Document 9J20oEnJYopBwvDE1aOG19xBD

RECYCLED 00 AR226-2696 AR226-2696 EMSER 70-03/4842 \ WP ' STUDY PROTOCOL Arnr,T U,DATED BIODEGRADATION OF 8-2 TELOMER B ALCOHOL SPONSOR E. I. du Pont de Nemours and Company DuPont Chemical Solutions Enterprise Wilmington, DE 19898, USA TESTING FACILITIES: E. T. du Pont de Nemours and Company Environmental and Microbiological Sciences & Engineering Corporate Center for Engineering Research Central Research & Development Glasgow, Building 300, P.O. Box 6101 Newark, DE 19714-6101 USA AND E. I. du Pont de Nemours and Company Haskell Laboratory for Health and Environmental Sciences Newark, DE 19714 USA STUDY DIRECTOR: PROPOSED STARTING DATE: PROPOSED COMPLETION DATE: STUDY NUMBER: Ning Wang, Ph.D. . 30-June-2003 September-2003 EMSER 70-03 / 4842 "CONFIDENTIAL. This document is subject to restriction by order of the Cir Court of Wood City, WV, Case No. 01-C-608, and may not be copied or disseminated except by Order of the Court." EID812304 EMSER 70-03/4842 STUDY PROTOCOL APPROVAL Study Director: Ning Wang, Ph.D. Date DuPont Central Research & Development Research Manager: John T. Gannon, Ph.D. Date DuPont Central Research & Development Analytical Director: S. Mark Kennedy DuPont Haskell Laboratory for Health and Environmental Sciences Date ) Page 2 of 18 "CONFIDENTIAL. This document is subject to restriction by order of the Cir Court of Wood City, WV, Case No. 01-C-608, and may not be copied or disseminated except by Order of the Court." EID812305 EMSER 70-03/4842 1.0 SUMMARY The biodegradability o f the test substance will be determined with activated sludge or sludge culture originated from POTW or industrial waste treatment facilities. The solution o f the 8-2 telomer B alcohol (8-2 TBA) in E2-BSMYE cell growth medium is inoculated with sludge or sludge culture and kept in closed bottles in the dark at room temperature. Degradation is followed by analysis of the concentration o f the test chemical and its possible metabolites (biotransformation products) during the test (28-90 days). The extent o f biodegradation o f the test chemical can be expressed as the percentage of the test chemical that has been converted and the accumulation of its major metabolites, including Fluoride. 2.0 OBJECTIVES 1. Determine the biodegradation potential of 8-2 Telomer B Alcohol. 2. Identify possible degradation products. 3. Determine the degree of defluorination. 3.0 TEST SUBSTANCE Name: Synonym: Active substancc(s) CAS Name: CAS Number(s): Structure: 8-2 Telomer B Alcohol (Perfluorooctyl)ethanol, 8-2 TBA 8-2 Telomer B Alcohol, + 99% 1-Decanol, 3,3,4,4,5,5,6,6,7,7,8,8,9,9,10,10,10heptadecafluoro- 678-39-7 F H Number Lot Number: H-24691 P.00/001 Page 3 of 18 "CONFIDENTIAL. This document is subject to restriction by order of the Cir Court of Wood City, WV, Case No. 01-C-608, and may not be copied or disseminated except by Order of the Court. EID812306 EMSER 70-03/4842 EMSE Sample Number: Concentration of a.s., nominal: Concentration of a.s., analyzed: Certificate of Analysis Date: Expiration Date: Date Received: Solubility at 25C.: Vapor pressure: Stability: Appearance/Color: Storage Conditions: Safety Precautions: Supplier: E93386-80 99% 99.2% 13-Sept-2001 Unknown 26-Mar-2002 -140 ggL'1 0.023 mm Hg Stable at ambient room temperature White solid Room temperature; keep tightly closed Wear lab coat, protective gloves, and safety glasses Clariant Co. TEST SYSTEM 4.1 Test System Justification The test system incorporated published EPA guideline OPPTS 835.3120 and OECD guideline 301D & 302D. 4.2 Description o f Test System The test system consists o f individually capped test vessels made o f material that does not significantly sorb the test substance and the sludge culture. All experiments will be conducted in triplicate (unless otherwise noted) in the test vessels at room temperature. A calibrated temperature chart-recorder or thermometer will be placed in proximity to the samples to monitor the temperature. All test samples will be uniquely identified and documented in the study records. 