Document emj7nQvjREVR07x7qe9aKERG
AR226-1892
81 PP
EPA -DuPont Telomers Degradation
Technical Meeting
9 AM - 4 PM 15 December 2004 Washington, D. C.
RECEIVED OPPT NCIC 2004 DEC 22 AM 11:20
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Page 1 December 15, 2004
Welcome & Introductions Meeting Purpose Desired Outcomes Agenda
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Page 2 December 15, 2004
Meeting Participants
DuPont Central Research & Development
- Environmental & Microbiological Sciences & Engineering : Bill Berti, John Gannon, Ning Wang
- Corporate Center for Analytical Sciences : Mary Kaiser
Haskell Laboratory : Robert Rickard, Bogdan Szosek Chemical Solutions Enterprise : Bob Buck, Steve Korzeniowski
EPA OPPT : Bob Boethling, Mary Dominiak , Cathy Fehrenbacher, Laurence Libelo,
David Lynch ORD : Carolyn Acheson, Tim Collette, Tom Holdsworth, Toni Krasnic, John
Washington
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Page 3 December 15, 2004
Meeting Purpose
To share approaches, learnings and knowledge about conducting degradation studies on Telomer substances to enhance ongoing research and future planned studies
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Page 4 December 15, 2004
Desired Meeting Outcomes
Sharing of research experience, knowledge and methods Establish a process for sharing and discussion of study
results going forward Group Input ?
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Page 5 December 15, 2004
Agenda
Test Substances Overview Analytical Practices & Know-how Overview Soil Biodegradation Studies DuPont Study Results Timeline Work Process Forward Meeting Reflections
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Page 6 December 15, 2004
Agenda
Test Substances
- Telomer B Alcohol, Telomer-Based Polymeric Products - Potential Transformation Products : Telomer Acids, etc. - Properties, isolation, purification and characterization of
test substances
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Page 7 December 15, 2004
Fluorotelomer-Based Products
CF2=CF2
TFE
F(CF2CF2)nI Telomer A Iodide
n = 2-8 Even Carbon Number, Straight Chain
F(CF2CF2)nCH2CH2I Telomer B Iodide
F(CF2CF2)nCH2CH2OH Telomer B Alcohol
F(CF2CF2)nCH2CH2OC(O)C(R)=CH2 Telomer Acrylate & Methacrylate
Surfactants
Anionic - Phosphate, Carboxylate, Sulfonate
Nonionic - Ethoxylate Betaine Primarily F(CF2)6- Based
Polymers
Acrylic Ester
> 80% of all Sales Products
Amide
Urethane
Urea
Primarily F(CF2)8- Based
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Page 8 December 15, 2004
Polymeric Product Dispersion Properties
Aqueous Dispersion Polymeric Particles 100-200 nm Hydrocarbon Surfactant(s) Characterization
- Wt.% Active Ingredient - pH - Wt.% Fluorine - "8-2" Residual Raw Materials
e.g. 8-2 Telomer B Alcohol
- Polymer Mw (if possible)
80% Water 20% Polymer
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Page 9 December 15, 2004
Chemical Standards
Isotopically - Labeled Standards
- M+5 : C7F1513CF2CD2CD2OH (internal standard) - M+2 : C6F1313CF213COOH
C8F1713CH213COOH C7F15CF=13CH13COOH - Wellington Labs has many M+2 standards available "Cold" Standards
- C8F17CH2COOH - C7F15CF=CHCOOH - CnF2n+1COOH; n = 4,5,6,8,9,10,11 - Others are being synthesized for observed transformation
products
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Page 10 December 15, 2004
Transformation Products
8-2 Telomer B Alcohol
F(CF2)8CH2CH2OH
8-2 Telomer B Acid
F(CF2)8CH2COOH
8-2 Telomer B Unsaturated Acid
F(CF2)7CF=CHCOOH
7-3 Telomer Acid
F(CF2)7CH2CH2COOH
Pentadeca-2-Nonanol
F(CF2)7CH(OH)CH3
F(CF2)7COOH
F(CF2)nCOOH
n = 4,5,6,8, 9, 10, 11
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Page 11 December 15, 2004
Agenda
Analytical Practices & Know-how Overview
- Data & Method Quality Approach - Test Substance Characterization Methods - Approach to Environmental Fate Studies - Including best laboratory practices for conducting this work
due to the unique nature of these compounds (e.g., contamination control practices).
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Page 12 December 15, 2004
EPA -DuPont Telomers Degradation Technical Meeting
Mary A. Kaiser DuPont Central Research & Development Corporate Center for Analytical Sciences
15 December 2004 Washington, DC
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Page 13 December 15, 2004
US Governmental Resources
WWW.FDA.gov/cder/guidance/index.htm
- Guidance for Industry, Bioanalytical Method Validation
- "...procedures such as gas chromatography (GC), highpressure liquid chromatography (LC), combined GC and LC mass spectrometric (MS) procedures such as LC-MS, LC-MS-MS, GC-MS, and GC-MS-MS..."
