&EPA
United States
Environmental Protection
Agency
EPA/600/R-08/079 I February 2008 I www.epa.gov/nhsrc
Testing and Quality Assurance
Plan for the Evaluation of Wipe
Sampling Methods for Collecting
Chemical Warfare Agents (CWAs),
CWA Degradation Products, and
Toxic Industrial Chemicals from
Various Surfaces
Office of Research and Development
National Homeland Security Research Center
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EPA/600/R-08/079 | February 2008 | vwvw.epa.gov/nhsrc
United States
Environmental Protection
Agency
Testing and Quality Assurance Plan for
the Evaluation of Wipe Sampling
Methods for Collecting Chemical
Warfare Agents (CWAs), CWA
Degradation Products, and Toxic
Industrial Chemicals from Various
Surfaces
Office of Research & Development
National Homeland Security Research Center
Technology Testing and Evaluation Program
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EPA/600/R-08/079
February 2008
Testing and Quality Assurance Plan
for the Evaluation of Wipe Sampling
Methods for Collecting Chemical
Warfare Agents (CWAs), CWA
Degradation Products, and Toxic
Industrial Chemicals from Various
Surfaces
Prepared by
Battelle
505 King Avenue
Columbus, Ohio 43201
Contract No. GS23F0011L-3
Task Order No. 1132
Prepared for
Stephen Billets, Ph.D.
Environmental Sciences Division
National Exposure Research Laboratory
Office of Research and Development
U.S. Environmental Protection Agency
Las Vegas, NV 89119
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DISTRIBUTION LIST
EPA Task Order Project Officer
Stephen Billets
EPA/ORD/NERL
EPA NHSRC QA Manager
Eletha Brady-Roberts
EPA/ORD/NHSRC
Technical Review Panel
Doug Anders
FBI
Sheri Bettis
FBI
Lisa Detter-Hoskin
Georgia Tech Research Institute
Ted Haigh
EPA/Region 5
Carolyn Koester
Lawrence Livermore National Laboratory
Lisa Jo Melnyk
EPA/ORD/NERL
Jeff Morgan
EPA/ORD/NERL
Jack Pretty
CEMB/DART/NIOSH
Terry Smith
EPA/OSWER
Lawrence Zintek
EPA/Region 5
Battelle
Hani Karam
Jane Chuang
Amy Dindal
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Karen Riggs
Zachary Willenberg
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Notice
This document was prepared for the U.S. Environmental Protection Agency (EPA) under
Contract No. GS23F0011L-3, Task Order No. 1132. The document has met the EPA's
requirements for peer and administrative review and has been approved for publication. Mention
of corporation names, trade names, or commercial products does not constitute endorsement or
recommendation for use.
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Abstract
Wipe sampling is an important technique for the estimation of contaminant deposition on
surfaces. Numerous wipe sampling methods exist, and each method has its own specification
for the type of wipe, wetting solvent, extraction procedure, and determinative step to be used,
depending upon the contaminant of concern. Wipe sampling methods for the purposes of
analytical determination of surface contamination largely do not exist for compounds of interest
to the homeland security community. The goal of the project is to provide a wipe sampling
method or methods and associated method performance data for collecting selected chemical
warfare agents (CWAs), CWA degradation products, and toxic industrial chemicals from five
types of surfaces (laminate, galvanized metal, bare wood, industrial carpet, and painted
concrete). The objective of this testing and quality assurance plan is to present procedures for
testing, which will include documenting the performance of the methods at or below residential
risk-based cleanup goals. Testing will be conducted under the U.S. Environmental Protection
Agency's National Homeland Security Research Center's Technology Testing and Evaluation
Program. Note that an addendum has been added to this document as an appendix which
addresses modifications to this plan which were made after the start of testing.
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Table of Contents
Notice ii
Abstract iii
Table of Contents iv
Abbreviations, Acronyms, and Symbols vi
Acknowledgements viii
A. Project Management 1
Al. Background 1
A2. Testing and Quality Assurance Plan Description 2
A3. Schedule 3
A4. Roles and Responsibilities 3
A4.1. Battelle 3
A4.2. EPA 5
B. Measurement and Data Acquisition 6
Bl. Experimental Plan 6
Bl.l. Target Analytes and Analyte Groups 8
B1.2. Concentration Levels for Test Coupons 9
B1.3. Description of Test Coupons 13
B1.4. Wipes 13
B1.5. Preparation of Spiked and Non-spiked Test Coupons 14
B1.6. Wipe Sampling Approach 17
B1.7. Wipe Sample Preparation and Analysis Methods 18
B2. Field Samples and Quality Control (QC) Samples 18
B2.1. Field Spiked Samples 19
B2.2. Method Blanks 19
B2.3. Field Blanks 19
B2.4. Method Spiked Samples 19
B2.5. Post-extraction Spiked Field Blank Sample Extracts 19
B2.6. 100 Percent Recovery Standard Samples 19
B2.7. Performance Evaluation Samples 20
B3. Sample Handling and Custody Requirement 20
B4. Inspection/Acceptance of Supplies and Consumables 20
B5. Instrumentation Calibration and Frequency 21
B6. Instrument Maintenance 22
C. Data Management 23
Cl. Documentation/Records 23
C2. Data Analysis 23
C2.1. Accuracy 24
C2.2. Precision 24
C2.3. Evaluation of Data Quality Objective Goals 25
C3. Reporting 25
D. Health and Safety 26
Dl. Special Facilities 26
D2. Staff Training and Health 26
D3. Standard/Test Sample Handling 26
D4. Sample Handling During Test 27
E. References 28
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List of Appendices
Appendix A: Chemical structures for the Compounds of Interest A-l
Appendix B: DRAFT GC-HRMS/SIM, GC-LRMS/SIM, and LC-MS/MS
Analytical Protocols for Target Analytes B-l
Appendix C: Addendum C-l
List of Tables
Table 1. Project Schedule 3
Table 2. Provisional Risk-Based Surface Cleanup Goals for Target Compounds 10
Table 3. Proposed Target Compounds, their Risk-Based Surface Cleanup Residential
Levels, Estimated Instrument Detection Limits, and Proposed Spiking Levels 11
Table 4. Proposed Target Compounds, their Risk-Based Surface Cleanup Residential
Levels, Proposed Ix Spiking Levels, and Concentration of Splicing Solution 12
Table 5. Wipe Samples to be Generated for Phase I Experiments for Multi-analyte
versus Single Analyte Spiking Comparison 15
Table 6. Wipe Samples to be Generated Following Multi-Analyte/Single Analyte
Comparison - Phase n Approach - Wiping Solvent Isopropyl Alcohol 16
Table 7. Wipe Samples to be Generated using Methanol as a Wetting Solvent for the
Polar Target Analytes - Phase II Approach 16
TableS. Data Quality Objective Goals for the Wipe Sampling Method(s) 25
List of Figures
Figure 1. Organizational Chart of Key Participants 4
Figure 2. Phase I and Phase II Experiments 7
Figure 3. Spiking of Individual or Multi-Analyte Cocktail on Test Coupon 17
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Abbreviations, Acronyms, and Symbols
%D
HL
ASE
ASTM
CAC
CCV
CHP
cm
cm2
CWA
DCM
EPA
ESI
GC
GD
HD
HN-3
HPLC
HRMS
IPA
LC
LRB
LRMS
mL
mm
MPA
MS
MS/MS
NERL
NHSRC
OP
PEG
PFK
PFTBA
PMPA
PSP
QA
QC
QMP
RDTE
SIM
SOP
percent difference
microliter
accelerated solvent extraction
American Society for Testing and Materials
Columbus Analytical Chemistry
Continuing Calibration and Verification
Chemical Hygiene Plan
centimeter
square centimeter
chemical warfare agent
dichloromethane
U.S. Environmental Protection Agency
electrospray ionization
gas chromatography
soman
distilled mustard
nitrogen mustard 3
high-performance liquid chromatography
high resolution mass spectrometry
isopropanol or isopropyl alcohol
liquid chromatography
laboratory record book
low resolution mass spectrometry
milliliter
millimeter
methyl phosphonic acid
mass spectrometry
tandem mass spectrometry
National Exposure Research Laboratory
National Homeland Security Research Center
organophosphate pesticide
polyethylene glycol
perfluorokerosene
perfluorotributylamine
pinacolylmethylphosphonic acid
Physical Security Plan
quality assurance
quality control
Quality Management Plan
Research, Development, Testing and Evaluation
selected ion monitoring
standard operating procedure
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TDG thiodiglycol
TEPP tetraethyl pyrophosphate
TL team leader
TOL task order leader
TOPO Task Order Proj ect Officer
TQAP testing and quality assurance plan
TTEP Technology Testing and Evaluation Program
VX O-ethyl-S-[2(diisopropylamino)ethyl] methylphosphonothiolate
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Acknowledaements
This testing and quality assurance plan (TQAP) was prepared by Battelle for the U.S.
Environmental Protection Agency (EPA) Technology Testing and Evaluation Program (TTEP)
under the direction and coordination of Stephen Billets of the EPA's National Exposure
Research Laboratory (NERL), Environmental Sciences Division, in Las Vegas, Nevada. Funding
support for this project was provided by EPA's National Homeland Security Research Center
(NHSRC). EPA NERL recognizes Oba Vincent, Rob Rothman, and Eric Koglin from EPA
NHSRC for their contributions. EPA NERL thanks the following peer reviewers for their review
of this TQAP: Doug Anders and Sheri Bettis of the FBI, Lisa Detter-Hoskin of Georgia Tech
Research Institute, Ted Haigh and Lawrence Zintek of EPA/Region 5, Carolyn Koester of
Lawrence Livermore National Laboratory, Lisa Jo Melnyk and Jeff Morgan of
EPA/ORD/NERL, Jack Pretty CEMB/DART/NIC-SH, and Terry Smith of EPA/OSWER.
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Project Management
A1. Background
Wipe sampling is one of the primary techniques for assessing surface contamination in a variety
of applications including monitoring in environmental, industrial hygiene, remedial, security, and
compliance scenarios (7). When implemented following a validated method, the technique is a
quick and easy means of assessing the level or degree of contamination that may reside on the
surface.
Procedures for the collection of contaminants from surfaces have several components in
common, including the wipe sampling media, the wetting solvent, and the collection
technique (7). However, wipe sampling procedures can vary widely, depending on the
contaminant(s) of interest and the surface to be sampled. Reliability of the sample results begins
with accurate and reproducible collection of a sample for analysis. Thus, the wipe sampling
procedures used for a particular analyte on a given surface, including the proper combination of
the wipe sampling components described above, are an integral aspect of whether or not the
results generated will be representative of the contamination (7).
A recent literature study completed by U.S. Environmental Protection Agency (EPA) found that
wipe sampling is widely used in industrial hygiene, drug enforcement, exposure risk assessment,
and other related applications. However, very little performance data for any of these
applications was located and no information on the compounds of interest to the homeland
security community was found (7).
Four of the 15 Department of Homeland Security Planning Scenarios address various forms of
a chemical attack on and in buildings, structures, and outdoor spaces (2). Those responsible for
the cleanup of contaminated structures must understand the nature and extent of the
contamination on various surfaces. However, collecting a sample of a contaminant of interest
from a surface that is representative of the concentration present at the location has proven
difficult and there is no one agreed upon or recognized method for sample collection. To address
this deficiency, EPA's National Homeland Security Research Center (NHSRC) intends to
provide a wipe sampling method or methods that can be used to collect selected chemical
warfare agents (CWAs), their degradation products, and other toxic industrial chemicals (TICs)
from a variety of non-porous and porous surfaces. In developing a wipe sampling method or
methods, two different scenarios are considered:
Emergency Response Scenario: Wipe sampling would be used immediately following an
incident. Sampling will need to occur quickly and it would be used to collect the parent agent or
agents because there would have been little time for degradation to occur. Also, the
concentration of the contaminants would be at their highest.
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Cleanup or Clearance Scenario: Wipe sampling will be more thorough and less hurried.
Degradation products may be more abundant than the parent compounds and at concentrations
far lower than were originally present immediately following an incident. Therefore, the
sampling method must be compatible with more sensitive and selective analytical methods.
The emergency response and consequence management communities need validated and reliable
wipe sampling methods to address the above scenarios, and this project intends to address this
need with emphasis on the Cleanup Scenario. Exposure to CWAs and TICs can occur through
routes of inhalation of the vapor and/or aerosol, dermal absorption of the aerosol, and ingestion
of contaminated food and non-food items. After a release incident, individuals may also become
exposed by living near accident sites, touching contaminated surfaces, or consuming
contaminated water or food. It is necessary to establish health-based benchmarks for the
contaminants of health concern in support of the cleanup efforts in case of a release of CWAs or
TICs. Provisional risk-based cleanup goals for selected CWAs and TICs have been established
by the Threat and Consequence Assessment Division of EPANHSRC based on the document
established by the Contaminants of Potential Concern (COPC) Committee of the World Trade
Center Indoor Air Task Force Working Group and an on-going effort to update the EPA Risk
Assessment Guidance for Superfund (RAGS), Part E, Dermal Guidance (3,4). Since the
objective in the Cleanup Scenario is ultra-low sensitivities to identify human exposure issues,
pre-cleaned wipes, which offer a lower chance of analytical interferences, and sensitive
analytical techniques (e.g., selected ion monitoring high resolution mass spectrometry and
tandem mass spectrometry) will be utilized.
This project represents the second phase of a four-phase effort planned by EPANHSRC. The
first phase was the literature review. This phase will focus on determining a method addressing
the Cleanup Scenario with a subset of the target analyte list. The third phase will involve a
single-laboratory validation of the method determined by this project with the full suite of target
analytes. The fourth phase will be a round-robin study of the validated method.
A2. Testing and Quality Assurance Plan Description
The objective of this testing and quality assurance plan (TQAP) is to evaluate a wipe sampling
method or methods that can be used to collect selected CWAs, their degradation products, and
toxic industrial chemicals (specifically organophosphorous pesticides or OPs) from a variety of
non-porous and porous surfaces. This study, which will be conducted under NHSRC's
Technology Testing and Evaluation Program (TTEP) in compliance with the program's quality
management plan (QMP) (5), will provide recovery and reproducibility data that can be used to
assess the efficiency of the sampling method for selected compounds. This TQAP describes the
procedures that will be used to conduct the wipe sampling method evaluation. The primary
sections of this TQAP include:
Section A, Project Management, describes project history and objectives, roles and
responsibilities of the participants, and documents project planning.
Section B, Measurement and Data Acquisition, covers the experimental aspects of the
project, including design, implementation, and quality control.
Section C, Data Management, details the data handling, evaluation, archival, and
reporting procedures.
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Section D, Health and Safety, highlights health and safety aspects of the study, including
handling of chemical agents.
Section E, References, includes citations for the documents referenced in this TQAP.
A3. Schedule
Table 1 describes the schedule for this project. The schedule details the major milestones for the
experimental work, which is anticipated to take four months to complete. A draft wipe sampling
method will be provided to EPA by June 23, 2008. The peer-reviewed report, which will include
the final method, will be submitted to EPA by September 30, 2008.
Table 1. Project Schedule
Activity | Target Completion Date
Perform Phase I Experiments
Perform Phase II Experiments
Compile and evaluate data
Provide draft method to EPA
First draft report to EPA
Revised draft report submitted to EPA for peer review
Final report submitted to EPA
March 14, 2008
May 30, 2008
June 16, 2008
June 23, 2008
July 11, 2008
August 14, 2008
September 30, 2008
A4. Roles and Responsibilities
The responsibilities of the key participants in this project are described in this section. Figure 1
is an organization chart showing the relationship between the key participants.