4.3 Sources o fInoculum 1. The inoculum used in this study will come either from Wilmington (DE) municipal waste treatment plant or an industrial waste treatment facility or other appropriate sources. 5.0 ANALYTICAL REFERENCE STANDARDS, REAGENTS, AND SOLVENTS Analytical reference standards that are used to aid the identification o f the test substance and its potential degradation products will be documented in the study records. All chemicals and solvents will be reagent grade or purer. Sources will be documented in the Page 4 of 18 ). "CONFIDENTIAL. This document is subject to restriction by order of the Cir Court of Wood City, WV, Case No. 01-C-608, and may not be copied or disseminated except by Order of the Court." EID812307 EMSER 70-03/4842 study records. Deionized water produced at the testing facility will be used throughout in the study. 6.0 APPARATUS AND BOTTLES AND CONTAINERS 6.1 N orm al laboratory apparatus and: (a) Biological safety cabinet or equivalent (b) Water bath, incubator or equivalent, for keeping test vessels or bottles at constant temperature with the exclusion o f light. 6.2 Bottles or Containers (a) Test vessels: 50-300 ml with stoppers or caps (b) 0.001 - 8 L Bottles for general purpose such as medium and sample preparations, centrifugation etc. 7.0 PREPARATION OF STOCK SOLUTIONS OF ORGANIC NUTRIENTS 7.1 If needed, dissolve in water of 20-400g/L of an organic nutrient (e.g.'yeast extract or glucose) for microorganism growth. Sterilize the medium and store it at room temperature or refrigerated. 8.0 PREPARATION OF E2-BSMYE GROWTH MEDIUM 8.1 E2 basal salts medium +yeast extract (E2-BSMYE) 8.1.1 Mix 5 ml o f 200 x P (phosphate) salts stock (pH to 7.2), 100 ml of Non-P salts stock solution, 5 ml ofE 2 trace elements, and 100 ml o f 20 % w/v (NH4)2S04 stock solution with 500 mL o f deionized water. (See Attachment 2-5 for stock solution preparations.) 8.1.2 Adjust pH to 6.5~7.5 with NaOH or HC1, add 2.5 - 100 mL o f organic nutrient stock solution or ethanol and bring the volume to 1.0 L with water. 8.1.3 Filter-sterilize the solution using a 0.2 pm Nalgene filter unit or equivalent. 8.1.4 Store at room temperature. Page 5 of 18 ' "CONFIDENTIAL. This document is subject to restriction by order of the Cir Court of Wood City, WV, Case No. 01-C-608, and may not be copied or disseminated except by Order of the Court." EID812308 EMSER 70-03/4842 9.0 PREPARATION OF 8-2 TELOMERB ALCOHOL SOLUTION 9.1 If needed, dissolve appropriate amount o f 8-2 TBA in a suitable solvent (Ethanol or methanol. Store the stock solution properly. 10.0 PREPARATION OF SODIUM CYANIDE STOCK SOLUTION 10.1 If necessary, weigh appropriate amount of NaCN in a container and add appropriate volume o f sterile water to a final concentration of 50 - 500 mM. Store the NaCN stock solution at room temperature or refrigerated. 11.0 INOCULUM PREPARATION 11.1 Collect a fresh sample o f activated sludge from a POWT or from other microbial sources. If necessary, settle the sludge for 5- 30 min and collect upper aqueous phase o f the sludge as the inoculum. Remove coarse particles if necessary by filtration with Whatman #1 filter paper and keep the sludge aerobic thereafter. The sludge or sludge filtrate will be used as an inoculum for sludge culture preparation. 11.2 If sludge has to be taken from a high rate treatment plant, or is thought contain inhibitors, it should be washed with E2-BSMYE medium. Settle oridfentrifiigetho ! re-suspended sludge after thorough mixing, discard the supernatant and again re suspend the washed sludge in a further volume o f E2-BSMYE medium. Repeat this procedure until the sludge is considered to be free from excess substrate or inhibitor. The cleaned-up sludge will be used as an inoculum for sludge culture preparation. 12.0 PREPARATION OF SLUDGE CULTURE 12.1 Add 0.050-5 mL o f sludge or sludge filtrate (inoculum) to 50-500 mL volume of sterilized test vessels that contained 4-100 mL o f E2-BSMYE growth medium. Add 8-2 TBA to a final concentration of 0.3-50 mg/L if necessary (Solvent concentration < 0.1% in test vessels). 