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Page 14 December 15, 2004
Method Validation
Critical to demonstrate:
- Accuracy - Precision - Specificity - Linearity - LLOQ - Recovery - Storage stability
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Page 15 December 15, 2004
Data Acceptability Criteria
Resolution of the chromatographic peak Defined acceptability criteria for recoveries Non-detects in instrument blanks Analyte response at LLOQ >5x matrix blank <20% deviation of standards from nominal concentration
at LLOQ (<15% above LLOQ) Quality control samples (QC's) within 15% of nominal
value
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Page 16 December 15, 2004
Criteria for Repeat Analyses
Poor chromatography Retention times drift more than +4% within an analytical
run Unacceptable calibration curve (correlation coefficient
<0.9925 i.e., R2 should be >0.985) QC's fail Samples outside assay range Inconsistent replicate analyses Sample processing errors; equipment failure
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Page 17 December 15, 2004
Fluorotelomer Alcohols: Physical Properties
CAS # Molar mass (g/mol) Melting point (C)
Boiling point (C)
Vapor Pressure (Pa)
Water solubility (mg/L at 25C) Log Kow Koc
4-2 Fluorotelomer
Alcohol
2043-47-2
6-2 Fluorotelomer
Alcohol
647-42-7
8-2 Fluorotelomer
Alcohol1
678-39-7
8-2 Fluorotelomer
Olefin
21652-58-4
10-2 Fluorotelomer
Alcohol
865-86-1
264
140-143* 1200 (at 50C)2; 992 (at 25C)4; 252 (at 25 C)5
364.1
75-80/14m m * 108 (at 35C)2; 713 (at 25C)4; 145.2 (at 25C)5
464.12
46-47* 112-114 at
10mm*
3 (at 21C); 254 (at 25C)4; 45.90
(at 25C)5
446.11 146-147* 14.9 (at 25C)**
564
92-93* 111-112/10
mm* 1.4 (at 35C)2; 144 (at 25 C)4; 13.27 (at 25C)5
0.71+0.04663 0.137+0.053
0.001**
= well understood
= somewhat understood
= not well understood
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Page 18 December 15, 2004
Physical Property References
(previous slide)
1 Data are taken from Kaiser, Cobranchi,Kao, Krusiv, Marchione, and Buck, J. Chem. Eng. Data, 49(4), 912-916, 2004, unless otherwise noted.
2 Krusic, Marchione, Davidson, Kaiser, Kao, Richardson, Botelho, Waterland, and Buck, submitted.
3 Cobranchi and Kaiser, EAS, 2001 4 Stock, Ellis, Deleebeeck, Muir, and Mabury, ES&T, 38, 1693-1699,
2004. 5 Lei, Wania, Mathers, and Mabury, J. Chem. Eng. Data, 49, 1013-
1022, 2004. * Oakwood Products, Inc., http://www.oakwoodchemical.com
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Page 19 December 15, 2004
Perfluorocarboxylic Acids:Physical Properties
CAS # Molar mass (g/mol)
Melting point (C)
C8 acid
335-67-1 414.07 451 ; 55-562,*, 56.4-57.93,4
Boiling point (C)
Vapor Pressure (Pa) Water solubility (mg/L at 25C) Koc
189-1921 133 (solid-state
VP of free acid)1
3700; 340095002 141
C9 acid
375-95-1 464.08 71-772; 67-70*; 68-73#, 59-62#
C10 acid 335-76-2 514.09
83-852,#; 77-81# 218 at 740 mm
(100 Pa)2
~10 mm (at 0C)#
C11 acid 2058-94-8
564.09
96-1012 160 at 60 mm
(8 Pa)2
C12 acid
307-55-1 614.1
107-1092,*,#; 105-108#
245 at 740 mm (100 Pa)2,*,#
= well understood
= somewhat understood
= not well understood
Shinoda et al.,(CF2)2CF(CF2)4COOH Melting Point, 13-14 C.
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Page 20 December 15, 2004
Physical Properties References
(previous slide)
1. US EPA Hazard Assessment of Perfluorooctanoic Acid and its Salts, OPPT, Risk Assessment Division, US EPA, 2002 and J. Franklin, "Screening Assessment of the Potential for Long-Range Transport of Perfluoro-octanoic Acid", Solvay Research & Technology, 2003.
2. Detecting and Quantifying Low Levels of Fuoropolymer Polimerization Aids", SPI, Washington, DC 2003.
3. Shinoda, Hato, and Hayashi, J. Phys. Chem., 7, 909, 1972.
4 Note: Shinoda et al.,(CF2)2CF(CF2)4COOH Melting Point = 13-14 C.
* Oakwood Products, Inc., http://www.oakwoodchemical.com # Sigma -Aldrich Co., https://www.sigmaaldrich.com
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Page 21 December 15, 2004
Guard Column
Useful in gradient measurements
Internal standard
From guard column
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Page 22 December 15, 2004
Analytical Issues
Standards
- Telomers (no certified standards)
Telomers come as even numbered mixtures)
- Perfluorocarboxylic acid
Telomer acids are even numbered Carboxylic acids are made either by the telomer process (even numbered)
or electrofluorination (up to 15 % branched)
- See: Martin et al., ES&T, 38(13), 248A-255A, (2004)
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Page 23 December 15, 2004
"Standards?" : Perfluorooctanoic Acid
Produced by Electrochemical Fluorination
IS 415>370
linear
C8 413>369 branching
linear
Branched distribution may vary considerably from lot to lot.
