PROGRESS REPORT: AUGUST 2010 BIODEGRADATION TESTING SUPPLEMEN TAL ENVIRONMENTAL PROJECT Submitted to E.I du Pont de Nemours and Company Wilmington, Delaware 19898 U.S.A. United States Environmental Protection Agency Washington, DC U.S.A. 

# Wildlife International, Ltd. 8598 Commerce Drive Easton, Maryland 21601 USA 1 (410) 822-8600 August 31, 2010

Page 1 of 266Does Not Contain Confidential Business Information (CBI) 2010_July_Progress Report 2 Table of Contents Title Page ..................................................................................................................... ..............1 Table of Contents.............................................................................................................. .........2 Introduction................................................................................................................... .............3 Progress Summary ............................................................................................................... ......3 Method Development............................................................................................................. ....4 Pilot Study Sample Analyses.................................................................................................... .5 Appendix A Invoice............................................................................................................. ......7 Appendix B Protocol, Protocol Amendments & Deviations for Initial Pilot Study ..................9 Appendix C Pilot Study Sludge Acclimation-Raw Data .........................................................41 Appendix D Pilot Study Biol ogical Phase-Raw Data..............................................................63 Appendix E Analysis of Acid Analytes in Styrene Trapping Media by LC/MS/MS............208 Appendix F Method Installation Trial Raw Data ..................................................................214 Appendix G Analytical Standard, Stock Solu tion and Reagent Preparation Information.....255Does Not Contain Confidential Business Information (CBI) 2010_July_Progress Report 3 Introduction This document presents the pr ogress of the technical work for the Biodegradation Testing Supplemental Environmental Project (“Biodegr adation SEP”) performed from 23 July 2010 up to 24 August 2010. Studies conducted under pr otocol will be examined by the Wildlife International Quality Assurance Unit for compliance with Good Laboratory Practices, Standard Operating Procedures and the specifie d protocol. Raw data that has been audited by the Wildlife International Quality Assurance will be included in the progress report, as appropriate. Progress Summary The total costs associated with the project for the period covered in this Progress Report are approximately $146,247.50 for labor and $18,900 for instrumentation. The invoice and an allocation of costs with associated tasks are presented in Appendix A. The protocol for the initial SCAS pilot st udy was signed by the Study Director, Laboratory Management and Sponsor’s Representative on 14 June 2010. The approved protocol, protocol amendments and deviati on are presented in Appendix B. The acclimation phase of the initial pilot st udy was started on 26 July 2010 and concluded on 02 August 2010. Data from the acclimation is presented in Appendix C. The sludge exhibited a clear supernatant after 30 minutes of settling and an effl uent dissolved organic carbon (DOC) concentration of le ss than 12 mg C/L throughout the acclimation phase. The DOC of the effluent was measured three ti mes and averaged approximately 7.5 mg C/L. The biological phase of the initial pilot study started on 02 August 2010 and concluded on 16 August 2010. Data from the biological phase is presented in Appendix D. Sampling of the aqueous supernatant, sludge solids, and gas pha se trapping media for analysis of study analytes was conducted on August 04, 06, 09, 11, 13 and 16. The technical work consisted of method development trials specific for LC/MS/MS determination of the telomer acids in styrene-trapping medi a, conduct of an analytical methods installation trial and analytical proces sing of samples from the initial pilot study.Does Not Contain Confidential Business Information (CBI) 2010_July_Progress Report 4 Bench processing and determination of study an alytes in the pilot study samples by GC/MS and LC/MS/MS continues. Method Development For the LC/MS/MS analysis of surrogate re covery