12.2 Incubate the vessels at room temperature or controlled temperature (20-30 C) for 1 - 1 5 days. 12.3 If needed, transfer 0.050-5 mL of cell culture from step 122 to sterilized test vessels that contained 4-100 mL of E2-BSMYE growth medium. Add test chemical to a final concentration of 0.3-50 mg/L if necessary. Page 6 of 18 "CONFIDENTIAL. This document is subject to restriction by order of the Cir Court of Wood City, WV, Case No. 01-C-608, and may not be copied or disseminated except by Order of the Court." EID812309 EMSER 70-03/4842 12.4 Repeat steps 1 1 2 and 12.3 as needed to acclimate the culture to 8-2 TBA. 12.5 Centrifuge the sludge culture at 2000-20000g for 5-15 min and decant the supernatant. Resuspend the pellet with E2-BSMYE growth medium. 12.6 Repeat the step 12.5 2-3 times. 12.7 Read Optical O.D. (600 ran) of the sludge or sludge culture. 13.0 PROCEDURES FOR DIFFERENT EXPERIMENTAL TREATMENTS Note: Add appropriate amount of different solutions according to different experimental treatments. The following table is provided as an example for filling 120 mL serum bottles with 40ml o f different test medium. The actual medium volume per bottle and bottle size may be varied and will be recorded in the study records. T reatm ent #i #2 #3 #4 #5 n** Solutions to be added (m L) 1 4 C -A n ilin e . F.2-BSM YF. 3 g 8-2 TBA /I S lu d g e or A utoclaved 0.2 M N aC N Spike w ith 3 g /l. o f HC1 (0.5-2g/L ) m edium fin ethanol) culture slu d g e or culture in w ater 8-2 TBA (m ethanol 4 40 0 .0 0 2 -0 .0 1 2 0.05 -0 .5 00 0 4 40 0 .0 0 2 -0 .0 1 2 0 0 .0 5 -0 .5 0.1 0 3 40 0 0 .0 5 -0 .5 0 0 0 .0 0 2 -0 .0 1 2 2 40 0 0.0 5 - 0.5 00 0 0 .0 2 -0 .0 6 40 0 0.05 - 0.5 00 0 Note: The glass serum bottles used in different treatments axe not coated. The volume calculation is>on a,-pernd bottle (40 mL) basis. The volume per bottle of the test medium can be varied to suit the study conditions. ' * f Number of replicates for each sampling time point 13.1 Treatm ent 1 - Biodegradation Test Vessels 13.1a Prepare enough solution to fill a total of 16 - 24 bottles (4 replicates * 4 6 sampling time points) or appropriate number of bottles. For example, to make 1 L o f the treatment solution, mix 1.25 -1 2 .5 mL o f the sludge or sludge culture and 987.5 - 998.8 mL o f E2-BSMYE medium. Make appropriate adjustments when making less than one liter or more than one liter of the treatment solution. 13. lb Transfer 40 mL o f the mixed solution to each of the individual serum bottles. Add 2 - 12 uL of3g/L o f 8-2 TBA stock solution to each of the serum bottles. Seal the bottles with pre-washed (with methanol and sterile water) septa or butyl rubber* stoppers, or equivalent. If needed, cover the mouth of each of the serum bottles with a piece o f pre-washed (with Page 7 of 18 "CONFIDENTIAL. This document is subject to restriction by order of the Cir Court of Wood City, WV, Case No. 01-C-608, and may not be copied or disseminated except by Order of the Court." EID812310 EMSER 70-03/4842 methanol and sterile water) aluminum foil before sealing the bottles with septa or stoppers. 13.2 Treatm ent2 -A b io tic Control 13.2a Prepare enough solution to fill a total o f 16 - 24 bottles (4 replicates x 4 6 sampling time points) or appropriate number of bottles. For example, to make 1 L o f the treatment solution, mix 1.25 --12.5 mL o f autoclaved sludge or sludge culture, 2.5 mL o f 0.2 M NaCN stock solution, and 985 996.3 mL o f E2-BSMYE medium. Make appropriate adjustments when making less than one liter or more than one liter of the treatment solution. 13.2b Transfer 40 mL o f the mixed solution to each o f the individual serum bottles. Add 2 - 12 uL o f 3g/L of 8-2 TBA stock solution to each o f the serum bottles. Seal the bottles with pre-washed (with methanol and sterile water) septa or butyl rubber stoppers, or equivalent. If needed, cover the mouth of each of the serum bottles with a piece of pre-washed (with methanol and sterile water) aluminum foil before sealing the bottles with septa or stoppers. 