Document Dv79q11Nrv1GKYrNvvj88amEB
A R 2 2 6 -1 892
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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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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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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Fluorotelomer-Based Products
c f 2=c f 2
tfe
1
F(CF2CF2)nI Telomer A Iodide
-\
n = 2-8 Even Carbon Number, Straight Chain
F(CF2CF2)nCH2CH2I Telomer B Iodide
-- ------------------------- 'l
F(CF2CF2)nCH2CH2OH Telomer B Alcohol
F(CF2CF2)nCH2CH2OC(O)C(R)=CH2
Telomer Acrylate & Methacrylate
J
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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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% W ater 20% Polymer
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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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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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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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EPA -DuPont Telomers Degradation Technical Meeting
Mary A. Kaiser DuPont Central Research & Development Corporate Center for Analytical Sciences
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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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Method Validation
Critical to demonstrate:
- Accuracy - Precision - Specificity - Linearity - LLOQ - Recovery - Storage stability
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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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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., R2should be >0.985) QC's fail Samples outside assay range Inconsistent replicate analyses Sample processing errors; equipment failure
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Fluorotelomer Alcohols: Physical Properties
CAS # M olar m ass (g/m ol) M elting point (C)
Boiling point (C)
Vapor Pressure (P a )
W a te r solubility (m g/L at 25C ) Log Kow Koc
4-2 Fluorotelom er
A lc o h o l
6-2 Fluorotelom er
A lc o h o l
8-2" Fluorotelom er
A lcohol1
8-2 Fluorotelom er
O lefin
10-2 Fluorotelom er
A lc o h o l
2 0 4 3 -4 7 -2
647-42-7
678-39-7
2 1 6 5 2 -5 8 -4
865-86-1
264
364.1
4 6 4 .1 2
446.11
140-143*
75-80/14m m *
4 6 -4 7 * 112-114 at
10mm *
146-147*
1 2 0 0 (a t 5 0 C ) 2; 1 0 8 (a t 3 5 C ) 2; 3 (a t 2 1 C ); 2 5 4
9 9 2 (a t 2 5 C ) 4; 7 1 3 (a t 2 5 C ) 4; (a t 2 5 C ) 4; 4 5 .9 0
2 5 2 (at 25 C )5 145.2 (at 2 5 C )5
(at 2 5 C )5
14.9 (at 2 5 C )**
564 92-93* 1 1 1 -1 1 2 /1 0
mm* 1 .4 (a t 3 5 C ) 2; 1 4 4 (a t 2 5 C )4; 1 3 .2 7 (a t 2 5 C )5
0.7 1+ 0 .0 46 63 0.137+0.053
0 .0 0 1 **
= well understood
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= somewhat understood
= not well understood
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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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Perfluorocarboxylic Acids:Physical Properties
C8 acid
C9 acid
C10 acid
CAS # Molar mass (g/mol)
335-67-1 414.07
375-95-1 464.08
335-76-2 514.09
451 ; 55-562*' 71-772; 67-70*;
Melting point (C)
56.4-57.934 68-73#, 59-62# 83-852#; 77-81# 218 at 740 mm
Boiling point (C)
189-1921
(100 Pa)2
133 (solid-state
VP of free
~10 mm (at
Vapor Pressure (Pa) W ater solubility (mg/L at 25C)
acid)1 3700; 3400
95002
0C)#
Koc 141
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 a/.,(CF2)2CF(CF2)4COOH Melting Point, 13-14 C.
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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
mu
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Guard Column
Useful in gradient measurements
r ^ Chromatogram - [0 5 1 0 2 0 0 4 -0 5 ]
I File Edit Display Process Window Tools Help
b % rs m
05102004-05 100-1
yw i ijI
G a a m , & Q.+ Q : o r TIC 4
QAA592
+X
Internal standard
m _ & XI
2: MRM of 1 Channel ES414.92 > 369.S 2.41 e4
%
0-=r=r=
05102004-05 100
-
1: MRM of 1 Channel ESTIC
2
From guard column
\
.. i .
..
5.00
.. .
...
...
..
10.00
15.00
..............m . . . ........................................... ...................... ..........
