4 HPLC/MS Analysis Results
Semi-quantitative analysis was conducted only for the SPE eluate 2 extracts from 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 Bio Analytical 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, S1000200.spl, and S100400.spl 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 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, analysis was 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 C6, C8, C 10, and C12-telomer alcohols, and moderate loss of the C 14-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 16-telomer alcohol was degraded very slowly and showed little loss.
Page 19 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
35000
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.
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 C u-TA was observed and little degradation of the C i 6-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
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, Ca-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 (C5 PFA), perfluorohexanoic acid (C6-PFA), perfluoroheptanoic acid (C7-PFA), perfluorooctanoic acid (Ca-PFA, PFOA), perfluorononanoic acid (C9-PFA), perfluorodecanoic acid (C 10-PFA), perfluoroundecanoic acid (C t1-PFA) and perfluorododecanoic acid (C12-PFA).
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 p-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
parent substrates.
HPLC/MS/MS Data (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 3i c g- 150000
| 100000
S0) Q.
50000
0
Figure 5. Integrated peak area responses for the perfluorinated fatty acids (PFAs) observed in test culture extracts. Even numbered carbon chain length carboxylic acids were the most abundant carboxylic acid peaks and were: perfluorohexanoate (C6-PFA), perfluorooctanoate (Cs-PFA) and perfluorodecanoate (C 10-PFA).
ro^24ctenoate (oleifnicaddX r \ .
2Hi3^ijoro-2^ecancate(d^inic add)`^
'.-j ''I
2H.2H-perfluofooctanoate, _
i
ik.J
< -s * i
if j
23
Incubation Time (days)
Figure 6. Transient polyfluorinated fatty acid intermediates observed. The 2Hperfluorinated olefinic fatty acids are suspected p-oxidation intermediates that formed rapidly upon initial exposure to telomer alcohols, and then were transformed to their corresponding perfluorinated carboxylic acids end products
Page 22 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
Analytes Observed
Chemical Formula
Retention Time
Co-telomer alcohol*
CF3(CF2)3CH2CH2O H ...C HaC O O '
7.63
C8-telomer alcohol* Cio-telomer alcohol*
CF3(CF2)5CH2CH2O H ...C HjCO O ` CF3(CF2)7CH2CH2O H ...C H 3C O O '
8.13 8.50
C i2-telomer alcohol* C i4-telomer alcohol*
CF3(CF2)9CH2CH2O H ...CHaCOO CF3(CF2)i 1CH2CH2O H ...CH3COO
8.96 9.53
C i6-telomer alcohol* 2H, 2H-perfluorooctanoate*
CF3(CF2)13CH2CH20 H ... CH3C O O ' CF3(CF2)5CH2COO
10.25 7.17
2H, 2H-perfluorodecanoate*
CF3(CF2)7CH2COO
7.38
2H, 2H Perfluorododecanoate*
CF3(CF2)9CH2COO
7.49
2H-perfluoro-2-octenoate*
CF3(CF2)4C F = C H C O O '
7.14
2H-perfluoro-2-decenoate*
CF3(CF2)6C F = C H C O O '
7.35
2H-Perfluoro-2-dodecenoate*
CF3(CF2)sCF=CHCO O
7.52
Perfluoropentanoate Perfluorohexanoate
CF3(CF2)3COO CF3(CF2)4C O O '
6.62 6.88
Perfluoroheptanoate Perfluorooctanoate Perfluorononanoate
CF3(CF2)5COO CF3(CF2)6COO CF3(CF2)7COO
7.07 7.22 7.30
Perfluorodecanoate Perfluoroundecanoate Perfluorododecanoate
CF3(CF2)8COO CF3(CF2)9COO CF3(CF2),oCOO
7.41 7.48 7.52
*; Only anions corresponding to even chain length molecules were observed.
LC/MS anions observed 323 423 523 623 723 823 377 477 577 357,293 457,393 557,493 263,219 313,269 363,319 413,369 463, 419 513, 469 563, 519 613, 569
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.