13.3 Treatment 3- Test Substance Spike Recovery Control 13.3a Prepare enough solution to fill a total of 12 - 18 bottles (3 replicates x 4 - 6 sampling time points) or appropriate number of bottles. For example, to ill make 1 L o f the treatment solution, mix 1.25 - 12.5 mL of the sludge or "'iatmenr >-nn y sludge culture and 987.5- 998.8 mL of E2-BSMYE medium. Make <:9S7.r -v> appropriate adjustments when making less than one liter or more than one liter of the treatment solution. 13.3b Transfer 40 mL o f the mixed solution to each of the individual serum bottles. Seal the bottles with pre-washed (with methanol and sterile water) septa or butyl rubber stoppers, or equivalent. If needed, cover the mouth of each of the serum bottles with a piece of pre-washed (with methanol and sterile water) aluminum foil before sealing the bottles with septa or stoppers. The bottles will be spiked with 8-2 TBA stock solution 3g/L in ethanol) at each sampling time point for 8-2 TBA spike recovery analysis, after 10-15 mL o f medium is withdrawn from the bottles to other containers.. If necessary, the 10-15 mL o f sample medium will be spiked with standard fluorinated acids stock solution at each sampling time point for fluorinated acids spike recovery analysis. 13.4 Treatment 4 Intrinsic (Growth M edium M atrix) Control Page 8 of 18 "CONFIDENTIAL. This document is subject to restriction by order of the Cir Court of Wood City, WV, Case No. 01-C-608, and may not be copied or disseminated except by Order of the Court. EID812311 EMSER 70-03/4842 13.4a Prepare enough solution to fill a total o f 8 - 12 bottles (2 replicates 4 - 6 sampling time points) or appropriate number of bottles. For example, to make 1 L of the treatment solution, mix 1.25 -1 2 .5 mL o f the sludge or sludge culture and 987.5 - 998.8 mL of E2-BSMYE medium. Make appropriate adjustments when making less than one liter or more than one liter o f the treatment solution. 13.4b Transfer 40 mL of the mixed solution to each o f the individual serum bottles. Seal the bottles with pre-washed (with methanol and sterile water) septa or butyl rubber stoppers, or equivalent. If needed, cover the mouth of each of the serum bottles with a piece o f pre-washed (with methanol and sterile water) aluminum foil before sealing the bottles with septa or stoppers. 13.5 Treatment # 5- Reference Compound Biodegradation Control (Positive Control) 13.5a Prepare enough solution to fill a total o f 8 - 12 bottles (2 replicates * 4 - 6 sampling time points) or appropriate number of bottles. For example, to make 1 L of the treatment solution, mix 1.25 - 12.5 mL of the sludge or sludge culture, 987.5 - 998.8 mL of E2-BSMYE medium, and 1.0 mL of 14C-aniline.HCl stock solution. Make appropriate adjustments when making less than one liter or more than one liter of the treatment solution. 13.5b Transfer 40 mL of the mixed solution to each o f the individual serum bottles. Seal the bottles with pre-washed (with methanol and sterile water) septa or butyl rubber stoppers, or equivalent. 13.6 At day zero, withdraw approximately 5 mL aliquot (or the appropriate volume) of test medium from each o f the four serum bottles for Treatments 1 & 2 and from the two bottles for Treatment 4 into appropriate containers for fluoride analysis. If necessary, freeze the sample containers and sample bottles until extraction and analysis. Also withdraw 5-10 mL aliquot o f the test medium from each o f the 4 serum bottles for Treatments 1 & 2 and from the two bottles for Treatment 4 into appropriate containers for extraction of biotransformation products (fluorinated acids) with acetonitrile. If necessary, freeze the 5-10 mL sample medium until extraction and analysis. After the medium withdrawing, Inject MTBE-H2S 04 solution into each of the sample bottles for extraction of 8-2 TBA. For Treatm ent3, withdraw 10-15 mL (or the appropriate volume) o f