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2011_April_Progress Report 1

PROGRESS REPORT: APRIL 2011 

BIODEGRADATION TESTING SUPPLEMENTAL 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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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 Solution, and Reagent Preparation Information ....14 

Appendix D. Current Analyte Inventory and Revised Certificates of Analysis .....................27 
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Introduction 

This document presents the progress of the technical work for the Biodegradation Testing 

Supplemental Environmental Project (“Biodegradation SEP”) performed from 28 March 

2011 up to 24 April 2011. Studies 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. 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 $9,820 for labor and $18,900 for instrumentation. The invoice and an 

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

Technical work conducted over the period covered by this progress report consisted of a 

method installation trial to demonstrate quantitative acid analyte procedural recovery using 

the revised activated sludge mixed-liquor procedure. Appendix 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 with 10 ng of M+8 PFOA surrogate recovery 

standard), a reagent fortification (polished water fortified with 10 ng of each acid analyte and 

M+8 PFOA), and duplicate matrix fortification samples (freshly collected and solids adjusted 

activated sludge mixed liquor each fortified with 10 ng of each acid analyte and M+8 PFOA). 

The samples were processed using the proposed revised activated sludge mixed liquor 

method (2011_April_12 Method Outline Mixed Liquor) that was recently presented and 

distributed to EPA and DuPont. The cited method outline was followed with the exception 

that all procedures pertaining to volatile analyte processing and determination were omitted 

for the purposes of the present trial. 
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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 procedural 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 nominal fortified mass) 

independent of matrix constituents and 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 overall (range 70%-73%). In contrast to the 

trends seen for the perfluorinated acids and 8-2 FTUCA, the reagent fortification sample had 

significantly better uncorrected mass recoveries for 7-3 Acid (91%) and 8-2 FTCA (77%) 

compared with those for the replicate fortified matrix samples (range 55%-62%). The 

presence of matrix constituents clearly impacts both the mass distribution across sequential 

extractions and overall mass balance for these two acid analytes. 

The summary sheet also presents mass balance with the data adjusted/corrected for both the 

M+8 PFOA and M+2 PFHxA surrogate recovery standards 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-labeled surrogates yielded acceptable results for 7-3 Acid, 8-2 

FTCA and 8-2 FTUCA (range 75%-98% in activated sludge mixed liquor), but resulted in an 

overcompensation of the perfluorinated acid recoveries (range 122%-127% in activated 

sludge mixed liquor). The high bias in the adjusted 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 rectified procedural error. 
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The mass recovery for M+2 PFHxA was consistently lower (by 8%-14%) in “C” extracts 

compared with the quantitative recoveries obtained 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. This would exonerate temporal processing 

differences between the “C” and “D” extraction rounds and any procedural bias due to 

differences in fortification technique or equipment. However, upon review of the 

ACN: MeOH extract nitrogen evaporation 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 located within the bath. With insufficient water 

ballast or movement, localized heating might have 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 proportion of analyte mass was extracted in the earlier MTBE 

extraction rounds compared with the perfluorinated acid analytes, non-perfluorinated acid 

analyte recoveries were not biased to the same degree. Therefore, the most appropriate data 

treatment in our judgment was to assume quantitative recovery of the perfluorinated 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 obtained for all seven acid analytes using the 

newly refined methodology. 

Records of analytical stock, standard solution, and reagent preparation performed during the 

review period are presented in Appendix C. An updated analyte inventory and revised 

certificates of analysis for specified manufactured lots for two of the acid analytes (PFNA 

and 8-2 FTUCA) are presented in Appendix D. 
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Appendix A 

Invoice 
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Allocation of Costs 

Month, Year Tasks 

 Design and 

Procurement of 

SCAS Units 

(Task A) 

Acquisition 

of Standards 

(Task B) 

Adaptation of 

Analytical 

Method (Task C) 

Pilot Studies 

Task (D) 

Refinement of 

Methods 

Task (E) 

Method 

Validation

s (Task F) 

Storage 

Stability 

(Task G) 

Biod

dat

Stu

(Tas

Dec 2009 $1,803 $1,085 $42,422 Jan 2010 $29,227 Feb 2010 $1,300 $36,745 Mar 2010 $14,350 Apr 2010 $570 $15,935 May 2010 $760 $71,860 June 2010 $57,870 $35,003 July 2010 $42,430 $15,130 August 2010 $32,530 $113,718 September 2010 $1,350 $115,228 October 2010 $88,823 $3,613 November 2010 $42,690 $855 $570 December 2010 $18,385 $3,530 $3040 January 2011 $15,570 February 2011 $2,945 March 2011 $8,075 April 2011 $9,820 Labor Total $1,803 $3,715 $344,719 $428,988 $44,588 $3,610 Materials $6,800 $10,500 $20,644 $1,724 Total Cost $8,603 $14,215 $365,363 $430,712 $44,588 $3,610 
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Appendix B 

Experimental Details and Summary Data 
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Appendix C 

Analytical Stock, Standard Solution, and Reagent Preparation Information 
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Appendix D 

Current Analyte Inventory and Revised Certificates of Analysis 
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