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Page 24 December 15, 2004
"Standards?": Perfluorononanoic Acid
Surflon S111
IS 415>370 C12 613>569
C11 563>519 C10 513>469 C9 463>419 C8 413>369
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Page 25 December 15, 2004
More Analytical Issues
Fluorotelomer-Based Polymer Samples
- Made from mixtures of mixtures - Not soluble in common solvents - Usually available as dispersion with surfactants, etc.
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Page 26 December 15, 2004
Fluorotelomer-Based Polymer Sample Issues
Difficult to remove polymer from dispersed system Difficult to determine sorbed residuals unless compete
dissolution is performed Difficult to get molecular weight distribution due to
solubility, "mixed composition," lack of standards (MALDI/MS may be required)
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Page 27 December 15, 2004
EPA -DuPont Telomers Degradation Technical Meeting
Bogdan Szostek DuPont Haskell Laboratory
15 December 2004 Washington, DC
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Page 28 December 15, 2004
Analytical options for 8-2 Telomer B Alcohol
Properties: Structure
- F(CF2)8-CH2-CH2-OH
Water solubility: 150 g/L Melting point: 44C Boiling point: 213C at 760
mm Hg Vapor pressure: 3 Pa at 21C UV: none for 225-400 nm
Low water solubility
Volatility of the analyte:
- proper sample extraction and storage
- limited option for sample processing (solvent exchange, concentration, clean-up)
Adsorption onto surfaces:
- especially septa, very hard to extract from septa; use Al foil;
- Glass or polypropylene OK;
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Page 29 December 15, 2004
Analytical options for 8-2 Telomer B Alcohol
Extraction :
Aqueous samples:
- MTBE (clean extracts, but partial extraction of acid-type metabolites); preferred for GC-MS based methods; can be handled by LC-MS methods)
- Acetonitrile (~ 60% ACN sufficient for aqueous samples; complete acid metabolite extraction; best for LC-MS methods);
Soil samples:
- Acetonitrile (100 %, recovers 8-2 TBA, but not all acid-type metabolites);
- 50-200 mM NaOH in 90/10 % acetonitrile/water
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Page 30 December 15, 2004
Analytical options for 8-2 Telomer B Alcohol
Analytical methods:
- GC-FID/ECD: limited sensitivity (ppm or sub-ppm levels) and selectivity;
GC-MS:
- Based on EI; various sample matrices; ~ 5 ppb LODs; - Based on CI: Martin et al., Anal. Chem., 74 (2002) 584;
LC/MS or LC/MS/MS:
- Desirable for compatibility with acid-type compounds - Higher sensitivity than GC/MS (sub-ppb LODs) - Simpler sample prep. procedures;
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Page 31 December 15, 2004
8-2 TBA - GC-MS methods
Abundance
4.97
Ion31.00-8-2TBA
1500
1000
500
0
Time-->
4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50 8.00 8.50 9.00 9.50 10.00
Abundance
15 g/L 8-2 TBA spiked into Sea water
Ion33.00D-8-2TBA
1500
4.95
1000
500
0
Time-->
4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50 8.00 8.50 9.00 9.50 10.00
Issues to Consider
Extraction solvent (MTBE worked best for us);
Simplicity and compatibility of sample preparation with other metabolites;
Cleanness of extracts; loss of sensitivity and peak shape;
Sensitivity of the method (LOQ ~ 10-20 ng/mL for extract);
Limited options for sample clean up (normal-phase SPE); solvent exchange not feasible;
Internal standards (D-8-2 TBA)
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Page 32 December 15, 2004
8-2 TBA - LC/MS and LC/MS/MS methods
8-2 TBA is amenable to analysis by LC-MS or LC/MS/MS
Telomer B Alcohols can be ionized by negative ion electrospray;
- Form deprotonated molecular ion (m/z: 463) - In presence of acetate 8-2 TBA forms predominantly
acetate adduct; - Deprotonated molecular ion is prone to in-source
fragmentation; need to be sampled at low cone voltage;
Mobile Phase
- Methanol is essential to formation of deprotonated molecular ions; does not work with acetonitrile
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Page 33 December 15, 2004
8-2 TBA - LC/MS and LC/MS/MS methods
Aqueous samples can be extracted with 60 % acetonitrile; Samples can be extracted with MTBE and run by LC/MS
after 1:10 dilution with acetonitrile; Methods work also with THF extracts
- THF is a good solvent for telomer based polymers;
LOQ
- < 3 ng/mL for LC/MS - Sub-ng/mL for LC/MS/MS;
Calibrations w/ Internal Standard
- Linear range up to 10 g/mL tested;
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Page 34 December 15, 2004
8-2 TBA - LC/MS and LC/MS/MS methods
100
LC/MS
8-2TBA 3.66 941
% 3 ng/mL 8-2 TBA
SIR of 5 channels,ES463, 523
1.026e+004
0
IS-8-2TBA
100
3.64
156 ng/mL IS 39515
%
min SIR of 5 channels,ES-
467, 528 3.451e+005
041904b16 100
MRM of 6 Channels ES-
5.69
563 > 503
1.29e4
0
min
LC/MS/MS 1.0 ng/mL 10-2 TBA
%
1.00
2.00
3.00
4.00
5.00
0 041904b16 100
%
MRM of 6 Channels ES-
3.38
463 > 403
4.41e3
1.0 ng/mL 8-2 TBA
0 041904b16 100
%
1.73
1.1 ng/mL 6-2 TBA
MRM of 6 Channels ES363 > 303 5.01e3
0
Time
0.50
1.00
1.50
2.00
2.50
3.00
3.50
4.00
4.50
5.00
5.50
6.00
6.50
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Page 35 December 15, 2004
Quantitation of 14C labeled 8-2 TBA and Metabolites by LC-ARC
Method 1: 2 mM ammonium acetate in water/methanol
x10^3
x10^3
2.9 8 2.9
1.9
ARC(CPM)
ARC(CPM)
1.9
0.9
-0.1
20.00
24.00
28.00
32.00
36.00
Min
7
2,3
0.9
V
1 6
5
4
Method 2: 0.15% acetic acid in water/methanol
x10^3 1.5
8 7
5
1.1
2
6
34
0.7
ARC(CPM)
0.3
V
1
-0.1
0.00
10.00
20.00
30.00
40.00
Min
-0.1 0.00
40.00
80.00
120.00 Min
LC-ARC Separation and Quantitation Samples originating from e-fate studies of 8-2 TBA contain low-levels of radioactivity (counts) and
substantial number of structurally similar metabolites. Quantitation of these metabolites is done with LC-ARC system. However, this requires a baseline
resolution of structurally similar metabolites. We developed various LC methods to efficiently separate metabolites and inject large volume of
samples extracted with acetonitrile (up to 2 mL).
