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BIODEGRADATION STUDY REPORT
R evision 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
A uthor
Cleston C. Lange, Ph.D.
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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
C f t M T A IM LAJrV I A ll'!
Wednesday, November 06, 2002
NO CBI 000003
Table of Contents
Title P a g e .................................................................................................................................................................................. 1 Table of C o n te n ts .................................................................................................................................................................. 2 List of Tables & F igures....................................................................................................................................................... 3 List of A p pen d ices................................................................................................................................................................. 4 Executive S um m ary...............................................................................................................................................................5 1.0 Project P e rs o n n e l........................................................................................................................................................... 6 2.0 Data Requirements andRevision Justification........................................................................................................ 6 3.0 Project O bjective............................................................................................................................................................. 6 4.0 Test Article.....................................................................................................................................................................7-8 5.0 Reference A rticles..........................................................................................................................................................9 6.0 Receipt/Generatlon of Sam ples.................................................................................................................................. 9 7.0 M eth od s............................................................................................................................................................................. 9
7.1 Sam ple P reparation...............................................................................................................................................9 7.1.1 Collection of S lud g e.............................................................................................................................. 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 P L C /M S )...................................................................................................................13 7.2.1 Instrument P aram eters...................................................................................................................... 13-14 7.2.2 Qualitative Analysis of Parents and Products.............................................................................14-15 7.2.3 Quantitative A nalysis................................................................................................................................16
7.3 Data Transformations and Calculations......................................................................................................... 16 7.3.1 Molar Calculations.....................................................................................................................................16 7.3.2 Conversion of ng/mL to micromolar and nan o m o lar.......................................................................16 7.3.3 Molar Mass Balance C alculations........................................................................................................ 17
7.4 Software V ersio n s................................................................................................................................................ 17 8.0 R esults............................................................................................................................................................................. 18
8.1 Sludge C haracterization......................................................................................................................................18 8.2 Quality Control/Sam ple Matrix Spike R esults...............................................................................................18 8.3 Analytical Blanks Results................................................................................................................................... 18 8.4 H PLC /M S Analysis............................................................................................................................................... 19
8.4.1 Qualitative H PLC /M S and H PL C /M S /M S R esults..................................................................... 19-24 8.4.2 Quantitative Analysis Results for P F O A .............................................................v...................... 24-25 9.0 C onclusions................................................................................................................................................................... 27 10.0 Literature C ite d ........................................................................................................................................................... 28 11.0 Sample and Data R eten tio n ................................................................................................................................... 29
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List of Tables & Figures
T ables
T ab le 1. Parent telomer alcohol expected ions......................................................................................................... 14 T ab le 2. Expected product fluorinated fatty acid products io n s ........................................................................... 15 T ab le 3. H PLC /M S Data Table for Parent and Product Ions o b s e rv e d ............................................................. 23 T ab le 4. H PL C /M S /M S D ata for p-Oxidation Product Ions o b served .................................................................24
Figures
Figure 1. Chemical Representation of Telom er A lcohols.........................................................................................8
Figure 2. Chemical Structure of perfluorooctanoate (P F O A ).................................................................................... 9
Figure 3. Plotted H P L C /M S IntegratedPeak Areas for C 8, C 10
and C 12 Telom er Alcohols....... 20
Figure 4. Plotted H P L C /M S IntegratedPeak Areas for C 6, C 14
and C 16 Telom er A lcohols.......20
Figure 5. Plotted H P L C /M S IntegratedPeak Areas for Perfluorinated Fatty Acids D e te c te d ............ 22
Figure 6. Plotted Peak Areas for Transient Polyfluorinated Fatty Acids D e tec ted .........................................22
Figure 7. Plotted PFO A Concentrations Measured in Biodegradation Samples (S P E eluate 2 ) .............. 25
Fig u re 8. Proposed Biodegradation Pathway for Telom er Alcohols................................................................... 26
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List of Appendices
Appendix A: Approval Signatures............................................................................................................................... 30 Appendix B: Final Quantitative Results for PFO A (ng/mL)