the test medium from the three or four scrum bottles into appropriate containers and spike the 10-15 mL solution with standard fluorinated acids and incubate for 10-120 min; followed by adding acetonitrile for extraction of fluorinated acids. Spike the rest o f 25-30 mL o f medium left in the serum bottles with 2 - 12 pL (or appropriate volume) o f 8-2 TBA stock solution (3g/L in ethanol) and incubate for 10-120 min; Inject MTBE-H2SO4 solution into each o f the sample bottles for extraction of 8-2 TBA. Page 9 of 18 "CONFIDENTIAL. This document is subject to restriction by order of the Cir Court of Wood City, WV, Case No. 01-C-608, and may not be copied or disseminated except by Order of the Court. PID 812312 EMSER 70-03/4842 For Treatment 5, withdraw approximately 0.01 - 0.5 mL (or appropriate volume) o f the 14C-aniline.HCl medium from the two serum bottles into each o f the scintillation vials. Alternatively, withdrawn 0 .5 - 2 mL of the air with a gas-tight syringe from inside the sealed serum bottles and inject the air slowly into 1-5 mL o f 0.5-1.5 Normality o f NaOH or Ba(OH>2 in scintillation vials (or other container) for 14C radioactivity measurement. Freeze the vials, if necessary, until analysis of 14C radioactivity. Freeze the serum bottles after sampling, if necessary. 13.7 Incubate the rest of sealed serum bottles at room temperature in an incubator/shaker or equivalent with shaking (approximately 50-300 RPM). 13.8 Periodically (approximately at day 7, day 14, and day 28, day 56, and day 90 sampling time points), repeat the Step 13.6 and Step 13.7, as needed. 13.9 Inject organic nutrient stock solution or sludge or sludge culture to each o f the sealed test vessels containing 40 mL of test solution at ~ day 30 and day 60, if the test lasts beyond 28 days. 14,0 ANALYTICAL METHODS GC/MS and LC/MS/MS analysis will be conducted in Haskell laboratory. The samples will be submitted for analysis only when the test vessels (Treatm ent 1) showed significant defluorination and at least 50% of 14C-aniline.HCl was mineralized in reference (positive) control vessels (Treatm ent 5) within 28 days o f the test period. The experiment will be * - terminated and repeated if above two criteria are not met. ' *nr Total samples to be submitted for GC/MS and LC/MS/MS analysis: 1) GC/MS quantification of 8-2 TBA: up to 78 samples (13 samples at each sampling time point - Days 0, 7, 14, 28, 56, and 90); 2) LC/MS/MS analysis o f fluorinated acids such as 8-2 acid, 8-2 unsaturated acid, PFOA, and PFHA: up to 78 samples (13 samples at each sampling time point - Days 0, 7, 14, 28, 56, and 90). 14.1 Fluoride (See Attachm ent 1) "" * For Treatment 1 & 2 and/or for other Treatments as appropriate, NaOH (or equivalent) will be added to the sample medium withdrawn from the serum bottles at each sampling time point (approximately at days 0, day7, day 14, day 28, day 56, and day 90). After incubation at room temperature for 0.2 - 4 h, the solution will be neutralized with H2S04(or equivalent). If necessary, centrifuge the sample solution after the neutralization. The neutralized solution will be used for F~ ion analysis by an appropriate analytical method or can be frozen until the analysis. The analytical method used will be documented in the study records and the final report. Page 10 of 18 "CONFIDENTIAL. This document is subject to restriction by order of the Cir Court of Wood City, W V , Case No. 01-C-608, and may not be copied or disseminated except by Order of the Court. EID812313 EMSER 70-03/4842 14.2 Biotransform ation products (Fluorinated acids) Appropriate volume o f test medium (5-15 mL) withdrawn from the test vessels at each sampling time point will be extracted with 10-40 mL o f acetonitrile for 1-6 h. The clear upper phase after the extraction will be filtered with a nylon filter and subject to LC/MS/MS identification and quantification. 