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Page 36 December 15, 2004
Acid-type metabolites by LC/MS/MS
15 ng/mL
F(CF2)5COOH
F(CF2)7COOH
F(CF2)7CF=CHCOOH F(CF2)8CH2COOH
Extracted from aqueous matrices with acetonitrile (~ 60% ACN sufficient);
Soil samples: require extraction with basic acetonitrile/water (90/10%);
Samples analyzed by LC/MS/MS, negative ion electrospray ionization;
Matrix effects can be severe, especially for soil extracts;
Developed a clean up method using Envi-carb to eliminate matrix effects;
Generally LOQs < 1 ppb
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Page 37 December 15, 2004
Analytical Publications / Presentations
B. Szostek, K.B. Prickett: "Determination of 8:2 fluorotelomer alcohol in animal plasma and tissues by gas chromatography-mass spectrometry", J. Chromatogr. B, 813 (2004) 313-321.
Szostek, B., Prickett, K.B., Kennedy, S.M., and Kaiser, M.A.: "Analytical challenges in determination of Telomer 8-2 Alcohol and Telomer-derived fluorosurfactants and polymers in biological and environmental matrices.", SETAC Europe 13th Annual Meeting, April 27-May 1, 2003, Hamburg, Germany;
Szostek, B., Prickett, K.B., Maslanka, J.C. and Kennedy, S.M.: "Development of analytical methodology for determination of Telomer B Alcohols, Telomer-derived anionic, nonionic fluorosurfactants, and polymers.", SETAC 24th Annual Meeting, November 9-13, 2003, Austin, TX.
B. Szostek, V. Capka, K. B. Prickett, S.M. Kennedy, R.C. Buck: "Application of LC-MS/MS methodology to determination of Telomer B alcohols, perfluorinated acids, and fluorosurfactants in environmental matrices.", SETAC Europe 14th Annual Meeting, April 18-22, 2004, Prague, Czech Republic.
B. Szostek, K.B. Prickett, R.C. Buck: "Determination of Telomer B Alcohols by Liquid Chromatography-Tandem Mass Spectrometry in Environmental Matrices", 52nd Annual Conference on Mass Spectrometry and Allied Topics; May 23-27, 2004, Nashville, TN.
Wang, N., Folsom, P., Szostek, B., Capka, V., Larsen, B., Gannon, J., and Buck, R.C.: "Biotransformation of the fluorotelomer 8-2 Telomer B Alcohol (8-2 TBA) in bacterial culture.", SETAC Europe 13th Annual Meeting, April 27-May 1, 2003, Hamburg, Germany
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Page 38 December 15, 2004
Analytical Publications / Presentations
Wang, N., Folsom, P.W., Berti, W.R., Szostek, B., Sulecki, L.M., Capka, V., Gannon, J.T. and Buck, R.C.: "Biotransformation of the 14C-labeled fluorotelomer 8-2 Telomer B Alcohol (8-2 TBA).", SETAC 24th Annual Meeting, November 9-13, 2003, Austin, TX.
Buck R.C., Wang N., Folsom P.W. , Szostek B., Capka V., and Gannon J.T.: "Perfluorooctyl Ethanol Biodegradation : Transformation Products and Pathway", SETAC Europe 14th Annual Meeting, April 18-22, 2004, Prague, Czech Republic.
N. Wang, B. Szostek, R.C. Buck, P. Folsom, L. Sulecki, J.T.Gannon: "Biotransformation of 14Clabeled Fluorotelomer substances by microorganisms from activated sludge or soil.", Fourth SETAC World Congress, November 14-18, 2004, Portland, OR
B. Szostek, V. Capka, K. B. Prickett, S.M. Kennedy, R.C. Buck: "Application of LC-MS/MS methodology to determination of Telomer B alcohols, perfluorinated acids, and fluorosurfactants in environmental matrices.", SETAC Europe 14th Annual Meeting, April 18-22, 2004, Prague, Czech Republic.