Table 1. Test Cultures Final Results .................................................................................................31 Table 2. Abiotic Cultures Final Results.............................................................................................. 31 Table 3. Blank Cultures Final R es u lts ................................................................................................31 Appendix C: Integrated Chromatogram Peak Area Data for the Telom er Alcohols Table 1. Telom er Alcohols Peak Areas ............................................................................................. 32 Table 2. Percentage of Peak Areas For Telom er Alcohols Remaining at Tim e Points........32
Appendix D: Table 1. Integrated LC/M S (SIR ) Peak Area Data for Perfluorinated Fatty A cids.............. 33 Appendix E: Table 1. P eak Area Data for Transiently Formed Polyfluorinated Fatty A c id s ................ 34 Appendix F: Table 1. Total Ion Chromatogram (TIC ) for SIR Data for Biodegradation S a m p le s ...... 35 Appendix G: Table 1. T IC s for SIR Data for Day 0 and Day 16 Biodegradation S am ples....................36
Appendix H: Table 1. T IC s for SIR Data for Day 0 and Day 16 Abiotic Control S am p les.....................37 Appendix I: Table 1. TIC s for Extracted Ion Data for Transient Polyfluorinated Fatty A cids.................... 38
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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 treatm ent 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 W astew ater Treatment Facility. Telom er 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 H PLC/M S analytical method was developed for analysis of the telomer alcohols and expected products. The HPLC/M S analysis of culture extracts provided strong evidence that the telomer alcohols were biodegraded. T he 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 C 5 to C 12 were observed in the test cultures. These compounds were not in controls or blank cultures. Perfluorooctanoate (PFO A ), 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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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
H PLC /M S Analyst Sample Custodian: Report Author
3M Dr. Jam es K. Lundberg
Dr. Cleston C. Lange Mr. Bruce E. W arden 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. T he 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 Telom er, or Zonyl type Telomer" with Telomer. T he 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 similar to earlier studies conducted for 3M by Pace as Pace projects C A 058 1 and C A 097 2 and CA104 3, C A 105 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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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, 2 00 0. The sponsor did not provide an M SD S, 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 (T C R ) number at Pace as C A -T C R 02 -0 09 and was stored at 4C . Material C A -T C R 02-009 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 (C F2)nC H 2C H 2OH where n equals approximately 7 to 8, and molecular weight was reported as M n ca. 4 43 .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 Septem ber 22, 2000. Upon receipt at Pace, the material was given tracability number C A -T N C 00-254 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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Molecular formula = C6H5 F9O Molecular Weight = 264.089 [M +CH3COO '] ' = 323
Molecular formula = C3H5F13 O Molecular Weight = 364.104 [M + CH3COO T = 423
Molecular formula = C 10H5Fl7O 10 Molecular Weight = 464.119
[M + CH3COO '] ' = 523
FFFFFFFFFF
Molecular formula = C12 H5F21 O C 12 Molecular Weight = 564.134
[M + CH3COO y = 623
FFFFFFFFFFFF F-
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FFFFFFFFFFFFFF F----------------------------------------------------------------------------------------------------
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Molecular formula = C14H6F250 C 14 Molecular Weight =664.149
[M + CH3COO '] ` = 723
Molecular formula = C16H5F29O C 16 Molecular Weight =764.164
[M + CH3COO '] ' = 823
Figure 1. The telomer alcohols. Based on the H PLC /M S 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 C 12, 7.2% + 1 .0% as C 14, and 2 .0% + 0 .2 % as C 16.
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5.0 Reference Materials
The sponsor provided reference material. The neat material was stored at -8 0C. NS: Expiration date not specified
5.1. Perfluorooctanoate Ammonium Salt (PFO A ) Purity: 95.2 %; 3M#: T C R -99131-37; Pace #: C A -TC R 02-001 ; Expires: NS
F F FFFF F F
0`
NH4+
F F FFF F F
Figure 2. Perfluorooctanoate ammonium sait
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 (S P E ) 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 H PLC /M S analysis was conducted for eluate 2 only, and the resulting data was evaluated to determine whether biodegradation occurred.
7.0 Methods 7.1 Sam ple Preparation
*v
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 3 1 ,2 0 0 0 and
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delivered with Pace chain of custody (C O C ) form 4 65254. The sludge for the second set of cultures was collected on Septem ber 18, 2000 and was accompanied with C O C # 528791. Sludge used for the second set of cultures in this study was also used in preparation of cultures for other fluorochemical biodegradation studies (C A 105 4 and C A 132 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 (M LSS) from the aeration units at the Twin Cites Metro W astewater Treatment Facility located in St. Paul, M N. Typically, Four liters of MLSS was collected by Pace laboratory personnel and delivered as four 1-liter Nalgene polypropylene bottles containing M LSS, 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" w ere 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.
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7.1.2. Culture Preparation.
The first set of cultures were prepared August 9, 2000 and incubation of those cultures continued with intermittent culture harvests until the final harvest on September 13, 2000, for a total incubation time of 35 days. The second set of cultures were prepared on Septem ber 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 (S O P ) C A G -S P-03 6.
Cultures were prepared using a mineral salts medium defined by EPA Guideline O P PTS 835.3200. The mineral salts medium pH was 7.4 and contained per liter, 0.334 g N a2H P 0 4-2 H 20 , 0.005 g N H 4CI, 0.2175 g K2H P 0 4, and 0.085 KH2P 0 4, 0.0275 g C aC I2-anhydrous, 0 .0225 g M g S 0 4-7H 20 , and 0.00025 g FeCI3-6H 20 .
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 C A 058-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 C A 085-S W -001 at 7,288 ng/mL in methanol), was added to the test cultures by transferring 5 and 8f.iL, respectively, to the appropriate test cultures. The final test concentration in cultures was 2.056 fjQ/rciL of Zonyl BA-N telomer alcohol for set one, and 2.332 |jg/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 (S O P ) C A G -S P -04 1.
All cultures and control cultures were prepared by solid-phase extraction methodology using SEP-VA C C 18 6cc SPE cartridges from W aters Corporation (Part No. W A T 036905). 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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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 H PLC /M S. The remaining volume of each eluate was stored at 4C + 2C.
The final SPE extractions of the day 7, 14 and 16 day cultures from set two occurred on September 24, 2001.
7.2 Instrumental Analysis (LC/MS)
7.2.1 Instrum ent Parameters The HPLC/MS method used was a modified version of Pace method CAGO R G -23 8, as described below.
Analysis of culture extracts from culture set one, prepared with Zonyl BA-N telomer alcohol, was conducted using instrument "S T IN G " which included an H P1100 HPLC pump with Gilson 215 liquid handling system in line with a Micromass Quattro II triple-quadrapole mass spectrometer detector.
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Analysis of culture extracts from culture set two, prepared with Zonyl BA-L telomer alcohol, were analyzed both on instrument "S T IN G " and instrument "10LCMS03" which consisted of a W aters 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 (mini.