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
Product Analytes Observed
2H,2H-perfluorooctanoate* 2H,2H-perfluorodecanoate* 2H,2H Perfluorododecanoate* 2H-perfluoro-2-octenoate* 2H-perfluoro-2-decenoate* 2H-Perfluoro-2-dodecenoate*
Chemical Formula
CF3(CF2)5CHzCOO CF3(CF2)7CH2COO CF3(CF2)9CH2C O O ' CF3(CF2),,CF=CHCOO CF3(CF2)sCF=CHCO O CF3(CF2)8CF=CHCOO
Retention Time 7.17 7.38 7.49 7.14 7.35 7.52
L C /M S /M S Parent anion
377 477 577 357 457 557
LC/MS/MS daughter anions observed 333,313,293 413,393 -
313,293,243,143,119 413,393,343 493
Table 4. H PLC /M S/M S data fo r 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: P FO A , PFO Sulfinate, PFO S, 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 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
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 of the telomer alcohols resulted in an increase in the measured levels of PFOA during the 16-day study (Figure 7).
180.0 160.0 140.0
120.0 O) 100.0
c, < 80.0 O
60.0 40.0
20.0
0.0 0 2 4 6 8 10 12 14 16 18 Incubation time (days)
Figure 7. The measured perfluorooctanoate (PFOA) in cultures containing sludge and fluorotelomer 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 Study for Telomer Alcohols" -Revision 1 Study Director: Cleston C. Lange, Ph.D. Pace Analytical Services, Bio-Analytical Services Group Wednesday, November 06, 2002
FFFFFFFF
I
FFFFFFFF
OH
C ,0 Telomer Alcohol
dehydrogenase
FFFFFFFF
dehydrogenase
lr F F l!
III F------------- 1----- 1-------
F F FF
T V -
Illb1
-Oxidation
Perfluorononanoate +
C02
Illb2 Illb3
FFFFFFFOH
FFFFFFFF
y*
I ! dehydrogenase
ketoacyf thiolase
FFFFFFF
0
FFFFFFF
O
Perfluorooctanoate
+
H,C. ^ 0
O
Acetate
Figure 8. Proposed biodegradation pathway for telomer alcohols, shown for Cio-telomer alcohol as example. The p-oxldation pathway branch (illb) involves primary formation of a fatty acyl-CoA thioester that is not shown, to clearly depict the products observed by HPLC/MS. The fatty acyl-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 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 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, III. The first, less utilized route (Figure 8, Ilia), involves oxidation of the ct-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 (Figure 8, lllb) is the more common route of fatty acid 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 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 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
8.0 Literature Cited
1 . Final Reports for Pace Project CA058 (3M # E00-2252), "2-W eek A/-EtFOSE Alcohol Biodegradation Screen Study Report" and "Aerobic Biodegradation of W-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. Pace method CAG-SP-03. "Culture Preparation for Assessment of Aerobic Biodegradability of Fluorochemicals Using Municipal or Industrial Sludge as Microbial Inoculum."
7. Pace method CAG-SP-04. "C18 Solid Phase Extraction Procedure for Fluorochemicals 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 28 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 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 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
Appendix A Approval Signatures
Project Title:
Biodegradation Screen Study for Telomer Alcohols
Client Project ID:
E01-0684
Contract Analytical Project Number: CA085
Report Revised by: Mourad Rahi
Laboratory Management: Bruce E Warden
f0 & Signature
ZilCO n ~ 7 - c i Date
Signature
ll/ljd l^
D ite/
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
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.
Samle
Dav 0 frodegradation samle. CA085-0927-SA-031, 62 Oav 1 btodeqradation samle. CA085-0927-SA-033, E2 Dav 2 b'odegradsbon samle, CA085-0927-SA-035, E2 Dav 5 btodeqradation samle, CA085-0927-SA-037, E2 Dav 7 txodearadation samle. CA085-0927-SA-039. E2 Dav 14 brodearadation samle. CAO85-0927-SA-O41. 2 Dav 18 b*odegradaton samle. CA085-0927-SA-043. E2
Concentration (ng/mU fEluate 2f PFOA
0.0 252 650 111.4 1396 15B.B 160.0
Table 2. The measured PFOA concentrations in the abiotic control cultures during the 16-day screening study for telomer alcohol aerobic biodegradability.
Samle Dav 0 no sludge control. CA085-0927-SA-053, E2 Dav 1 no sludoe control. CA085-0927-SA-055, E2 Dav 2 no sludoe control. CA085-0927-SA-056, E2 Dav 5 no sludoe control. CA085-0927-SA-059, E2 Dav 7 no sludoe control, CA085-0927-SA-060, E2 Dav 14 no sludge control. CA085-0927-SA-062 E2 Dav 16 no sludoe control. CA085-0927-SA-064, E2
Concentration (nfl/mL) fEluate 21
PFOA 0.0 0.0 0.0 0.0 0.0 0.0 0.0
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
Oav O sludae blank. CA085*0927-SA-045. E2 Dav 1 sludge blank. CA085-0927-SA-048, E2 Oav 2 Sludae blank. CA085-0927-SA-047. 62 Dav 5 sludge blank. CA085-0927-SA-048, E2
Dav 7 sludge blank. CA085-0927-SA-049. E2 Dav 14 sludge blank, CA085-0927-SA*050. E2 Dav 16 sludge blank. CA085-0927-SA-051. E2
Concentration fng/mL) fEluate 21
PFOA 0.0 00 0,0 0.0 0.0 00 0.0
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
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. Data acquired from sequence run CA085_092701a.spl as SIR data.