C. Powley, T. Ryan, S. George: "Matrix-effect free analytical methods for determination of perfluorinated carboxylic acids in environmental and biological samples.", Fourth SETAC World Congress, November 14-18, 2004, Portland, OR
V. Capka, R.F. Rossi, N. Wang, P.W. Folsom;"Determination of biotransformation products of fluorotelomer 8-2 Telomer B alcohol in environmental matrices by liquid chromatography-tandem mass spectrometry", 51st Annual Conference on Mass Spectrometry and Allied Topics; June 8-12, 2003, Montreal, Canada
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Page 39 December 15, 2004
Agenda
Soil Biodegradation Studies
- 8-2 Telomer B Alcohol Studies
Methods, Protocols, Analytical Methods (as a basis for polymeric product studies)
- Telomer-based Polymeric Products
Study Protocol
- test objectives, duration, number, types and sources of soil
Sampling and Analysis
- media for analysis (air, water, soil) & analytes
Analytical Methods
- Development and validation of soil-extraction and analysis procedures
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Page 40 December 15, 2004
EPA -DuPont Telomers Degradation Technical Meeting
Ning Wang DuPont Central Research & Development Environmental & Biological Sciences & Engineering
15 December 2004 Washington, DC
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Page 41 December 15, 2004
14C-8-2 TBA Study Outline
F(CF2)714CF2CH2CH2OH
Transformation in Sludge - Mixed Bacterial Culture
- 28-Day Ready Biodegradation - 90-Day Accelerated Biodegradation
Transformation in Soil
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Page 42 December 15, 2004
I. Aerobic Ready Biodegradation
14C-8-2 TBA Experimental Design
Treatment
Replicate (n)
[14CF2]-8-2 TBA
(mg L-1)
NaCN (mM)
14Caniline (mg L-1)
Fluorinated acid
standards* (mg L-1)
1
4
300-340
0
(Live sludge test vessels)
2
4
300-340
0.5
(Abiotic control; sterile
sludge)
3
3
~ 407
0
(Spike recovery vessels; )
0
0
0
60-70
4
2-4
0
(14C-aniline biodegradation
measurement)
0
1.2
0
5
2
0
(Matrix blank soil )
0
0
0
* F(CF2)8CH2COOH (8-2 acid), F(CF2)7CF=CHCOOH (8-2 unsaturated acid), F(CF2)7COOH (PFOA), and F(CF2)5COOH (C6, PFHA)
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Page 43 December 15, 2004
Ready Biodegradation Methods
OECD 301 D as Guideline
Study Materials
- F(CF2)714CF2CH2CH2OH; radiochemical purity > 99%
Experimental System
- Closed bottles (30 mL of test substance solution in 120-mL glass serum bottles crimp-sealed with PTFE septa).
Concentration of the Test Substance
- 300 - 340 mgL-1 dispersed in mineral medium. - Additional organic carbon source: ~13 mgL-1 of ethanol
(as co-solvent).
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Page 44 December 15, 2004
Ready Biodegradation Methods
OECD 301 D as Guideline
Inoculum
- Fresh sludge from Municipal POTW (Non-acclimated, 5 mL sludgeL-1 test solution or 0.5%).
Experimental Duration and Sampling frequency
- 28 days at room temperature and sampled at days 0, 7, 14, and 28.
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Page 45 December 15, 2004
Ready Biodegradation Methods -Continued
Analytical methods:
- Sample extraction:
Acetonitrile (25 mL sample medium plus 45 mL of CH3CN) for 14C-labeled parent and transformation products; alkaline lysis for fluoride ion.
- Parent [F(CF2)8CH2CH2OH and F(CF2)714CF2CH2CH2OH]
Quantified by GC/MS and fluoride ion by ion selective electrode.
- LC/ARC (On-line liquid chromatography/accurate radioisotope counting)
Separation and quantification of 14C-labeled parent and transformation products (LOQ = 0.5% of 14C applied).
- Transformation Products Identification was conducted by Q-TOF-MS. - Spike-Recovery
14C-labeled parent : Liquid scintillation counting Standard fluorinated acids from the sample matrices : LC/MS/MS.
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Page 46 December 15, 2004
II. Accelerated Biodegradation : 14C-8-2 TBA Experimental Design
Treatment
Replicate (n)
[14CF2]-8-2 TBA
(mg L-1)
NaCN (mM)
14Caniline (mg L-1)
Fluorinated acid
standards* (mg L-1)
1
4
950 - 1100
0
(Live sludge test vessels)
2 (Abiotic control; sterile
sludge)
4
950 -1100
0.5
3
3
~ 407
0
(Spike recovery vessels; )
0
0
0
0
0
60-70
4
2-4
0
(14C-aniline biodegradation
measurement)
0
1.2
0
5
2
0
(Matrix blank soil )
0
0
0
* F(CF2)8CH2COOH (8-2 acid), F(CF2)7CF=CHCOOH (8-2 unsaturated acid), F(CF2)7COOH (PFOA), and F(CF2)5COOH (C6, PFHA).