%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
15.00
97.0 3.0
HM Resolution: 14
Flow: 1 mLVmin, 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 C 8 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 (W aste:M S)
7.2.2 Qualitative Parent Analyte and Products Analysis.
Parent Analyte C8 telomer alcohol acetate adduct C 10 telomer alcohol acetate adduct C 12 telomer alcohol acetate adduct C 14 telomer alcohol acetate adduct C 16 telomer alcohol acetate adduct
Expected Hons (m/z) 423 523 623 723 823
Table 1. Parent telomer alcohols analyzed.
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To conduct the analysis of parent compounds and expected biodegradation products, the H PLC /M S system was set up with the same chromatographic configuration as that described in method C A G -O R G 23 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
Peril uorobutyrate (C 4)
CF3(CF2)2COO
Perfluoropentanoate (C 5)
CF3(CF2)3C O O '
Perfluorohexanoate (C6)
CF3(CF2)4COO
Perfluoroheptanoate (C 7)
CF3(CF2)5COO
Perfluorooctanoate (C 8, PFOA)
CF3(CF2)6COO
Perfluorononanoate (C 9)
CF3(CF2)7COO
Perfluorodecanoate (C 10)
CF3(CF2)8COO
Perfluoroundecanoate (C-n)
CF3(CF2)9COO
Perfluorododecanoate (C 12)
CF3(CF2)ioCOO
2H, 2H-perfluorooctanoate (C 8)
CF3(CF2)5CH2COO '
2H, 2H-perfluorodecanoate (C 10)
CF3(CF2)7CH2COO
2H, 2H-perfluorododecanoate (C 12) CF3(CF2)9CH2C O O '
Expected anions (m/z)
213,169 2 6 3 ,2 1 9 3 1 3 ,2 6 9 3 6 3 ,3 1 9 4 1 3 ,3 6 9 463, 419 513, 469 5 6 3 ,5 1 9 6 1 3 ,5 6 9
377 477 577
Table 2. Expected products, and expected anions for each.
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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/M S 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 C 8 column used for the quantitation of extracts from culture set two had serial number 1 101567H 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 mole conversion values for each analyte are as follows:
PFO A 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/mL)*(1 nmole / 434.7 ng) = 5.135 nmole/mL = 5.135 pmole/liter = 5.135 pM-- assuming 100% purity.
150 ng/mL PFOA (NH 4+ salt) = (150 ng/mL) * (1 nmole / 431.1 ng) = 0.3479 nmole/mL = 0.3479 pmole/L = 0.3479 pM = 347.9 nM
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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, PFO A 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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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 C A 0 5 8 1. The sludge for samples prepared on Septem ber 27, 2000 was not characterized, but was obtained from the Twin Cities Municipal waste treatment facility in a m anner consistent with the sludge collected and used for project CA058.
8.2 Quality Control/Sample Matrix Spike Results.
The determination of the analyte recoveries from sample matrix spikes was not included as part of this screening study. Recoveries are only reflected in the ability to achieve molar mass balance based on expected parent and product yields from samples and by the use of a sludge-extracted curve for semiquantitative analysis of PFOA.
8.3 Analytical Blanks
Methanol solvent blanks were injected onto the H PLC /M S 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.
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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 Septem ber of 2001 as sequence C A 085_092401b.spl (with full scan MS data), and CA085_092701a.spl (SIR data) on instrument 10LC M S 03 in the BioAnalytical services group at Pace. The qualitative H PLC /M S analyses and H P L C /M S method development for this study were conducted in Septem ber 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
T he first set of test cultures containing sludge and BA-N telomer alcohol substrate provided evidence that biodegradation occurred. T he H PLC /M S data showed near total loss of all of the telomer alcohol peaks in the H PLC /M S 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 C5, C8, C 10, and C 12-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 i6-telomer alcohol was degraded very slowly and showed little loss.
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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
000021
35000
30000
25000 20000 15000
C8 TA peak area AC10 TA peak area C12 TA peak area
10000
5000
0 0 5 10 15 20
Figure 3. HPLC/MS integrated peak areas for the most abundant telomer alcohols (TA). Near complete degradation of all three was observed. Data plotted with the best manual fit of the data.
Figure 4. HPLC/MS integrated peak areas for the less abundant telomer alcohols (TA). Near complete degradation was observed for the C6-TA. Moderate degradation of the C14-TA was observed and little degradation of the 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 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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Concomitant with the loss in telomer alcohols was the formation of compounds with H PLC /M S ions and retention times consistent with perfluorinated acids (PFAs), including perfluorooctanoate (PFO A , C8-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 (F ig u re 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 (C 5 PFA), perfluorohexanoic acid (C 6-PFA), perfluoroheptanoic acid (C /-P FA ), perfluorooctanoic acid (C 8-PFA, PFOA), perfluorononanoic acid (C9-PFA), perfluorodecanoic acid (C 10-PFA), perfluoroundecanoic acid (C-n-PFA) and perfluorododecanoic acid (C 12-PFA).
Transiently formed intermediate compounds were also observed at early time points following the initial exposure of the sludge to the telom er alcohol substrate (Figure 6). The transient compounds were suspected to be: 2H, 2H-perfluorooctanoate; 2H, 2H -perfluorodecanoate; 2H, 2H Perfluorododecanoate and the possible (3-oxidation pathway intermediates: 2H-perfluoro-2-octenoate, 2H-perfluoro-2-decenoate, and 2H-perfluoro-2dodecenoate. T ab le 3 shows the MS ions and retention times observed for the polyfluorinated and perfluorinated acid products and the telomer alcohol parent substrates. H PL C /M S /M S Data (Tab le 4) support the identification of the suspected metabolites..