20.00
25.00
30.00
Time
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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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"Standards?" : Perfluorooctanoic Acid
Produced by Electrochemical Fluorination
F C95 A ssav-1 -2 Quat t r o LC Q24-3719
100 "I
SDherisorbOD S 200X2.1mm 20% 2m MA M M AC80% MeOH
IS 415>370
linear
12-Jun-20041 0:45:40 larsen
5: MR M of 1 C h an ne l E S TIC
3.25e4
oQ24-
C8 413>369
linear
Branched
1: M R M of 1 C h an ne l ES8.98eb
distribution may vary
branching .
considerably from lot to lot.
' I 1 1 1 ' I 1 1 1 1 I 1 1 1 1 I 1 1 1 1 I 1 1 1 1 I 1 1 1 1 I 1 1 1 1 I 1 1 1 1 I 1 1 1 1 1 1 1 1 I .............i i i i i " i i i " i i i i m i i i N i i i i i i i i i i i i i i i i i i i i i i i i i i i N i i i i i i i i i i i i i i i N i i i i i i N i i i N i i i i i i i 'i T i n
1.00
2.00
3.00
4.00
5.00
5.00
7.00
8.00
9.00
10.00
11.00
12.00
13.00
14.00
15.00
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"Standards?": Perfluorononanoic Acid
Surflon S111
S111 A Assav-1-1 Quattro LC Q24-3692
IS 415>370 %
SpherlsorbODS 200X2.1mm 20%2m MAMM AC80% MeOH 6.09 4759
12-Ju n-200403:27:31
lars en
6: MRM of1 Channel ESTIC
6 .3 1e4 Area
1I 111 1I 11111111111111 I 11 11I 111111111 I 111 11111111111 I 1 Q24-3692
111111 I 1111I 11 11111111111 1I 111 11111111111I 11 11I 11 1111111 I 111 111111111111111 1I 11 11I ........ 5: MRM of 1 Channel ES-
100:Q24-3692 C10 513>469
7.68_ 1344
3 MRM of 1 Channel ESTIC
1.60e4
Area
O'
Q24-3692
100:
C9 463>419
6.97 100873
2: MRM of1 Channel ESTIC
1.33e6
Area
O'
Q24-3692 10On
C8 413>369
6.08 _ 3293
1: MRM of 1 Channel ESTIC
4.35e4
Area
O' Time
1.00
2 00
3 00
4 00
5 00
6 00
7 00
8 00
9.00
10 00
11 00
12 00
13.00
14 00
15 00
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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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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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EPA -DuPont Telomers Degradation Technical Meeting
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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 pg/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;
(site
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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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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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8-2 TBA - GC-MS methods
Abide
4& Ian31(0-8Z1B\
Tn>
Abundanc
15 pg/L 8-2 TBA spiked into Sea water
km3DG8ZB
HD 49
HE)
33D
Tne>
0 4(0 430 00 500 600 600 70) 75) 8 0 8 3 0 9 0 9 0 1 0 0
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
mu
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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 pg/mL tested;
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8-2 TBA - LC/MS and LC/MS/MS methods
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Quantitation of 14C labeled 8-2 TBA and _______ Metabolites by LC-ARC_______
Method 1: 2mMammoniumacetateinwater/methanol
Method 2: 0.15% acetic acid in water/methanol
x 10n3
1.1 0.7
- 0.1
LC-ARC Separation and Quantitation
Samplesoriginatingfrome-fatestudiesof8-2TBAcontainlow-levelsof radioactivity(counts) and substantial numberofstructurallysimilarmetabolites.
QuantitationofthesemetabolitesisdonewithLC-ARCsystem. However, thisrequiresabaseline resolution of structurallysimilar metabolites.
WedevelopedvariousLCmethodstoefficientlyseparatemetabolitesandinject largevolumeof samplesextractedwithacetonitrile(upto2mL).