A4.1. Battelle
Ms. Amy Dindal is the Battelle's Task Order Leader (TOL) for this project. In this role,
Ms. Dindal will have overall responsibility for ensuring that the technical, schedule, and cost
goals established for the project are met. Specifically, Ms. Dindal will:
Contribute to preparation of the TQAP, wipe sample collection method(s), and project
report.
Revise the draft TQAP, wipe sample collection method(s), and project report in response
to reviewers' comments.
Coordinate distribution of the final TQAP, wipe sample collection method(s), and project
report.
Manage staff to ensure the budget is not exceeded and schedule is met.
Ensure that necessary Battelle resources, including staff and facilities, are committed to
the verification test.
Assist Battelle team leaders and technical staff as needed in performing the project in
accordance with this TQAP.
Serve as the primary point of contact for EPA.
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K. Riggs
Battelle TTEP
Manager
Battelle
Management
E. Koglin
EPA TTEP
Program Manager
A. Dindal
Battelle TOL
Z. Willenberg
Battelle
QA Manager
S. Billets
EPATOPO
E. Brady-Roberts
EPA NHSRC
QA Manager
H. Karam
Team Leader
for CWAs
I
J. Chuang
Team Leader for
OP pesticides
Technical
Staff
I
Technical
Staff
Figure 1. Organizational Chart of Key Participants
(dotted line indicates indirect reporting)
Dr. Hani Karam is Battelle's team leader (TL) for the CWA analyses with the responsibility for
coordinating and overseeing sample analyses, data interpretation, and data reporting for CWAs
and their degradation products. As such, Dr. Karam will:
Contribute to the TQAP, wipe sample collection method(s), and project report, with
primary responsibility for the sections related to CWA and CWA degradation product
analyses.
Oversee the execution of the Battelle technical staff performing the CWA and CWA
degradation product analyses.
Compile and evaluate data generated from the CWA testing.
Maintain communication with Battelle's TOL.
Ms. Jane Chuang is Battelle's TL with the responsibility for coordinating and overseeing
sample analyses, data interpretation, and data reporting for OPs. As such, Ms. Chuang will:
Contribute to the TQAP, wipe sample collection method(s), and project report, with
primary responsibility for the sections related to OP pesticide analyses.
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Oversee the execution of the Battelle technical staff performing the OP pesticide
analyses.
Compile and evaluate data generated from the OP pesticide testing.
Maintain communication with Battelle's TOL.
Mr. Zachary Willenberg is Battelle's QA Manager for this project. Mr. Willenberg will:
Review and approve the draft and final TQAP.
Conduct a technical systems audit at least once during the technology evaluation.
Audit at least 10% of the evaluation data.
Notify Battelle's TTEP Manager to issue a stop work order if internal audits indicate that
data quality is being compromised.
Review and approve the draft and final wipe sample collection method(s).
Review and approve the draft and final project report.
Ms. Karen Riggs is Battelle's TTEP Manager. As such, Ms. Riggs will:
Review and approve the draft and final TQAP.
Ensure that necessary Battelle resources, including staff and facilities, are committed to
the project.
Provide the TOPO with monthly technical and financial progress reports.
Monitor adherence to budgets and schedules in this work.
Review and approve the draft and final wipe sample collection method(s).
Review and approve the draft and final project report.
Issue a stop-work-order if internal audits indicate that data quality is being compromised.
A4.2. EPA
Dr. Stephen Billets is EPA's Task Order Project Officer (TOPO) for this project. Dr. Billets will:
Review and approve the draft and final TQAP, wipe sample collection method(s), and
project report.
Provide technical guidance as appropriate to address the needs of EPA.
Make technical decisions regarding the direction of the work such as implementing
options.
Oversee the EPA review process, including securing reviewers, for the TQAP and project
report.
Ms. Eletha Brady-Roberts is EPA NHSRC QA Manager. Ms. Brady-Roberts will:
Review and approve the draft and final TQAP.
Review the draft and final wipe sample collection method(s).
Review the draft and final project report.
Notify the EPA TOPO to contact the Battelle TTEP Manager to issue a stop work order if
an external audit indicates that data quality is being compromised.
Mr. Eric Koglin is the EPA TTEP Program Manager who directs Battelle's activities on the
contract, "Testing and Investigation of Homeland Security-Related Technologies for the
Measurement, Sampling, Removal, and Decontamination of Chemical and Biological Agents"
under which TTEP has been established.
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Measurement and Data Acquisition
The section covers the experimental aspects of the project including experimental design,
implementation, and quality control.
B1. Experimental Plan
A recent literature review study (7) indicated that virtually no wipe sampling method
performance information is available for collecting CWAs and CWA degradation products from
various types of surfaces. The American Society for Testing and Materials (ASTM) has a wipe
sampling method for organic compounds (e.g., pesticides, Aroclors) from non-porous surfaces
(6) and a recent study discussed wipe sampling method for pesticides from porous and non-
porous surfaces (7). However, there is no precision and accuracy data for CWAs and CWA
degradation products reported in the literature to document the performance of the wipe
sampling method(s). The main objective of this project is to prepare a robust and reproducible
standard method for the collection of target analytes including CWAs, CWA degradation
products, and selected OPs from five types of surfaces (i.e., laminate, galvanized metal, bare
wood, industrial carpet, and painted concrete) at or below residential risk-based cleanup goals.
The general approach for evaluating the wipe sampling method(s) to be established consists of:
Applying a known amount of target compound(s) onto test coupons made from different
surface materials,
Wiping test coupons with a pre-cleaned wiping material wetted with solvent to remove and
collect the spiked target compounds,
Determining the recovery of the spiked target compounds using established analytical
method(s), and
Incorporating quality assurance (QA)/quality control (QC) measures to monitor method
performance in each phase of the process.
A two-phase experimental design (Figure 2) will be used to carry out experiments to accomplish
this objective. Phase I will establish a robust, reproducible, and reliable wipe sampling method
for the target analytes on a non-porous surface (i.e., laminate) at a single (mid-range or 5x)
concentration level. Phase II will evaluate the performance of the method established in Phase I
on all five types of surfaces at three concentration levels and modify the method if necessary.
The key hypotheses to be tested in Phase I are:
1. Comparable results are obtained between a streamlined multi-analyte spiking method and a
straight forward single-analyte spiking method
2. Quantitative and reproducible recoveries of the target analytes from spiked non-porous test
coupons (i.e., laminate) are obtained
3. Consistent results are obtained between horizontal and vertical wiping methods.
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Phase I
Investigate a selected spiking
and wipe sampling method on
one type test coupon
(non-porous surface)
Phase II
Evaluate the wipe sampling
method(s) on five types of test
coupons (non-porous and
porous surfaces)
Two Phase
Experimental
Design
Phase I
Test coupon: laminate
Spiking method: single- and
multi-analyte at 5X (mid level)
Wipe material: cotton gauze
Wetting solvent: IPA
Wiping method: horizontal and
vertical
Do recovery/precision
results for single and
multi-analyte spiking
methods meet Data
Quality Objectives?
Discuss outcome with
EPATOPOand modify
Phase II approach
accordingly
Phase II
Testcoupon: laminate
galvanized metal, bare wood,
painted concrete, and industrial
carpet
Spiking method: multi-analyte
at 1X and 10X (laminate), and
at1X, 5X, and 10X for other
four types of test coupons
Wipe material: cotton gauze
Wetting solvent: IPA (Groups I,
II, III) and methanol (Group III)
Figure 2. Phase I and Phase II Experiments
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Note that the term "horizontal wiping method" used in the TQAP means to place the test coupon
flat (e.g., representing floor surfaces) in the hood for wiping, and the term "vertical wiping
method" means to place the test coupon vertically with a back support (e.g., representing wall
surfaces) for wiping.
According to the literature review results and the ASTM method (1,6), cotton gauze is a
commonly used wiping material and isopropanol (IPA) is a commonly used wetting solvent for
similar compounds, so when considering the nature of the target analytes, this combination was
chosen as a starting point for the Phase I study.
The recovery data obtained from Phase I will be evaluated in terms of data quality objective
goals. These goals are:
The percent difference (%D) values of the mean recovery data of field spiked coupon
samples derived from single- versus multi-analyte spiking as well as horizontal versus
vertical methods are within ±10%
The average recovery data of triplicate field spiked coupon samples range from 70 to
110% and % relative standard deviation (% RSD) values of triplicate field spiked
samples are within ±20%.
Battelle will discuss the results of Phase I with the EPA TOPO and determine whether it is
feasible to move forward to Phase n experiments as outlined in Figure 2. If the results from
Phase I experiments do not meet all the criteria as stated above, Battelle will consult with the
EPA TOPO on revising the approach for the Phase II full-scale study accordingly.
The Phase II experiments outlined in Figure 2 are based on the assumption that satisfactory
results are achieved for the Phase I hypotheses. Thus, multi-analyte spiking and cotton gauze
wetted with IPA are planned for the Phase II full-scale study. In addition, Battelle will also
examine a second wetting solvent (e.g., methanol) for the more polar target analytes in Group ITI
(as defined below) to determine if improved performance can be achieved with methanol for
these target analytes.
Technical challenges are expected in Phase n experiments when different types of surfaces are
tested with the wipe sampling method(s). For example, the galvanized-metal surfaces may react
with selected CWAs such as VX and cause reduced recoveries of the target analyte from the
surface. Painted concrete surface could present potential matrix interference for the analytical
methods. Relatively lower recoveries for the spiked field samples are expected for porous
surfaces (i.e., bare wood and industrial carpet) as compared to non-porous surfaces because the
porous surface materials tend to absorb the spiked compounds and lower the sample wiping
efficiency. Battelle's experimental design takes into consideration these technical challenges.
The CWA degradation products, 1,4-dithiane and 1,4-thioxane, will be monitored for CWA-
spiked test coupons in an attempt to determine if degradation of HD occurs on the spiked
surfaces. Six types of QC samples (described in Section B2.7) are included to document
precision and accuracy of the analytical methods employed and potential sample matrix effect.
Detailed discussion of the experimental design is described below.
B1.1. Target Analytes and Analyte Groups
A list of compounds of interest to the EPA with the associated Provisional Risk-based Surface
Cleanup Goals (3,4) is presented in Table 2. The wipe sampling method(s) to be evaluated will
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be developed for test coupons challenged with target analytes at or below the Provisional Risk-
based Surface Cleanup Goals to ensure the adequacy of the wipe sampling method(s) to address
the detection of such low levels of contaminants, for public health safety reasons. A subset of
the compounds listed in Table 2 will be studied in this project while all the Table 2 compounds
will be challenged in a future EPA NHSRC single laboratory validation study using the
optimized wipe sampling method(s) established here. As shown in Table 3, a total often target
analytes, including five selected by EPA marked with an asterisk and five selected by Battelle
marked with two asterisks, will be studied in this project.
Table 3 summarizes the estimated instrument detection limits in terms of micrograms per sample
(lag/sample) and micrograms per square centimeter (|ig/cm2) for these 10 target analytes, the
analytical methods to be used for their analysis, the proposed spiking levels, and Battelle's
rationale for the inclusion of a second target compound in each class. Chemical structures for
the 10 compounds of interest are presented in the Appendix A.
According to the chemical/physical properties and established analytical methods, the 10 target
compounds will be split into three groups. Group I will consist of the target CWAs, namely HD,
HN-3, GD, and VX. Group II will contain CWA degradation product (1,4-dithiane) and OP
pesticides (dichlorvos and tetraethylpyrophosphate (TEPP). The more polar CWA hydrolysis
products thiodiglycol (TDG), pinacolylmethylphosphonic acid (PMPA), and methylphosphonic
acid (MPA) will be in Group III.
Based on Battelle's experience with these target compounds from other previous studies (5), the
interactions of the target compounds in each group should be negligible. As outlined in Figure 2
and described in section Bl, multi-analyte spiking method is proposed for the Phase II full-scale
study. However, as a quality assurance measure, spike recovery experiments will be performed
using both the single- and multi-analyte spiking methods on laminate surface at 5X spiking
levels in triplicate in Phase I experiments. One target analyte from each of the three groups will
be used (GD from Group I, dichlorvos from Group II, and PMPA from Group III) for the single-
analyte spiking method. Each of the three analyte groups containing multi-analytes as discussed
above will be used in the multi-analyte splicing method.
B1.2. Concentration Levels for Test Coupons
Three concentration levels (i.e., Ix, 5x, and lOx) will be used to prepare test coupons (Table 3).
The lowest concentration level (Ix) for all the target analytes to be spiked onto the test coupons
is set at or below residential Risk-Based Surface Cleanup Goals. As shown in Table 2,
residential risk-based surface cleanup goals are available for six target analytes (HD, GD, VX,
1,4-dithiane, dichlorvos, MPA) but not for HN-3, TDG, PMPA, and TEPP. An assumption is
made that compounds in the same compound class would have similar risk-based surface
cleanup goal. Thus, the Risk-Based Surface Cleanup Goals assigned for HN-3 and TEPP are the
same as HD and dichlorvos, respectively (Table 3). Target analytes with fairly high Risk-Based
Surface Cleanup Goals (1,4-dithiane and MPA) or no known Risk-Based Surface Cleanup Goals
(TDG and PMPA) will be spiked at three concentration levels of multiples of the established
instrument detection limits for these analytes (Table 3). Table 4 compares the proposed Ix
concentration spiking level of target analytes to the Risk-Based Surface Cleanup Goal and
summarizes the concentrations of target analytes in the spiking solutions required to accomplish
the Ix spiking level in both single- and multi-analyte spiking methods
-------
Table 2. Provisional Risk-Based Surface Cleanup Goals for Target Compounds
Risk-based Surface Cleanup
Compound Goals (jig/cm2)
CWA - Blister
Agents
CWA - Blister
Agent
Degradation
Products
Nitrogen
Mustards
Degradation
Products
CWA - Nerve
Agents
CWA - Nerve
Agent
Precursors and
Degradation
Products
OP Pesticides
*Distilled Mustard (HD)
Mustard (T)
Mustard, nitrogen (HN-1)
Mustard, nitrogen (HN-2)
Mustard, nitrogen (HN-3)
*l,4-Dithiane
1,4-Thioxane
Thiodiglycol
N-Ethyldietnanolamine
N-Methyldiethanolamine
Triethanolamine
Sarin (GB)
Soman(GDlandGD2)
Tabun (GA)
*VX
Cyclohexyl Sarin (GF)
Diinethvlphosphite
Diisopropyl methyl phosphonate
Dimetliylpliosphorainidic acid
EA 2 192(8-2-
diisopropylaminoethyl
methylphosphonotliionic acid)
Ethyluiethyl phosplionic acid
Isopropyl methylphosphonic acid
*Methylphosphonic acid
Cyclohexylmethylphosphonic acid
Pinacolylmetliylphosphonic acid
*Dichlorvos
Dicrotophos
Fenamiphos
Methyl parathion
Mevinphos
Phorate
Tetraethylpyrophospliate
Crimidine
505-60-2
172672-28-5
538-07-08
51-75-2
555-77-1
505-29-3
15980-15-1
111-48-8
139-87-7
105-59-9
102-71-6
107-44-8
96-64-0
77-81-6
501782-69-9
329-99-7
868-85-9
1445.75-6
33876-51-6
73207-98-4
1832-53-7
1832-54-8
993-13-5
1932-60-1
616-52-4
62-73-7
141-66-2
22224-92-6
298-00-0
7786-34-7
298-02-2
107-49-3
535-89-7
0.00022
d
e
e
e
6.0
d
d
t
f
t
0.012
0.0024
0.024
0.00036
d
d
48.0
d
d
d
60.0
15.0
d
d
0.0058
0.06
0.15
0.15
d
0.12
d
d
.000081
d
e
e
e
2.1
d
d
f
f
t
0.0043
0.00086
0.0086
0.00013
d
d
17.0
d
d
d
21.0
5.2
d
d
0.0022
0.021
0.054
0.054
d
0.043
d
d
a * denotes compounds designated by EPA to be included in this project
b Occupational Exposures for non-porous surfaces. Exposure assumptions are for adults for 25 years, 8 horn's/day, 250 days/year; exposure
pathways include dermal and oral associated with hand-to-mouth activity. Inhalation exposures are not included in this evaluation. Values are
provisional only.
c Residential - Exposures for non-porous surfaces. Exposure assumptions are for adults for 24 years, 16 hours/day. 250 days/year: exposure
pathways include dermal and oral associated with hand-to-mouth activity. Inhalation exposures are not included in this evaluation. Values are
provisional only.
d no toxicity value available to derive cleanup goal.
e Using only a comparison of acute lethality data (LD50), the nitrogen mustards appear to be somewhat less toxic man sulfur (distilled) mustard
(HD). Therefore, the cleanup goal for HD can be used for the nitrogen mustards and would be sufficiently protective.