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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 (CS-PFA) perfluorooctanoate (Cs-PFA) and perfluorodecanoate (C10-PFA).
ir
2H-perfluoro-2'OCtenoate (otafirucaciojl f ^
H-pffluoro-2-decenoate (dfinie acid)"' 'I
2H.2H-perfluorooctanoate
, ,y; !
2H-perfluoro-2-dodecenoate (olefinic acid) '
2H,2H-perfluorodecanoate ; i
2H-perfluorododecanoate
" V l u
23 Incubation Time (days)
Figure 6. Transient polyfluorinated fatty acid intermediates observed. The 2Hperfluorinated olefinic fatty acids are suspected (5-oxidation intermediates that formed rapidly upon initial exposure to telomer alcohols, and then were transformed to their corresponding perfluorinated carboxylic acids end products
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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
Analytes Observed
Chemical Formula
Retention Time
C6-telomer alcohol*
CF3(CF2)3CH2CH2OH...CH3COO
7.63
Ca-telomer alcohol*
CF3(CF2)5CH2CH2O H...CH3COO
8.13
Cio-telomer alcohol*
CF3(CF2)7CH2CH2OH...CH3COO
8.50
C i2-telomer alcohol*
CF3(CF2)9CH2CH2OH...CH3COO '
8.96
Cu-telomer alcohol*
CF3(CF2)i i CH2CH2OH...CH3COO
9.53
Ci6-telomer alcohol*
CF3(CF2)13CH2CH2OH...CH3COO
10.25
2H, 2H-perfluorooctanoate*
CF3(CF2)sCH2COO
7.17
2 H, 2 H-perfluorodecanoate*
CF3(CF2)7CH2COO
7.38
2H, 2H Perfluorododecanoate*
CF3(CF2)9CH2COO
7.49
2H-perfluoro-2-octenoate*
CF3(CF2)4CF=CHCOO
7.14
2H-perfluoro-2-decenoate*
CF3(CF2)6C F = C H C O O '
7.35
2H-Perfluoro-2-dodecenoate*
CF3(CF2)aCF=CHCOO `
7.52
Perfluoropentanoate
CF3(CF2)3COO
6.62
Perfluorohexanoate
CF3(CF2)4COO
6.88
Perfluoroheptanoate
CF3(CF2)sCOO
7.07
Perfluorooctanoate
CF3(CF2)6C O O '
7.22
Perfluorononanoate
CF3(CF2)7COO
7.30
Perfluorodecanoate
CF3(CF2)8C O O '
7.41
Perfluoroundecanoate
CF3(CF2)9C O O '
7.48
Perfluorododecanoate
CF3(CF2)10COO
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.
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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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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)5CH2C O O ' CF3(CF2)7CH2COO CF3(CF2)9CH2COO CF3(CF2)4CF=CHCOO CF3(CF2)6CF=CHCOO CF3(CF2)8CF=CHCOO
Retention Time 7.17 7.38 7.49 7.14 7.35 7.52
LC/MS/MS Parent anion
377 477 577 357 457 557
LC/MS/MS daughter anions observed 333,313,293 413,393 -
313,293,243,143,119 413,393,343 493
Table 4. HPLC/MS/MS data for suspected -oxidation products. Parent and daughter anions from the HPLC/MS/MS analysis of the SPE eluate 2 extract from the 1-day biodegradation sample (CA0850927-SA-033 E2). The parent anions were observed as transiently formed anions in early sample point biodegradation samples only, with the most intense signals for the Ions observed at day one. The MS/MS data collected for each product was consistent with the predicted chemical formula. Common mass loss from the parent anion, attributed to loss of CO2 (mass 44) and HF (mass 20), were observed.
8.4.2 Quantitative Analysis of PFOA
The H P L C /M S multi-component calibration standards used in this study for product quantitation contained the target analytes: PFOA, PFOSulfinate, PFOS, FOSA, AZ-MeFOSA, M 556, M 570, and A/-MeFOSE alcohol. Five calibration standards were prepared by addition of a known amount ot. perfluorinated analytes to a culture medium with sludge, and then extraction by solid phase extraction in manner identical to the treatment of samples. The PFO A 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.
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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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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 H P L C /M S runs. The Instrument calibrations were performed for PFO A by use of a five point calibration with quadratic fit of the data. The LLOQ was 11.0 ng/mL PFO A , and the coefficient of determination (r) for PFO A 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 m easured levels of PFO A during the 16-day study (Figure 7).
180.0 160.0 140.0 3 120.0 E 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%.
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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
000027
FFFFFFFF FFFFFFFF
Cl0 Telomer Alcohol
dehydrogenase
FFFFFFFF FFFFFFFF
dehydrogenase
FFFFFFFF
Illb1
-Oxidation
Peril uorononanoate CO,
IHb2 Hlb3
FFFFFFFOH
FFFFFFFF
4dehydrogenase
ketoacyi thioiase
Flilil
FFFFFFF
O
Perfluorooctanoate
0I . Acetate
Figure 8. Proposed biodegradation pathway for telomer alcohols, shown for Cio-telomer alcohol as example. The p-oxidation pathway branch (lllb) 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.
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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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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 m ajor end products. This observation suggests that, following the initial oxidation of the hydroxyl-carbon to form the primary carboxylic acid (telomer carboxylate), two oxidation m echanisms 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 (Figure 8, lllb) is the more common route of fatty acid (3-oxidation, as evidenced by the formation of detectable transient (3-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.
