EPA 600/R-12/033 | January 2013 | www.epa.gov/ord
United States
Environmental Protection
Agency
Evaluation Report
Enzymatic Decontamination
of Chemical Warfare Agents
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National Homeland Security Research Center
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EPA 600-R-12-033
January 2013
Evaluation Report
Enzymatic Decontamination
of Chemical Warfare Agents
UNITED STATES ENVIRONMENTAL PROTECTION AGENCY
RESEARCH TRIANGLE PARK, NORTH CAROLINA 27711
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ERRATA Sheet
For the document: Enzymatic Decontamination of Chemical Warfare Agents (EPA/600/R-
12/033) April 2012
Updated December 2012 with the following modifications:
• Section 2.6.2 (Page 11), the following text was added to clarify the procedure that was
used:
The test coupons were spiked with VX or TGD and allowed to weather for 30 minutes;
then the DEFENZ VX-G enzyme was added for the specified contact time for the
decontamination test. The positive control coupons were spiked with VX or TGD and
allowed to weather for 30 minutes plus the specified contact time used for the test
coupons.
• Section 2.6.3 (Page 13), the following text was added:
The test coupons were spiked with HD and allowed to weather for 30 minutes; then the
DEFENZ B-HD enzyme was added for the specified contact time for the
decontamination test. The positive control coupons were spiked with HD and allowed to
weather for 30 minutes plus the specified contact time used for the test coupons.
• A new Appendix A contains additional experimental data and discussion thereof that are
directly related to the main body of the report. The data include the measurement of the
enzymatic decontamination efficacy of the same enzyme containing decontamination
products described in the main body of this report as derived from solution chemistry
experiments without the presence of coupon surfaces. The additional data provides
insights on the observed efficacies during bench scale coupon testing described in the
main body of this report.
in
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ACKNOWLEDGMENTS
The following individuals and organizations are acknowledged for review of this document:
United States Environmental Protection Agency
Office of Solid Waste and Emergency Response, Office of Emergency Management
Jeanelle Martinez
Leroy Mickelsen
Office of Research and Development, National Homeland Security Research Center
Worth Calfee
Contributions of the following organization are acknowledged:
Battelle
IV
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EXECUTIVE SUMMARY
The U.S. Environmental Protection Agency (EPA) is the primary federal agency
responsible for remediation in the aftermath of a terrorist release of chemical warfare agent
(CWA). The imminent threat of release in a building or transportation hub resulted in EPA
research on methods for effective neutralization/cleanup. As one of the potential tools/methods,
EPA is systematically evaluating the effectiveness of enzyme-based decontamination
technologies: DEFENZ™ VX-G (for decontamination of VX and G-type nerve agents) and
DEFENZ™ B-HD (for decontamination of sulfur mustard [FID]). In addition, the extent to which
the efficacy of the enzyme solutions changed after preparation and storage was evaluated.
DEFENZ™ VX-G contains granulated organophosphorus acid anhydrolase (OPAA) and
organophosphorus hydrolase (OPH) enzymes while DEFENZ™ B-HD contains an arylesterase
enzyme that catalyzes a chemical reaction to produce peracetic acid.
Efficacy results, i.e., the difference in CWA recovered from positive controls and
CWA recovered from decontaminated test coupons as a percentage of CWA recovered from
positive control coupons, are summarized in Table ES-1. DEFENZ™ VX-G exhibited a
statistically significant efficacy (Student's t-test p < 0.05) against VX on all materials tested
except vinyl, with a 15-minute (min) contact time. Statistically significant efficacy means that
the average measured amount of agent recovered from the test coupons after decontamination
was statistically significantly lower than the average amount recovered from the positive control
coupons (i.e., those without decontamination application). Tests on galvanized metal showed
that efficacy against VX increased with increasing contact time and when higher concentrations
of the enzymes were employed. DEFENZ™ VX-G exhibited a statistically significant efficacy
against thickened soman (TGD) on carpet (but not against TGD on the other four test materials)
with a 15-min contact time. DEFENZ™ VX-G enzymes applied to TGD on laminate showed
that although no statistically significant efficacy was observed with a 15 min contact time, there
was a higher efficacy with a 30-min contact time. After a 45-min contact time, less soman (GD)
was recovered from laminate treated with enzyme than from positive controls, but the difference
between the treated laminate and the positive controls was not statistically significant. Tests on
galvanized metal showed that efficacy against TGD increased when higher concentrations of the
enzymes were employed.
DEFENZ™ B-HD exhibited a statistically significant efficacy against HD on all five
materials tested with 15-min contact time. Efficacy was increased by using a longer contact time
(60 min, but not 30 min) for both vinyl and carpet.
No toxic byproducts were found to be produced by use of the DEFENZ™ VX-G or
DEFENZ™ B-HD enzymes and no damage to the test material coupons was visually observed
from the use of the enzymes.