14.3 8-2 TBA For Treatm ent 1 - 4 , 25-50 mL o f MTBE-H2SO4 (50 mM final in MTBE) will be injected into each o f the sample bottles after the medium withdrawing (for Fluoride and fluorinated acid analysis) at each sampling time point (approximately at days 0, day7, day 14, day28, day 56, and day 90). After 1-6 h incubation, the settled upper MTBE phase will be transferred to 50-mL polypropylene tubes and centrifuged at ~2000 rpm for 5-20 min in a clinical centrifuge. After centrifugation, the clean upper MTBE phase will be used for GC/MS quantification of 8-2 TBA. 14.4 u C-aniline.HCl For Treatment 5, concentrated HC1 (or equivalent) will be added to the sample medium (0.01 - 0.5 mL) in scintillation vials from Step 13.6. The final concentration o f HC1 in the vials is approximately 10-150 mM. The vials are incubated for 0.1- 6 h at room temperature before adding scintillation cocktail for 14C radioactivity measurement with a scintillation counter. 15.0 DATA ANALYSIS ) ' `-N* LYSIS 15.1 Tim e course o f the concentration changes ofparent compound 8-2 BA. 15.2 Time course o f the accumulation o f the major stable metabolites. 15.3 Mass Balance (Mb) Calculation (theoretical Mb equals 1.0) Mh = [Z (D ,+ D 2 + - + D n)]/Pc Di --Concentration o f degradation product 1; D2--concentration o f degradation product 2; D,, - concentration of degradation product n. Pc - Concentration o f parent compound. 15.4 Time course o f the accumulation of the F~ ion. 15.5 Tabulate all the stable metabolites identified during the course of the experiment. Page 11 of 18 1 "CONFIDENTIAL. This document is subject to restriction by order of the Cir Court of Wood City, WV, Case No. 01-C-608, and may not be copied or disseminated except by Order of the Court. EID812314 EMSER 70-03/4842 16.0 STATISTICAL METHODS AND CONTROL OF BIAS Statistical methods including means, standard deviations, and regression lines will be used as appropriate. Bias will be effectively controlled through duplicate sampling, replicate analysis, sample spiking, and maintenance o f material balance. 17.0 REPORTING O F DATA Interim reports may be written and submitted to the study sponsor during the various phases of the preliminary and definitive studies. A draft of the final report will be submitted to the sponsor approximately one month after the conclusion o f all of the definitive studies. The final report will include, but not be limited to: a. Study information containing the identification o f testing facility, location of the raw data, study dates, and name o f the study director. b. Names o f principal study personnel and the management. c. Information on the test substance and information on analytical standards. Method of preparation o f dosing solution, the amount and identity of any cosolvents or extractants used, and the amount of test substance used. d. Description o f experimental design and experimental procedures and any deviations from the procedures stated in the protocol. e. Description of analytical methods and recoveries. f. Results and discussion to include, but not be limited to: ) 1) Time course of the concentration changes o f parent compound 8-2 BA. 2) Time course o f the accumulation of the major stable metabolites. 3) Mass balance (Mb) calculation o f stable metabolites versus parent compound. 4) Time course of the accumulation o f the F" ion. 5) A list o f all stable metabolites identified during the course of the test. 6) Possible biodegradation pathways of 8-2 Telomer B Alcohol. 18.0 STUDY RECORD MAINTENANCE AND RETENTION All original paper data generated during the study, the original signed final report and all original facility-specific raw data will be retained by in the DuPont CR&D Environmental & Microbiological Sciences and Engineering Laboratory (EMSE) archive, excluding the original analytical data and related original facility-specific raw data regarding the analysis of 8-2 TBA and possible biotransformation products conducted by participating analytical facility. The original analytical data for 8-2 TBA and metabolite analysis and all original facility-specific raw data regarding the 8-2 TBA quantification and biotransformation products analysis by the participating analytical facility will be retained in DuPont Haskell laboratory for Health and Environmental Sciences. Page 12 o f 18 "CONFIDENTIAL. This document is subject to restriction by order of the Cir Court of Wood City, WV, Case No. 01-C-608, and may not be copied or disseminated except by Order of the Court. EID812315 EMSER 70-03/4842 Alternatively, the original data may be retained by Iron Mountain Records Management, Wilmington, DE 19802, U.S.A. At least one copy of the signed final report will be provided to the sponsor. 