standards (M+8 PFOA and M+2 PFHxA) and acid analytes (PFOA, PFNA, PFDA, PFUA, 7-3 Acid, 8-2 FTCA and FTUCA adsorbed onto sytrene ( e.g, Xad-2) trapping media, experiments were conducted to further investigate the matrix suppression effects noted in the July 2010 Progress Report. Methanol (MeOH) acidified with lower stre ngth nitric acid and MeOH acidified with alternative acids were investigated for use in extracting acid analyt es from Xad-2 trapping media. The objective was finding an extrac tion solvent system that maintained the quantitative extractability of 0.1% HNO 3 in MeOH while minimizing the LC/MS/MS instrumental response suppression previously no ted when employing this solvent system to extract styrene-trapping media. The use of lower strength (0.01%) nitric or equi valent strength (0.1%) hydrochloric acid (less oxidative than HNO 3 ) in MeOH maintained the quantitat ive extractability of 0.1% HNO 3 but did not provide a significant impact in reduci ng the severity of the suppression effects (Appendix E). As seen for formic acid, a weaker acid additive, such as acetic acid did not meet the extractability criterion. (Note: 7-3 Acid data are pr esented without application of the M+2 8-2 FTCA matrix standard). In the methods installation trial, appropriate amounts of all study analytes were fortified into SCAS mixed-liquor and trapping media. The samples were then processed according to the applicable method outlines and the analytes were determined by LC/MS/MS or GC/MS as appropriate. Mass balance was demonstrated fo r all acid analytes a nd quantitative recovery was calculated for the acid analyte surrogate recovery standards in the SCAS matrices (Appendix F). For volatile anal ytes, consistent and low ma ss balance was achieved while quantitative recovery of the su rrogate recovery standard (M+4 8-2 FTOH) from each matrix was calculated. This finding suggested that eith er a systematic procedural error occurred upon fortification into the mixed li quor, or that the analytes were lost via volatilization (testDoes Not Contain Confidential Business Information (CBI) 2010_July_Progress Report 5 system not maintained gas-tight) or proc edural losses due to adsorption onto handling/transfer apparatus ( e.g. , syringe plungers) during th e dosing and initial processing steps. Experiments are planned to conduct th e same experiment under controlled conditions ( e.g ., using reagent water). During the conduct of the installatio n trial, an experiment was also completed to evaluate any potential loss of MTBE upon frozen storage of samples taken through initial processing and then held. The potential for losses due to multiple punctures through the original sample septa and the integrity of the septa seals used were the objectives of this exercise. Quantitative MTBE recovery was obtained for al l experimental storage conditions evaluated (see end of Appendix F). Pilot Study Sample Analysis Study samples have been processed for the analys is of the test system, domestic sewage and Intervals 1, 2 and 3. Alt hough it is premature at this j uncture to summarize surrogate recovery standard recoveries, analyte concentrat ions and analyte concentration trends in the SCAS test systems, several analytical observations are worth noting here. A small but reversible decrease in telomer alcohol response wa s evident with processing of study samples for GC/MS determination. The phe nomenon appeared to be a substantially mitigated version of the non-specific matrix effect that mandated use of a back-partition MTBE extract clean-up step during method de velopment. While of concern, the current observed sensitivity changes are far more subtle than the complete and irreversible response loss noted in previous Progress Reports. The current decrease was only evident after hundreds of sample injections on the same GC/M S platform and as stated above, does appear reversible at this time. Subsequent to the analysis of polymer trea tment extracts from the first sampling event (Interval 1) of sludge, an interf erence associated with the tail of the 8-2 FTOH peak (primary and confirmatory ions) was evid ent in the chro matography that appeared column related and persistent. The interference was present in the 8-2 FTOH peaks for trailing calibration standards from the same sequence and did ca rryover into the initial standards of theDoes Not Contain Confidential Business Information (CBI) 2010_July_Progress Report 6 subsequent sequence. The