'Using only a comparison of acute lethality data (LD50), these nitrogen mustard degradation byproducts appear to be somewhat less toxic than
sulfur (distilled) mustard (HD). Therefore, the cleanup goal for HD can be used for these chemicals and would be sufficiently protective.
-------
Table 3. Proposed Target Compounds, their Risk-Based Surface Cleanup Residential Levels,
Estimated Instrument Detection Limits, and Proposed Spiking Levels
Risk-Based
Surface Cleanup
Instrument
Detection Limits'
Spiking Levels
(jig/wipe)
Battelle Goals - ^^^^H
Compound Rationale for Residential ^^H^^H
Class Compound a Selection (usi/cm2) ^R^m^l
CWA- Blister
Agents
CWA-Blister
Agent
Degradation
Products
CWA- Nerve
Agents
CWA-Nerve
Agent
Precursors and
Degradation
Products
OP Pesticides
* Distilled Mustard (HD)
**Mustard , nitrogen (HN-3)
*l,4-Dithiane
**Thiodiglycol (TDG)
**Soman (GDI and GD2)
*VX
* Methylphosphonic acid
(MPA)
* *Pinacorylmethylphosphonic
acid (PMPA)
* Dichlorvos
**Tetraethyl pyrophosphate
(TEPP)
Different types of
blister agents
Inclusion of
degradation
products for
different CWAs
Compounds with
relatively low risk
levels (in ug/cm2)
Inclusion of
degradation
products for
different CWAs
TEPP. a relatively
unstable OP, is
included to
challenge the
method
0.000081
0.000081 c
2.1
Not Available
0.00086
0.00013
5.2
Not Available
0.0022
0.0022 c
0.0011
0.00002
0.010
0.020
0.00009
0.0022
0.018
0.020
0.010
0.010
^^H^l^l Analytical ^^^1
^^m^^^^H Method ^R^H
0.000011
0.0000002
0.00010
0.00020
0.0000009
0.000022
0.00018
0.00020
0.00010
0.00010
GC/HRMS-
SEVI
GC/LRMS-
SDVI
LC/MS/MS
GC/HRMS-
SDVI
LC/MS/MS
GC/LRMS-
SIM
0.008
0.008
0.040
0.400
0.008
0.012
0.400
0.400
0.040
0.040
5x
0.040
0.040
0.200
2.0
0.040
0.060
2.0
2.0
0.200
0.200
lOx
0.080
0.080
0.400
4.0
0.080
0.120
4.0
4.0
0.400
0.400
a * denotes compounds designated by EPA to be included in this project and ** denotes compounds selected by Battelle.
b Instrument Detection Limit for each target analyte is based on a 10:1 S/N of the selected qualifier ion of the analyte by gas chromatography/low resolution mass spectrometry-
selected ion monitoring (GC/LRMS-SIM), gas chromatography/high resolution mass spectrometry-selected ion monitoring (GC/HRMS-SIM), or liquid chromatography/mass
spectrometry/mass spectrometry (LC-MS/MS) analysis. This assumes a wiped surface area of 100 cm2. Final extract volume is 1 ml for GC/MS and 10 ml for LC/MS/MS.
c An assumption is made that compounds in the same chemical class would have similar risk-based surface cleanup goal.
-------
Table 4. Proposed Target Compounds, their Risk-Based Surface Cleanup Residential Levels,
Proposed Ix Spiking Levels, and Concentration of Spiking Solution
Risk-Based Surface Concentration of
Cleanup Ix Spiking Target Analyte in
Analyte Goals - Residential Analytical Concentration Spiking Cocktail
Compound Class Compound " Group (ug/wipe) b Method (ug/wipe) (ug/mL) d
CWA- Blister
Agents
CWA-Blister
Agent Degradation
Products
CWA- Nerve
Agents
CWA-Nerve Agent
Precursors and
Degradation
Products
OP Pesticides
* Distilled Mustard (HD)
**Mustard , nitrogen (HN-
3)
*l,4-Dithiane
**Thiodiglycol (TDG)
**Soman (GDI and GD2)
* VX
* Methylphosphonic acid
(MPA)
**Pinacolylrnethyl-
phosphonic acid (PMPA)
* Diclilorvos
**Tetraethyl pyrophosphate
(TEPP)
I
I
n
m
I
I
ni
ni
n
n
0.0081
0.0081 c
210
Not Available
0.086
0.013
520
Not Available
0.22
0.22 c
GC/HPJVIS-SIM
GC/LRMS-SIM
LC/MS/MS
GC/HRMS-SIM
LC/MS/MS
GC/LRMS-SIM
0.008
0.008
0.040
0.400
0.008
0.012
0.400
0.400
0.040
0.040
0.008
0.008
0.040
0.40
0.008
0.012
0.40
0.40
0.040
0.040
a * denotes compounds designated by EPA and ** denotes compounds selected by Battelle.
b Based on wiped surface of 10cm x 10 cm.
c An assumption is made that compounds in the same chemical class will have the same risk-based surface cleanup goal.
d Concentration of target analyte in single or multi-analyte spiking cocktail. To accomplish 1x spiking level, 1000 pL of each cocktail will be spiked onto test coupons
-------
B1.3. Description of Test Coupons
Five types of test materials, namely, laminate, galvanized metal, bare wood, industrial carpet,
and concrete will be evaluated. Laminate sheets (48"x 96", A'Jack Inc., Columbus, Ohio),
premium eastern bare pine wood (96"x 120", Home Depot, Canal Winchester, OH), and
industrial-grade carpet squares (24"x 24", Carpet Corporation of America, Rome, GA) will be
purchased in bulk and cut into 6"x 6" (-15 cm x 15 cm) test coupons. The 24 gauge galvanized
sheet metal (Adept Products Inc., West Jefferson, OH) and concrete which conforms to ASTM
C90 (Wellnitz Company, Columbus, OH) will be purchased as pre-cut coupons in 6" by 6" size.
The concrete coupons will be primed with one coat of latex primer (Kilz 2, Home Depot, Canal
Winchester, OH), followed by one coat of white latex (American Tradition Interior 100%
Acrylic Ultrawhite, Lowe's, Canal Winchester, OH). A 10 x 10 cm square will be marked on
each coupon to indicate the spiking area (Figure 3). A scriber will be used to mark the 10x10
area on all surfaces except carpet. As for the carpet coupons, a 10 x 10 cm will be marked using
masking tape. The 15 cm x 15 cm size coupon is a workable size for performing wiping in a
fume hood and for disposal afterward.
It is anticipated that porous surfaces such as bare wood and industrial carpet will tend to absorb
the spiked target analytes, and thus result in lower recoveries and poorer reproducibilities of
these analytes than the non-porous surfaces (such as laminate) when wiped with IPA-wetted
wipe. Painted concrete surfaces may present a challenging matrix background (especially for
low-spike levels) that could impact the analysis of target analytes by the selected analytical
method. The galvanized-metal surfaces may also react with selected CWAs such as VX and
cause reduced recoveries of the target analyte from the surface.
B1.4. Wipes
Cotton gauze will be used as the wiping material. Cotton gauze was chosen because it is
commonly used for surface wiping, easily transported, readily wetted, and convenient for
sampling most surfaces. IPA is selected as the primary wetting solvent for the cotton gauzes
because of its ability to dissolve the target analytes and for its low toxicity. A second wetting
solvent more polar than IPA (e.g., methanol) or a combination, dual-solvent system, will be
evaluated for the more polar Group DI analytes.
Because the cotton gauze wipe material is known to potentially present matrix interferences, the
cleanliness of the cotton gauze varies by brand and even by lot, and this effort is focused on
achieving low-level risk-based surface cleanup goals under the Cleanup Scenario, the wipes will
be pre-cleaned. The procedure for pre-cleaning will involve extracting them with acetone
followed by dichloromethane (DCM) using accelerated solvent extraction (ASE), following the
procedures used in an on-going EPA study (8). The pre-cleaned wipes will be dried in a drying
chamber under nitrogen. Two dried and clean wipes, each wetted with 2-mL of IPA, will be
placed in a clean jar. The jar containing the pre-cleaned wipe will be sealed with Teflon tape and
stored in a refrigerator for up to 1 month until it is ready to use. Note that two precleaned and
wetted wipes will be used for each test coupon. Wipe sampling procedure is described in
SectionB 1.6.
The cotton gauze/IPA wiping approach may not be effective on non-porous surfaces such as
industrial carpet and bare wood. In the event that the sampling method is suspected of being
ineffective in recovering the target analyte(s) from the surface, Battelle will explore the
modification and/or refinement of the sampling procedure to improve recoveries. If the
-------
evaluation of a second wipe material or a third wetting solvent is necessary during the
performance of this project, Battelle will discuss the related issues with the EPA TOPO and
propose the alternative approach for method improvement. If necessaiy, a modification of the
project in terms of scope, and budget will be needed to implement these changes.
B1.5. Preparation of Spiked and Non-spiked Test Coupons
As shown in Figure 2, experiments for the comparison of single analyte versus multi-analyte
spiking method on the laminate surface coupons will be carried out first (Phase I). Table 5
summarizes the number of wipe samples that will be generated for the Phase I experiments.
At 5x level, two types of spiking solutions will be prepared, namely single-analyte and multi-
analyte in either acetone, for Groups I and II analytes, and acetone or methanol for Group III
analytes. The target analytes will be purchased individually, then stock solutions will be
prepared individually for single-analyte spiking and combined accordingly for multi-analyte
spiking. Tables 6 and 7 summarize the number of samples that will be generated under Phase II
experiments using IPA-wetted wipes and methanol-wetted wipes, respectively. Multi-analyte
spiking and horizontal wiping methods are proposed for the Phase II experiments. As shown in
Table 6, two spiking levels (Ix and 10 x) will be used for laminate test coupons and three spiking
levels (Ix, 5x, and lOx) will be used for the other four types of test coupons. A different wetting
solvent, methanol, will be tested with Group III analytes to determine if improved recoveries
could be achieved.
All spiking procedures will be carried out in a fume hood. A group of four test coupons (3 to be
used as field spikes and 1 to be used as a field blank) will be placed in a clean container inside
the hood. An aliquot (1000 uL) of the spiking solution will be spiked onto each coupon at 5
spots, at a rate of 200 jaL/spot (Figure 3). For each field blank, same amount of solvent (1000
jaL) used for the preparation of the individual spiking solution will be spiked in the same manner
as the field spike test coupons. After spiking, the coupons will be left in the hood for an
additional five minutes for drying. The drying time for the spiked coupons may be adjusted as
necessary, after discussion with the EPA TOPO.
-------
Table 5. Wipe Samples to be Generated for Phase I Experiments for
Multi-analyte versus Single Analyte Spiking Comparison
Laminate Surface
Sample Types and Number of Samples to be Collected in Phase I Approach -
Multi-Analyte Spiking + Limited Single Analyte Spiking (5x Level)a
Wiping Solvent Isopropyl Alcohol
Spiked Coupon
Horizontal
Wiping
Vertical
Wiping
Non-
Spiked
Coupon
Post-
Extraction
Spikes
Spiked
Wipes
Non-
Spiked
Wipesf
Solvent
Spikes a
(100%
Recovery)
Total
Group I'
3
12
Group II'
12
Group in'
12
GD
12
Dichlorvos
12
PMPA
1
1
I1
1
12
Total
18
12
27
81
3 Spiking level as defined in Table 3
b Group I consists of HD, HN-3, VX, and GD
c Group II consists of 1,4-dithiane, dichlorvos, and TEPP
d Group III consists of TDG, MPA, and PMPA
e Three of the spiked wipes for each group will be refrigerated for 48 hours along with field spike and field blank samples
(Section B1.6), while the other 3 spiked wipes will be freshly prepared prior to sample extraction (Spiked wipes are defined
in Section B2.4).
f Non spiked wipes are method blanks (as defined in Section B2.2.)
9 Each non-spiked wipe will be stored for 48 hours along with the field spiked, field blank, and the associated spiked wipes.
h Each non-spiked wipe will be freshly prepared, extracted with the respective field spike and field blank samples, along with
the stored wipe method spikes, wipe method blank, and freshly prepared wipe method spikes, and analyzed for all the
target analytes in that group.
-------
Table 6. Wipe Samples to be Generated Following Multi-Analyte/
Single Analyte Comparison a Phase n Approach - Wiping Solvent Isopropyl Alcohol
Laminate Galvanized Metal Bare Wood Industrial Carpet Painted Concrete
Coupons Coupons Coupons Coupons Coupons Post- Non-
Target ^^^^|
KJIMkW^^BB Spikes
Group I A
Group E e
Group III '
Total
6
6
6
18
Non-
Spikes
1
1
1
3
Spikes
9
9
9
27
Non-
Spikes
1
1
1
3
Spikes
9
9
9
27
Non-
Spikes
1
1
1
3
Spikes
9
9
9
27
Non-
Spikes
1
1
1
3
Spikes
9
9
9
27
^E^^^l Extraction Spiked Spiked
B^n^ffH Spikes c Wipes a Wipes g Total
1
1
1
3
5
5
5
15
9
9
9
27
3
3
3
9
64
64
64
192
3 Triplicates at spiking levels of 1x, 5x, and 10x, for each surface type, except where noted otherwise (see section B2.4). Spiking levels are defined in Table 3.
b Triplicates at spiking levels of 1x and 10x
c A single replicate for each surface type and for each Group of analytes at spiking level of 5x only (post-extraction spike is defined in Section B2.5)
" Group I consists of HD, HN-3, VX, and GD
e Group II consists of 1,4-dithiane, dichlorvos, and TEPP
f Group III consists of TDG, MPA, and PMPA
9 Non spiked wipes are method blanks (as defined in Section B2.2.)
Table 7. Wipe Samples to be Generated using Methanol as a
Wetting Solvent for the Polar Target Analytes a Phase II Approach
Target
Analyte
or Group
Group
ffld
Laminate Galvanized Bare Wood
Coupons Metal Coupons Coupons
Industrial Carpet Painted Concrete
Coupons Coupons
1
1
1
1
Solvent
Spikes
Post- Non- (100%
Extraction Spiked Spiked Recovery)
Spikes'
Wipes * Wipes '
1
68
3 Triplicates at spiking levels of 1x, 5x, and 10x (spiked wipes are defined in section B2.4). Spiking levels are defined in Table 2.
b A single replicate for each surface type at spiking level of 5x only (post-extraction spike is defined in Section B2.5)
c A single replicate at spiking level of 5x only
d Group III consists of TDG, MPA, and PMPA
e Non spiked wipes are method blanks (as defined in Section B2.2.)