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Table ES- 1. Summary of Decontamination Efficacy Results
CWA DEFENZ™ Enzyme Material
VX
vx
VX
vx
vx
vx
vx
vx
vx
TGD
TGD
TGD
TGD
TGD
TGD
TGD
TGD
TGD
HD
HD
HD
HD
HD
HD
HD
HD
HD
VX-G
VX-G
VX-G
VX-G
VX-G
VX-G
VX-G
VX-G
VX-G
VX-G
VX-G
VX-G
VX-G
VX-G
VX-G
VX-G
VX-G
VX-G
B-HD
B-HD
B-HD
B-HD
B-HD
B-HD
B-HD
B-HD
B-HD
Laminate
Wood
Carpet
Vinyl
Galvanized metal
Galvanized metal
Galvanized metal
Galvanized metal
Galvanized metal
Galvanized metal
Wood
Carpet
Vinyl
Laminate
Laminate
Laminate
Laminate
Laminate
Galvanized metal
Laminate
Wood
Carpet
Vinyl
Vinyl
Vinyl
Carpet
Carpet
Contact Time,
mm*
15
15
15
15
15
30
45
15
15
15
15
15
15
15
30
45
15
15
15
15
15
15
15
30
60
30
60
Concentrationf
IX
IX
IX
IX
IX
IX
IX
2X
3X
IX
IX
IX
IX
IX
IX
IX
2X
3X
IX
IX
IX
IX
IX
IX
IX
IX
IX
Mean Efficacy
On Test Coupons
12% (p <
50% (p =
19% (p <
19% (p =
11% (p =
23% (p <
26% (p <
29% (p <
39%(p<
-5% (p =
-9% (p =
42% (p <
30% (p =
-37% (p =
48% (p <
24% (p =
68% (p <
51% (p<
24% (p <
27% (p <
29% (p <
16% (p =
24% (p =
7% (p =
35% (p =
15%(p =
30% (p =
0.01)
0.05)
0.01)
0.09)
0.04)
0.01)
0.01)
0.01)
0.01)
0.82)
0.77)
0.01)
0.19)
= 0.27)
0.01)
0.41)
0.01)
0.01)
:0.04)
0.01)
0.01)
<0.01)
<0.01)
0.22)
0.01)
0.09)
<0.01)
* Manufacturer recommends 15-min contact time.
f IX is enzyme diluted with deionized water per manufacturer's recommendation; 2X is diluted with half the recommended
water; 3X is diluted with one-third of the recommended water.
While the DEFENZ™ VX-G and DEFENZ™ B-HD enzymes demonstrate efficacy, a
substantial portion of the chemical agents (VX, TGD, and FID) can be extracted from the test
VI
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materials even after the longest contact times and using the highest enzyme concentrations
evaluated. Longer contact times, or repeated applications, may be necessary to reduce the CWA
to acceptable levels. Higher concentrations of DEFENZ™ VX-G than the manufacturer's
recommendation may increase efficacy against VX and TGD. Likewise, use of longer contact
times than the manufacturer's recommendation of 15 min for both DEFENZ™ VX-G and
DEFENZ™ B-HD appears to increase efficacy. No loss of efficacy was observed for the
DEFENZ™ VX-G and DEFENZ™ B-HD when prepared and stored according to
manufacturer's recommendations.
Caution should be used in extrapolating from bench testing to field application of the
enzymes. However, given the observed efficacies of the DEFENZ™ VX-G enzyme against VX
and TGD and the DEFENZ™ B-HD enzyme against HD and the lack of visible damage to a
range of indoor building materials, the enzymes appear to be technologies that might be
considered for use against these CWA on indoor building materials after a terrorist release.
Activity of enzymes depends strongly on the manufacturer's production process. Hence,
the results obtained for this report reflect solely on the commercially available DEFENZ™
enzyme decontamination products rather than the associated (OPAA and OPH) enzymes.
vn
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DISCLAIMER
The U.S. Environmental Protection Agency (EPA), through its Office of Research and
Development, funded and managed the research described here under EPA Contract Number EP-
C-10-001, Work Assignment Number 2-04 to Battelle. This document has been subjected to the
Agency's review and has been approved for publication. Note that approval does not signify that
the contents necessarily reflect the views of the Agency.
Mention of trade names or commercial products in this document or in the methods referenced in
this document does not constitute endorsement or recommendation for use. EPA does not
endorse the purchase or sale of any commercial products or services.
Questions concerning this document or its application should be addressed to:
Lukas Oudejans
National Homeland Security Research Center
Office of Research and Development
U.S. Environmental Protection Agency
Mail drop: E343-06
Research Triangle Park, NC 27711
(919)541-2973
oudejans.lukas@epa.gov
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FOREWORD
Following the events of September 11, 2001, EPA's mission was expanded to address critical
needs related to homeland security. Presidential directives identify EPA as the primary federal
agency responsible for the country's water supplies and for decontamination following a
chemical, biological, and/or radiological (CBR) attack.