Sample
Day 0 biodegradation sample. CA085-0927-SA-03t. E2 Day i biodegradation sample, CA085-0927SA-Q33. E2 Oay 2 biodegradation sample CA085-0927-SA-3S, E2 Day $ biodegradation sample. CA085-0927-SA-037. E2 Day 7 biodegradation sample. CA08S-0927-SA-039, E2 Day 14 biodegradation sample. CA085-0927-SA-041. E2 Day 16 biodegradation sample. CA085-0927-SA-043. E2
Fil ID CA085 0927013 ooa CA085 092701a 009 CA085 092701a 010 CA085 092701a 011 CA085 092701a 012 CA085 092701a 013 CA085 092701a 014
Day 0 no sludoe control. CA085-0927.SA-053, E2 Day 1 no sludge control, CA085-0927-SA-055. E2 Day 2 no sludge control. CA085-0927-5A-056, E2 Day 5 no sludge control. CA085-0927-SA-059. E2 Day 7 no sludge control. CA065-0927-SA-060, E2 Day 14 no sludqe control, CA085-0927-SA-062 E2 Day 16 op sludge control. CA065-0927-SA-064, E2
CA085 092701a 016 CAO85 092701a 017 CA085 092701a 018 CA085 092701a 019 CA085 092701a 020 CA085 092701a 021 CA085 092701a 022
Day 0 sludqe blank. CA085-0927-SA-04S E2 Day 1 sludge blank. CA085-0927-SA-04Q. E2 Day 2 sludge blank. CA085-Q927-SA-047, E2 Day 5 sludge blank. CA085-0927-SA-048. E2 Day 7 sludoe blank. CA085-0927-SA-049, E2 Day 14 sludge blank. CA085-0927-SA-050. E2 Oay 16 sludge blank. CA085-0927-SA-051. E2
CA085 092701a 044 CA085 092701a 045 CA085 092701a 046 CA085 092701a 047 CA085 092701a 048 CA085 092701a 049 CA085 092701a 050
C6 3503 NO NO 12 NO ND NO
5072 4891 5198 4383 3515 3319 4602
NO ND ND NO 37 ND 17
C8 21704
91 268 690 631 594 643
30841 31305 31862 27486 21460 21284 27329
47 ND ND ND 162 211 68
Telomer Alcohol Integrated Peak Areas
C10 C12 C14
28940
18446
5556
1309
8633
4766
262 6677 4483
103 3070 3701
ND
1283
3248
ND 670 1324
117 885 1276
22594 20373 24414 22470 13621 14366 17948
ND ND ND ND ND ND NO
13734 11273 13866 12856 10429 10226 12215
NO NO ND NO ND ND 269
5014 4712 5379 4941 4252 4992 5230
NO ND ND NO ND ND ND
C16 1326 1340 1213 1140 1343 1093 1137
1492 1477 1462 1393 1095 1265 1476
NO NO ND NO ND NO NO
SUM 1326 1340 1213 1140 1343 1093 1137
1492 1477 1462 1393 1095 1265 1478
NO ND NO NO ND ND ND
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 C 10 TA, which was 79.4%.
Sample Description
Day % TA Remaining versus Day 0
C TA Ca TA Cm TA Cm TA C-u TA Ci TA
Day 0 biodegradation sample, CA085-0927-SA-031, E2 Day 1 biodegradation sample, CA085-0927-SA-033, E2
0 100.0% 100.0% 100.0% 100.0% 100.0% 100.0%
1
0.0%
0.4%
4.5% 46.8% 85.8% 101.1%
Day 2 biodegradation 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%
Day 7 biodegradation sample, CA085-0927-SA-039, E2
7
0.0%
2.9% 0.0% 6.8% 58.4% 101.3%
Day 14 biodegradation sample, CA085-0927-SA-041, E2 14
0.0%
2.7% 0.0% 3.6% '23.8% 82.4%
Day 16 biodegradation sample, CA085-0927-SA-043, E2 16
0.0%
3.0% 0.4%
3.7% 23.0% 85.7%
Day 0 no sludge control, CA085-0927-SA-053, E2 Day 1 no sludge control, CA085-0927-SA-055, E2 Day 2 no sludge control, CA085-0927-SA-056, E2 Day 5 no sludge control, CA085-0927-SA-059, E2 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, CA085-0927-SA-064, E2
0 100.0% 100.0% 100.0% 100.0% 100.0% 100.0% 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% 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'1-Revision 1 Study Director: Cleston C. Lange, Ph.D. Pace Analytical Services, Bio-Analytical Services Group Wednesday, November 06, 2002
Appendix 0 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.