-------
B1.6. Wipe Sampling Approach
For Phase I experiments, the spiked laminate coupons will be placed in clean trays (16" x 16" x
1") in both horizontal and vertical positions for wipe sample collection in the fume hood. Note
that all experiments will be performed in the fume hood under a constant laboratory temperature
(~71±5°F). The temperature will be measured with a National Institute of Standards and
Technology-traceable thermometer daily while coupon spiking is occurring. The designated
wiping area (10 by 10 cm) on the test coupon shown in Figure 3 will be wiped with two pre-
cleaned cotton gauze pads (3" x 3"-12 ply), each wetted with 2-mL IPA. The first IPA-wetted
wipe will be used to wipe the coupon area in a single direction from top to bottom, while the
second wetted wipe will be used to wipe the same coupon area in a single direction from left to
right, with three strokes each. After the first stroke, the exposed surface of the first cotton gauze
will be folded inward for the second stroke, and then folded again for the third stroke. The
cotton gauze pad is folded again with the exposed surface inside; then placed in the original jar.
The test coupon will then be wiped again with the second wipe using the same procedures as
described above. The second wiped cotton gauze pad will be placed in the same container as the
first wiped cotton gauze pad; then the container sealed with Teflon tape and refrigerated for 48
hours prior to extraction and analysis. The purpose of this 48 hour storage time is to simulate
field handling and storage conditions of the wipe samples as well as the elapsed time between
sampling and extraction. Wipe samples spiked with the target analytes (Groups I, II, or III),
along with non-spiked wipes, will also be stored under the same conditions as the field samples.
For Phase II experiments, all spiked test coupons will be placed in the horizontal position for
wiping, using the same procedures as described above. Storage conditions of Phase II samples
will be determined, following discussion of Phase I results with the EPA TOPO.
200 j.iL Spot
10 cm x 10 cm Wiped
Area
o
o
o
o
o
15 cm x 15 cm Coupon
Figure 3. Spiking of Individual or Multi-Analyte Cocktail on Test Coupon
-------
B1.7. Wipe Sample Preparation and Analysis Methods
For Groups I and II analytes, the surface wipe samples collected from spiked test coupons (i.e.,
field spiked samples) will be extracted with 50% DCM in acetone using Accelerated Solvent
Extraction (ASE), based on the EPA Method 3 545A, with minor modifications intended to
enhance recoveries of the target analytes from the wipe matrix (9). The modifications consist of
increasing static extraction cycle from one to two and the static extraction time from 5 minutes to
10 minutes. Note that each wipe storage jar will be rinsed with 50% DCM in acetone and the
rinsates combined with their respective wipe sample for processing. The QC samples for each
group (i.e., stored wipe method blanks, field blanks, freshly-prepared wipe method spikes, and
stored wipe method spike samples) will be extracted by the same procedure as the field spiked
samples. After extraction, each sample extract will be concentrated to 1 mL using Kuderna
Danish (KD) evaporating technique and spiked with known amount of the internal standards.
The internal standard mixture will consist of l,4-dichlorobenzene-d4, naphthalene-dg,
acenaphthene-dio, phenanthrene-dio, chrysene-di2, and perylene-di2, which is based on an EPA
Method 8270D (10). The concentrated sample extracts will then be analyzed by either
GC/HRMS (Group I analytes) or GC/LRMS methods (Group II analytes) based on analytical
methods developed under other studies (8). According to the retention time of the individual
target analyte, appropriate internal standard compounds will be assigned to each target analyte
for quantification. Note that all 6 internal standards may not be needed for quantification. The
finalized analytical methods will specify which internal standards are used. However, the other
unused internal standards can provide useful information for future validation study. The final
concentration for the internal standards will be 5 ng/mL for GC/HRMS analysis and 100 ng/mL
for GC/LRMS analysis. Draft analytical protocols for GC/HRMS (Group I analytes) and
GC/LRMS (Group II analytes) methods are included in Appendix B.
For Group III analytes, the wipe samples will be extracted with Milli-Q, deionized water using a
syringe extraction procedure. The QC samples (i.e., stored wipe method blanks, field blanks,
freshly-prepared wipe method spikes, and stored wipe method spike samples). Each wipe will be
extracted using a total of 6 mL of water. The extract volume will be adjusted to 10 mL with
Milli-Q water, then filtered. An aliquot of each extract for Group HI analytes will then be spiked
with the appropriate isotopically-labeled internal standard (13C4-TDG, 13Ci, D3-MPA, 13C6-
PMPA) and analyzed by Liquid Chromatography (LC)-MS-MS for TDG, MPA, or PMPA,
respectively, using existing protocols. Minor optimization of Battelle's current LC-MS/MS
method for MPA will be required to enable the incorporation of the 13C, Ds-MPA internal
standard. The method optimization for MPA is described in the draft analytical protocol for the
LC-MS/MS method for Group in analytes, which is included in Appendix B.
The final analytical protocol for Groups I, n, and III target analytes will be included in the final
report.
B2. Field Samples and Quality Control (QC) Samples
Field spiked samples and six types of QC samples will be collected and analyzed concurrently to
document data quality. The types of QC samples include (1) method blanks, (2) field blanks, (3)
analytical method spiked samples, (4) post-extraction spiked sample extracts, (5) 100% recovery
standard samples, and (6) performance evaluation samples.
-------
B2.1. Field Spiked Samples
Field spiked samples (spiked coupons) will be generated from the spiked test coupons from
Phase I and II experiments. One type of spiked test coupons (laminate) will be generated in
Phase I and all five types of spiked test coupons (laminate, metal, concrete, bare wood, industrial
carpet) will be generated in Phase H Triplicate samples at each spiking level from each type of
surface will be generated. Tables 5, 6, and 7 summarize the number of field samples to be
generated. Recovery data generated from the field spiked samples will be used to evaluate
overall wipe sampling method precision and accuracy.
B2.2. Method Blanks
The method blank (non-spiked wipe), which will consist of a pre-cleaned cotton gauze, will be
used to determine if any contamination occurred during the analytical sample storage,
preparation, concentration, and analysis procedures. The method blank will be carried through
the same sample storage and preparation procedures as the field spiked and field blank samples
using the same lot number of extraction solvent. Two sets of wipe method blanks will be
created; one set will be stored with the field spiked samples, field blanks, and wipe method
spikes (one for each group and analytical method), and another set to be freshly prepared prior to
extraction of all stored samples and freshly prepared wipe method spikes (also one for each
group and analytical method). Method blank result will be used to demonstrate that all
glassware, reagents, and instruments are free of interferences and that the wipe material, storage,
and/or sample preparation processes are not contributing a background level of each target
analyte.
B2.3. Field Blanks
The field blank (non-spiked coupon) is the wipe sample collected from a non-spiked test coupon.
Any potential contamination resulting from sample handling will be addressed by the field blank
samples. The field blank result will be used to determine whether other chemical residues
extracted on or within the wiped surface interfere with the analysis of the target analytes.
B2.4. Method Spiked Samples
The method spiked sample (spiked wipe) is a clean wipe that is spiked with known amounts of
target analyte(s). Two types of wipe method spikes will be generated; one set to be refrigerated
with their respective field spikes, field blank, and wipe method blank samples for 48 hours
(Phase I only), and another set, freshly prepared prior to extraction. The spiked wipe is then
prepared and analyzed by the same procedures as the field samples. The method spike result will
be used to document effect of storage on target analytes and analytical method precision and
accuracy.
B2.5. Post-extraction Spiked Field Blank Sample Extracts
The post-extraction spiked sample extract is prepared by spiking an aliquot of the field blank
sample extract with the target analyte such that the concentration of target analyte in the spiked
sample is at 5x. Recovery results of the post-extraction spiked field blank sample extracts will
be used to document any sample matrix interference (e.g., matrix enhancement or suppression
effects) and could be used as a correction factor for potential sample matrix interference.
B2.6. 100 Percent Recovery Standard Samples
The 100% recovery standard sample (solvent spike) is an aliquot of extraction solvent that is
fortified with the same spiking solution used to spike method spikes. It will be used both to
-------
evaluate the ability of the analytical system to detect the analytes and to test the spiking solution.
Note that 100% recovery standard samples will only be prepared and analyzed for Phase IIPA-
wetted wipes, and Phase II methanol-wetted wipes.
B2.7. Performance Evaluation Samples
Performance Evaluation (PE) samples will be prepared and analyzed in Phase I to confirm
method performance, and as such the PE samples will not be repeated in Phase II. PE samples
will be prepared for Groups II and III target analytes only and will be used to challenge the
analytical instruments (GC/LRMS and LC/MS/MS) and to document the performance evaluation
for standard preparation and analysis. The standards will be from a second source, and will be
prepared in the same manner and concentration as the CCV (5x or mid point on the cal curves).
The PE sample will not be available for Group I target CWAs because there is a single source for
these chemicals (Edgewood Chemical and Biological Center). PE acceptance criteria will be
percent difference of ± 10%. If the PE fails, the standard will be run again and a second failure
will require the instrument(s) to be recalibrated. There is no SOP for preparation of PE samples
since this is considered a standard laboratory dilution method.
B3. Sample Handling and Custody Requirement
The wipe samples will be generated, extracted, and analyzed in Battelle's Columbus Analytical
Chemistry (CAC) laboratories The preparation of all samples will be documented in project
specific laboratory record books (LRBs) to document internal sample chain-of custody. Sample
transfers, retrievals, and storage will be documented in the LRBs throughout the laboratory
activities, so that the location of a sample can be determined at any time. Documentation will
include date and time of activity; name of person retrieving, transferring, or storing the samples;
and location and conditions of storage. If not in the physical custody of the laboratory staff,
samples will be returned to appropriate storage, and the storage location and conditions
documented in the LRBs.
The sample codes will be assigned a unique nine-digit identification (ID) number (XXXXX-XX-
XX). The first five digits of this ID number will correspond to the LRB number in which
generation of the sample is being documented, the sixth and seventh digits to the page in the
LRB, and the last two digits to the line on the page assigned to an individual sample. The ID
number will be used in documenting all laboratory activities to reference individual samples.
The field test coupons will be decontaminated with bleach then disposed of as hazardous waste,
following collection and extraction of the wipe samples. Extracted wipes will be disposed of as
hazardous waste. Upon completion of the laboratory analyses, all sample extracts will be
returned to archival storage (freeze at -20°C±10°C or refrigerate at 4°C ±3°C). The transfer to
storage will be documented in the LRB. One month after the final data package of the sample
set has been submitted to and accepted by the EPA, the all sample extracts will be disposed of by
Battelle following established laboratory procedures, unless requested otherwise by the EPA.
B4. Inspection/Acceptance of Supplies and Consumables
The purity of all target CWAs will be checked at Battelle West Jefferson Laboratory. Dilutions
of all neat CWAs at levels below RDTE (Research, Development, Testing and Evaluation)
together with the purity results will be transferred to Battelle Columbus laboratory where all the
experimental activities will be performed. The purity for the commercially available standards
for target analytes and internal standards will be based on the Certificate of Analysis records to
-------
be provided by vendors. Bulk materials for preparation of test coupons will be purchased from
the same lot numbers. Consumables (solvents, neat chemicals, and standard solutions) will be
labeled with the expiration dates suggested by the manufacturer. These expiration dates will be
adhered to by the laboratory. Purity, expiration dates, and lot numbers of standards, solvents,
coupons, and other consumables will be recorded in the project specific LRBs.
B5. Instrumentation Calibration and Frequency
The GC/LRMS system will be tuned with the calibration gas perfluorotributylamine (PFTBA)
prior to set up of each analysis sequence following the standard instrument-specific protocol
(11). Mass spectral intensities for PFTBA will be generated and these intensities will be used to
verify that the mass tuning of the mass spectrometer has not varied significantly during analysis
of the samples. The calibration results and GC/LRMS maintenance records will be kept in the
GC/LRMS facility LRBs.
The GC/FfRMS system calibration will be accomplished using perfluorokerosene (PFK). The
manufacturer supplied software will be utilized to calibrate the mass analyzer with the accurately
known exact masses of PFK ions produced in the source. Instrument sensitivity tuning of the
GC/HRMS system will be completed using an appropriate reference (lock-mass) compound
(e.g., PFTBA, PFK, decalin-di8, etc.).
Instrument sensitivity of the GC/HRMS system will be optimized using the auto-tune program
supplied by the manufacturer (72). Once the source sensitivity optimization is complete, the slits
will be adjusted to achieve the desired resolving power (e.g., R > 10,000 when measured at 10%
peak valley). Results of the auto tune program and complete instrument settings prior to each
acquisition will be printed and included in the data package. Also included will be printouts
(oscilloscope captures) of the lock mass and calibration mass ions at both the beginning and
ending of the data of record acquisition to demonstrate appropriate instrument resolving power.
The LC-MS-MS system will be mass-calibrated, in accordance with the manufacturer
specifications (73), prior to sample analysis by infusing Poly Ethylene Glycol (PEG) 400
solution in Electrospray ionization (ESI) Positive mode to assure that the proper mass-to-charge
ratios (m/z) have been assigned. The first quadrupole mass analyzer (MSI) and the second
quadrupole mass analyzer (MS2) are tuned between 40-400 m/z. The mass accuracy after mass
calibration is performed should be ±0.2 Da.
For each analysis sequence and each analysis group, multi-point calibration curves (0.5x, Ix, 5x,
lOx, and 15x; relative to the Ix spiking level, as defined in Table 3) which include target
analytes and internal standard(s) will be generated. Calibration standard solutions consist of
target analytes and internal standards. In addition, for GC/HRMS, 1,4-dithiane and 1,4-thioxane
will be monitored during the analysis of wiped surfaces or wipe samples spiked with Group I
target analytes. For GC/LRMS and GC/HRMS, an average response factor (RF) method will be
used for the quantification, if the % RSD of the RF values for the target analyte is < 15%. If
%RSD is > 15%, regression (either linear fit or quadratic fit, depending on the best curve fit)
method will be used. If regression method is used, the correlation coefficient (r) will be greater
than 0.99. For LC-MS-MS analysis, the recalculated concentrations of the standards used to
generate each calibration curve should be within 15 percent of the theoretical value for that
standard, except in the case of the lowest standard (0.5x), which may be within 25 percent. The
calibration curves will be linear, with coefficient of determination (r2) >0.99 and with the origin
-------
excluded. One of the calibration points other than the Ix may be dropped if needed to meet these
requirements.
For GC/LRMS, GC/HRMS, and LC-MS-MS, a solvent analysis (system blank) will be
performed after the injection of the highest level of the standard solution (15x) to document that
there is no carry over from the instrument. A mid-level standard solution (5x) will be used as the
continuous calibration verification (CCV) solution and will be analyzed after every 10 samples.
Each analysis sequence will end with a CCV and a sensitivity check standard (the Ix standard) to
document the performance of the instrument. The percent deviation will be within ±25% for
each target analyte in the CCV as compared to the expected values.
If CCV values fail the acceptance criteria, corrective actions will be implemented accordingly.
The corrective actions may include cleaning MS source, cutting the front end of a GC column,
changing gold seal and injector inlet, changing GC or LC column.
B6. Instrument Maintenance
Preventive maintenance will be performed on GC/LRMS, GC/HRMS, and LC-MS/MS systems
according to the schedule defined in the appropriate facility standard operating procedures
(SOPs). Preventive maintenance and calibration will also be conducted on micropipettes and
balances, according to the schedule specified by the manufacturers and as defined by the
established laboratory procedures. As for refrigerators and freezers, the temperature of each unit
is checked and recorded daily to ensure that it is within the specified range (4°C - 7°C for
refrigerators and -20°C±10°C for freezers). If the temperature does not meet the specifications,
even after minor adjustments, the refrigerator or freezer will be serviced and in extreme cases
replaced. In addition, the temperature of each unit in Battelle's RDTE analytical laboratories
will be recorded continuously using a temperature data logger, and the data downloaded to a PC
and evaluated once every 3 months. When applicable, the following information will be
recorded:
Results of performance tests
Instrument calibration information and calibration checks
Dates on which routine maintenance is performed and a detailed account of what was
done
Instances of instrument failure
Record of all changes in location, instrument repairs, changes, and modifications
Description of any problems encountered and steps taken to rectify them.
