PROGRESS REPORT : JANUARY 2011

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

January 26, 2011

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2011_January_Progress Report 2 Table of Contents

Title Page ..................................................................................................................... ..............1
Table of Contents.............................................................................................................. .........2
Introduction................................................................................................................... .............3 Progress Summary ............................................................................................................... ......3 Appendix A Invoice............................................................................................................. ......7 Appendix B Experimental Details and Results, Task E, Item 1 ................................................9 Appendix C Summary Data Adjust ed for Procedural Recovery .............................................49 Appendix D Analytical Stock, Standard Soluti on, and Reagent Preparation Information......55Does Not Contain Confidential Business Information (CBI)

2011_January_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 27 December 2010 up to 21 January 2011. Studies conducted unde r protocol will be examined by the Wildlife International Quality Assurance Unit for compliance with Good Laboratory Practices, Standard Operating Procedures and the specified protocol. Ra w 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 $15,570 for labor and $18,900 for in strumentation. Th e invoice and an
allocation of costs with associated tasks are presented in Appendix A.

Work conducted over the period covered by th is progress report consisted of conduct of
method refinement trials.

The technical work consisted of three method de velopment trials to investigate the low mass balance obtained for all volatile analytes upon fortifying and sampling SCAS activated sludge that was first reported in the August 2010 Progress Report.

The first and second method development trials summarized in the present Progress Report followed experimental design outlines as presented in the document titled “Task E: Refinement of Methods” (items 1A and 1B, respectively). The document was previously distributed to DuPont and EPA (referenc e file 2010_November_02_Tas k E_detail.doc). A third method development trial was indicated after reviewing findings from the first two trials. The additional trial focused specifically on the effect of processing delay (time from fortification to initial MTBE addition) on volatile analyte mass balance data.

Appendix B provides a compilation of raw data including method outlines, sample preparation records and initial data summary sheets for each of the experimental trials described herein. In summary sheets found in Appendix B, surrogate standard proceduralDoes Not Contain Confidential Business Information (CBI)

2011_January_Progress Report 4 recoveries and analyte mass balance have be en calculated without use of a procedural recovery correction factor (i.e ., using data obtained for M+ 4 8-2 FTOH surrogate recovery standard). In Appendix C, analyte mass balan ce adjusted for surrogate recovery standard procedural recovery has been appended to each of the original summary sheets.

The results from the first tr ial in reagent water indicate d that personnel and mode of fortification were not significant factors to explain the original results. In the original trial, fortifications were performed below the surface of the activated sludge. A different staff member had performed fortifications above th e surface of a given matrix in prior method development trials where acceptable procedural r ecoveries for the volatile analytes had been demonstrated for fortifications performed in se gregated test compartments (sludge solids and aqueous supernatant). In this first trial, the same staff member performed analyte
fortifications both above and below the su rface of reagent water in duplicate.

Mass balance for the alcohol analytes (6-2 FTOH, 7-2 sFTOH, 8-2 FTOH and 10-2 FTOH) in reagent water were significantly improved over th ose obtained in the original sludge trial. Mass balance for the alcohol analytes was co mparable to that calculated for the M+4 8-2 FTOH surrogate recovery standard. In co ntrast, mass balance for the iodide (PFOI, PFDI, 8-2 FTI and 10-2 FTI) and acrylate (8-2 FTAC) analytes in reagent water were low and very similar to those calcula ted in activated sludge for these same analytes in the original mass balance experiment. Mass recoveries in replicate preparations did appear more consistent using the below surf ace fortification procedure, but overall differences in mass balance when compared with the above surface fortification approach were not very significant.

Mass balance trends as a functi on of compound class for these vol atile analytes suggests that wholesale mass losses due to a compromised s ub-sampling event (e.g. fa ilure to maintain a gas-tight test system during phase separation st eps) was unlikely the cause of the uniformly low mass balance in the original trial. Given the absence of a SCAS matrix in this first trial, it is suggested that either non-reversible adsorption to the sampling/phase separation apparatus, or an abiotic transformation m echanism (e.g., hydrolysis ) might be plausible explanations for the poor mass balances obtained for the i odide and acrylate analytes.Does Not Contain Confidential Business Information (CBI)

2011_January_Progress Report 5 Although mass balance had not yet been demonstrated for all vo latile analytes, the decision was made to proceed with the second trial, which now incorporated the SCAS activated sludge matrix into the experime ntal design. The objective was to see whether or not the data trends obtained for fortified reagent water coul d be replicated in fortified activated sludge. The same individual who performed the analyte fortifications in the first trial also performed the analyte fortifications in the second trial using the below surface procedure.

