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2011_April_Progress Report 1 PROGRESS REPORT: APRIL 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

April 29, 2011

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2011_March _Progress Report 2 10 Table of Contents

Title Page .................................................................................................................... ...............1
Table of Contents ............................................................................................................. ..........2
Introduction .................................................................................................................. ..............3 Progress Summary .............................................................................................................. .......3 Appendix A. Invoice .......................................................................................................... .......6 Appendix B. Experimental Details and Summary Data ...........................................................8 Appendix C. Analytical Stock, Standard Solu tion, and Reagent Preparation Information ....14 Appendix D. Current Analyte Inventory a nd Revised Certificates of Analysis .....................27Does Not Contain Confidential Business Information (CBI)

2011_March _Progress Report 3 10 Introduction This document presents the pr ogress of the technical work for the Biodegradation Testing Supplemental Environmental Project (“Biode gradation SEP”) performed from 28 March 2011 up to 24 April 2011. Stud ies conducted under 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 $9,820 for labor and $18,900 for in strumentation. The invoice and an
allocation of costs with associated tasks are presented in Appendix A.

Technical work conducted over the period cove red by this progress report consisted of a method installation trial to demonstrate quantita tive acid analyte proced ural recovery using the revised activated sludge mi xed-liquor procedure. Appendi x B provides a compilation of raw data including the method outline and data summary sheets for the experimental trial
described herein.

The method trial consisted of preparation and LC /MS/MS analysis of acid analytes (PFOA, PFNA, PFDA, PFUA, 7-3 Acid, 8-2 FTCA and 8-2 FTUCA) in each of four sample preparations. The sample set included a matrix blank (freshly collected and solids adjusted activated sludge mixed liquor fortified w ith 10 ng of M+8 PFOA surrogate recovery standard), a reagent fortificati on (polished water fortified with 10 ng of each acid analyte and M+8 PFOA), and duplicate matrix fortification sa mples (freshly collected and solids adjusted activated sludge mixed liquor each fortified wi th 10 ng of each acid analyte and M+8 PFOA). The samples were processed using the propo sed revised activated sludge mixed liquor method (2011_April_12 Method Outline Mixed Li quor) that was recently presented and
distributed to EPA and DuPont. The cited me thod outline was followed with the exception that all procedures pertaining to volatile an alyte processing and determination were omitted for the purposes of the present trial.Does Not Contain Confidential Business Information (CBI)

2011_March _Progress Report 4 10 As noted in our earlier method work, the distribution of acid analyte mass across the four sequential extractions employed (“A” and “B” using MTBE and “C” and “D” using 1:1 (v/v) ACN: MeOH) was a function of the presence or absence of sludge solids in the matrix and acid analyte carbon chain length.

Data that has not been adjusted for procedur al recovery using mass- labeled surrogates shows that the perfluorinated acid analytes (PFOA, PFNA, PFDA and PFUA) had a consistent measured mass (recovered mass ranged between 84%-88% of the nomi nal fortified mass) independent of matrix constituents a nd the presence of sludge solids.

The mass recovery trend for 8-2 FTUCA mirrored that for the perfluorinated acids, although 8-2 FTUCA mass recovery was slightly lower ov erall (range 70%-73%). In contrast to the trends seen for the perfluorinated acids and 8- 2 FTUCA, the reagent fortification sample had significantly better unco rrected mass recoveries for 7-3 Acid (91%) and 8-2 FTCA (77%) compared with those for the replicate fort ified matrix samples (range 55%-62%). The presence of matrix constituents clearly impacts both the mass distribution across sequential
extractions and overall mass bala nce for these two acid analytes.

The summary sheet also presents mass balance w ith the data adjusted/corrected for both the M+8 PFOA and M+2 PFHxA surrogate recovery st andards and with the data adjusted for M+8 PFOA only. Again, M+8 PFOA reflects mass losses across the overall procedure while M+2 PFHxA applies strictly to procedural losses during the ion-pairing cleanup step following individual ACN: MeOH (“ C” or “D”) extractions.

Application of both mass-labele d surrogates yielded acceptabl e results for 7-3 Acid, 8-2 FTCA and 8-2 FTUCA (range 75%-98% in activat ed sludge mixed liquor), but resulted in an overcompensation of the perfl uorinated acid recoveries (r ange 122%-127% in activated sludge mixed liquor). The high bias in the ad justed perfluorinated acid recoveries was attributed to the comparatively higher volatility of M+2 PFHxA compared to the longer chain perfluorinated acid analytes in combination with an easily rectifie d procedural error.Does Not Contain Confidential Business Information (CBI)

2011_March _Progress Report 5 10 The mass recovery for M+2 PFHxA was consiste ntly lower (by 8%-14%) in “C” extracts compared with the quantitative recoveries ob tained for the “D” extracts. M+2 PFHxA was added (5 ng fortified mass) at the same time to all “C” and “D” ACN: MeOH extracts using
the same stock and fortification method. Th is would exonerate temporal processing
differences between the “C” and “D” extracti on rounds and any procedural bias due to differences in fortification technique or equipment. However, upon review of the ACN: MeOH extract nitrogen ev aporation step (using a newly procured manifold), it was noted that the “C” extracts had been arrayed very close to one of the bath heating elements while the “D” extracts were more centrally loca ted within the bath. With insufficient water ballast or movement, localized heating might ha ve occurred in the immediate vicinity of the heating element. This may have resulted in an upward deviation from the target 35oC bath temperature with an attendant loss of the relatively more volatile M+2 PFHxA.

Since a relatively greater pr oportion of analyte mass was ex tracted in th e earlier MTBE extraction rounds compared with the perfluorinat ed acid analytes, non- perfluorinated acid analyte recoveries were not biased to the same degree. Therefore, the most appropriate data treatment in our judgmen t was to assume quantitative recove ry of the perflu orinated acid analytes during both extract evaporation steps and to apply only the overall method surrogate M+8 PFOA to this particular data set. Review of the data corrected only for M+8 PFOA shows that acceptable mass recoveries were obtai ned for all seven acid analytes using the newly refined methodology.

Records of analytical stock, standard soluti on, and reagent preparation performed during the review period are presented in Appendix C. An updated an alyte inventory and revised certificates of analysis for speci fied manufactured lots for tw o of the acid analytes (PFNA and 8-2 FTUCA) are presented in Appendix D.Does Not Contain Confidential Business Information (CBI)

2011_March _Progress Report 6 10 Appendix A
InvoiceDoes Not Contain Confidential Business Information (CBI)2011_April_Progress Report7Allocation 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 $18,900February 2011 $2,945 $2,945 $18,900March 2011 $8,075 $8,075 $18,900April 2011 $9,820 $9,820 $18,900Labor Total $1,803 $3,715 $344,719 $428,988 $44,588 $3,610 $827,422Materials $6,800 $10,500 $20,644 $1,724 $39,669Total Cost $8,603 $14,215 $365,363 $430,712 $44,588 $3,610 $867,091 $264,600Does Not Contain Confidential Business Information (CBI)

2011_April_Progress Report 8 Appendix B Experimental Details and Summary DataDoes Not Contain Confidential Business Information (CBI)

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2011_March _Progress Report 14 10 Appendix C Analytical Stock, Standard Solution, and Reagent Preparation InformationDoes Not Contain Confidential Business Information (CBI)

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2011_March _Progress Report 27 10 Appendix D Current Analyte Inventory and Revi sed Certificates of AnalysisDoes Not Contain Confidential Business Information (CBI)

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