19.0 GLP COMPLIANCE This study will be Non-GLP. 20.0 REFERENCES 1. Organization for Economic and Cooperative Development (OECD) Guidelines for Testing of Chemicals; Section 3: Degradation and Accumulation; Ready Biodegradability: 301 A Doc Die-Away Test; 301 D Closed Bottle Test (1993). 2. Organization for Economic and Cooperative Development (OECD) Guidelines for Testing o f Chemicals; Section 3: Degradation and Accumulation; Proposed 302D Inherent Biodegradability - Concawe Test (October, 2001) 3. U.S. Environmental Protection Agency, Office of Prevention, Pesticides, and Toxic Substances (OPPTS), Fate, Transport and Transformation Test Guidelines, OPPTS 835.3120, Sealed Vessel C02 Production Test. EPA 712-C-98-311, January 1998. 4. King Wang, DuPont EMSE Report No. 15-03, Accelerated Biodegradation o f 8-2 Telomer B Alcohol - A Preliminary Screening Study (2003). 5. King Wang, DuPont EMSER 14-03 study protocol: Aerobic Ready Biodegradation of 14C-labeled 8-2 Telomer B alcohol (2003). Page 13 o f 18 "CONFIDENTIAL. This document is subject to restriction by order of the Cir Court of Wood City, WV, Case No. 01-C-608, and may not be copied or disseminated except by Order of the Court. EID812316 EMSER 70-03/4842 ATTACHMENT 1 F- Ion Extraction 1. Withdraw 5 mL from the sealed serum bottles at each sampling time points into individual 15-mL polypropylene tubes that already contained 50 pL o f 5N sodium hydroxide; shake the bottles at about 300 rpm for 0.2-4 h. 2. Add 41.7 pL o f concentrated (6 N) sulfuric acid to the 15-mL tubes and mix well and store the samples refrigerated for several days or in a freezer for further treatments in Step 3. 3. If necessary, centrifuge the sample solution from Step 2 after thawing the samples and transfer 4-5 mL supernatant into 50-mL polypropylene tubes and add 4-5mL o f TTSAB II (Thermo Orion; cat#: 940909) solution. 4. Measure F~ ion concentration with F_ selective electrode. Fluoride Ion Analysis with Ion-Selective Electrode 1. Turn on the Thermo Orion 710A plus pH/ISE meter (Serial #___________ ) at least 15 min before the measurement. 2. Flush out old reference electrode filling solution and fill the reference chamber o f the Fion selective electrode (Thermo Orion cat#: 9609BN; Lot # ______ ; Received________ ) with the fresh solution (Thermo Orion Cat# 90061; Lot #_______ ; Received__________ ). 3. Insert the F- ion selective electrode into each sample solution to be measured or into Fstandard solution made in Vt. strength TISAB II buffer. 4. Periodically, gently shake the measurement solution. 5. Record the conductivity in mV over tim e:______ min after the incubation (usually 3-7 min). 6. Rinse the electrode with deionized water (mega fi-cm = 17-18) and wipe the electrode tip to absorb the water with soft tissue between each measurement. Page 14 o f 18 "CONFIDENTIAL. This document is subject to restriction by order of the Cir Court of Wood City, WV, Case No. 01-C-608, and may not be copied or disseminated except by Order of the Court." EID812317 EMSER 70-03/4842 ATTACHMENT 2 CCER / EMSE Notebook: E100633-AC P age:_______ E2 Basal Media P-Salts Stock Solution P-Saits KH2P 0 4 NaH2P 0 4*H20 lx Stock 0.70 g/1 0.395g/l 200x Stock 140 g/1 79g/l Make a lOOx stock of phosphate salts (P-Salts) and adjust to pH 7.2 with NaOH Record Lot numbers of chemicals and weights used in making solution. Label as P-Salts lOOx Stock with Lot # and store at room temp, non