The findings from the second method development trial essentially rep licated and confirmed the original experimental results from July 2010 (summary results from th e original trial have been reproduced in Appendices B and C for comparative purposes). Again, alcohol mass balances were reduced to levels comparable to those obtained for the iodide and acrylate analytes while surrogate recovery standard procedural recoveries in the segregated compartments remained acceptable. Perhaps a biotic factor was responsible for the significantly lowered alcohol analyte mass bala nce (relative to mass balance results in reagent water) in the presen ce of the activated sludge.

The objective of the third and final method development trial was to focus on potential
abiotic and biotic kinetics. Tw o sample sets were prepared and each was further processed without phase separation. One set of sample s consisting of reagen t water and replicate activated sludge samples was fortified and then immediately processed. A second set of samples consisting of reagent water and repli cate activated sludge samples was fortified at the same time as the first set, but was left on the bench top at ambien t conditions for 2 hours prior to the initial addition of MTBE extracti on solvent (containing M+4 8-2 FTOH surrogate recovery standard). The sele cted time delay bracketed thos e estimated in previous method development trials (>1 hour) with phase sepa ration. The fortification procedure was as described for the previous (second) trial. The results from the third method development trial (Appendices B and C) indicate d that the time delay from analyte fortification until
introduction of extraction solvent could have be en the major factor to explain the low mass balances obtained in the origin al activated sludge experiment.

In reagent water, mass balance did not appear to be a function of processing time. The mass
balance for the iodide and acr ylate analytes was still low upon immediate processing and didDoes Not Contain Confidential Business Information (CBI)

2011_January_Progress Report 6 not significantly change thereaf ter. In contrast, mass balanc e for the alcohol analytes was acceptable upon immediate processi ng and, like the iodides and acrylate analytes, did not significantly change thereafter. The finding implies that the iodi de and acrylate analytes are irreversibly bound to glass under these MTBE ex traction conditions. Adsorption to glass was clearly evident for all volatile analytes in each of the reagent water fortification trials performed in the present work. Specifically , the analyte mass present in the residual (approximately 1-mL) water split (pseudo solids) always exceeded that for the same analyte in the approximately 14-mL water split (pseudo s upernatant). For the alcohol analytes, these MTBE extraction conditions (i.e., solvent-to-m atrix proportions, and extraction times and extraction action used in the current methods) a ppeared adequate to qua ntitatively remove the
materials from the walls of the glass vessel. The su rrogate procedural recovery remained acceptable in each case.

In activated sludge, all analyte mass balance was deteriorated w ith processing time delay and more significantly so for the alcohol analyt es. Again, surrogate procedural recovery remained acceptable in each case. We specula te that the presence of the sludge solids resulted in sorptive losses due to irreversible binding for all of the volatile analytes under
these MTBE extraction conditions. The abundance of solids in the slud ge matrix may very well mitigate any influence of the glass test vessels on sorptive mass losses.

The reproducibility of these final trial findings for both reagent water and activated sludge with results obtained using physical phase se paration of the same corresponding matrices provided additional evidence that the interactive chemical properties of the analytes, MTBE extraction solvent, glass test vessels and th e SCAS sludge matrix, as opposed to randomized procedural errors or an inherent experimental design flaw in the phase separation procedure, were the most likely reason for the low mass balances obtained in the original trial.

Records of stock, standard and reagent prepara tions required to perf orm the experimental trials summarized in this Progress Report are provided in Appendix D.Does Not Contain Confidential Business Information (CBI)

2011_January_Progress Report 7 Appendix A
InvoiceDoes Not Contain Confidential Business Information (CBI)2011_January_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 $18,900September 2010 $1,350 $115,228 $116,578 $18,900October 2010 $88,823 $3,613 $92,478 $18,900November 2010 $42,690 $855 $570 $44,115 $18,900December 2010 $18,385 $3,530$3040 $24,955 $18,900January 2011 $15,570 $15,570 $18,900Labor Total $1,803 $3,715 $344,719 $428,988 $23,748 $3,610 $806,582Materials $6,800 $10,500 $20,644 $1,724 $39,669Total Cost $8,603 $14,215 $365,363 $430,712 $23,748 $3,610 $846,251 $207,900Does Not Contain Confidential Business Information (CBI)

2011_January_Progress Report 9 Appendix B
Experimental Details and Results, Task E, Item 1Does Not Contain Confidential Business Information (CBI)

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2010_ September May _Progress Report 10 Appendix C
Summary Data Adjusted for Procedural RecoveryDoes Not Contain Confidential Business Information (CBI)

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2010_ September May _Progress Report 10 Appendix D
Analytical Stock, Standard Solution, and Reagent Preparation InformationDoes Not Contain Confidential Business Information (CBI)

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