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Page 47 December 15, 2004
Accelerated Biodegradation Methods
OECD 302 B as Guideline Study Materials
- 14C-8-2 TBA. - Additional Carbon Source: 0.1% yeast extract and ~ 110
mgL-1 of ethanol (bacterial cell density: O.D.600 nm = 2; NH4 and ethanol as potential electron donors).
Experimental System
- Closed bottles - (30 mL of test substance solution in 120-mL bottles).
Concentration of the Test Substance:
- 950 - 1100 mgL-1 dispersed in growth medium.
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Page 48 December 15, 2004
Accelerated Biodegradation Methods
OECD 302 B as Guideline Inoculum:
- From industrial waste water treatment facility; grown with ~ 10 mgL-1 of 8-2 Telomer B alcohol prior to the test (mixed bacterial culture).
Experimental Duration and Sampling Frequency:
- 90 days at room temperature and sampled at ~ days 0, 7, 14, 28, 56, and 90.
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Page 49 December 15, 2004
Accelerated Biodegradation Methods - Continued
Sample Extraction:
- Acetonitrile (25 mL sample medium plus 45 mL of CH3CN) for 14C-labeled parent and transformation products; alkaline lysis for fluoride ion.
Analysis:
- fluoride selective electrode for fluoride ion - LC/ARC,Q-TOF-MS, GC/TOF, and GC/MSD for 14C-
transformation products, - LC/MS/MS for PFHA and spiked standardard fluorinated
acids.
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Page 50 December 15, 2004
Accelerated Biodegradation Methods - Continued
14C-Volatile Trapping:
- 10 mL of headspace gas were passed through Two C18 cartridges (0.6 g each) mounted in tandem to trap organic volatiles and the cartridges were eluted with 5 mL of CH3CN.
14CO2 Trapping:
- The headspace gas after passing through two C18 cartridges was trapped in 5 mL of 1 N of NaOH.
In Situ Monitoring of Headspace O2 Content:
- A 26-gauge miniature O2 probe was pierced through the septa of sealed sample bottles for the measurement.
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Page 51 December 15, 2004
III. Aerobic Soil Biodegradation : 14C-8-2 TBA Experimental Design
Treatment
1 (Live soil test vessels)
Replicate (n)
4
[14CF2]-8-2 TBA
(mg kg-1 soil)
0.2 - 1.5
Chloramphe nicol
(mg kg-1 soil)
0
14Cbenzoic
acid (mg kg-1
soil)
Fluorinated acid
standards* (mg kg-1 soil)
0
2
4
0.2 - 1.5
~ 200
0
(Abiotic control; sterile soil)
3
3
(Spike recovery vessels;
sterile soil)
4
2-3
(Vessels for soil respiration
measurement)
5
2-4
(14C-benzoic acid
biodegradation
measurement)
6
2
(Matrix blank soil )
0.2 - 1.5 0 0 0
~ 200 0 0 0
10-200
0.01-0.2 0 0
0
0
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Page 52 December 15, 2004
Soil Biodegradation Methods
OECD 307
Soil:
- Sassafras Soil : pH = 5.5, OM = 2.9% (Organic carbon = 1.7%), Sandy loam
- Microbial Biomass = 154.4 mg organic carbong-1 dry soil.
Study Material:
- 14C-8-2 TBA - Additional Carbon Source: ~50 mg ethanolkg-1 soil (as
cosolvent).
Concentration of the Test Substance:
- ~ 0.2 mgkg-1 soil (except day 1 & day 7 at ~ 0.85 mgkg-1 soil).
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Page 53 December 15, 2004
Soil Biodegradation Methods
OECD 307 as Guideline
Experimental System:
- Closed bottles (10 g soil plus substance solution in 127-mL bottles).
- The experimental duration is up to one year and the O2 content and the respiration rate of the soil were monitored during the study.
Experimental Duration and Sampling Frequency:
- Up to one year. Sampled at approximately days 0, 1, 2, 7, 14, 28, 56, 84, and 148.
Soil Respiration Measurement:
- Measured with a Micro-Oxymax respirometer at days 0, 7, 14, 28, 56, 84, and 148.
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Page 54 December 15, 2004
Soil Biodegradation Methods - Continued
Extraction:
- 1st: CH3CN (acetonitrile) at room T for several days; - 2nd: 90% CH3CN + 20 mM NaOH at 50 C overnight. - The 1st and 2nd extract solutions were analyzed by LC/ARC.
Analysis:
- LC/ARC (On-Line Chromatograph/Accurate Radioisotope Counting) and quadrupole time of flight mass spectrometry (Q-TOF-MS) for 14Cmetabolite quantification and identification,
- LC/MS/MS for PFHA [F(CF2)5COOH] quantification. - The spiked (dosed) standard fluorinated acids were quantified by
LC/MS/MS.
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Page 55 December 15, 2004
Soil Biodegradation Methods - Continued
14C-Volatile Trapping:
- 10 mL of headspace gas were passed through - Two C18 cartridges (0.6 g each) mounted in tandem to
trap organic volatiles and the cartridges were eluted with 5 mL of CH3CN.
14CO2 Trapping:
- The headspace gas after passing through two C18 cartridges was trapped in 5 mL of 1 N of NaOH.
In Situ Monitoring of Headspace O2 Content:
- A 26-gauge miniature O2 probe was pierced through the septa of sealed sample bottles for the measurement.