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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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8.0 Literature Cited
1 . Final Reports for Pace Project CA058 (3M # E 00-2252), "2-W eek N -EtFOSE Alcohol Biodegradation Screen Study Report" and "Aerobic Biodegradation of AZ-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 Perfiuorooctanesulfonyl-based Chemistries.'' Author: Cleston C. Lange, Ph.D.
3. Final report for Pace Project CA104 (3M # E 0 1-0444), "The 35-D ay Aerobic Biodegradation Study of Perfluorooctanesulfonate (P F O S )." 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 CA G -SP-03. "Culture Preparation for Assessm ent of Aerobic Biodegradability of Fluorochemicals Using Municipal or Industrial Sludge as Microbial Inoculum."
7. Pace method CA G -SP-04. "C 18 Solid Phase Extraction Procedure for Fluorochemicals Recovery from Aqueous/Sludge Matrices."
8. Pace method C A G -O R G -23 "Quantitative Analysis of Fluorochemicals by High Performance Liquid Chromatography with Mass Spectrometric Detection."
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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 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):
T h e original signed analytical report and one copy of the signed original. T he 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 C D 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 Chem ical and solvent traceability logs Standard Operating Procedures Methods pertaining to the conduct of this project
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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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Appendix A Approval Signatures
Project Title:
Biodegradation Screen Study for Telom er Alcohols
Client Project ID:
E01-0684
Contract Analytical Project Number: CA085
Report Revised by: Mourad Rahi
Laboratory Management: Bruce E W arden
/ Signature v Signature
Date
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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
0000a 32
Appendix B Final Q uantitative Results for PFOA in samples, controls and blanks
Table 1. The m easured PFO A concentrations in test cultures during the 16-day screening study for telomer alcohol aerobic biodegradability.
Sam ple Dav 0 biodegradation samle. CA085-0927-SA-031, E2 Oav i biodegradation samle. CA085-O927-SA-O33. E2 Dav 2 biodegradation samle. CA085-0927-SA-035, E2 Oav 5 biodegradation samle. CA085-0927-SA-037, E2 Day 7 biodegradation sample. CA085-0927-SA-039, E2 Dav 14 biodegradation samle. CA085-0927-SA-041, E2 Oav 16 biodegradation samle. CA085-0927-SA-043, E2
Concentration (ng/m L) fEluate 2] PFOA 00 25 2 650 111.4 1398 158.6 160.0
Table 2. The m easured PFO A concentrations in the abiotic control cultures during the 16-day screening study for telom er alcohol aerobic biodegradability.
S a m p le Dav 0 no sludge control. CA085-0927-SA-053, E2 Dav 1 no sludge control. CA085-0927-SA-055, E2 Dav 2 no sludge control. CA085-0927-SA-056, E2 Dav 5 no sludge control. CA085-O927-SA-059, E2 Dav 7 no sludae control. CA085-0927-SA-060, E2 Dav 14 no sludae control. CA085-0927-SA-062 E2 Dav 16 no sludae control. CA085-0927-SA-064. E2
C o n cen tratio n <ng/mL) fE luate 2] PFOA 0.0 0.0 00 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. PFO A was not detected in any of the sludge blanks.
S a m p le Dav 0 sludae blank. CA085-0927-SA-045. E2 Dav 1 sludge blank. CA085-0927-SA-046. E2 Dav 2 sludae blank. CA085-0927-SA-047. E2 Dav 5 sludae blank, CA085-0927-SA-048, E2 Dav 7 sludge blank. CA085-0927-SA-049, E2 Dav 14 sludge blank. CA085-0927-SA-050, E2 Oav 16 sludge blank. CA08S-0927-SA-051. E2
C oncentration fng/m L) fEluate 2] PFOA 0.0 00 00 00 00 00 00
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Appendix C Integrated Chromatogram Peak Area Data for telomer alcohols
Table 1. Integrated LC /M S peak area raw data for the parent telomer alcohols substrates. Data acquired from sequence run C A 085_092701a.spl as SIR data.