Sam ple Day 0 biodeqradation sample. CA085*0927-SA*031. E2 Day i biodeqradation sample. CA085-0927-SA-033, E2 Dav 2 biodeqradation sample. CA085-0927-SA-035. E2 Day 5 oiodeqradauon sample. CA085-0927-SA-037, E2 Dav 7 oiodeqradauon sample. CA085-0927-SA-039, E2 Dav 14 biodeqradation sample. CA085-0927-SA-041, E2 Dav 16 biodegradation sample. CA085-0927-SA-043, E2
File ID CA085 092701a 008 CA085 092701a 009 CA085 092701a 010 CA085 092701a 011 CA085 092701a 012 CA085 092701a 013 CA085 092701a 014
P e rflu o ro o e n ta n o a te
276 13796 46384 88648 72870 78978
79826
Perfluorohexanoata
3576 46183 127207 204221 210569 221543 215870
Perfluoro* h e p ta n o a ta
12127 15464 25641 35747 43227 47751 47315
Integrated Peak Areas
P e rflu o ro o c ta n o a te
2715 41821
P e rflu o ro nonaanoate
6818 6386
88508
8287
140409
9375
170923
13751
190903
15382
192075
15669
P e rflu o ro decanoate
253 6428 17566 44419 75377 92168 96834
Perfluoro* Perfluoro* undecanoate dodecanoate
1569
375
1281
395
1378
1192
1641
2865
2644
0047
3071
11078
3276
11603
Day 0 no sludae control. CA085-0927-SA-053. E2
CA085 092701a 016
193
861
9244
1057
4362
184 1283
Oay 1 no sludqe control. CA085-0927-SA-055. E2
CA085 092701a 017
288
1722
16685
1695
5516
233
1692
Day 2 n o sludae control. CA085-0927'SA>056. E2
CA085 092701a 018
351
1437
17394
1649
5958
164
1178
Day 5 no sludge control, CA085-0927-SA-059, E2
CA085 092701a 019
475
1744
17948
1683
7420
278
1928
Dav 7 no sludae control. CA085-0927-SA-060. E2
CA08S 092701a 020
457
1843
15212
1583
6328
173
1015
Day 14 no sludqe control, CA0&5-0927-SA-062 E2
CA085 092701a 021
581 '
1731
16978
1680
7129
214
1675
Day 18 no sludqe control, CA085-0927-SA-064, E2
CA085 092701a 022
611
1851
17910
1837
7203
233
1342
49 SI 21 61 25 66 34
Oav 0 sludqe blank. CA085-0927-SA-045, E2 Day t sludqe blank. CA085-0927-SA-048, E2 Day 2 sludqe blank, CA085-0927-SA-047. E2 Dav 5 sludqe blank. CAQ85-0927-SA-048, E2 Day 7 sludqe blank, CA085-0927-SA-049. E2 Day 14 sludqe blank. CA085-0927-SA-050, E2 Day 16 sludqe blank, CA085-0927-SA-051. 62
CA085 092701a 044
618
525
31
117 18
0
CA085 092701a 045
683
152
76
166 262
0
CA085 092701a 046
885
510
51
226 722
0
CA085 092701a 047
672
320
68
223 392
0
CA085 092701a 048
737
248
62
581 247
0
CA085 092701a 049
676
237
86
731 314
0
CA085 092701a 050
634
285
81
645 397
0
00 00 0 48 00 24 42 24 29 21 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
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.