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Page 56 December 15, 2004
EPA -DuPont Telomers Degradation Technical Meeting
Bill Berti DuPont Central Research & Development Environmental & Biological Sciences & Engineering
15 December 2004 Washington, DC
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Page 57 December 15, 2004
Preliminary Assessment of Aerobic Transformation in Soil
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Page 58 December 15, 2004
Preliminary Assessment of Aerobic Transformation in Soil
Contract laboratory
- Wildlife International, Ltd., Easton, MD USA
Study Director
- Edward Schaefer, Wildlife International, Ltd.
DuPont Representatives:
- Bill Berti, Central Research & Development - Bogdan Szostek, HaskellSM Lab for Health & Environ.
Sciences
Current status:
- Protocols signed, study commenced week of 13 Dec 2004
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Page 59 December 15, 2004
Objectives
- Optimize experimental and analytical procedures based on OECD 307 for subsequent definitive tests to assess transformation potential of fluorotelomer-based polymers in soils under aerobic and anaerobic conditions.
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Page 60 December 15, 2004
Selected Soil
One soil for preliminary study
- Entisol from Northwood, North Dakota USA - Collected from surface to approx. 15 cm depth - Pine tree forest - Not treated with pesticides or fertilizers for at least 10
years - Soil Characteristics (determined on previously collected
sample) Sandy loam: 65% sand, 18% silt, 17% clay pH 6.4 Organic carbon 2.0% Microbial biomass 306 gg-1 soil dry wt.
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Page 61 December 15, 2004
Treatments
Experimental design, each in triplicate:
- Background Blank Control: Soil with no test substance added - Test Chemical: Soil with added test substance concentration of 200
mg kg-1 DW soil. - Abiotic Control: 60Co-sterilized soil with added test substance
concentration of 200 mg kg-1 DW soil.
Spike Recovery Controls: 60Co-sterilized soil spiked with analytes of interest spiked with the following analytes:
- 250 g 8-2 TBAkg-1 sterile soil (dw) + 10 g 8-2 TB Acidkg-1 sterile soil (dw) + 10 g 8-2 TB Unsaturated Acidkg-1 sterile soil (dw) + 10 g APFOkg-1 sterile soil (dw)
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Page 62 December 15, 2004
Analytes of Interest
Treatment Groups 1) Background Blank Control 2) Test Substance 3) Abiotic Control 4) Spike Recovery Controls
Samples
Headspace & Soil analytes
Headspace & soil
8-2 TBA
Headspace & soil
8-2 TBA
Headspace & soil
8-2 TBA
Headspace & soil
8-2 TBA
Soil analytes
8-2 Telomer B Acid, 8-2 Telomer B Unsaturated
Acid and PFOA
8-2 Telomer B Acid, 8-2 Telomer B Unsaturated
Acid and PFOA
8-2 Telomer B Acid, 8-2 Telomer B Unsaturated
Acid and PFOA
8-2 Telomer B Acid, 8-2 Telomer B Unsaturated
Acid and PFOA
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Page 63 December 15, 2004
Test Apparatus & Conditions
Test chambers: glass serum bottles (160 mL)
- Foil lined closures
Approx. 25 g (dw) test soil to each test chamber Incubated statically at 20C; no active ventilation Minimum incubation of 28 days
- Sample on days 0, 14, and 28
Soil sterilized using 60Co irradiation Target soil moisture content
- 50% of water holding capacity (WHC)
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Page 64 December 15, 2004
Soil Dosing
Test substance mixed directly into each soil aliquot Acids dissolved in water and 8-2 TBA dissolved in
ethanol
- Injected into each soil using glass microsyringe
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Page 65 December 15, 2004
Sampling
Headspace
- Microsyringe through septum prior to opening test chambers
- Pass through C18 SPE tube, elute with acetonitrile - Analyze using LC/MS-MS
Soil Extraction
- Acetonitrile added to each test chamber to extract analytes - Analyze using LC/MS-MS
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Page 66 December 15, 2004
Test Summary
Treatment Groups 1) Background Blank Control
Replicates 3
Sampling Days
0, 14, 28
Total Test Chambers
9
2) Test Substance
3
0, 14, 28
9
3) Abiotic Control
3
0, 14, 28
9
4) Spike Recovery Controls: 8-2 TBA and 3 3 fluorinated acids in Sterile Soil
TOTAL TEST CHAMBERS
0, 28
6
33
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Page 67 December 15, 2004
Draft Protocol Highlights
Definitive Studies for Transformation Potential of Fluorotelomer-Based Polymers in Aerobic and Anaerobic Soils
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Page 68 December 15, 2004
Objectives
Conduct definitive tests using GLP to assess potential for transformation of fluorotelomer-based polymers in four (4) soils under aerobic and anaerobic conditions over a test period of 1 year.