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Acid-type metabolites by LC/MS/MS
100102x13 Smooth(Mn,2x2)
100-1
15 ng/mL
%-
F(CF2)5COOH
PFHA_ 3.27 440.30
IVRM of 4 channols.ES313 : 200
6.1250+003
100102x13 Smooth(Mn.2x2)
100i
i ii |i i M111M11M1111M11111111 i'i 111 li 11111111 T fw p fftT i mm
WRMof4 channols.ES-
PFOA
413 > 360
3.45 1.1670+004
857.07
%- F(CF2)7COOH
100102x13 Smooth(Mn,2x2)
10O-i
---------------------
WEMof4 channels.ES-
2H-HDF-2-DA_ 3.58
457 393 1.8180+004
1307.93
%-
F(CF2)7CF=CHCOOH
0- l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l
100102x13 Smooth(Mn,2i2j
100-i
min 4 oh anno Is.ES-
477 =393 1.1110+003
F(CF2)8CH2COOH
o-C nn"''"'V.LM:i''l" V.no',"'2.uo"
mm
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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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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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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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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EPA -DuPont Telomers Degradation Technical Meeting
Ning Wang DuPont Central Research & Development Environmental & Biological Sciences & Engineering
mu
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Page 41 December 15, 2004
14C-8-2 TBA Study Outline
F(C F2)714C F2CH2C H 2OH
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)
[14C F2]-8-2 TBA
(mg L-1)
NaCN (mM)
14Caniline (mg L-1)
Fluorinated acid
standards* (mg L-1)
1 (Live sludge test vessels)
2 (Abiotic control; sterile
sludge)
3 (Spike recovery vessels; )
4 4 3
300-340 300-340
~ 407
0 0.5 0
00 0
60-70
4 (14C-aniline biodegradation
measurement)
2-4
0
0 1.2
5 (Matrix blank soil )
2
0
00
*F(CF2)8CH2COOH(8-2acid), F(CF2)7CF=CHCOOH(8-2unsaturatedacid), F(CF2)7COOH(PFOA), andF(CF2)5COOH(C6,PFHA)
0 0
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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 mg^L-1 dispersed in mineral medium. - Additional organic carbon source: ~13 mg^L-1 of ethanol
(as co-solvent).
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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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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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II. Accelerated Biodegradation : 14C-8-2 TBA Experimental Design
Treatment
Replicate (n)
[14C F2]-8-2 TBA
(mg L-1)
NaCN (mM)
14Caniline (mg L-1)
Fluorinated acid
standards* (mg L-1)
1 (Live sludge test vessels)
2 (Abiotic control; sterile
sludge)
3 (Spike recovery vessels; )
4 950 - 1100 4 950 -1100 3 ~ 407
0 0.5 0
00 00 0 60-70
4 (14C-aniline biodegradation
measurement)
2-4
0
0 1.2 0
5 (Matrix blank soil )
2
0
0 00
*F(CF2)8CH2COOH(8-2acid), F(CF2)7CF=CHCOOH(8-2unsaturatedacid), F(CF2)7COOH(PFOA), andF(CF2)5C2OOH(C6,PFHA).
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Accelerated Biodegradation Methods
______________ OECD 302 B as Guideline____________
Study Materials
- 14C-8-2 TBA.
- Additional Carbon Source: 0.1% yeast extract and ~ 110 mg^L-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 mg^L-1 dispersed in growth medium.
mu
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Accelerated Biodegradation Methods
_____________ OECD 302 B as Guideline____________ Inoculum:
- From industrial waste water treatment facility; grown with ~ 10 mg^L-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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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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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.
14CO2Trapping:
- 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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III. Aerobic Soil Biodegradation : 14C-8-2 TBA Experimental Design
Treatment
1 (Live soil test vessels)
2 (Abiotic control; sterile soil)
Replicate (n)
4 4
[14C F2]-8-2 TBA
(mg kg-1 soil)
0.2 - 1.5
0.2 - 1.5
Chloramphe nicol
(mg kg-1 soil) 0
~ 200
14Cbenzoic
acid (mg kg-1
soil)
Fluorinated acid
standards* (mg kg-1 soil)
0
0
3 (Spike recovery vessels;
sterile soil)
4 (Vessels for soil respiration
measurement)
5 (14C-benzoic acid
biodegradation measurement)
6' (Matrix blank soil )
tan?
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0.2 - 1.5
~ 200
2-3 0
0
2-4 0
0
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10-200
0.01-0.2 0 0
00
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 carbomg-1 dry soil.
Study Material:
- 14C-8-2 TBA - Additional Carbon Source: ~50 mg ethanol^kg-1 soil (as
cosolvent).
Concentration of the Test Substance:
- 0.2 mg^kg-1 soil (except day 1 & day 7 at ~ 0.85 mg^kg-1 soil).
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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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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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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 O2Content:
- A 26-gauge miniature O2 probe was pierced through the septa of sealed sample bottles for the measurement.
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EPA -DuPont Telomers Degradation Technical Meeting
Bill Berti DuPont Central Research & Development Environmental & Biological Sciences & Engineering
mu
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Page 57 December 15, 2004
Preliminary Assessment of Aerobic Transformation in Soil
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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, HaskellSMLab for Health & Environ.