sterile. KH2P 0 4 Potassium Phosphate Monobasic Crystals FW 136.09 / CAS # 7778-77-0 NaH2P 0 4*H20 Sodium Phosphate Monobasic Monohydrate Crystals FW 137.99 / CAS # 10049-21-5 Additional Comments / Notes : Witnessed by : __________________ Date : Page 15 o f 18 "CONFIDENTIAL. This document is subject to restriction by order of the Cir Court of Wood City, WV, Case No. 01-C-608, and may not be copied or disseminated except by Order of the Court." EID812318 EMSER 70-03/4842 'I ATTACHMENT 3 CCER / EMSE Notebook: E10Q633-AC Page: E2 Basal Media Non P-Salts Stock Solution Non P-Salts KC1 M gS04*7H20 CaCL2*2H20 NaCl lx Stock 0.5 g/1 0.5 g/1 0.025 g/1 1.0 g/1 lOx Stock 5.0 g/1 5.0 gn 0.25 g/1 10.0 g/1 Make a 1Ox stock o f non-phosphate salts (Non P-Salts) with no pH adjustment. Record Lot numbers of chemicals and weights used in making solution. Label as Non P-Salts lOx Stock with Lot # and store at room temp. Store as non-sterile stock. KCL Potassium Chloride Crystals FW 74.56 / CAS # 7447-40-7 M gS04*7H20 Magnesium Sulfate 7-Hydrate Crystals FW 246.48 / CAS # 10034-99-8 CaCl2*2H20 NaCl Calcium Chloride Dihydrate Crystals FW 147.02/CA S # 10035-04-8 Sodium Chloride FW 58.45 / CAS # 7647-14-5 Additional Comments / Notes : Witnessed by : ________________ Date : Page 16 o f 18 - 1 "CONFIDENTIAL. This document is subject to restriction by ' order of the Cir Court of Wood City, WV, Case No. 01-C-608, j and may not be copied or disseminated except by Order of the Court." EID812319 EMSER 70-03/4842 ATTACHMENT 4 C C ER /EM SE Notebook: E100633-AC Page: E2 Basal Media Trace Element Stock Solution (200x) Non P-Salts F e S 0 4*7H20 C u S 0 4*5H20 CoC12*6H20 MnCl2*4H20 Na2M o02*2H20 C6H5Na3O7*2H20 NiCl2*6H20 ZnCl2 gms /100 mis 1.0 0.1 0.2 0.02 0.05 2.0 0.2 0.01 Dissolve sodium citrate first and adjust to pH 5.0 with HC1, then dissolve metals. Label as Trace Element 200x Stock with Lot # and store at room temp. Store as non-sterile stock. F e S 0 4*7H20 Ferrous Sulfate Heptahydrate CAS #7782-63-0 CuS 0 4*5H20 Cupric Sulfate Pentahydrate CA S# 7758-99-8 CoC12*6H20 Cobalt Chloride Hexahydrate CA S# 7791-13-1 MnCl2*4H20 Manganese Chloride Tetrahydrate CA S# 13446-34-9 Na2M o02*2H20 Sodium Molybdate Dihydrate CA S# 10102-40-6 C6H5Na3O7!i'2II20 Sodium Citrate Dihydrate Tribasic CAS # 6132-04-03 NiCl2*6H20 Nickel Chloride Hexahydrate CAS # 7791-20-0 ZnCl2 Zinc Chloride CAS # 7646-85-7 Additional Comments / Notes : Witnessed by :. Date : Page 17 o f 18 "CONFIDENTIAL. This document is subject to restriction by order of the Cir Court of Wood City, WV, Case No. 01-C-608, and may not be copied or disseminated except by Order of the Court." EID812320 EMSER 70-03/4842 ATTACHMENT 5 C C E R /E M SE Notebook: E10Q633-AC Page: E2 Basal Salts Media + Yeast Extract (E2-BSMYE) Lot ID # __________________ D a te : _____ Amount M ade__________________ Made By : Component Stock Solutions 200x P Salts Stock (pH 7.2) lOx Non P-Salts Stock E2 Trace Elements Solution 200x stock 20 % (NH4)2 SO4 Stock Solution 10 40% Yeast Extract Stock mis / liter 5 mis 100 mis 5 mis 100 mis *Variable Combine stock solutions an adjust pH to 7.2 with NaOH * Yeast Extract added as need to reach specific final concentration. Filter sterilize using 0.2 um Nalgene filter unit or equivalent. Label with lot ID and store sterile at room temperature. lOOx P Salts Stock (pH 7.2) Lot # ____________ mis added ____________ lOx Non P-Salts Stock Lot # ____________ mis added ____________ E2 Trace Elements Solution 200x stock Lot # ____________ mis added ____________ 20% (N H 4)2 SO4 L o t# ____________ mis added ____________ 10 % Yeast Extract Stock final cone, of Y E ______ Lot # ____________ mis added ____________ Final p H ____ Adjusted w ith_____ mis NaOH Lot # _______ Additional Comments / Notes : ______________________________ Page 18 of 18 "CONFIDENTIAL. This document is subject to restriction by order of the Cir Court of Wood City, WV, Case No. 01-C-608, and may not be copied or disseminated except by Order of the Court." EID812321