- Tests based on OECD 307 - 9 sampling points
days 0, 7, & 14 and months 1, 2, 4, 6, 9, & 12
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Page 69 December 15, 2004
Selected Soils for DuPont Biodegradation Studies
Soil Orders
Soil order represents _% of total land in US
Comments
Entisol Mollisol Inceptisol
8
To be used in preliminary
soil test studies at Wildlife
International
25
To be used in TRP
studies at Noack
Laboratory (Germany)
16
Alfisol
13
Sassafras Soil
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Page 70 December 15, 2004
Analytes of Interest : Soil & Headspace
8-2 Telomer B Alcohol
CAS# 678-39-7
- F(CF2)8CH2CH2OH
8-2 Saturated Acid
CAS# 27854-31-5
- F(CF2)8CH2COOH
8-2 Unsaturated Acid
CAS# 161094-76-4
- F(CF2)7CF=CHCOOH
PFOA
CAS# 335-67-1
- F(CF2)7COOH
Other Fluorinated Acids for which standards and analytical methods
are available, such as :
- F(CF2)nCOOH where n = 8, 9, 10, 11
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Page 71 December 15, 2004
Treatments
Background Blank Control:
- Soil with no test substance added will be tested for analytes of interest at least in duplicate to check for background concentrations of analytes prior to starting the test.
Test Chemical:
- Soil with added test substance concentration of 100 to 1000 mg kg-1 DW soil will be tested for analytes of interest at least in duplicate at all time points.
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Page 72 December 15, 2004
Treatments
Abiotic Control:
- 60Co-sterilized soil with added test substance concentration of 100 to 1000 mg kg-1 DW soil will be tested for analytes of interest at least in duplicate at all time points.
Spike Recovery Control:
- 60Co-sterilized soil will be tested at least in duplicate at all time points by spiking with analytes of interest spiked at day 0 at a concentration range of from 10 to 300 g/kg dry weight soil. (any sequestration?)
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Page 73 December 15, 2004
Treatments
Positive (reference) Control:
- Soil to which a biodegradable reference substance will be added monthly to check the metabolic activity of the test soil.
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Page 74 December 15, 2004
Evaluation
Statistical methods will be applied.
Concentration of transformation products in soil and headspace will be given as mg kg-1 (dry weight) and as molekg-1 (dry weight) for each sampling interval.
Plot transformation products (analytes of interest) in soil & headspace samples against time.
If applicable, transformation half-lives will be determined from the graphs and by applying appropriate kinetic model calculations.
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Page 75 December 15, 2004
Reports
Interim Report following analysis of 6-month samples Final Report
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Page 76 December 15, 2004
Soil Studies : Discussion Questions
Methods for analysis of reactor-vessel headspace for the 8-2 telomer alcohol and aldehyde (LOD, LOQ)
Feasibility of isolating polymer beads from soil after incubation and performing 19F NMR, and/or surface analysis by AFM, FTIR spectrometry/attenuated total reflectance or similar methods.
Determination of the stability of frozen extracts for analysis of PFOA and related acids
Mass balance, Determination of rate constants, Methods for dosing/mixing polymers in soil, Sampling frequency , Data reporting/interpretation of results
Effect of loss of volatile degradation products to headspace on overall rates and extent of conversion to acids.
Methods to assure sealed headspace, maintenance of oxic conditions and proper soil moisture, simultaneously.
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Page 77 December 15, 2004
Agenda
DuPont Study Results Timeline Work Process Forward Meeting Reflections
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Page 78 December 15, 2004
DuPont Biodegradation Program Timeline
T iming
Atmospheric Fate of Perfluoroaldehydes
Atmospheric Partitioning of 8-2 Telomer B Alcohol, Hetergeneous Uptake
Atmospheric Transformation Pathways of Alcohols and Al de hyde s
Atmospheric Partitioning of Telomer Alcohols and Olefins
Long-range Transport Modeling of 8-2 Alcohol
Biodeg in Sludge of 14C 8-2 Telomer B Alcohol
Biodeg in Soil of 14C 8-2 Telomer B Alcohol
Biodeg in Sediment of 14C 8-2 Telomer B Alcohol
Biodeg of DuPont Surfactants in Sludge
Biodeg in Sludge of Fluorotelomer-based Polymers
Biodeg in Soil, Sediment, and Sludge of Fluorotelomerbased Polymers
Biodeg of DuPont Fluorotelomer-based Polymers in Sludge
Biodeg of DuPont Fluorotelomer-based Polymers in Soil/Sediment - Screening
Biodeg of DuPont Fluorotelomer-based Polymers in Soil
Biodeg of DuPont Fluorotelomer-based Polymers in Sediment
Biodeg of DuPont Fluorotelomer-based Polymers in Anaerobic Sludge
Biodeg in Soil &Sediment of 14C 8-2 Telomer B Alcohol Based Urethane Polymer
1Q'04
2Q'04 3Q'04
TRP TRP TRP
TRP OECD 302B
4Q'04
1Q'05 2Q'05
Final Re port
Final Re port
Final Re port
Final Re port
Interim Re port
Interim Re port
Final Re port
Final Re port
TRP
Final Re port
Interim Re port
OECD 307
OECD 308
OECD 311
OECD 307/308
3Q'05
Final Re port Final Re port Final Re port
Interim Re port
Interim Re port
Interim Re port Interim Re port Final Re port Interim Re port
4Q'05
1Q'06
Final Re port
Final Re port Final Re port Final Re port
2Q'06
Final Re port
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Page 79 December 15, 2004
Agenda
Work Process Forward Meeting Reflections
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Page 80 December 15, 2004
EPA/DuPont Telomer Research Meeting
December 15, 2004
NAME
ORGANIZATION
TELEPHONE #
E-MAIL ADDRESS
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