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Data Management
A variety of records will be generated for this project. The records will include the TQAP, LRBs,
electronic files (both raw data and spreadsheets of sample results or statistical calculations), and
the final project report. This section will describe how the project records will be generated,
compiled, reviewed, maintained, reported, and archived.
C1. Documentation/Records
All preparation and analysis activities will be recorded in LRBs. Data will be generated by
GC/LRMS, GC/HRMS, or LC/MS analysts. All data will be electronically transferred by
analysts to validated Excel spreadsheets. All data will be thoroughly reviewed first by analysts
then by the appropriate Battelle TLs.
Data packages will include any of the following elements that are applicable to the analysis:
Instrument tuning (GC/LRMS, GC/HRMS, and LC/MS methods)
Calibration data
Calibration verifications
Internal standard response and retention times (GC/MS methods)
All QC data required by the analytical method or the TQAP
Run logs
Recovery data for field spiked and QC samples.
All records received by the Battelle TLs will be maintained in the Battelle TL's office until the
completion of the report at which time the records will be transferred to permanent storage at
Battelle's Records Management Office. All Battelle LRBs are stored indefinitely, either by
Battelle's Records Management Office or the Battelle TLs. One month after the final report is
approved by the EPA TOPO, all files associated with the test including project management files
and the draft data summary, will be sent to Battelle's Records Management Office and archived
for at least three years. EPA will be notified before disposal of any files.
All written records must be in ink. Any corrections to notebook entries, or changes in recorded
data, must be made with a single line through the original entry. The correction is then to be
entered, initialed, reason for the change, and dated by the person making the correction.
C2. Data Analysis
The following section describes the data analysis to be performed. Any calculations done in
addition to those discussed below will be described in detail in the final report.
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C2.1. Accuracy
Accuracy is a measure of how close the measurements are to spike values. The analytical
method accuracy will be reported as percent recovery from the spiked samples using the
following equation:
C - C
% Recovery = '" f' x 1 00
.s
where Cm, CM, and Cs are the concentration of each target analyte measured in the spiked sample,
in the un-spiked sample, and the spike concentration, respectively. Analytical method accuracy
and overall sampling and analysis accuracy will be calculated in this manner.
C2.2. Precision
Precision is the reproducibility of the replicate measurements. The standard deviation (S) of the
results for the replicate analyses of the same sample will be calculated as follows:
where n is the number of replicate samples, Mk is the measurement for the kth sample, and M
is the average measurement of the replicate samples. The precision for each sample will be
reported in terms of the percent relative standard deviation (RSD), which will be calculated as
follows.
RSD(%) =
M
xlOO
Analytical method precision and overall sampling and analysis precision will be calculated.
Comparisons of method performance will be reported as percent difference (%D) between the
field spiked samples generated by the two types of methods.
C - C
%D= '* 2 .xlOO
where Ci and 2 are the concentrations of the mean values of the target analyte measured from
replicate samples in the single- and multi-analyte spiking and/or horizontal and vertical wiping
methods, respectively; Cavg is the average of Ci and 2.
Analytical and overall method precision will be documented in term of %RSD values in
triplicate method spiked and field spiked samples, respectively. Method comparisons will be
expressed as %D between the two methods employed. In Phase I experiments, two spiking
(single- vs. multi-analyte) and two wiping (horizontal vs. vertical) methods will be evaluated.
Comparisons of the method performance between each of the two methods evaluated will be
expressed as % D values of the mean values derived from the triplicate samples generated from
the two methods evaluated.
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C2.3. Evaluation of Data Quality Objective Goals
The criteria for Phase I method performance is set at ±10% D values for the results obtained
between single- versus multi-analyte spiking, and horizontal versus vertical wiping methods.
Phase I criteria must be met in order for Battelle to proceed with Phase II. If one or more target
analytes fail Phase I criteria, then Battelle will contact the TOPO immediately to discuss the
implications and the course of action for the rest of the project.
Data quality objectives for the measurement data resulting from the project will be expressed in
terms of precision and accuracy goals. Analytical method precision and accuracy will be monitored
through the analysis of QC samples (i.e., wipe method spikes). The data quality objective goals for
the analytical method are at < 10% for analytical method precision (%RSD of the triplicate method
spikes) and 80-105% (% recovery of method spikes) for analytical method accuracy.
The data quality objectives for the wipe sampling method(s) to be established under this project,
which are inclusive of the analytical method data quality objectives, are summarized in Table 8.
The focus of this project is to evaluate the wipe sampling method and document the performance
of the method. As indicated in Section B above, non-porous surfaces such as laminate are
expected to provide relatively higher recoveries (accuracy) and tighter reproducibility (precision)
of target analytes than non-porous surfaces (bare wood and industrial carpet). These
expectations are reflected in the overall method performance for the various surfaces in the table
below. At the completion of Phase n, Battelle will determine which target analytes meet the
goals, exceed the goals and do not meet the goals for each type of test coupon. Battelle will
discuss the results with EPA TOPO and determine if additional experiments would be needed
under a modification of the project.
Table 8. Data Quality Objective Goals for the Wipe Sampling Method(s)
Data Quality Objective Goal
Wipe Sampling Method
Surface Type
Precision"
Laminate
Metal
Painted Concrete
Bare wood
Industrial Carpet
<20%
<20%
<30%
<30%
<30%
Accuracy
70-110%
70-110%
30-70%
30-70%
30-70%
a Wipe sampling method precision is the average %RSD values of triplicate field spiked samples
b Wipe sampling method accuracy is the average % recovery data of field spiked samples.
C3. Reporting
The data obtained in the project will be compiled in an EPA report. The report will describe the
purpose of the project, a summary of the experimental design, the interpretation of the data, and
the conclusions. The report will also contain a wipe sampling method as an appendix. A draft of
the wipe sampling method will be delivered to EPA by June 23, 2008. Deviations from the
TQAP will be noted in the report. The report will be approximately 30 pages.
A draft report will be submitted for review by the EPA TOPO, EPA Quality Manager, and peer
reviewers. Comments on the draft report will be addressed in revisions of the report. The peer
review comments and responses will be tabulated to document the peer review process. A final
report will be delivered to EPA no later than September 30, 2008.
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D1. Special Facilities
Battelle is certified to work with chemical surety material through a Bailment Agreement with
U.S. Army Research, Development, and Engineering Command. The Army regularly sends an
Inspector General team to conduct on-site chemical surety inspections, thereby ensuring that
Battelle is operating in accordance with the terms of the Bailment Agreement.
Battelle facilities available for the project are in compliance with all applicable Federal, state,
and local laws and regulations, including U.S. Army regulations. Battelle's facilities meet or
exceed all requirements for the safe use, storage, decontamination, and accountability of
chemical agent as defined by Army regulation AR50-6. Battelle's CAC (RDTE dilute)
Laboratory and Hazardous Material Research Center (HMRC) (neat and RDTE dilute) are
certified by Underwriters Laboratory, Inc., in accordance with ISO 9001-2000.
D2. Staff Training and Health
Each staff member working with RDTE solutions is required to take monthly RDTE quizzes, and
an annual refresher RDTE training. Prior to working with RDTE solutions, all staff members are
required to read and sign off on all relevant CAC RDTE and facility SOPs (14) as well as the
Chemical Hygiene Plan (75) and Physical Security Plan (16), then annually or every time these
SOPs are revised. All staff members worldng with RDTE solutions are also required to have an
annual physical exam with a physician on site, and once every three years during this visit a
blood sample is withdrawn to monitor the background cholinesterase level.
D3. Standard/Test Sample Handling
All handling of test items, spiking solutions of contaminants and possible interferences will be
done inside of a laboratory fume hood with hood sash set to the lowest height that still allows for
safe manipulation of materials. All CAC RDTE spiking solutions are stored in a limited access
freezer which is locked at all times and only accessed by authorized personnel on the day of
spiking. The following guidelines will be adhered to:
Personal protective equipment will include safety glasses with side shields, a fully-
fastened laboratory coat, and nitrile laboratory gloves. Gloves shall be changed every
5 minutes during which RDTE solutions are being handled inside the hood and
immediately changed if they become contaminated.
All contaminated wastes will be decontaminated with the appropriate decontamination
solution (e.g., 5.25% bleach) in accordance with RDTE SOPs and handled as hazardous
waste and disposed of according to facility regulations.
-------
D4. Sample Handling During Test
Laboratory and field handling of any solutions used during the test will be accomplished by
taking the following precautions:
All containers shall be stored and transported in double containment.
Safety goggles, nitrile gloves with long cuffs, and a chemical resistant laboratory coat
shall be worn when handling all chemicals. Gloves shall be immediately changed if they
become contaminated.
All CAC RDTE spiking solutions are single use only and will be decontaminated with
bleach immediately at the conclusion of the spiking session of the test coupons.
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1. U.S. Environmental Protection Agency, 2007. A Literature Review of Wipe Sampling
Methods for Chemical Warfare Agents and Toxic Industrial Chemicals. EPA/600/R-07/004.
National Exposure Research Laboratory, Las Vegas, NV (January 2007).
2. U.S. Homeland Security Department: National Preparedness Guideline, September, 2007.
http://www.dhs.gov/xlibrary/assets/National Preparedness Guidelines.pdf.
3. The Contaminants of Potential Concern (COPC) Committee of the World Trade Center
Indoor Air Task Force Working Group, May 2003. World Trade Center Indoor Environment
Assessment: Selecting Contaminants of Potential Concern and Setting Health-Based
Benchmarks, www.epa.gov/WTC.
4. McKean, D.L. and Sonich-Mullin, C. Risk-based Cleanup Goals for Indoor Surfaces,
International Workshop on Decontamination of Buildings and Facilities after Chemical
Terrorism, Moscow, Russia, 2006.
5. U.S. Environmental Protection Agency, January 2006. Quality Management Plan National
Homeland Security Research Center Technology Testing and Evaluation Program (TTEP),
Version 2.
6. American Society for Testing and Materials, 2001. D6661-01 Standard Practice for Field
Collection of Organic Compounds from Surfaces Using Wipe Sampling, May 2006.
Annual Book of Standards. Vol. 11.04.
7. Bernard, C.E. Berry, M.R. Wymer, L.J. Melnyk, L.J. Sampling Household Surfaces for
Pesticides: Comparison between a Press Sampler and Solvent-Moistened Wipes, Sci. of
Total Environ. In press, 2008.
8. U.S. Environmental Protection Agency, 2006. Evaluation of Sample Preparation and
Analytical Methodologies, Work Plan and Quality Assurance Project Plan for the Scientific,
Technical, Research, Engineering, and Modeling Support (STREAMS) Contract (#EP-C-05-
057), Task Order 1.
9. US EPA Method 3545A, Pressurized Fluid Liquid Extraction (PLE), Revision 1 (January
1998).
10. US EPA Method 8270D, Semivolatile Organic Compounds by Gas Chromatography/Mass
Spectrometry (GC/MS), Revision 4 (Feb 2006).
11. Hewlett Packard 5973 Mass Selective Detector Hardware Manual, Manual Part Number
G2589-90001, Agilent Technologies (November 1999).
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12. High Resolution Mass Spectrometer 119_5600_DFS_Operating Manual. Version
#2.0.1.0468 Draft 1195600. Thermo Electron Corporation (June 2006).
13. "Micromass Quattro II Mass Spectrometer User's Guide," Issue 2, and "Waters
Micromass Quattro Premier Mass Spectrometer Operator's Guide", Revision B.
14. Guidelines for Managing a Research, Development, Testing, and Evaluation (RDTE) Dilute
Solution Laboratory - September 2005, Edgewood Chemical and Biological Center
(ECBC) - Risk Reduction Office
15. Chemical Hygiene Plan for the Columbus Analytical Chemistry Group (October 2007).
16. Physical Security Plan (PSP) for the Battelle Columbus Analytical Chemistry (CAC),
Research, Development, Test & Evaluation (RDTE) Solution Laboratory (April 2007).
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Appendix A
CHEMICAL STRUCTURES FOR THE
COMPOUNDS OF INTEREST
-------
0
1,4-Dithiane
rf.
0 P C- d
f I
Dichlorvos
Distilled Mustard (HD)
if
H,C P OH
OH
Methylphosphonic
acid (MPA)
CH,
r\
/
HN-3
CH, ^
f
Soman (GD)
\
H,C CH,
Pinacolylmethylphosphonic
acid (PMPA)
HD-
C«
Thiodiglycol
C'
II
0
fl
.F,
o o
Tetraethyl pyrophosphate
(TEPP)
0
II
-t s
{
vx
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Appendix B
DRAFT GC-HRMS/SIM, GC-LRMS/SIM, AND LC-MS/MS
ANALYTICAL PROTOCOLS FOR TARGET ANALYTES
-------
Appendix B: DRAFT Analytical Protocol For Group I Analytes
Analysis of 1,4-Dithiane, 1,4-Thioxane, HN-3, HD, GD, and VX in Organic Extracts by Gas
Chromatography High Resolution Mass Spectrometry (GC/HRMS)
Revision No.: 00 Effective Date: Page 1 of 16
Scope and Applicability
This protocol describes the general procedures implemented at Battelle Columbus for the
determination of 1,4-Dithiane, GD, HD, HN-3, 1,4-Thioxane, and VX by Gas
Chromatography (GC) High Resolution Mass Spectrometry. A subset of isotopically
labeled polycyclic aromatic hydrocarbons (PAHs) used as internal standards in EPA Method
8270D will be used as internal standards in this analytical protocol. The methodology has
yet to be validated; procedures herein may be modified prior to the start of test sample
analysis.
Analytical Procedure
Reagent Preparation
Gas Chromatography
Helium: Ultra-high purity.
High Resolution Mass Spectrometry
Perfluorotetrabutylamine (PFTBA): Mass Spectrometry tuning grade or equivalent.
Standards and Test Sample Preparation
Intermediate and Calibration Standard Solutions
1.4-Dithiane (PITH) Working Standard Solution (40 ue/mL): Dilute 400 jiL of 1,4-
dithiane stock solution (Cerilliant, 1000 |ag/mL solution in methanol) to 10 mL final
volume with acetone. Solution is stored in a freezer for up to 6 months.
O-Pinacolyl methylphosphonofluoridate (GD, Soman) Working Standard Solution (8.0
|ag/mL): Dilute appropriate volume of GD RDTE stock solution to 10 mL final volume
with acetone. Solution is stored in a freezer for up to 6 months.
Bisfc-chloroethvO sulfide (HD) Working Standard Solution (8.0 ug/mL): Dilute
appropriate volume of HD RDTE stock solution to 10 mL final volume with acetone.
Solution is stored in a freezer for up to 6 months.
Tris(2-chloroethyl)amine (HN-3) Working Standard Solution (8.0 fig/mL): Dilute
appropriate volume of HN-3 stock solution to 10 mL final volume with acetone. Solution
is stored in a freezer for up to 6 months.
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Appendix B: DRAFT Analytical Protocol For Group I Analytes
Analysis of 1,4-Dithiane, 1,4-Thioxane, HN-3, HD, GD, and VX in Organic Extracts by Gas
Chromatography High Resolution Mass Spectrometry (GC/HRMS)
| Page 2 of 16
Re vision No.: 00
Effective Date:
1,4-Thioxane (THIOX) Working Standard Solution (8.0 fig/tnL): Dilute appropriate
volume of THIOX stock solution to 10 mL final volume with acetone. Solution is stored
in a freezer for up to 6 months.
