PROGRESS REPORT: JANUARY 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 

January 26, 2011 

Page 1 of 62 
Does Not Contain Confidential Business Information (CBI)

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 Adjusted for Procedural Recovery .............................................49 

Appendix D Analytical Stock, Standard Solution, and Reagent Preparation Information......55 
Does Not Contain Confidential Business Information (CBI)

2011_January_Progress Report 3

Introduction 

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

Supplemental Environmental Project (“Biodegradation SEP”) performed from 27 December 

2010 up to 21 January 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 $15,570 for labor and $18,900 for instrumentation. 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 conduct of 

method refinement trials. 

The technical work consisted of three method development 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 (reference file 2010_November_02_Task 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 procedural 
Does Not Contain Confidential Business Information (CBI)

2011_January_Progress Report 4

recoveries and analyte mass balance have been 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 balance adjusted for surrogate recovery standard 

procedural recovery has been appended to each of the original summary sheets. 

The results from the first trial in reagent water indicated 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 the surface of a given matrix in prior method 

development trials where acceptable procedural recoveries for the volatile analytes had been 

demonstrated for fortifications performed in segregated test compartments (sludge solids and 

aqueous supernatant). In this first trial, the same staff member performed analyte 

fortifications both above and below the surface 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 those obtained in the original sludge trial. 

Mass balance for the alcohol analytes was comparable to that calculated for the M+4 

8-2 FTOH surrogate recovery standard. In contrast, 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 calculated 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 surface 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 function of compound class for these volatile analytes suggests that 

wholesale mass losses due to a compromised sub-sampling event (e.g. failure to maintain a 

gas-tight test system during phase separation steps) 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 mechanism (e.g., hydrolysis) might be plausible 

explanations for the poor mass balances obtained for the iodide 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 volatile analytes, the decision 

was made to proceed with the second trial, which now incorporated the SCAS activated 

sludge matrix into the experimental design. The objective was to see whether or not the data 

trends obtained for fortified reagent water could 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 replicated and confirmed 

the original experimental results from July 2010 (summary results from the 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 balance (relative to mass balance results in 

reagent water) in the presence of the activated sludge. 

The objective of the third and final method development trial was to focus on potential 

abiotic and biotic kinetics. Two sample sets were prepared and each was further processed 

without phase separation. One set of samples consisting of reagent water and replicate 

activated sludge samples was fortified and then immediately processed. A second set of 

samples consisting of reagent water and replicate activated sludge samples was fortified at 

the same time as the first set, but was left on the bench top at ambient conditions for 2 hours 

prior to the initial addition of MTBE extraction solvent (containing M+4 8-2 FTOH surrogate 

recovery standard). The selected time delay bracketed those estimated in previous method 

development trials (>1 hour) with phase separation. The fortification procedure was as 

described for the previous (second) trial. The results from the third method development trial 

(Appendices B and C) indicated that the time delay from analyte fortification until 

introduction of extraction solvent could have been the major factor to explain the low mass 

balances obtained in the original 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 acrylate analytes was still low upon immediate processing and did 
Does Not Contain Confidential Business Information (CBI)

2011_January_Progress Report 6

not significantly change thereafter. In contrast, mass balance for the alcohol analytes was 

acceptable upon immediate processing and, like the iodides and acrylate analytes, did not 

significantly change thereafter. The finding implies that the iodide and acrylate analytes are 

irreversibly bound to glass under these MTBE extraction 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 supernatant). For the alcohol analytes, these 

MTBE extraction conditions (i.e., solvent-to-matrix proportions, and extraction times and 

extraction action used in the current methods) appeared adequate to quantitatively remove the 

materials from the walls of the glass vessel. The surrogate procedural recovery remained 

acceptable in each case. 

In activated sludge, all analyte mass balance was deteriorated with processing time delay and 

more significantly so for the alcohol analytes. Again, surrogate procedural recovery 

remained acceptable in each case. We speculate 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 sludge 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 separation of the same corresponding matrices 

provided additional evidence that the interactive chemical properties of the analytes, MTBE 

extraction solvent, glass test vessels and the 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 preparations required to perform 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 

Invoice 
Does Not Contain Confidential Business Information (CBI)

2011_January_Progress Report 

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 Labor Total $1,803 $3,715 $344,719 $428,988 $23,748 $3,610 Materials $6,800 $10,500 $20,644 $1,724 Total Cost $8,603 $14,215 $365,363 $430,712 $23,748 $3,610 
Does Not Contain Confidential Business Information (CBI)

2011_January_Progress Report 9

Appendix B 

Experimental Details and Results, Task E, Item 1 
Does Not Contain Confidential Business Information (CBI)

2010_SeptemberMay_Progress Report 10
Does Not Contain Confidential Business Information (CBI)

2010_SeptemberMay_Progress Report 10
Does Not Contain Confidential Business Information (CBI)

2010_SeptemberMay_Progress Report 10
Does Not Contain Confidential Business Information (CBI)

2010_SeptemberMay_Progress Report 10
Does Not Contain Confidential Business Information (CBI)