S am p le Day 0 biodegradation sample, CA085-0927-SA-031. E2 Day i biodegradation sample, CA085-0927-SA-033. E2 Day 2 biodegradation sample. CA085-0927-SA-035, E2 Day 5 biodegradation sample. CA085-0927-SA-037. E2 Day 7 biodegradation sample. CA085-0927-SA-039, E2 Day 14 biodegradation sample. CA065-0927-SA-041, E2 Day 16 biodegradation sample. CA0&5-0927-SA-043, E2
File ID
CA0B5 092701a 008 CA085 092701a 009 CA085 092701a 010 CA085 092701a 011 CA085 092701a 012 CA085 092701a 013 CA085 092701a 014
Day 0 no sludge control, CAO85-O027-SA-O53. E2 Oay 1 no sludge control. CA085-0927-SA-055, E2 Day 2 no sludge control, A085-0927-SA-056, E2 Day 5 no sludge control. CA085-0927-SA-059. E2 Day 7 no sludge control, CAO8S-O027-SA-O6O. E2 Day 14 no stuOQe control, CAO85-O627-SA-O02 E2 Day 16 no sludge control. CA085-0927-SA-064, 2
CA085 092701a 016 CA085 092701a 017 CA085 092701a 018 CA085 092701a 019 CA085 092701a 020 CA085 092701a 021 CA085 092701a 022
Day 0 sludge blank. CA085-0927-SA-045 E2 Day 1 sludge blank CA085-0927-SA-046 E2 Day 2 sludge blank. CA085-0927-SA-047. E2 Day 5 sludge blank, CA085-0927-SA-Q48 E2 Day 7 sludqe blank, CA085-0927-SA-04. E2 Dav 14 sludge blank, CA085-0927-SA-050, E2 Day 16 sludge blank, CA085-0927-SA-051, E2
CA08S 092701a 044 CA085 092701a 045 CA085 092701a 046 CA085 092701a 047 CA085 092701a 048 CA085 092701a 049 CA085 092701a 050
C6 3503 ND ND
12 ND ND ND
5072 4691 5198 4383 3515 3319 4802
ND NO ND ND 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
T e lo m e r A lco h o l In te g rated P eak A reas
C10
C12
C14
28940
18446
5558
1309
8633
4766
262
6677
4463
103 3070 3701
ND
1263
3248
NO 670 1324
117 685 1278
22594 20373 24414 22470 13821 14366 17948
13734 11273 13866 12856 10429 10226 12215
5014 4712 5379 4941 4252 4992 5230
NO ND
NO
ND ND ND
ND ND ND
ND ND
ND
ND NO NO
ND ND ND
ND 269 ND
C16 1326 1340 1213 1140 1343 1093 1137
1492 1477 1462 1393 1095 1265 1478
NO NO ND ND NO NO NO
SUM 1328 1340 1213 1140 1343 1093 1137
1492 1477 1462 1393 1095 1265 1478
NO NO NO ND NO ND NO
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. N ear 90% remained in control cultures for all except C 10 TA, which was 79.4% .
Sam ple D escription
Day
Day 0 biodegradation sample, CA085-0927-SA-031, E2 Day 1 biodegradation sample, CA085-0927-SA-033, E2 Day 2 biodegradation sample, CA085-0927-SA-035, E2 Day 5 biodegradation sample, CA085-0927-SA-037, E2 Day 7 biodegradation sample, CA085-0927-SA-039, E2 Day 14 biodegradation sample, CA085-0927-SA-041, E2 Day 16 biodegradation sample, CA085-0927-SA-043, E2
0 1 2 5 7 14 16
C T A
100.0% 0.0% 0.0% 0.3% 0.0% 0.0% 0.0%
% TA R em aining versus Day 0
C ,T A
Cm TA c 12t a
100.0% 100.0% 100.0% 100.0%
0.4% 4.5% 46.8% 85.8%
1.2% 0.9% 36.2% 80.7%
3.2% 0.4% 16.6% 66.6%
2.9% 0.0% 6.8% 58.4%
2.7% 0.0% 3.6% 23.8%
3.0% 0.4% 3.7% 23.0%
p -1
>
C 16TA
100.0% 101.1% 91.5% 86.0% 101.3% 82.4% 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 sludqe 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%
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Appendix D Integrated Peak Area Data for Perfluorinated Acid End Products
Table 1. Integrated LC /M S peak area raw data for the end products formed. Data acquired from sequence run C A 085_092701a.spl as SIR data.
S a m e l* Day 0 biodegradation sample. C A 085-0927-SA-031 2 Day t biodegradation sample. CA085-0927-SA-033. E2 Day 2 biodegradation sample. CA085-0627-SA-035. E2 Day 5 biodegradation sample. CA085-0927-SA-037. E2 Day 7 biodegradation sample, CA085-0927-SA-039. E2 Day 14 biodegradation sample. CA08S-0927-SA-041. E2 Day 16 CHOdearadation samle. CA085-0927-SA-043, E2
F lte lD CA08S 092701a 008 CA06S 092701a 009 CA085 092701a 010 CA065 092701a 011 CA08S 092701a 012 CA085 092701a 013 CA0BS 092701a 014
P erflu o ro p e n ta n o a te
276 13798 48364 68648 72870 78970 79628
P erflu o ro h e x a n o a te
3576 46183 127207 204221 210569 221543 215670
P erflu o ro * h e p ta n o a te
12127 15464 25641 35747 43227 47751 47315
Integrated Peak Areas
P erflu o ro o c ta n o a te
2715 41621 B8S08 140406 170923 190903 192075
P arflu o ro n o n a a n o a ta
8818 6388 8267 9375 13751 15382 15669
P erflu o ro * d ecanoate
253 6428 17566 44419 75377 62168 66834
P erflu o ro *
P erflu o ro -
undecanoate dodecanoate
1569
375
1281
395
1376
1192
1641
2865
2644
6047
3071
11078
3276
11603
Day 0 no sludge control. CA085-0927-SA-053, E2
CA065 092701a 018
193
861
9244
1057
4382
184 1283 49
Day 1 no sludge control. CA085-0927-SA-055, E2
CA085 092701a 017
268
1722
16685
1695
5516
233
1892
51
Day 2 no sludge control. CA085-0927-SA-056. E2
CA085 092701a 018
351
1437
17394
1649
5958
164
1178
21
Day 5 no sludge control. CA085-0927-SA-059. E2
CA08S 092701a 019
475
1744
17948
1683
7420
278
1928
61
Day 7 no sludge control. CA085-0927-SA-080. E2
CA085 092701a 020
457
1643
15212
1S83
6328
173 1015
25
Day 14 no sludge control. CA085-0627-SA-082 E2
CA085 092701a 021
561
1731
16978
1680
7129
214
1675
68
Day 16 no sludge control. CA085-0927-SA-084, E2
CA085 092701a 022
611
1851
17910
1637
7203
233
1342
34
Day 0 sludae blank, CA085-0927-SA-045. E2
CA065 092701a 044
618
525
31
117 18
0
0
3
Day i sludge blank. CA065-0927-SA-046. E2
CA085 092701a 045
683
152
76
166 262
0
0
D
Day 2 sludge blank. CA085-0927-SA-047. E2
CA085 0927013 046
665
510
51
226 722
0
0 48
Day 5 sludqe blank. CA085-0927-SA-048, E2
CA085 092701a 047
872
320
66
223 392
0
0
0
Day 7 sludqe blank, CA085-0927-SA-049. E2
CA085 092701a 048
737
248
62
581 247
0
24 42
Dav 14 sludge blank. CA085-0927-SA-050. E2
CA085 092701a 049
876
237
86 731 314
0
24 29
Day 16 sludge blank. C A 085-0627-SA-051, E2
CA085 092701a 050
634
285
81
845 397
0
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
000035
Appendix E
Integrated Peak Area Data for Transiently formed Polyfluorinated Acid Products
Table 1. Integrated LC /M S peak area data for transient products. Data from sequence C A 085_092401b.spl as full scan MS data from m /z 100 to m /z 1000.