Sam ple
i
DavO biodegradation sample, CA085-0927-SA-031, E2
Dav 1 biodeoradation sample. CAQ85-O927-SA-033, E2
Day 2 biodegradation sample. CA085-0927-SA-035, E2
Day S biodegradation sample. CA085-0927-SA-037, E2
Day 7 biodegradation sample. CAG65-0927-SA-039. E2
Dav 14 biodegradation sample. CA085-0927-SA-041. E2
Day 18 biodegradation s a m p le . C A 0 B 5 -0 9 2 7 -S A -O 4 3 , E2
Day 0 n o sludge c o n tro l. CA085-0927-SA-053, E2 Oav 1 no sludge control. CA085-0927-SA-QS5, E2 Day 2 no sludge control. CA085-0927-SA-OS8, E2 Day 5 no sludge control, CA085-0927-SA-059. E2 Oav 7 no sludge control, CA065-0927-SA-060. E2 Day 14 no sludge control. CA085-0927-SA-062 E2 Day 16 no sludge control. CA085-0927-SA-084. E2
Day 0 sludoe blank. CA085-0927-SA-045, E2 Dav 1 sludoe blank, CA085-0927-SA-046. E2 Day 2 sludge blank. CA085-0927-SA-047. E2 Dav 5 sludoe blank. CA085-0927-SA-04S. E2 Dav 7 sludoe blank. CA085-0927-SA-049. E2 Day 14 studae blank, CA065-0927-SA-050. B 2 Day 16 sludoe blank. CA085-0927-SA-051. E2
File ID CA085 092401b 008 CA065 092401b 009 CA085 092401b 010 CA085 092401b 011 CA065 092401b 012 CA085 092401b 013 CA085 092401b 014
CA085 092401b 016 CA085 092401b 017 CA065 092401b 018 CA085 092401b 019 CA085 092401b 020 CA085 092401b 021 CA085 092401b 022
CA085 092401b 030 CA085 092401b 031 CA085 092401b 032 CA065 092401b 033 CA085 092401b 034 C A Q 85 0 9 2 4 0 1 b 035 CA085 092401b 036
2Hperfluoro-2-
octenoate 1040 3429 223 NO
NO NO NO
Integrated Peak Areas
2H- 2Hperfluoro-2- perfluoro-2-
decenoate dodecenoate NO NO 780 249 NO NO NO NO NO NO NO NO NO NO
1 H .1 H perfluorooctanoate
3085 15825 8020 236
157 68 128
NO NO NO NO NO NO NO NO NO NO NO NO NO NO NO NO NO NO NO NO NO NO NO NO NO NO NO NO
NO NO NO NO NO NO NO NO NO NO NO NO NO NO NO NO NO NO NO NO NO NO NO NO NO NO NO NO
1 H .1 H perftuorodecanoate
211 7055 1802 NO NO NO NO
NO NO NO NO NO NO NO
NO NO NO NO NO NO NO
1 H .1 H p e ril u o ro dodecanoate
NO 958 263 NO NO NO NO
NO NO NO NO NO NO NO
NO NO NO NO ND NO NO
4
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
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.
Perfluorinated Fatty Acid Peaks
CA085_C92701a_01t 10CF
Day-5
Day-2
Day-1
AC**5_09270 a__L-Ofi
100
Day-0 *: 0:-
2S?
6 91 7 22
I'
SIR of 17 Channels ES TIO
1.53e6
ivR o' *7 O h a n r* * r.S t
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
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 biodag sample
CA0a5_OJ2701 djJOt
100
%
o-------- --------------- ---------------
CAC85.00270 t aj) 0 8
100
%
0
CA085_092701a_008
100
CA085_092701a_008 100:
%
CA085_092701a_008 100
%'
0 - - CA085_09?70la_008 100
%\
0 ' CA085- 092701a_008 100:
%: i
" 1.00
290 2.00
3.00
4 23 4.00
5.00
CA085-0927-SA-043 E2, day 16 blodeg sample
CA065.0927013_014 100
%:
a ---------------------------------------------------------------------------CA05_027C1a_0M 100
% 0:--------------
C A085_092701a_0)4 fQG:
,- ,
8.96 , .A .
8.50
A i?
6.13
(A y?
7.83 8J3
6.00 7.00 8 00 9.00
SIR of 17 Channels E3<123.00 i OODa
5 00e5
SiR of 17 Channels ES?23 00 i OODa 5 0e5
SiR of 17 Channels ES623.00 1.000a 5.00e5
SIR of t7 Channels ES523.00 1.000a 5.00e5
SIR of 17 Channels ES423.00 I 000a 5 0Oe5
SiR of 17 Channels ES323.00 1 0003
S.OQcS
_ _____ 11 ___
1000
11.00
12.00
SiR of 17 Channels ESTiC
5.00e5
13.00
1400
SIR of 17 Channels E5-
823.00 1.000a S.OGe
SiR of 17 Channels 5 723 00 1 OOC-a 5.00e5
SiR of 17 Channels ES6 2 3 .0 0 I .OODa
S.OOeS
CAOa5.O9270fa_O14 100
% 0......... .....
