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PROGRESS REPORT: AUGUST 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

August 31, 2011

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Table of Contents

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

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

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

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

Appendix A. Invoice.................................................................................................................5

Appendix B. Comparison of Analytical Bias in Pilot Study and Centrifugation Trial.............7

Appendix C. Records of Sample, Stock, Standard and Reagent Preparation .........................13
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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 24 July 2011 

up to 27 August 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 $11,663 for labor, $1,813 for supplies 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 preparation of a 

draft definitive study protocol, efforts towards finalization of the pilot study report and of a 

centrifugation method trial (under Task E) as presented on pages 2-3 of the document 

Refinements Modifications to Methods 18 July 2011. The objective of the trial was 

determining if increasing the centrifugation force applied to MTBE extracts of SCAS 

matrices would suffice in removing Group A1 polymers from the extract. The results of the 

trial clearly indicated that (1) the analytical bias (e.g. calibration curve shift, carry-over and 

higher-than-expected concentrations, etc.) for telomer alcohol analytes documented for the 

latter sampling intervals of the initial pilot study for Group A1 polymers in MTBE extracts of 

SCAS sludge solids (see document Task E: Refinement of Methods

(2010_November_02_Task E_detail.doc), Item 3), was very well reproduced using reagent 

water as the matrix and (2) centrifugation at 45,000 x g for 30 minutes had no impact in 

reducing the analytical bias (Appendix B). These results were presented to EPA and DuPont 

at the monthly teleconference meeting on held on August 24, 2011.

To overcome the analytical bias, the laboratory now recommends to install LC/MS/MS 

methodology originally developed by DuPont for analysis of 6-2 FTOH, 8-2 FTOH and 10-2 

FTOH in MTBE extracts as detailed on pages 1-2 of the document “Refinements 
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Modifications to Methods 18 July 2011. An accurate assessment for the number of

sequential MTBE extractions required to quantitatively recover all of these analytes in an 

activated sludge mixed-liquor matrix in the presence of the polymer cannot be completed 

until an acceptable method is in place.

Centrifugation trial documentation to include specific sample preparation and processing 

procedures, a worksheet to calculate the concentration of polymer to be expected following 

12 sampling intervals in a definitive SCAS trial (for estimating polymer test substance 

fortification levels – 600 mg/L was selected), GC/MS sequence information, and records of 

stock, standard and reagent preparation is presented in Appendix C.
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Appendix A

Invoice
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Allocation of Costs

Month, Year Tasks Total

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 

Validations 

(Task F)

Storage 

Stability 

(Task G)

Biodegra

dation 

Studies 

(Task H)

Instrument

Lease

Dec 2009 $1,803 $1,085 $42,422 $45,310

Jan 2010 $29,227 $29,227

Feb 2010 $1,300 $36,745 $38,045

Mar 2010 $14,350 $14,350 $18,900

Apr 2010 $570 $15,935 $16,505 $18,900

May 2010 $760 $71,860 $72,620 $18,900

June 2010 $57,870 $35,003 $92,873 $18,900

July 2010 $42,430 $15,130 $57,560 $18,900

August 2010 $32,530 $113,718 $146,248 $18,900

September 2010 $1,350 $115,228 $116,578 $18,900

October 2010 $88,823 $3,613 $92,478 $18,900

November 2010 $42,690 $855 $570 $44,115 $18,900

December 2010 $18,385 $3,530 $3040 $24,955 $18,900

January 2011 $15,570 $15,570 $18,900

February 2011 $2,945 $2,945 $18,900

March 2011 $8,075 $8,075 $18,900

April 2011 $9,820 $9,820 $18,900

May 2011 $5,770 $5,770 $18,900

June 2011 $2,850 $190 $3,040 $18,900

July 2011 $2,965 $2,965 $18,900

August 2011 $950 $10,713 $11,663 $18,900

Labor Total $1,803 $3,715 $344,719 $428,988 $66,705 $3,800 $849,730

Materials $6,800 $10,500 $20,644 $1,724 $1,813 $41,482

Total Cost $8,603 $14,215 $365,363 $430,712 $68,518 $3,800 $891,212 $340,200

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Appendix B

Comparison of Analytical Bias in Pilot Study and Centrifugation Trial
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Appendix C

Records of Sample, Stock, Standard and Reagent Preparation
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