O-Ethyl S-2-diisopropvlaminoethyl methyl phosphonothiolate (VX) Working Standard
Solution (12 ^g/mL): Dilute appropriate volume of VX RDTE stock solution to 10 mL
final volume with acetone. Solution is stored in a freezer for up to 6 months.
Calibration Curve Standard Solutions: Prepare separate calibration curve standards as
shown in Table B-l using a solvent system composition consistent with the final field
sample extracts. Store refrigerated. Solutions are stable for 1 month.
Table B-l. Calibration Curve Solutions
Calibration
Level
(CL)
Volume of
Working
Standard
Solution
(uL)
Final DITH GD HD HN-3 THIOX VX
Volume Cone Cone Cone Cone Cone Cone
(mL) (ng/mL) (ng/mL) (ng/mL) (ng/mL) (ng/mL) (ng/mL)
Cl (IDL)
C2
C3
C4
C5
C6
5
10
25
50
100
150
10
10
10
10
10
10
20
40
100
200
400
600
4
8
20
40
80
120
4
8
20
40
80
120
4
8
20
40
80
120
4
8
20
40
80
120
6
12
30
60
120
180
Internal Standards
Semivolatile Internal Standard Mix (EPA Method 8270D) Intermediate Internal Standard
Solution (IISS): Stock Internal Standard (IS) Mix (Supelco, 2000 ug/mL solution in
methylene chloride/benzene) contains each of the following isotopically labeled analytes:
Acenaphthene-dio, Chrysene-^b, Naphthalene-^, perylene-^b, Phenanthrene-t/io,
l,4-Dichlorobenzene-£/4. Dilute 100 uL of the Stock IS Mix (2000 ug/mL) to 10 mL with
acetone (pesticide residue grade). The concentration of this solution is 20 ug/mL.
Solution is stored in a freezer for up to 6 months.
Semivolatile Working Internal Standard (WIS): Dilute 50 jiL of the Intermediate Internal
Standard Solution (20 ug/mL) to 10 mL in acetone. This will produce a WIS solution
with a concentration of 100 ng/mL. Solution is stored in a freezer for up to 6 months.
Test Sam pies
Following extraction and concentration of sample extracts to 1 mL, each Field Spike,
Field Blank, Method Blank, and Method Spike test sample extract will be fortified with
50 |iL of the WIS. The sample extract will be mixed and transferred to a GC vial for
-------
Appendix B: DRAFT Analytical Protocol For Group I Analytes
Analysis of 1,4-Dithiane, 1,4-Thioxane, HN-3, HD, GD, and VX in Organic Extracts by Gas
Chromatography High Resolution Mass Spectrometry (GC/HRMS)
| Revision No.: 00 | Effective Date: | Page 3 of 16
subsequent GC/HRMS analysis. The 100% Recovery Standard sample (1 mL) will also
be fortified with 50 jaL of the WIS. The post-extraction spiked field blank sample extract
will be prepared by removing an aliquot (500 jiL) of the field blank sample extract to
another GC vial and adding an aliquot (2.5 jiL) of the working analyte standard for GC-
HRMS analysis. The concentration of the target analyte in the post-extraction spiked field
blank sample extract will be at the C4 level.
Instrument Operation
The GC/HRMS system will be tuned according to the manufacture's instructions in order to
verify that acceptable performance criteria are achieved. For sensitivity, the spectrometer will be
tuned using either autotune or manual tune. Typically, PFTBA is bled into the instrument
through the reference inlet system. When using autotune, the final optimization should show an
intensity change of ± 5% relative to the previous attempt. The resolving power (m/Am, 5% peak
height) will be adjusted to > 10,000 and documented for all lock and calibration masses in each
scan function prior to the start of the analysis. Likewise, the ending resolving power must be
>9,000 and must be documented.
At a minimum, two ion transitions (quantitation and qualifier ions) will be monitored for each
target analyte. The exact mass of each monitored ion (precursor or fragment), as calculated on
the HRMS data acquisition system, will be used in the acquisition method. The analytical
response (peak area and/or height) will be determined. The ratio of the analytical response of the
two ion transitions will be calculated. Prior to the start of the analytical sequence, the scan
window time functions will be set and verified using a calibration standard of an appropriate
concentration.
All chromatographic peaks must have signal-to-noise ratio >3 to be considered detected.
Typical GC-HRMS Operating Conditions
Typical GC/HRMS operating conditions are listed in Table B-2. Other conditions may
be used but all minimum performance criteria must be met.
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Appendix B: DRAFT Analytical Protocol For Group I Analytes
Analysis of 1,4-Dithiane, 1,4-Thioxane, HN-3, HD, GD, and VX in Organic Extracts by Gas
Chromatography High Resolution Mass Spectrometry (GC/HRMS)
| Page 4 of 16
Re vision No.: 00
Effective Date:
Table B-2. Typical GC/HRMS Operating Conditions
GC/HRMS System
Gas Chromatograph
Mass Spectrometer
MS Source
GC Column
GC Temperature Program
Carrier Gas Flow Rate
Transfer Line Temperature
Injection Volume
Injection Type
Acquisition Mode
Run Time
lonization Energy
Dwell Time
Ion Source Temperature
Trap Current
Thermo Fisher DPS (or equivalent)
Thermo Fisher Trace GC Ultra (or equivalent)
Thermo Fisher DPS High Resolution Mass Spectrometer (or equivalent)
Electron impact, positive ion mode
Varian CP-Sil 5CB, 30 m, 0.25 mm ID, 1.0 urn film
50 °C hold for 2.0 min
20 °C/min to 80 °C, hold for 3.0 nun
4 °C/min to 250 °C, hold for 0 min
30 °C/min to 300 °C. hold for 3 min.
1 to 2 mL/min
250 °C
2.0 uL
Splitless (Split at 1.0 min at 30 mL/min)
Multiple ion detection (MID), equivalent to SIM
55 min
30 to 70 eV
25 ms for Lock and Cali Mass, >50 uis for analyte
250 °C
600 uA
Monitored Ions
Ions typically monitored for the target analytes are shown in Table B-3. The exact masses
will be calculated using the system's data processing software. Other ions may be added
or substituted to these but the elemental composition must be known and documented.
Regardless of the ions monitored, all performance criteria must be met.
-------
Appendix B: DRAFT Analytical Protocol For Group I Analytes
Analysis of 1,4-Dithiane, 1,4-Thioxane, HN-3, HD, GD, and VX in Organic Extracts by Gas
Chromatography High Resolution Mass Spectrometry (GC/HRMS)
Re vision No.: 00
Effective Date:
Page 5 of 16
Table B-3. Elemental Compositions for Ions Typically Monitored by GC/HRMS
Analyte Elemental Composition (nominal m/z)
DITH
GD
HD
HN-3
THIOX
VX
1.4-Dichlorobenzene-rf4 (IS)
Acenaphthene-c/u, (IS)
Chrysene-t/^
Naphthalene-ds
Perylene-c/12
Phenanthrene-du,
C2H4S2(92);C4H8S2(120)
CHSPO2F (99); C,H8PO2F (126)
C3H6S35C1 (109); C3H6S37C1 (111)
C,HK,N35C12 (154); C,H10N3;1C137C1 (156)
C4H8SO (104); C3H6S (74)1
C7H16N(114);C8H17N(127)
C6D435C12 (150); C6D435C137C1 (152)
C12D9(162);C12D10(164)
C18D10 (236)2; C18D12 (240)
C10D6(132)2;C10D8(136)
C20DK, (260)2; C20D12 (264)
C14D8 (184)2; C14D10 (188)
1. Secondary ion (m/z 74) for THIOX is optional due to large mass range ratio.
2. Secondary ion for internal standard is optional.
Quality Control
System Blanks
A system blank, prepared using a solvent system composition consistent with the final
field sample extracts and spiked with the IS, will be analyzed at the beginning of the
analysis to confirm system cleanliness.
A system blank will also be analyzed after the highest calibration standard; if analyte
carryover > 0.5x Cl level exists, a second system blank will be analyzed before test
samples are analyzed. Analyte carryover will be calculated and noted in the data package.
Calibration curves
A set of calibration curve solutions will be analyzed at the start of each test sample batch.
The calibration curve for each target analyte will be constructed by plotting the relative
response of the quantitation ion of each analyte with respect to its internal standard
(Acenaphthene-c/io, Chrysene-t/i2, Naphthalene-t/g, perylene-t/i2, Phenanthrene-^io, or
l,4-Dichlorobenzene-t/4) against the concentration of the target analyte. The recalculated
concentrations of the standards used to generate each calibration curve should be within
15% of the theoretical value for that standard, except in the case of the lowest standard,
which may be within 25%. The calibration curves should be linear, with coefficients of
determination > 0.99, with the origins excluded. Weighted models may be applied but any
activity must be noted in the data package. One of the calibration points, other than the Cl
standard, may be excluded if needed to meet these requirements.
-------
Appendix B: DRAFT Analytical Protocol For Group I Analytes
Analysis of 1,4-Dithiane, 1,4-Thioxane, HN-3, HD, GD, and VX in Organic Extracts by Gas
Chromatography High Resolution Mass Spectrometry (GC/HRMS)
| Revision No.: 00 | Effective Date: | Page 6 of 16
Continuing Calibration and Verification (CCV) standards
A standard at C3 concentration level will be analyzed at least every 10 test samples and at
the end of the analytical sequence. The concentration of the target analyte should be ±
25% of the theoretical concentration. Both ion transitions should be detected.
If a CCV fails the ± 25% accuracy criterion, all samples between the previous successful
laboratory check standard and the next successful laboratory check standard must be
reanalyzed.
Ion Ratios
Ion ratios will be determined using the average ratio of all calibration standards (>C2) used
in the analysis sequence.
Sensitivity check standards
A sensitivity check standard at Cl level will be analyzed near the end of the sequence.
Both ion transitions should be detected with a signal-to-noise ratio >3.
Test Sample Concentrations Outside Calibration Curve Range
If a target analyte is detected in a test sample and the concentration is below 0.5x Cl level,
the concentration will be flagged as estimated. If a target analyte is detected and the
concentration is at or above 0.5x Cl level, then the actual concentration will be reported.
If the concentration of a target analyte in a test sample exceeds the upper calibration limit,
the sample will be diluted and reanalyzed to bring the concentration within the calibration
range.
Reporting
The analyst will assemble a data package containing the date of analysis, instalment identity, and
quantitative results. Excel spreadsheets will be used to determine precision results. A brief
report that discusses the analytical results and any anomalies will be provided.
Sample Disposal
Following analysis, data review, reporting, and acceptance of the analytical results, any
remaining aqueous sample extracts from a given trial will be decontaminated with bleach.
-------
Appendix B: DRAFT Analytical Protocol for Group III Analytes
Analysis of TDG, PMPA, and MPA in Aqueous Solutions by Liquid
Chromatography/Mass Spectrometry (LC-MS/MS)
| Revision No.: 00 | Effective Date: | Page 7 of 16
Scope and Applicability
This protocol describes the general procedures implemented at Battelle Columbus for the
determination of 1,4-dithiane, dichlorvos, and tetraethylpryophosphate (TEPP) in organic
solutions by gas chromatography/mass spectrometry (GC/MS). A subset of isotopically
labeled polycyclic aromatic hydrocarbons (PAHs) used as internal standards in EPA Method
8270D will be used as internal standards in this analytical protocol. The methodology is yet
to be finalized; procedures herein may be modified prior to the start of test sample analysis.
Analytical Procedure
Standard Solutions and Test Sample Extract Preparation
Intermediate and Calibration Standard Solutions
1,4-Dithiane Working Standard (10 |ig/mL): Dilute 100 jiL of 1,4-dithiane stock solution
(Cerilliant, 1000 |ag/mL solution in methanol) to 10 mL final volume with acetone.
Solution is stored in a freezer for up to 6 months.
Dichlorvos Working Standard (10 |ag/mL): Dilute 100 |aL of dichlorvos stock solution
(Cerilliant, 1000 |ag/mL solution in methanol) to 10 mL final volume with acetone.
Solution is stored in a freezer for up to 6 months.
TEPP Working Standard (10 ng/mL): Dilute 100 jiL of TEPP stock solution (Absolute
Chemical, 1000 |ag/mL solution in hexane) to 10 mL final volume with acetone. Solution
is stored in a freezer for up to 6 months.
Calibration Curve Standard Solutions: Prepare separate calibration curves as Table B-4
below using IPA as diluent. Store refrigerated. These solutions are stable for 1 month.
Internal Standards
Working EPA Method 8270D Internal Standard (WIS): Dilute 50 jiL of EPA Method
8270D internal standard stock solution (Supelco, 2000 ng/mL solution in 1: 1 mixture of
dichloromethane/benzene) to 10 mL final volume with acetone. Stock Internal Standard
(IS) Mix contains each of the following isotopically labeled analytes: Acenaphthene-t/io,
Chrysene-t/12, Naphthalene-t/8, perylene-t/i2, Phenanthrene-c/io, and 1,4-Dichlorobenzene-
6/4. The final concentration for the WIS is 10 jag/mL. Solution is stored in a freezer for up
to 6 months.
-------
Appendix B: DRAFT Analytical Protocol for Group III Analytes
Analysis of TDG, PMPA, and MPA in Aqueous Solutions by Liquid
Chromatography/Mass Spectrometry (LC-MS/MS)
Re vision No.: 00
Effective Date:
Page 8 of 16
Table B-4. Calibration Curve Standard Solutions
CalStdID
Volume of
Working
Analyte
Standard
(uL)
Volume of
Working
Internal
Standard3
(ML)
Final
Volume
(mL)
1,4-Dithiane
Cone (ng/mL)
Dichlorvos
Cone
(ng/mL)
TEPP Cone
(ng/mL)
Cl(orlDL)
C2
C3
C4
C5
C6
20
40
100
200
400
600
100
100
100
100
100
100
10
10
10
10
10
10
20
40
100
200
400
600
20
40
100
200
400
600
20
40
100
200
400
600
See Section 2.1.2 for the preparation of working internal standard solution.
2.1.3 Test Sample Extracts
Following extraction and concentration of sample extracts to 1 mL, each Field Spike,
Field Blank, Method Blank, and Method Spike test sample extract will be fortified with
10 jiL of the WIS. The sample extract will be mixed and transferred to a GC vial for
subsequent GC/MS analysis. The 100% Recovery Standard sample (1 mL) will also be
fortified with 10 jiL of the WIS. The post-extraction spiked field blank sample extract
will be prepared by removing an aliquot (500 |aL) of the spiked field blank sample extract
to another GC vial and adding an aliquot (10 |iL) of the working analyte standard for
GC/MS analysis. The concentration of the target analyte in the post-extraction spiked
field blank sample extract will be at the C4 level.
Instrument Operation
Automated Hewlett-Packard gas chromatograph/mass selective detector (6890/5973A
GC/MSD) equipped with an autosampler or equivalent GC/MS system. The instrument
will be operated in the full mass scan (FMS) mode first to establish parameters (e.g.,
dwelling times for monitored ions, times for switching monitored ions) to be used in the
selected ion monitoring (SIM) mode. In the SCAN mode, the detector scans all masses
repeatedly during the GC run between a lower and an upper mass limit, typical from
35 to 550 atomic mass unit (amu). A project specific acquisition method for the SIM
mode
will be established according to the retention time and mass spectral information from the
FMS mode. All calibration standards and sample extracts will be analyzed in the SIM
mode. Peaks must have signal-to-noise ratios > 3:1 to be considered detected.
The GC/MS system must be tuned according to the manufacturer's instructions, to verify
that acceptable performance criteria are achieved. If the tune criteria are not met,
-------
Appendix B: DRAFT Analytical Protocol for Group III Analytes
Analysis of TDG, PMPA, and MPA in Aqueous Solutions by Liquid
Chromatography/Mass Spectrometry (LC-MS/MS)
Re vision No.: 00
Effective Date:
Page 9 of 16
corrective actions will take place immediately (e.g., clean MS source, change GC
column, etc.).
