PROGRESS REPORT: AUGUST 2010 

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, 2010 

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Table of Contents 

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

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

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

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

Method Development.................................................................................................................4 

Pilot Study Sample Analyses.....................................................................................................5 

Appendix A Invoice...................................................................................................................7 

Appendix B Protocol, Protocol Amendments & Deviations for Initial Pilot Study ..................9 

Appendix C Pilot Study Sludge Acclimation-Raw Data .........................................................41 

Appendix D Pilot Study Biological Phase-Raw Data..............................................................63 

Appendix E Analysis of Acid Analytes in Styrene Trapping Media by LC/MS/MS............208 

Appendix F Method Installation Trial Raw Data ..................................................................214 

Appendix G Analytical Standard, Stock Solution and Reagent Preparation Information.....255 
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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 23 July 2010 

up to 24 August 2010. 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 $146,247.50 for labor and $18,900 for instrumentation. The invoice and an 

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

The protocol for the initial SCAS pilot study was signed by the Study Director, Laboratory 

Management and Sponsor’s Representative on 14 June 2010. The approved protocol, 

protocol amendments and deviation are presented in Appendix B. 

The acclimation phase of the initial pilot study was started on 26 July 2010 and concluded on 

02 August 2010. Data from the acclimation is presented in Appendix C. The sludge 

exhibited a clear supernatant after 30 minutes of settling and an effluent dissolved organic 

carbon (DOC) concentration of less than 12 mg C/L throughout the acclimation phase. The 

DOC of the effluent was measured three times and averaged approximately 7.5 mg C/L. 

The biological phase of the initial pilot study started on 02 August 2010 and concluded on 16 

August 2010. Data from the biological phase is presented in Appendix D. Sampling of the 

aqueous supernatant, sludge solids, and gas phase trapping media for analysis of study 

analytes was conducted on August 04, 06, 09, 11, 13 and 16. 

The technical work consisted of method development trials specific for LC/MS/MS 

determination of the telomer acids in styrene-trapping media, conduct of an analytical 

methods installation trial and analytical processing of samples from the initial pilot study. 
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Bench processing and determination of study analytes in the pilot study samples by GC/MS 

and LC/MS/MS continues. 

Method Development 

For the LC/MS/MS analysis of surrogate recovery standards (M+8 PFOA and M+2 PFHxA) 

and acid analytes (PFOA, PFNA, PFDA, PFUA, 7-3 Acid, 8-2 FTCA and FTUCA adsorbed 

onto sytrene (e.g, Xad-2) trapping media, experiments were conducted to further investigate 

the matrix suppression effects noted in the July 2010 Progress Report. 

Methanol (MeOH) acidified with lower strength nitric acid and MeOH acidified with 

alternative acids were investigated for use in extracting acid analytes from Xad-2 trapping 

media. The objective was finding an extraction solvent system that maintained the 

quantitative extractability of 0.1% HNO3 in MeOH while minimizing the LC/MS/MS 

instrumental response suppression previously noted when employing this solvent system to 

extract styrene-trapping media. 

The use of lower strength (0.01%) nitric or equivalent strength (0.1%) hydrochloric acid (less 

oxidative than HNO3) in MeOH maintained the quantitative extractability of 0.1% HNO3 but 

did not provide a significant impact in reducing the severity of the suppression effects 

(Appendix E). As seen for formic acid, a weaker acid additive, such as acetic acid did not 

meet the extractability criterion. (Note: 7-3 Acid data are presented without application of 

the M+2 8-2 FTCA matrix standard). 

In the methods installation trial, appropriate amounts of all study analytes were fortified into 

SCAS mixed-liquor and trapping media. The samples were then processed according to the 

applicable method outlines and the analytes were determined by LC/MS/MS or GC/MS as 

appropriate. Mass balance was demonstrated for all acid analytes and quantitative recovery 

was calculated for the acid analyte surrogate recovery standards in the SCAS matrices 

(Appendix F). For volatile analytes, consistent and low mass balance was achieved while 

quantitative recovery of the surrogate recovery standard (M+4 8-2 FTOH) from each matrix 

was calculated. This finding suggested that either a systematic procedural error occurred 

upon fortification into the mixed liquor, or that the analytes were lost via volatilization (test 
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system not maintained gas-tight) or procedural losses due to adsorption onto 

handling/transfer apparatus (e.g., syringe plungers) during the dosing and initial processing 

steps. Experiments are planned to conduct the same experiment under controlled conditions 

(e.g., using reagent water). 

During the conduct of the installation trial, an experiment was also completed to evaluate any 

potential loss of MTBE upon frozen storage of samples taken through initial processing and 

then held. The potential for losses due to multiple punctures through the original sample 

septa and the integrity of the septa seals used were the objectives of this exercise. 

