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2011_July_Progress Report 1 PROGRESS REPORT: JULY 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

July 29, 2011

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2011_March _Progress Report 2 Table of Contents

Title Page ..................................................................................................................... ..............1

Table of Contents.............................................................................................................. .........2

Introduction................................................................................................................... .............3

Progress Summary ............................................................................................................... ......3

Appendix A. Invoice........................................................................................................... ......4

Appendix B. Task E: Modifications and Refinements to Test Methods .................................6Does Not Contain Confidential Business Information (CBI)

2011_March _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 25 June 2011

up to 23 July 2011. Studies c onducted under protocol 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 $2,965 for labor and $18,900 for in strumentation. The invoice and an

allocation of costs with associated tasks are presented in Appendix A.

Work conducted over the period covered by this progress report consisted of the review and

discussion of data from test substance characterization and di scussion of Task E items with

stakeholders. Work included revision of the Scope of Work (SOW) presented in the June

2011 Progress Report. The SOW was composed to investigate approaches to eliminate or

avoid carryover and enhancement effects observed and documented for GC/MS

determinations of 6-2 FTOH, 8-2 FTOH and 10-2 FTOH in MTBE extracts during the initial

pilot study with Group A1 polymer. The in itial SOW document presented an outline for

developing an LC/MS/MS method for these alcohol s as a potential solution to the problem.

Another approach presented in th e interim is use of increased centrifugation force and time to

minimize or eliminate co-extracted polymer in th e MTBE extract as a prelude to the original

GC/MS determination method. The SOW was theref ore revised to include this experimental

option (Appendix B).Does Not Contain Confidential Business Information (CBI)

2011_March _Progress Report 4 Appendix A

InvoiceDoes Not Contain Confidential Business Information (CBI)2011_July_Progress Report5Allocation of CostsMonth, Year Tasks TotalDesign andProcurement ofSCAS Units(Task A)Acquisitionof Standards(Task B)Adaptation ofAnalyticalMethod (Task C)Pilot StudiesTask (D)Refinementof MethodsTask (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 $18,900February 2011 $2,945 $2,945 $18,900March 2011 $8,075 $8,075 $18,900April 2011 $9,820 $9,820 $18,900May 2011 $5,770 $5,770 $18,900June 2011 $2,850 $190 $3,040 $18,900July 2011 $2,965 $2,965 $18,900Labor Total $1,803 $3,715 $344,719 $428,988 $55,993 $3,800 $839,017Materials $6,800 $10,500 $20,644 $1,724 $39,669Total Cost $8,603 $14,215 $365,363 $430,712 $55,993 $3,800 $878,686 $321,3009Does Not Contain Confidential Business Information (CBI)

2011_July_Progress Report 6 Appendix B. Task E: Modifications and Refinements to Test Methods

From the document Task E: Refinement of Methods (2010_November_02_Task E_detail.doc), Item 3 is the only remaining analytical method issu e identified by Wildlife International, Ltd. following the initial pilot study not yet resolved following consultation with DuPont/EPA. The original stat ement is reproduced below (with minor

clarifications/corrections indicated in parenthesis):

3) After injection of sludge extracts from poly mer treatment SCAS chambers, an enhanced

(GC/MS) signal for 6-2 FTOH, 8-2 FTOH, and 10-2 FTOH (was) observed and the

enhanced signal persisted for subsequent injections of standards.

The consensus of the stakeholders is that the MTBE activated sludge extracts contain polymer co-extractives, which are degrading under the thermal conditions of the GC/MS inlet to form the alcohols. One approach is to migrate from GC/MS to LC/MS/MS for determination of 6-2 FTOH, 8-2 FTOH, and 10-2 FTOH in MTBE extracts.

A. Fortify replicate negative control sludge matrices with 1. 6-2 FTOH, 8-2 FTOH and 10-2 FTOH 2. Fluorotelomer Product Group A1 polymer 3. A1 polymer and 6-2 FTOH, 8-2 FTOH and 10-2 FTOH

B. Process following the sample preparat ion and LC/MS/MS instrumental conditions presented in Biodegradation SEP, Attachment D , Analytical Methods : Szostek, B.; Prickett, K.B.; Buck, R.C., Determina tion of fluorotelomer alcohols by liquid chromatraphy/tandem mass spectrometry in water. Rapid Communications in Mass Spectrometry 2006, 20, (19), 2837-2844.

1. Perform infusion experiments using LC/MS or LC/MS/MS to evaluate the feasibility of performing adduct fr ee analyses in our facilities. 2. If it is not viable to monitor deproton ated parent ions using LC/MS/MS due to the prevalence of formic/acetic acid residuals in our systems, develop alternative strategies using appropriate adducts of the th ree target alcohol analytes. 3. Use LC/MS or LC/MS/MS depending on the requisite sensitivity and selectivity requirements to produce an appropriate and robust method. 4. Investigate a combination of deprotonated parent ion and adduct monitoring in order to minmize instrumental setup time for future pilot and definitive SCAS

trials.

Estimated budget: $30,000

Estimated time frame: 4 weeks

Another approach is to centrifuge the MTBE: reagent water solvent partion created during the back-partition MTBE extract clean-up step in the proposed activated sludge mixed liquor method at a higher relative centrif ugal force and for a longer durat ion than the settings usedDoes Not Contain Confidential Business Information (CBI)

2011_March _Progress Report 7 in the initial pilot study for sludge solids and s upernatant SCAS matrices . The objective is to physically remove polymer co-extractives from the final cleaned MTBE extract prior to the

GC/MS determination.

A. Fortify replicate reagent water samples with variable amounts of Fluorotelomer Product Group A1 polymer corresponding to the range of polymer mass calculated to have been theoretically pr esent in 10-mL mixe d-liquor subsamples during the latter stages of th e initial pilot study. Prepar e replicate fortifications and perform sequential “A” and “B” MT BE extractions for each of the combinations of polymer mass and centr ifugation force (during back-partition step) to be investigated per the most current activa ted sludge mixed liquor method

outline.

B. Transfer replicate aliquots from each MTBE extract into separate plastic centrifuge tubes containing reagent water for a final solvent ration of 1 part MTBE: 2 parts H 2 O and centrifuge at se lected centrifugation force settings (3800 xg and greater) for 30 minutes. Note: pre-weigh the empty plastic centrifuge tubes and use a common centrifugation for ce for any given pairing of “A” and

“B” MTBE extracts.

C. For each sample, make physical observations for evidence any polymer precipitate and take aliquouts of the supernatant for GC/MS determination of 6-2 FTOH, 8-2 FTOH and/or 10-2 FTOH to measure any potential relative differences in alcohol response. Prep are post-spike samples of the desired telomer alcohol(s) as needed.

D. Weigh the containers after processing for potential quantitation of any precipitate that may or may not be apparant in the plastic vessels.

Estimated budget: $8,000

Estimated time frame: 1 week