Typical GC-LRMS Operating Conditions
Typical GC/LRMS operating conditions are listed in Table B-5. Other conditions may be
used but all minimum performance criteria must be met.
Table B-5. Typical GC/LRMS Operating Conditions
GC/LRMS System
Gas Chromatograph
Mass Selective Detector
MS Source
GC Column
GC Temperature Program
Carrier Gas Flow Rate
Injector Temperature
Transfer Line Temperature
Injection Volume
Injection Type
Acquisition Mode
Run Time
lonization Energy
Dwell Tune
MS Source Temperature
Hewlett-Packard GC/MSD(or equivalent)
Hewlett-Packard 6890(or equivalent)
Hewlett-Packard 5973 A (or equivalent)
Electron impact mode
RTX-5MS GC column, 0.25 mm x 30 m with 0.25
jim film thickness
100 °C hold for 2.0 min
8 °C/min to 290 °C, hold for 10 min.
1 to 2 mL/min
270 °C
290 °C
1.0 uL
Splitless (Split at 1.0 min at 30 mL/min)
Multiple ion detection (MID), equivalent to SIM
~36 min
70 eV
>50 ms for target analytes and >30 ms for internal
standards
230 °C
Monitored Ions
Ions typically monitored for the target analytes are shown in Table B-6. The monitored
ions may be changed as necessary and will be recorded in the final protocol.
Table B-6. Ions for Target Analytes/IS Typically Monitored by GC/LRMS
Analyte
1,4-Dithiane
Dichlorvos
TEPP
1.4-Dichlorobenzene-^4 (IS)
Acenaphthene-t/io (IS)
Chrysene-t/i2
Naphtiialene-o's
Perylene-ob
Phenanthrene-c/io
Monitored Ions (m/z)
120, 61, 46
220, 185, 145, 109
263, 235, 179, 161
152
164
240
136
264
188
-------
Appendix B: DRAFT Analytical Protocol for Group III Analytes
Analysis of TDG, PMPA, and MPA in Aqueous Solutions by Liquid
Chromatography/Mass Spectrometry (LC-MS/MS)
| Revision No.: 00 | Effective Date: | Page 10 of 16
Quality Control
System Blanks
A system blank (IP A) fortified with the IS will be analyzed at the beginning of the
analysis to confirm system cleanliness.
A system blank will also be analyzed after the highest calibration standard; if carryover
above 0.5x Cl level exists, a second system blank will be analyzed before test samples
run.
Calibration curves
A set of calibration curve solutions will be analyzed at the start of each test sample batch.
The calibration curve for each target analyte will be constructed by plotting the relative
response of the quantitation ion of each analyte with respect to its internal standard
(Acenaphthene-t/io, Chrysene-t/i2, Naphthalene-t/g, perylene-t/i2, Phenanthrene-^io, or
l,4-Dichlorobenzene-t/4) against the concentration of the target analyte. The calibration
curves can be generated using an average response factor (Rf) method, if the % RSD of
the Rf values for the target analyte is < 15%. If %RSD is > 15%, regression (either linear
fit or quadratic fit, depending on the best curve fit) method will be used. If regression
method is used, the correlation coefficient (r) should be greater than 0.99. If these criteria
are not met, the GC/MS system will be checked to determine the sources for this
variation. Corrective actions (e.g., clean source or change column) will be taken and all
samples in the sequence will be reanalyzed.
Continuing Calibration and Verification Standards
A C3 standard will be analyzed at least every 10 test samples and at the end of the
analytical sequence. The concentration of the target analyte should be ± 25% of the
theoretical concentration. If a check standard fails the ± 25% accuracy criterion, all
samples between the previous successful laboratory check standard and the next successful
laboratory check standard must be reanalyzed.
Sensitivity Check Standards
A sensitivity check standard at (i.e. Cl standard) will be analyzed near the end of the
sequence. Monitored ions should be detected with > 3:1 signal-to-noise ratio.
-------
Appendix B: DRAFT Analytical Protocol for Group III Analytes
Analysis of TDG, PMPA, and MPA in Aqueous Solutions by Liquid
Chromatography/Mass Spectrometry (LC-MS/MS)
| Revision No.: 00 | Effective Date: | Page 11 of 16
Test Sample Concentrations Outside Calibration Curve Range
If a target analyte is detected in a test sample and the concentration is below 0.5x Cl level,
the concentration will be flagged as estimated. If a target analyte is detected and the
concentration is at or above 0.5x Cl level, then the actual concentration will be reported.
If the concentration of a target analyte exceeds the upper calibration limit, the sample will
be diluted and reanalyzed to bring the concentration within the calibration range.
Reporting
The analyst will assemble a data package containing the date of analysis, instalment identity, and
quantitative results. Excel spreadsheets will be used to determine precision results. A brief
report that discusses the analytical results and any anomalies will be provided.
Sample Disposal
Following analysis, data review, reporting, and acceptance of the analytical results, any
remaining sample extracts from a given trial will be disposed of following established laboratory
procedures.
Scope and Applicability
This protocol describes the general procedures implemented at Battelle Columbus for the
determination of thiodiglycol (TDG), pinacolylmethylphosphonic acid (PMPA), and
methylphosphonic acid (MPA) in aqueous solutions by Liquid Chromatography-Mass
Spectrometry (LC-MS/MS). Isotopically labeled TDG, PMPA, and MPA are used as
internal standards. The MPA methodology is yet to be developed; procedures herein may be
modified prior to the start of test sample analysis.
-------
Appendix B: DRAFT Analytical Protocol for Group III Analytes
Analysis of TDG, PMPA, and MPA in Aqueous Solutions by Liquid
Chromatography/Mass Spectrometry (LC-MS/MS)
| Revision No.: 00 | Effective Date: | Page 12 of 16
Analytical Procedure
Reagent Preparation
Liquid Chromatography Mobile Phase
TDG Mobile Phase: Into a 500 mL volumetric flask place 50 mL of methanol, 1 mL of 1 M
formic acid, and 1 mL of 1 M ammonium formate. Adjust to 500 mL final volume with
Milli-Q water. Proportional volumes may be used as needed.
PMPA Mobile Phase: Into a 500 mL volumetric flask place 150 mL of acetonitrile and
0.5 mL of formic acid (approximately 99%). Adjust to 500 mL final volume with Milli-Q
water. Proportional volumes may be used as needed.
MPA Mobile Phase: To be determined. May be binary gradient composition.
Standards and Test Sample Preparation
Intermediate and Calibration Standard Solutions
TDG Working Standard Solution (4000 ng/mL): Dilute 40 |aL of TDG stock solution
(Cerilliant, 1000 |ag/mL solution in methanol) to 10 mL final volume with Milli-Q water.
Store refrigerated up to 3 months.
PMPA Working Standard Solution (4000 ng/mL): Dilute 40 |iL of PMPA stock solution
(Cerilliant, 1000 |ag/mL solution in methanol) to 10 mL final volume with Milli-Q water.
Store refrigerated up to 3 months.
MPA Working Standard Solution (4000 ng/mL): Dilute 40 |iL of MPA stock solution
(Cerilliant, 1000 jag/mL solution in methanol) to 10 mL final volume with Milli-Q water.
Store refrigerated up to 3 months.
Calibration Curve Standard Solutions: Prepare separate calibration curves as tabled
below using Milli-Q water as diluent. Store refrigerated. TDG and PMPA solutions are
stable up to 60 days; stability of MPA solutions is to be determined.
-------
Appendix B: DRAFT Analytical Protocol for Group III Analytes
Analysis of TDG, PMPA, and MPA in Aqueous Solutions by Liquid
Chromatography/Mass Spectrometry (LC-MS/MS)
| Page 13 of 16
Re vision No.: 00
Effective Date:
CalStdID
Table B-7. Calibration Curve Solutions
Volume of
Working
Standard
Solution
OiL)
Final Volume
(mL)
TDG Cone
(ng/mL)
PMPA Cone
(ng/mL)
MPA Cone
(ng/mL)
Cl(orlDL)
C2
C3
C4
C5
C6
50
100
250
500
1000
1500
10
10
10
10
10
10
20
40
100
200
400
600
20
40
100
200
400
600
20
40
100
200
400
600
Fortify 190 jaL of calibration standard with 10 uL of appropriate WIS (see below), and
vortex prior to analysis.
Internal Standards
TDG-13C4 Working Internal Standard (WIS): Dilute 200 |aL of TDG-13C4 stock solution
(Cerilliant, 100 jag/mL solution in methanol) to 10 mL final volume with Milli-Q water.
Cone = 2000 ng/mL. Store refrigerated up to 3 months.
13,
13,
PMPA- Cfi Working Internal Standard (WIS): Dilute 200 jiL of PMPA- C6 stock
solution (Cerilliant, 100 |ig/mL solution in methanol) to 10 mL final volume with Milli-Q
water. Cone = 2000 ng/mL. Store refrigerated up to 3 months.
13,
13/
MPA-1JC. D. Working Internal Standard (WIS): Dilute 200 jiL of MPA- C, D3 stock
solution (Cerilliant, 100 |ig/mL solution in methanol) to 10 mL final volume with Milli-Q
water. Cone = 2000 ng/mL. Store refrigerated up to 3 months.
Test Samples
Each Field Spike, Field Blank, Post-extraction Spiked Field Blank, Method Blank, Method
Spike, and 100% Recovery Standard test sample extract received with the analytical batch
will be fortified with the appropriate IS. Add 10 jaL of appropriate WIS to 190 ^L of test
sample. Vortex prior to analysis.
Instrument Operation
The spectrometer will be mass-calibrated, or have its calibration verified, prior to the start of
analysis. The mass accuracy (residuals) after calibration must be ± 0.2 Da.
Two ion transitions (quantitation and qualifier ions) will be monitored for each target analyte.
Peak areas will be determined. The ratio of the peak areas of the 2 transitions will be calculated.
Peaks must have signal-to-noise ratios > 3:1 to be considered detected.
-------
Appendix B: DRAFT Analytical Protocol for Group III Analytes
Analysis of TDG, PMPA, and MPA in Aqueous Solutions by Liquid
Chromatography/Mass Spectrometry (LC-MS/MS)
Re vision No.: 00
Effective Date:
Page 14 of 16
LC-MS/MS Operating conditions for TDG and PMPA are summarized in Table B-8.
Table B-8. Operating Conditions for TDG and PMPA
LC-MS/MS System
HPLC Mass Spectrometer
Mass Spec Source
HPLC Column
Column Temperature
Mobile Phase (Isocratic)
Flow Rate
Injection Volume
Run Time
Retention times
MRM Transitions
Waters 2695 (or equivalent)
Micromass Quattro II with Z-spray source (or equivalent)
Electrospray, positive ion mode
TDG: Restek Allure PFP propyl, 2.1 x 150 mm, 5 urn
PMPA: Phenomenex Columbus C8, 2 x 50 mm, 5 um
Ambient
TDG: 10% aqueous methanol containing 2 mM each of formic acid and
ammonium formate
PMPA: 30% aqueous acetonitrile containing 0.1% of formic acid
0.3 mL/min (no split to MS)
20 ML
5 min for TDG and 2.5 mm for PMPA
Approximately 3.3 min for TDG and 1.3 min for PMPA
TDG: 123>105 and 123>87
TDG-13C4: 127>109 and 127>91
PMPA: 181>97and 181>79
PMPA-13C6: 187>79 and 187>97
MPA Operating Conditions
Infuse mass-labeled MPA (MPA-13C, D3) solution into the spectrometer to collect precursor
and product ions. Optimize cone and collision energy settings for found transitions.
Determine the linear calibration range of non-labeled MPA using a developed
chromatographic method.
Check for the presence of non-labeled MPA in the MPA-13C, D3 internal standard:
chromatograph a solution of MPA-13C, D3 that is prepared near the mid-point (or lower) of
the non-labeled MPA linear calibration range. If the MPA-13C, D3 is free of non-labeled
MPA indications, the tested MPA-13C, D3 may be tried as the concentration of internal
standard for a calibration curve. If non-labeled MPA is found, analyze lower concentrations
of MPA-13C, D3 to find a concentration where the MPA level is below one-half of the lowest
MPA calibration standard; the determined MPA-13C, D3 concentration which is free of MPA
indications may then be tried as the concentration of internal standard for a calibration curve.
The LC-MS/MS system used to analyze the test samples and associated controls for MPA
will be the same as before (Waters 2695 or equivalent HPLC system coupled with a
Micromass Quattro II or equivalent with Z-spray source mass spectrometer operated in the
Electrospray positive ion mode). The MRM transitions for non-labeled MPA will be 97>79
and 97>47. The MRM transitions for MPA-13C, D3 and all other operating conditions (HPLC
column, column temperature, mobile phase, flow rate, injection volume, run time, and
retention times) are to be determined.
-------
Appendix B: DRAFT Analytical Protocol for Group III Analytes
Analysis of TDG, PMPA, and MPA in Aqueous Solutions by Liquid
Chromatography/Mass Spectrometry (LC-MS/MS)
| Revision No.: 00 | Effective Date: | Page 15 of 16
Quality Control
System Blanks
A system blank (Milli-Q water) will be analyzed at the beginning of the analysis to
confirm system cleanliness.
A system blank will also be analyzed after the highest calibration standard; if carryover
above 0.5 Cl exists, a second system blank will be analyzed before test samples run.
Blank + IS
The Blank + IS sample will be analyzed before the calibration curve to verify that the IS
contributes less than 0.5 Cl of the target analyte's response.
Calibration curves
A set of calibration curve solutions will be analyzed at the start of each test sample batch.
The calibration curve for each target analyte will be constructed by plotting the relative
response of the quantitation ion of each analyte with respect to its internal standard (TDG
relative to TDG-13C4; PMPA relative to PMPA-13C6; MPA relative to MPA-13C, D3)
against the concentration of the target analyte. The recalculated concentrations of the
standards used to generate each calibration curve should be within 15 percent of the
theoretical value for that standard, except in the case of the lowest standard, which may be
within 25 percent. The calibration curves should be linear, with coefficients of
determination > 0.99, with the origins excluded. One of the calibration points, other than
the Cl standard, may be excluded if needed to meet these requirements.
Continuing Calibration and Verification (CCV) standards
A standard at C3 concentration will be analyzed at least every 10 test samples and at the
end of the analytical sequence. The concentration of the target analyte should be ± 25% of
the theoretical concentration. Both ion transitions should be detected.
If a CCV fails the ± 25% accuracy criterion, all samples between the previous successful
laboratory check standard and the next successful laboratory check standard must be
reanalyzed.
Sensitivity check standards
A sensitivity check standard at Cl will be analyzed near the end of the sequence. Both ion
transitions should be detected with > 3:1 signal-to-noise ratio.
-------
Appendix B: DRAFT Analytical Protocol for Group III Analytes
Analysis of TDG, PMPA, and MPA in Aqueous Solutions by Liquid
Chromatography/Mass Spectrometry (LC-MS/MS)
| Revision No.: 00 | Effective Date: | Page 16 of 16
Test Sample Concentrations Outside Calibration Curve Range
If a target analyte is detected in a test sample and the concentration is below 0.5x Cl level,
the concentration will be flagged as estimated. If a target analyte is detected and the
concentration is at or above 0.5x Cl level, then the actual concentration will be reported.
If the concentration of a target analyte in a test sample exceeds the upper calibration limit,
the sample will be diluted and reanalyzed to bring the concentration within the calibration
range.
Reporting
The analyst will assemble a data package containing the date of analysis, instrument identity, and
quantitative results. Excel spreadsheets will be used to determine precision results. A brief
report that discusses the analytical results and any anomalies will be provided.