Quantitative MTBE recovery was obtained for all experimental storage conditions evaluated 

(see end of Appendix F). 

Pilot Study Sample Analysis

Study samples have been processed for the analysis of the test system, domestic sewage and 

Intervals 1, 2 and 3. Although it is premature at this juncture to summarize surrogate 

recovery standard recoveries, analyte concentrations and analyte concentration trends in the 

SCAS test systems, several analytical observations are worth noting here. 

A small but reversible decrease in telomer alcohol response was evident with processing of 

study samples for GC/MS determination. The phenomenon appeared to be a substantially 

mitigated version of the non-specific matrix effect that mandated use of a back-partition 

MTBE extract clean-up step during method development. While of concern, the current 

observed sensitivity changes are far more subtle than the complete and irreversible response 

loss noted in previous Progress Reports. The current decrease was only evident after 

hundreds of sample injections on the same GC/MS platform and as stated above, does appear 

reversible at this time. 

Subsequent to the analysis of polymer treatment extracts from the first sampling event 

(Interval 1) of sludge, an interference associated with the tail of the 8-2 FTOH peak (primary 

and confirmatory ions) was evident in the chromatography that appeared column related and 

persistent. The interference was present in the 8-2 FTOH peaks for trailing calibration 

standards from the same sequence and did carryover into the initial standards of the 
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subsequent sequence. The interference may be eliminated by using enough MTBE rinse 

injections following the minimum extract dilutions (“-X1”) of the polymer treatments in a 

given sequence or prior to commencing the next sequence. The impact of the matrix effect 

on the study data has been mitigated in large measure through judicious choice of integration 

parameters. 

The levels of 8-2 FTOH and 7-2 sFTOH were so elevated in the sludge and primary styrene 

trapping compartments of the positive control test chamber that quantitation of all volatiles in 

these specific sampling compartments was not feasible using the current methods. The 

completion of these analyses will require modification of current GC/MS processing and 

instrumental determination methods and will need to be conducted subsequent to all other 

analyses. Specifically, two separate GC/MS methods will be required to adequately quantify 

volatile analytes in these selected sample extracts. The first method will allow for 

determination of 8-2 FTOH (and perhaps other relatively high concentration volatile analytes 

such as 7-2 sFTOH) using volumetric dilutions so high that use of the matrix internal 

standard will not be required (e.g., perform secondary and/or tertiary serial dilutions with 

MTBE only). The second method will exclude 8-2 FTOH as a SIM window in the method to 

allow LOQ level determinations of other volatile analytes present at trace levels. 

Records of stock and standard preparations and all reagent preparations since the July 2010 

Progress Report are provided in Appendix G. 
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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) 

Biodeg

dation 

Studie

(Task H

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 Labor Total $1,803 $3,715 $343,369 $163,850 Materials $6,800 $10,500 $20,644 $1,724 Total Cost $8,603 $14,215 $364,4013 $165,574 
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Appendix B 

Protocol, Protocol Amendments & Deviations for Initial Pilot Study 
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Appendix C 

Pilot Study Sludge Acclimation – Raw Data 
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Appendix D 

Pilot Study Biological Phase – Raw Data 
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Appendix E 

Analysis of Acid Analytes in Styrene Trapping Media by LC/MS/MS 
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Appendix F 

Method Installation Trial Raw Data 
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Does Not Contain Confidential Business Information (CBI)

2010_July_Progress Report 249
Does Not Contain Confidential Business Information (CBI)

2010_July_Progress Report 250
Does Not Contain Confidential Business Information (CBI)

2010_July_Progress Report 251
Does Not Contain Confidential Business Information (CBI)

2010_July_Progress Report 252
Does Not Contain Confidential Business Information (CBI)

2010_July_Progress Report 253
Does Not Contain Confidential Business Information (CBI)

2010_July_Progress Report 254
Does Not Contain Confidential Business Information (CBI)

2010_July_Progress Report 255

Appendix G 

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

2010_July_Progress Report 256
Does Not Contain Confidential Business Information (CBI)

2010_July_Progress Report 257
Does Not Contain Confidential Business Information (CBI)

2010_July_Progress Report 258
Does Not Contain Confidential Business Information (CBI)

2010_July_Progress Report 259
Does Not Contain Confidential Business Information (CBI)

2010_July_Progress Report 260
Does Not Contain Confidential Business Information (CBI)

2010_July_Progress Report 261
Does Not Contain Confidential Business Information (CBI)

2010_July_Progress Report 262
Does Not Contain Confidential Business Information (CBI)

2010_July_Progress Report 263
Does Not Contain Confidential Business Information (CBI)

2010_July_Progress Report 264
Does Not Contain Confidential Business Information (CBI)

2010_July_Progress Report 265
Does Not Contain Confidential Business Information (CBI)

2010_July_Progress Report 266