Sciences
Current status:
- Protocols signed, study commenced week of 13 Dec 2004
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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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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 pg^g-1 soil dry wt.
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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 TBA^kg-1 sterile soil (dw) + 10 ^g 8-2 TB Acid*kg-1 sterile soil (dw) + 10 ^g 8-2 TB Unsaturated Acid*kg-1 sterile soil (dw) + 10 ^g APFO*kg-1 sterile soil (dw)
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Analytes of Interest
Treatment Groups 1) Background Blank Control 2) Test Substance 3) Abiotic Control 4) Spike Recovery Controls
Headspace & Samples Soil analytes
Headspace & soil
8-2 TBA
Soil analytes
8-2 Telomer B Acid, 8-2 Telomer B Unsaturated
Acid and PFOA
Headspace & soil
8-2 TBA
8-2 Telomer B Acid, 8-2 Telomer B Unsaturated
Acid and PFOA
Headspace & soil
8-2 TBA
8-2 Telomer B Acid, 8-2 Telomer B Unsaturated
Acid and PFOA
Headspace & soil
8-2 TBA
8-2 Telomer B Acid, 8-2 Telomer B Unsaturated
Acid and PFOA
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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)
IK
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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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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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Test Summary
Treatment Groups 1) Background Blank Control
Sampling Total Test Replicates Days Chambers
3 0, 14, 28 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 fluorinated acids in Sterile Soil
TOTAL TEST CHAMBERS
3
0,28
6 33
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Draft Protocol Highlights
Definitive Studies for Transformation Potential of Fluorotelomer-Based Polymers in Aerobic and Anaerobic Soils
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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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Selected Soils for DuPont Biodegradation Studies
Soil Orders
Soil order represents _% of total land in US
Comments
Entisol Mollisol Inceptisol
8
Tobeusedinpreliminary soil test studiesatWildlife
International
25 To be used in TRP
studies at Noack Laboratory (Germany)
16
Alfisol
13 Sassafras Soil
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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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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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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 pg/kg dry weight soil. (any sequestration?)
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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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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 mole^kg-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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Reports
Interim Report following analysis of 6-month samples Final Report
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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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Agenda
DuPont Study Results Timeline Work Process Forward Meeting Reflections
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DuPont Biodegradation Program Timeline
Timing
Atmospheric Fate of Perfluoroaldehydes
Atm ospheric Partitioning of 8-2 T e lo m e r B Alcohol, Hetergeneous Uptake
Atmospheric Transformation Pathways of Alcohols and Aldehydes
Atmospheric Partitioning of Telom er Alcohols and Olefins
Long-range Transport M odeling of 8-2 Alcohol
Biodeg in Sludge o f 14C 8-2 T e lo m e r B Alcohol
Biodeg in S oil o f 14C 8-2 T e lo m e r B Alcohol
Biodeg in S e d im e n t o f 14C 8-2 T e lo m e r B Alcohol
Biodeg o f D uPont Surfactants in Sludge
Biodeg in Sludge o f Fluorotelom er-based Polym ers
Biodeg in So il, S e d im e n t, and Sludge o f Flu o ro telo m erbased Polymers
Biodeg o f DuPont Fluorotelom er-based Polym ers in Sludge
Biodeg o f DuPont Fluorotelom er-based Polym ers in Soil/Sedim ent - Screening
Biodeg o f D uPont F luorotelom er-based Polym ers in Soil
Biodeg o f DuPont Fluorotelom er-based Polym ers in Sedim ent
Biodeg o f DuPont Fluorotelom er-based Polym ers in Anaerobic Sludge
Biodeg in Soil & S e d im en t o f 14C 8-2 T e lo m e r B Alcohol Based Urethane Polym er
1Q'04
2Q'04
TRP TRP TRP
3Q'04
4Q'04
1Q'05
Final Report Final Report Final Report Final Report
2Q'05
3Q'05
Final Report
Final Report
Final Report
4Q'05
1Q'06
2Q'06
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Page 79 December 15, 2004
Agenda
Work Process Forward Meeting Reflections
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j)PA/DuPont Telomer Research Meeting________________________________________________________________________ December 15,2004
NAME
ORGANIZATION
TELEPHONE #
E-MAIL ADDRESS
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