2010_SeptemberMay_Progress Report 10
Does Not Contain Confidential Business Information (CBI)

2010_SeptemberMay_Progress Report 10
Does Not Contain Confidential Business Information (CBI)

2010_SeptemberMay_Progress Report 10
Does Not Contain Confidential Business Information (CBI)

2010_SeptemberMay_Progress Report 10
Does Not Contain Confidential Business Information (CBI)

2010_SeptemberMay_Progress Report 10
Does Not Contain Confidential Business Information (CBI)

2010_SeptemberMay_Progress Report 10
Does Not Contain Confidential Business Information (CBI)

2010_SeptemberMay_Progress Report 10
Does Not Contain Confidential Business Information (CBI)

2010_SeptemberMay_Progress Report 10
Does Not Contain Confidential Business Information (CBI)

2010_SeptemberMay_Progress Report 10
Does Not Contain Confidential Business Information (CBI)

2010_SeptemberMay_Progress Report 10
Does Not Contain Confidential Business Information (CBI)

2010_SeptemberMay_Progress Report 10
Does Not Contain Confidential Business Information (CBI)

2010_SeptemberMay_Progress Report 10
Does Not Contain Confidential Business Information (CBI)

2010_SeptemberMay_Progress Report 10
Does Not Contain Confidential Business Information (CBI)

2010_SeptemberMay_Progress Report 10
Does Not Contain Confidential Business Information (CBI)

2010_SeptemberMay_Progress Report 10
Does Not Contain Confidential Business Information (CBI)

2010_SeptemberMay_Progress Report 10
Does Not Contain Confidential Business Information (CBI)

2010_SeptemberMay_Progress Report 10
Does Not Contain Confidential Business Information (CBI)

2010_SeptemberMay_Progress Report 10
Does Not Contain Confidential Business Information (CBI)

2010_SeptemberMay_Progress Report 10
Does Not Contain Confidential Business Information (CBI)

2010_SeptemberMay_Progress Report 10
Does Not Contain Confidential Business Information (CBI)

2010_SeptemberMay_Progress Report 10
Does Not Contain Confidential Business Information (CBI)

2010_SeptemberMay_Progress Report 10
Does Not Contain Confidential Business Information (CBI)

2010_SeptemberMay_Progress Report 10
Does Not Contain Confidential Business Information (CBI)

2010_SeptemberMay_Progress Report 10
Does Not Contain Confidential Business Information (CBI)

2010_SeptemberMay_Progress Report 10
Does Not Contain Confidential Business Information (CBI)

2010_SeptemberMay_Progress Report 10
Does Not Contain Confidential Business Information (CBI)

2010_SeptemberMay_Progress Report 10
Does Not Contain Confidential Business Information (CBI)

2010_SeptemberMay_Progress Report 10
Does Not Contain Confidential Business Information (CBI)

2010_SeptemberMay_Progress Report 10
Does Not Contain Confidential Business Information (CBI)

2010_SeptemberMay_Progress Report 10
Does Not Contain Confidential Business Information (CBI)

2010_SeptemberMay_Progress Report 10
Does Not Contain Confidential Business Information (CBI)

2010_SeptemberMay_Progress Report 10
Does Not Contain Confidential Business Information (CBI)

2010_SeptemberMay_Progress Report 10
Does Not Contain Confidential Business Information (CBI)

2010_SeptemberMay_Progress Report 10
Does Not Contain Confidential Business Information (CBI)

2010_SeptemberMay_Progress Report 10
Does Not Contain Confidential Business Information (CBI)

2010_SeptemberMay_Progress Report 10

Appendix C 

Summary Data Adjusted for Procedural Recovery 
Does Not Contain Confidential Business Information (CBI)

2010_SeptemberMay_Progress Report 10
Does Not Contain Confidential Business Information (CBI)

2010_SeptemberMay_Progress Report 10
Does Not Contain Confidential Business Information (CBI)

2010_SeptemberMay_Progress Report 10
Does Not Contain Confidential Business Information (CBI)

2010_SeptemberMay_Progress Report 10
Does Not Contain Confidential Business Information (CBI)

2010_SeptemberMay_Progress Report 10
Does Not Contain Confidential Business Information (CBI)

2010_SeptemberMay_Progress Report 10

Appendix D 

Analytical Stock, Standard Solution, and Reagent Preparation Information 
Does Not Contain Confidential Business Information (CBI)

2010_SeptemberMay_Progress Report 10
Does Not Contain Confidential Business Information (CBI)

2010_SeptemberMay_Progress Report 10
Does Not Contain Confidential Business Information (CBI)

2010_SeptemberMay_Progress Report 10
Does Not Contain Confidential Business Information (CBI)

2010_SeptemberMay_Progress Report 10
Does Not Contain Confidential Business Information (CBI)

2010_SeptemberMay_Progress Report 10
Does Not Contain Confidential Business Information (CBI)

2010_SeptemberMay_Progress Report 10
Does Not Contain Confidential Business Information (CBI)

2010_SeptemberMay_Progress Report 10