Sample
Day 0 biodegradation sample. CA085-0Q27-SA-031, E2 Day 1 biodegradation sample. CA085-0927-SA-033, 2 Day 2 biodegradation sample. CA085-0927-SA-035, 2 Day 5 biodegradation sample. CA085-0927-SA-037. E2 Day 7 biodegradation sample. CA085-0927-SA-039. 2 Oav 14 biodegradation sample. CA086-0927-SA-041, 2 Oav 16 biodegradation sample. CA065-0927-SA-043. 2
Oav 0 no siudoe control. CA085-0927-SA-053. E2 Day 1 no sludoe control. CA085-0927-SA-055. E2 Day 2 no sludge control. CAO85-O927-SA-O50, E2 Oav 5 no sludoe control. CA085-0827-SA-059, E2 Oav 7 no sludge control. CA065-0927-SA-060. E2 Oav 14 no sludae control. CA065-0927-SA-062 E2 Oav 16 no sludoe control. CA065-0927-SA-064. E2
Oav 0 sludae blank. CA085-0927-SA-D45, E2 Oav 1 sludge blank. CA065-0927-SA-046. E2 Oav 2 sludge blank. CA065-0927-SA-047. E2 Oav 5 sludae blank. CA085-0927-SA-048, E2 Day 7 sludoe blank. CA065-0927-SA-040. E2 Day 14 sludge blank. CA085-0927-SA-050 E2 Oav 16 sludae blank. CA085-0927-SA-051 E2
File ID
CA0B5 082401b 008 CA085 092401b 009 CA0B5 092401b 010 CA08S 092401b 011 CA065 092401b 012 CA085 092401b 013 CA085 092401b 014
CA08S 092401b 016 CA085 092401b 017 CA085 092401b 016 CA085 092401b 019 CA085 092401b 020 CA06S 092401b 021 CA08S 092401b 022
CA065 092401b 030 CA085 092401b 031 CA085 092401b 032 CA085 092401b 033 CA08S 092401b 034 CA065 092401b 035 CA08S 092401b 036
2Hperfluoro-2octenoate
1040 3429 223 NO
ND NO ND
Integrated Peak Areas
2H- 2H- 1H.1Hperfluoro-2- perfluoro-2- perfluoro-
decenoate dodecenoate octanoate
ND NO 3085
780 249 15025
ND ND 8020 NO ND 236
ND ND 157
NO ND
66
NO ND 128
ND NO ND ND ND ND NO ND ND ND ND ND ND NO ND ND ND ND ND ND ND NO ND ND NO NO ND ND
ND NO NO ND ND ND ND NO ND ND ND ND ND ND ND ND ND ND NO NO ND ND ND NO ND ND ND ND
1H.1Hperfluorodecanoate
211 7055 1802 ND ND ND NO
NO ND ND ND ND ND ND
ND ND ND ND ND ND ND
1H.1Hperfluorododecanoate
NO 958 263 ND ND ND ND
ND ND ND ND NO NO NO
ND NO ND ND NO NO NO
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
000036
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.
E2, day 1C biodeg la m p Je CA08SJ3W/01B_C14 100-,
Day-16*i________
100-.
Day-14*
_
CA085_0S2 701a_012 100
,J? ............. __
Day-7 %
0 -- ------- ------------CAG85_092701a_011
100-
% Day-5
....VgC
CA085_092701a_Q1Q
100
Day-2
0-:---------------;AG5.0C70ia
100
1.J
Day-1 0 . A ?:i 100
Day-0
3s?,,
2.00
^_4 23 4 00
Perfluorinated Fatty Acid Peaks
rL Jr e - n 'if ^ \ . .. *62 6?1 ^7 2t i ifiy 6 63 :: j . A
6 877 22 Jj. 7 40
6 61 1* ' L*
,t__. JP
JU L*7" T>,
1 722
SiR of 1/ Channels ES-
1.66TaIC6
SiR of 17 Channels ES-
1.6STe:Cf>.
SIR o f 17 C ha nn els ESTiC
1 5L'e6
SIR of 17 Channels ESTIC
1 53e6
SIR of 17 Channels ESTIC
1 02e6
SiR o r '-7 C hannels E tv
Sir o f : 7 i'-na ^n eis F S-
1300
....... ......... T im e 1400
Telomer Alcohol Peaks
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
00003
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
CA03S 09 2 7 0 U 00 100 *
0:--------------
100
%
0 ................ CA085_092701a_008 100
%: 0:-------------
CA085_082701a_008 100
% 0........ .......