CA085_092701a_Ol4 100
SIR of 17 Charnels ES523.00 1.000a 5.00e5
SIR of 17 Channels ES423 CO 1.000a 5 00e5
'>:i^S_0927<)1a_Ql4 100
SiR Of 17 Channels cS S
2AI.15.002701a_014 100
^7.41
SiR of 17 Channels $-
TiC 5 OCt
%
1 99 4 30
0*
100
2.00
3.00
4.00 5.00
6.00
7.00
8 00
9.00
10.00
1100
12.00
13 00
14 00
Time
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
Appendix H
SIR data Showing Ions for Perfluorinated Fatty Acids at Day-0 and Day-16 from Analysis of the Biodegradation Culture SPE eiuate 2.
CA085-0927-SA-031 E2, day 0 blodeg sample
CA0a5_092701a_008
too %
CA006_09270 ta_008
too %;
CAOfl5_OO27Ola.OO0
too.
SIR of 17 Channels ES* 613 00 i 000a 2.00e6
SIR of 17 Channels ES* 563 00 1 000a 2 00e6
SIR ot 17 Channels ES* 513 00 i X D a 2 OOeS
CA005_0y270la_fjCa
too V
CAO85_09270la_O0e 100
%i
SIR of '7 Channels ES-
462 00 ' OOCa
2 0CeS
850
SIR of 17 Channels ES* 413 00 l 000a 2.00e6
CAO05_O927O1a_OO8
too
7 07
SIR of 17 Channels ES-
363 00 1.000a
2.00e6
CAO85_OS270ta_00a
too
V
7.52 10.13
SIR of 17 Channels ES* 313 00 l OODa 2 OOeo
CA0B5_i>9270ta_00fl
too %.
S IR o f IT Channels ES263 00 i oooa
2 0Ge5
C A X 5.X 270fa_00fl
too
7J4746 8_09 8j50^ 896
10.13
SIR o f 17CxanneisES-
TiC
2 00e5
t.00
2.00
3.00
4.00
5 .X
6.00
7.00 ' 8.00 ' 900
to.oo t t . X
12.X
13.X
14.X
TUTUJ
CAO05-O927-SA-O43 E2. day 16 blodeg sample
CAOe5_092701a_014
100
%
CAO05_OD27O1a_Ot4
too
%\
CA065_092701a_014
too.
%! 0i
.... .
CA065_002701a.0t4
too
%;
CA0S5_092701a_014
too. %
CA085_092701a_014
too %:
O' --
CAO05._O627O1a_014
too
%
CAQBS ..092701a. 014
100 . %
0 CAOHi..Oy2701a..0l4
too
%
1 99
1 00 2 0 0 3.00 4.00
5 00
7.52
741 ,, A ..
7.30 7 22
7 07 6 88
6 62
6 /S ? 7? 7 . l 627.
. - . .i6,2
6 .X 7 00 8.00 3.00
Page 37 of 38
10.00
11.X
12.X
SIR of 17 Channels ES-
2006613 00 1 000a .e
SIR of 17 Channels ES-
563 00 1000 a
2 00e6
1SIR or 7 Channels ES-
513 00 i OOCa 2 OCeS
1SIR of 17 Channels ES463.00 OOCa 2 OOeS
SIR of 17 Channels ES* 4 1 3 .X i OOOa
" 2 00e6
SIR of 17 Channels ES363 0 0 f OOOa 2 OOee
SIR of 17 Chanters ES313 X 1 300s 2 3C6
!SIR of 7 Chanr-nid ES*263 OC JOCj
2 Xe6
SIR of 7 Chanr1*S-
2 Xe6
13 X 14 X
*
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
Appendix I Chromatograms of Extracted ions m/z 377, 477, and 577 and Mass Spectra for Each Peak for Polyfluorinated Acid Intermediates Formed Transiently in Biodegradation Cultures at
Day-0, Day-1, Day-2 and Day-5. Suspected (3-Oxidation olefinic acid intermediates.
C tM UtlT-tA-aiJ 3.4*1 I M A | MM
Day-0
714
,4
1 00 2.00 100 4 00 5.00 8.00 7.00 8.00 9.00 10.00 11 00 12.00 13 00 14.00
^
Page 38 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