interference ma y be eliminated by using enough MTBE rinse injections following the minimum extract diluti ons (“-X1”) of the polymer treatments in a given sequence or prior to commencing the next sequence. The impact of the matrix effect on the study data has been mitigated in large measure through judicious choice of integration parameters. The levels of 8-2 FTOH and 7-2 sFTOH were so elevated in the sludge and primary styrene trapping compartments of the positive control test chamber that quantitation of all volatiles in these specific sampling compartments was not feasible using the current methods. The completion of these analyses will require mo dification of current GC/MS processing and instrumental determination methods and will ne ed to be conducted subsequent to all other analyses. Specifically, two sepa rate GC/MS methods will be re quired to adequately quantify volatile analytes in these selected sample extracts. The first method will allow for determination of 8-2 FTOH (and perhaps other rela tively high concentrat ion volatile analytes such as 7-2 sFTOH) using volumetric dilutions so high that use of the matrix internal standard will not be required ( e.g. , perform secondary and/or ter tiary serial dilutions with MTBE only). The second method will exclude 8- 2 FTOH as a SIM window in the method to allow LOQ level determinations of other volatile analytes present at trace levels. Records of stock and standard preparations an d all reagent preparations since the July 2010 Progress Report are provided in Appendix G.Does Not Contain Confidential Business Information (CBI) 2010_July_Progress Report 7 Appendix A InvoiceDoes Not Contain Confidential Business Information (CBI)2010_July_Progress Report8Allocation of CostsMonth, Year Tasks TotalDesign andProcurement ofSCAS Units(Task A)Acquisitionof Standards(Task B)Adaptation ofAnalyticalMethod (Task C)Pilot StudiesTask (D)Refinement ofMethodsTask (E)MethodValidations (Task F)StorageStability(Task G)BiodegradationStudies(Task H)InstrumentLeaseDec 2009 $1,803 $1,085 $42,422 $45,310Jan 2010 $29,227 $29,227Feb 2010 $1,300 $36,745 $38,045Mar 2010 $14,350 $14,350 $18,900Apr 2010 $570 $15,935 $16,505 $18,900May 2010 $760 $71,860 $72,620 $18,900June 2010 $57,870 $35,003 $92,873 $18,900July 2010 $42,430 $15,130 $57,560 $18,900August 2010 $32,530 $113,718 $146,248 $18,900Labor Total $1,803 $3,715 $343,369 $163,850 $512,737Materials $6,800 $10,500 $20,644 $1,724 $39,669Total Cost $8,603 $14,215 $364,4013 $165,574 $552,406 $113,400Does Not Contain Confidential Business Information (CBI) 2010_July_Progress Report 9 Appendix B Protocol, Protocol Amendments & Deviations for Initial Pilot StudyDoes Not Contain Confidential Business Information (CBI) 2010_July_Progress Report 10Does Not Contain Confidential Business Information (CBI) 2010_July_Progress Report 11Does Not Contain Confidential Business Information (CBI) 2010_July_Progress Report 12Does Not Contain Confidential Business Information (CBI) 2010_July_Progress Report 13Does Not Contain Confidential Business Information (CBI) 2010_July_Progress Report 14Does Not Contain Confidential Business Information (CBI) 2010_July_Progress Report 15Does Not Contain Confidential Business Information (CBI) 2010_July_Progress Report 16Does Not Contain Confidential Business Information (CBI) 2010_July_Progress Report 17Does Not Contain Confidential Business 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41 Appendix C Pilot Study Sludge Acclimation – Raw DataDoes Not Contain Confidential Business Information (CBI) 2010_July_Progress Report 42Does Not Contain Confidential Business Information (CBI) 2010_July_Progress Report 43Does Not Contain Confidential Business Information (CBI) 2010_July_Progress Report 44Does Not Contain Confidential Business Information (CBI) 2010_July_Progress Report 45Does Not Contain Confidential Business Information (CBI) 2010_July_Progress Report 46Does Not Contain Confidential Business Information (CBI) 2010_July_Progress Report 47Does Not Contain Confidential Business Information (CBI) 2010_July_Progress Report 48Does Not Contain Confidential Business Information (CBI) 2010_July_Progress Report 49Does Not Contain Confidential Business Information (CBI) 2010_July_Progress Report 50Does Not Contain Confidential Business Information (CBI) 2010_July_Progress Report 51Does Not Contain Confidential Business Information (CBI) 2010_July_Progress Report 52Does Not 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