CA085_0927013_008 100
%
0 --------------------------C A 0 8 f> _ 0 9 2 7 0 1 a _ 0 0 8 100
%;
C A 08?_092701a_008 100;
%;
0 - -----------------------1.00
2.00 2.00
CA085-0927-SA-043 E2, day 16 biodeg sample C A O a S .. 0 9 2 7 0 1 a ..p !4
100
0 ------------------------ --------
CA>36_Oil2701 a _ 0 14 100
%
CA085J)S2701a_014 100
%.
C A085_092701a_014 100
%
CA05_O927O1a_0l4 100
%
100
%
0 ...................... - 1~ 'A i.i8 S jJ 9 2 7 ijia _ 0 !4
100
- - ................. ----------- ...........
%
JJQ
__4.30
1 00 2.00 3.00 4 00 5 00
8.96 850
JBj
850 714740 1 h : r
8.00
9 .0 0
6 62,.
.,7 41
! :jil ,
8 82
6 00
7.00
8 00
9.00
SIR o f 17 Channels ES*21 Of) i OODii 5 '.\0e5
SIR of i 7 Channels ES^21 OO i 000a 5 00e5
SIR of 17 Channels ES623 00 1 OODa 500e5
SIR o f 17 Channels ES523.00 1000a 5 00e5
SIR o f 17 Channels ES23 00 1 OODa 5 00e5
SIR of 17 Channels E$323 00 1 0003 5.00c5
SIR of 17 Channels ESTfC
fj.OOeS
SIR -.'>f ` 7 Channels ESS23 OC 1 OOCa 5 00e5
SIR of 17 Channels E3723.OC I.OOCa 5 OOefi
SIR o f 17 Channels ES623 00 1.000a 5 00e5
SIR of 17 Channels ES523.00 1.000a 5 00e5
SIR o f 17 Channels E5i Z l 30 1 OODa 5 GOeS
SlR of 17 Channels S-
SlR of 17 Channels STiC
5 0Ce5
10 00
11 00
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
000038
Appendix H
SIR data Showing Ions for Perfluorinated Fatty Acids at Day-0 and Day-16 from Analysis of the Biodegradation Culture SPE eluate 2.
CAO05- 0927-SA -031 E2, day 0 b io d e g am p le CA085 09270la_008
% 0.................
C A O 0 5 J 2 7 O la J 3 O 0
100
%:
o ..........- - .......... ... CAOC5_O927Oia_OO0
100
% 0 ...............
C A 0fl5_0{J27O la_008 100
%
O ' ------------------------CA085_092701a_008
100
%;
Q1----------- -~-
C A 0 8 5 _ 0 9 2 7 0 la _ 0 0 6
CA085_092701a_008
100.
% 0;------ -------
C A 085.09270la.008
100
%
0 -----------------------------
CA085 092701a,008
100
%; o-
1.00
200
300
4.00
5.00
SIR of 17 Channels ES613 00 1 000a 200e6
SIR of 17 Cnannels ES563.00 1 000a 2 00e6
SIR of 17 Channels ES513 00 1 OODa 2 OOeO
8 SO
SIR at i ' Cnannels ES463 uO i 000a 2 OOefl
_______ _______ _________ ^ ................. .................................
SIR of 17 Channels ES413 00 1 OODa 2 .0 0 e 6
7 07 7 52
10 13
SIR of 17 Channels ES363.00 1.000a 2 .0 0 e 6
SIR of 17 Channels ES* 313 00 1.000a 2 00e6
SIR o f 17 Channels S263 00 1 000 a 2 DOeS
SIR o f 17 Channels ES-
7 147 48 8 09 8 50 8 96 -, .<r`,?w.* 7 7 > nf^Km, ,f
7.00
8.00
9.00
1Q13 -, ............--
10.00
11.00
.......... -- --- -- -- .-- Time
12.00
13.00
14 00
2 0Ce3
C A 085-0927-S A -043 E2. day 16 b iodeg sam ple CA085_092701a.014
100
SIR of 17 Channels ES613 00 1 OODa 2 00e6
C A085_09270la_0l4 100
%'
C A 0 fl5 _ 0 9 2 7 0 1 a _ 0 M
100 ,
%: 0' -----------
CAQG5_092 701a _ 0 l4 100
% 0 ........... - ........ -- CA065_092701a_014 100
%
CA085_092701a_014 100
7 41
JL
SIR ot 17 Channels ES553 00 1 OOCa 2 OOefi
SIR of 17 Channels ES513 <X- 1 OOCa 2 OOef;
SIR o f 17 Channels S463.00 1 OODa
SIR of 17 Channels ES413.00 i OODa 2 OOeo
SIR of 17 Channels S363 00 l.OOOa 2 Q0e6
CA085 ,09270la_0l4
100
SIR o f ' 7 Cnannels ES313 00 1 0003 2 OCe
CA085 ,092701a ,014 100
SIR of 17 Chafineis ES-
63 ')0 '2OJOCeD-aJ
CA0ft:> 092701a ,014 100
1_99_ 2.00
5 00
6 62, ., ,,
6 .0 0
7 00
Page 37 of 38
10.00
11.00
12.00
TiCSIR of 17 C h a rn e s ES-
2 COe6
13 00
14 00
Time
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
000039
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.
CrtH-OW-SA-OUE?.
Day-0
7 14
A
0
1 00
2.00
3.00
4 00 ' 5.00 0.00 7 00
8.00
9 00
1000
1100
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
000040