Sample Disposal
Following analysis, data review, reporting, and acceptance of the analytical results, any
remaining aqueous sample extracts from a given trial will be decontaminated with bleach.
-------
Appendix C: Addendum
Appendix C: Addendum
Testing and Quality Assurance Plan for the Evaluation of Wipe
Sampling Methods for Collecting Chemical Warfare Agents (CWAs),
CWA Degradation Products, and Toxic Industrial Chemicals from
Various Surfaces
Addendum July 2, 2008
Approved hy:
,--
-
-j
7- -?-o?
Stephen Bfllets
L J5, EPA Task Order Project Officer
-------
Appendix C: Addendum
Section Bl, Experimental Plan of this document states: "Battelle will discuss the results
of Phase I with the EPA TOPO and determine whether it is feasible to move forward to
Phase n experiments as outlined in Figure 2. If the results from the Phase I experiments
do not meet all the criteria as stated above, Battelle will consult with the EPA TOPO on
revising the approach for the Phase II full-scale study accordingly."
Completion of Phase I experiments showed that the method for the laminate was
reproducible (mostRSDs < 20%; several < 10%); multi-analyte splicing was comparable
to single-analyte spiking (most < 10% D); vertical wiping was comparable to horizontal
wiping (most <10% D); and 48-hour holding of the wipes prior to extraction had a
negligible effect on recovery (few percent lower recoveries from stored wipes versus
freshly extracted wipes). However, only three of the 10 target compounds (VX, tetraethyl
pyrophosphate, and pinacolylmethylphosphonic acid) had recoveries that met the primary
data quality objective of greater than 70%. To address the findings, Phase II will be
modified from what was originally planned by adding: a new wetting/extraction solvent,
a new surface (vinyl tile), analysis by low resolution mass spectrometry, and two
phosphonic acids. Two surfaces (carpet and bare wood) were removed due to their
porosity. The project will continue to use the same pre-cleaned wipes, surface spiking
procedure, wiping procedure, and quality control samples that were used in Phase I. This
addendum describes the changes to the original plan that now will be incorporated into
Phase n of the experimental design. These modifications are summarized in Tables 1, 2,
and 3.
Target analytes: The original 10 target analytes included four CWAs (HD, HN-3, GD,
and VX), four degradation products [1,4-dithiane, thiodiglycol (TDG), methylphosphonic
acid (MPA), and pinacolylmethylphosphonic acid (PMPA)], and two organophosphorous
pesticides [dichlorvos and tetraethyl pyrophosphate (TEPP)]. In Phase I, 1,4-dithiane,
dichlorvos, and TEPP were measured by low resolution mass spectrometry-selected ion
monitoring (LRMS-SEVI) and the CWAs were measured by high resolution mass
spectrometry-selected ion monitoring (HRMS-SEVI). Both HRMS-SEVI and LRMS-SEVI
analytical techniques will be evaluated in modified Phase II for all analytes. For
efficiency, we will combine these seven analytes into one gas chromatograph (GC)
analysis. Of the remaining three analytes, the phosphonic acids and TDG will continue to
be analyzed by liquid chromatography-mass spectrometry (LC-MS-MS). In addition, we
will add ethylmethylphosphonic acid (BMPA) and isopropylmethylphosphonic acid
(IMPA) to the list of acid degradation products to be evaluated by LC-MS-MS in
modified Phase n. The current LC-MS-MS acid method will be modified to add EMPA
and IMPA. A commercially-available isotopically-labeled IMPA will be used as an
internal standard for IMPA. There is no isotopically-labeled EMPA standard available,
which requires that EMPA be quantified against the isotopically-labeled IMPA. In the
absence of an isotopically-labeled EMPA to use as an internal standard, the quantitation
of EMPA in this method may not be as accurate. The method, however, should still
provide useful information on the sampling and analytical performance of this compound
of interest.
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Appendix C: Addendum
Concentration levels: The risk-based surface cleanup goals (Table 2) continue to be the
driver. Consequently, HRMS-SIM must continue to be used to achieve these ultra-low
levels. As shown in Tables 1 and 2, two concentration levels (Ix and lOx of the risk-
based surface cleanup levels) will be evaluated for the GC and LC analytes, respectively.
An addition will be to evaluate and determine at what sensitivity level the LRMS-SIM
could be utilized instead of HRMS-SIM since most typical analytical laboratories will not
have HRMS-SIM capability. Table 3 summarizes the estimated detection limits of target
analytes by GC-HRMS-SIM, GC-LRMS-SIM, and the proposed spiking levels. If
detection for the seven GC analytes is achieved by LRMS-SIM from the lOx spiking
level but not from the Ix spiking level, the detection capability of the LRMS-SIM for
these target analytes will be extrapolated from the lOx spiking level.
Wetting solvent(s): Isopropyl alcohol (IPA) has been the only wetting solvent utilized to
this point. In the modified Phase II, we will evaluate IPA and 1:1
acetone:dichloromethane (ACE:DCM) for the GC analytes and IPA and methanol for the
LC analytes. The volume of wetting solvent for each wipe will continue to be 2 mL.
Wiping surfaces: Three of the five types of original test surfaces will be evaluated.
Laminate, galvanized metal, and painted concrete will be tested. Bare wood and
industrial carpet will be eliminated due to significant porosity and anticipated poor
recoveries so that we can invest remaining resources into better understanding the
performance of the method for other surfaces. We will add vinyl tile as a surface
(Armstrong commercial flooring, Standard Excelon vinyl composition tiles, Pattern
51858, Imperial Texture, sandrift white, 1/8 inch thick). Coupons will continue to be
prepared according to the procedure in Section B1.3 of the test/QA plan. We will also
continue to evaluate smaller sized coupons (3.2 cm x 3.2 cm =10 cm2 surface) which can
be wholly extracted by sonication for comparison of surface retention and wiping
efficiency. Pre-treatment of the surfaces will involve pre-cleaning prior to use by wiping
them with the pre-wetted cotton gauze wipes (with whatever wetting solvent is being
used) and allowing the surface to air dry prior to spiking.
Surface spiking procedure: We will continue to use the same liquid spiking procedure
as described in Section B1.5 of the test/QA plan. Briefly, all spiking procedures will be
carried out in a fume hood. A group of four test coupons (triplicate test samples and one
field blank) will be placed in a clean container inside the hood. An aliquot (1000 jiL) of
the spiking solution will be spiked onto each coupon at 5 spots, at a rate of 200 jaL/spot.
For each field blank, same amount of solvent (1000 jiL) used for the preparation of the
individual spiking solution will be spiked in the same manner as the field spike test
coupons. After spiking, the coupons will be left in the hood for no more than five
minutes for drying. The smaller sized coupons will be spiked in the same manner but the
volume of the spike will be 0.1 mL of a 10-fold more concentrated spiking solution of
GC target analytes. The GC and LC analytes will be in separate spiking mixtures.
Wipe sampling procedure: We will continue to use the same wiping procedure, as
described in Section B 1.6 of the test/QA plan. Briefly, the designated wiping area (10 cm
by 10 cm) on the test coupon will be wiped with 2 pre-cleaned cotton gauze pads, each
wetted with 2-mL of the wetting solvent (IPA, ACE:DCM, or methanol). The first wipe
will be used to wipe the coupon area in a single direction from top to bottom, while the
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Appendix C: Addendum
second wetted wipe will be used to wipe the same coupon area in a single direction from
left to right, with three strokes each. After the first stroke, the exposed surface of the first
cotton gauze will be folded inward for the second stroke, and then folded again for the
third stroke. The cotton gauze pad is folded again with the exposed surface inside; then
placed in the original jar. The test coupon will then be wiped again with the second wipe
using the same procedures as described above. The second wiped cotton gauze pad will
be placed in the same container as the first wiped cotton gauze pad; then the container
sealed with Teflon tape and refrigerated for 48 hours prior to extraction and analysis.
The purpose of this 48 hour storage time is to simulate field sample handling and storage
conditions of the wipe samples as well as the elapsed time between sampling and
extraction. Wipe samples spiked with the target analytes, along with non-spiked wipes,
will also be stored under the same conditions as the field samples. All spiked test
coupons will be placed in the horizontal position for wiping.
Sample preparation: Preparation procedures for the GC and LC analyses are described
in Section B1.7 of the test/QA plan. We will continue to use ACE:DCM by ASE as the
extraction procedure for the GC analytes. Each smaller coupon will be sonicated with
3x10 mL of 1:1 acetone/DCM for 10 minutes each time then extracts combined and
concentrated to a final volume of 1 mL. The LC analytes will be extracted with 10 mL of
Milli-Q deionized water instead of 7 mL to improve the extraction efficiency of target
analytes from the wipe matrix.
Sample analysis: All of the GC samples will be analyzed by HRMS-SIM and LRMS-
SEVI (following Appendix B of the test/QA plan). We will do some optimization of the
GC-LRMS-SIM method to accommodate all seven GC analytes. All of the LC samples
will be analyzed by LC-MS-MS following the protocol described in Appendix B of the
test/QA plan. The LC-MS-MS protocol for the acid target analytes will be optimized to
accommodate EMPA and IMPA in the same analysis.
QC samples: We will continue to use surface-coupon non-spikes, post-extraction
spikes, spiked wipes, non-spiked wipes, and solvent spikes as QC controls for GC-
MS-SIM (both HRMS and LRMS) and LC-MS-MS analyses.
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Appendix C: Addendum
Type of
Surface
Laminate,
Galvanized
Metal,
Painted
Concrete,
and
Vinyl Tile
Table 1. Summai
Multi-
analyte
Spike
1,4-
Dithiane
of Groups I and n GC-MS-SIMa - Modified Phase n Approach
Number of Samples
Spiking Spiking
Level 1 Level 2 Residential
(lOx) (Ix) Risk Based
/ fig/ Cleanup
Sample I Sample
Surface Coupon
Spikes
GD
HD
HN-3
VX
Dichlorvos
TEPP
0.400
0.080
0.080
0.080
0.120
0.400
0.400
0.040
0.008
0.008
0.008
0.012
0.040
0.040
Cleanup
Goals
us/Sample
210
0.086
0.0081
0.0081
0.013
0.220
0.220
12
Surface Coupon
Non-Spikes
Non-
Post- Spiked Spiked
extraction Wipes Wipes Solvent
Spikes8 J'h **' Spike'
a Samples in Table 1 will be generated for each of the four surfaces (laminate, galvanized metal, painted concrete, and vinyl tile) and analyzed by GC-
LRMS-SDVI and GC-HRMS-SHVI with the exception of the spiked wipes, non-spiked wipes, and solvent spikes which are independent of the surface so
they will only be generated for each batch.
b Spiking level is for the 10 cm x 10 crn coupons, the 3.2 cm x 3.2 cm coupons, and the spiked and stored wipes. The lOx is equivalent to 20x first
calibration (Cl) level.
0 Two wipes will be used for each sample. One wipe will be used to wipe the coupon from top to bottom and the second one from left to right. Both
wipes will be extracted/analyzed as a single sample. Half the wipes are wetted with 2 inL of IPA each and the other half are wetted with 1:1 ACE/DCM.
d Each extract will be concentrated to a final volume of 1 mL.
e Three replicates will be prepared at two spiking levels. For wipes, two wetting solvents (IPA and 1:1 ACE/DCM) will be evaluated.
'Each coupon is sonicated with 3x10 mL of 1:1 ACE/DCM for 10 min each time then extracts combined and concentrated to a final volume of 1 mL.
8 Post-extraction spike is only conducted on one non-spiked sonicated surface extract of each surface at each spiking level
h Two replicates at each spiking level and each wetting solvent
1 One non-spiked wipe for each wetting solvent evaluated.
' One solvent spike will be prepared at each spiking level in 100% acetone.
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Appendix C: Addendum
Type of
Surfacea
Laminate,
Galvanized
Metal,
Painted
Concrete,
and
Vinyl Tile
Multi-
analyte
Spike
MPA
EMPA
IMPA
PMPA
TDG
Table 2. Summary of Group m LC-MS-MS - Modified Phase n Approach
Number of Samples
Spiking
Level 1
(lOx)
Jig/
Sampleb
4.0
Spiking Residential
Level 2 Risk Based
(Ix) Cleanup
jig/ Goals
Samplec tig/Sample
Surface Coupon
Spikes
Methanol-
Wetted
Wipe
d,e,f
4.0
4.0
4.0
4.0
0.40
0.40
0.40
0.40
0.40
520
NA
2100
NA
5400
Surface
Coupon
Non-Spikes
Methanol-
Wetted
Wiped'r
Post- Spiked Non-
extraction Wipes Spiked
Spikes8 d'h Wipes Solvent
d-' Spikej
1
a Samples in Table 2 will be generated for each of the four surfaces (laminate, galvanized metal, painted concrete, and vinyl tile) and analyzed by LC-
MS-MS with the exception of the spiked wipes, non-spiked wipes, and solvent spikes which are independent of die surface so they will only be
generated for each batch.
b Since the final extract volume is 10 mL for Group IE wipe samples, the theoretical concentration of target analytes is 0.4 ug/mL which is 20 x Cl cal level
c Since the final extract volume is 10 mL for Group IE wipe samples, the theoretical concentration of target analytes is 0.04 ug/mL which is 2 x Cl cal level
d Two wipes will be used for each sample. For surfaces, one wipe will be used to wipe the coupon from top to bottom and the second one from left to
right. Both wipes will be extracted together and analyzed as a single sample. A wipe is wetted with either 2 mL of IP A or 2mL of methanol.
e Three replicates will be prepared at each spiking level
1 A different wetting solvent may be attempted instead, if another EPA NHSRC study suggests that methanol is not a good wetting solvent.
g Two post-extraction spikes will be conducted on each non-spiked surface extract, one at each spiking level
hTwo replicates at each spiking level and for each wipe wetting solvent
1 One replicate for each wipe wetting solvent
J Two solvent spikes will be prepared for each wetting solvent, one at each spiking level
NA = Cleanup goal is not available
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Appendix C: Addendum
Table 3. Comparison of Estimated Instrument Detection Limits (H>L) for Target Analytes by GC-
HRMS-SIM and GC-LRMS-SIM to Residential Risk Based Cleanup Goal and Proposed lOx and Ix
Spiking Level
Target Analyte
Residential Risk
Based Cleanup
Goals
ng/Sample
GC-HRMS-SIM
Estimated IDL
us/Samplea
GC-LRMS-SIM lOx Spiking Level Ix Spiking Level
Estimated IDL
ng/Sample"'b ng/Sample ng/Sample
1,4-Dithiane
GD
HD
HN-3
VX
Diclilorvos
TEPP
210,000
86
8.1
8.1
13
220
220
0.066
0.030
0.330
0.006
0.660
0.45
1.1
5.0
10
4.0
20
66
10
10
400
80
80
80
120
400
400
40
8.0
8.0
8.0
12
40
40
Detection limit for each analyte is based on a 3:1 S/N of the qualifier ion. Minimum of 2 ions per analyte are monitored by GC-
HRMS and up to 3 ions by GC-LRMS.
Note that a sample is a 10 cm x 10 cm coupon that is wiped, or a 3.2 cm x 3.2 cm coupon that is sonicated.
b GC-LRMS-SIM IDLs are based on other Battelle projects.
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EPA/600/R-08/079 I February 2008 I wvw.epa.gov/nhsrc
$EPA
United States
Environmental Protection
Agency
PRESORTED STANDARD
POSTAGES FEES PAID
EPA
PERMIT NO. G-35
Office of Research and Development
National Homeland Security Research Center
Cincinnati, OH 45268
Official Business
Penalty for Private Use
$300
Recycled/Recyclable
Printed with vegetable-based ink on
paper that contains a minimum of
50% post-consumer fiber content
processed chlorine free
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