As part of this expanded mission, the National Homeland Security Research Center (NHSRC)
was established to conduct research and deliver products that improve EPA's capability to carry
out its homeland security responsibilities. One specific focus area of our research is on
decontamination methods and technologies that can be used in the recovery efforts resulting
from a CBR contamination incident. In recovering from an incident and decontaminating the
area, it is critical to identify and implement appropriate decontamination technologies. The
selection and optimal operation of an appropriate technology depends on many factors including
the type of contaminant and associated building materials, temperature, relative humidity,
decontaminant concentration, contact time, and others. This document provides information on
how a commercially available enzyme containing decontamination product performed in
treatment of chemical warfare agents (CWAs) deposited on interior industrial building materials
at various operational conditions.
These results, coupled with additional information in separate NHSRC publications (available at
www.epa.gov/nhsrc) can be used to determine whether a particular decontamination technology
can be effective in a given scenario. With these factors in consideration, the best technology or
combination of technologies can be chosen that meets the cleanup, cost and time goals for a
particular decontamination scenario.
NHSRC has made this publication available to assist the response community to prepare for and
recover from disasters involving chemical contamination. This research is intended to move EPA
one step closer to achieving its homeland security goals and its overall mission of protecting
human health and the environment, while providing sustainable solutions to environmental
challenges.
Jonathan Herrmann, Director
National Homeland Security Research Center
IX
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TABLE OF CONTENTS
ACKNOWLEDGMENTS Ill
EXECUTIVE SUMMARY V
DISCLAIMER VIII
FOREWORD IX
LIST OF TABLES XII
LIST OF FIGURES XIII
ABBREVIATIONS/ACRONYMS XIV
1.0 INTRODUCTION 1
1.1 Purpose 1
1.2 Test Facility Description 1
1.3 Project Objectives 1
2.0 PROCEDURES 3
2.1 Technology Descriptions 3
2.2 Chemical Warfare Agents 3
2.3 Building Material Coupons 4
2.4 Coupon Spiking 4
2.5 Preparation of Enzyme-Based Decontamination Technologies 5
2.5.1 Preparation Procedure for DEFENZ™ VX-G 5
2.5.2 Preparation Procedure for DEFENZ™ B-HD 7
2.6 Test Matrices 9
2.6.1 Spray Application Demonstration to Select Enzyme Application Rate 9
2.6.2 DEFENZ™ VX-G Test Matrices 11
2.6.3 DEFENZ™ B-HD Test Matrices 13
2.7 Observation of Surface Damage 14
2.8 Extraction and Analysis 15
2.9 Method Demonstration 16
2.9.1 Recovery of CWA from Test Coupons 16
2.9.2 MDL for VX, GD, and HD Extracted from Vinyl 17
2.9.3 Quench of Decontamination Reaction 18
2.9.4 EA 2192 LC/MS Analysis 18
2.10 Efficacy Determination 21
2.11 Analysis of By-Products 22
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3.0 QUALITY ASSURANCE/QUALITY CONTROL 25
3.1 Control of Monitoring and Measuring Devices 25
3.2 Chemical Analysis Equipment Calibrations 26
3.3 Technical Systems Audit (TSA) 26
3.4 Performance Evaluation Audits 27
3.5 Data Quality Audit 27
3.6 Spike Control Data 27
3.7 Amendments 28
3.8 Deviations 28
4.0 RESULTS/DISCUSSION 30
4.1 Method Demonstration Results 30
4.2 Decontamination Results 30
4.2.1 Measurement of pH of Enzyme Solution on Coupons 31
4.2.2 Measurement of Peracetic Acid in DEFENZ™ B-HD Enzyme Solution 31
4.2.3 VX Decontamination 32
4.2.4 TGD Decontamination 36
4.2.5 HD Decontamination 40
4.3 By-Product Analysis 42
4.4 Observations of Damage to Coupons 43
5.0 CONCLUSIONS 47
6.0 REFERENCES 49
XI
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LIST OF TABLES
Table ES- 1. Summary of Decontamination Efficacy Results vi
Table 1. Chemical Warfare Agents Used 3
Table 2. Test Materials 4
Table 3. Enzyme-Based Decontamination Technology Concentrations 6
Table 4. Weight of Enzyme Aliquots and Volume of Make-up Water Required 8
Table 5. Enzyme Application Amounts for Bench-Scale Testing 11
Table 6. Test Matrix for Decontamination of CWA with DEFENZ™ VX-G Prepared per
Manufacturer's Recommendations and 15-Min Contact Time 11
Table 7. Test Matrix for Increased DEFENZ™ VX-G Enzyme Concentration 12
Table 8. Test Matrix for Effect of Storage of Activated 3 X DEFENZ™ VX-G Enzyme Solution
on VX Decontamination Results 13
Table 9. Test Matrix for Decontamination of CWA with DEFENZ™ B-HD 13
Table 10. Test Matrix for Longer Contact Times with DEFENZ™ B-HD 14
Table 11. Test Matrix for Delayed Application of DEFENZ™ B-HD Enzyme Solution 14
Table 12. Gas Chromatography/Mass Spectrometry Parameters for VX and GD Analysis* 15
Table 13. Pertinent Parameters for Target Chemicals 16
Table 14. Recovery of Agent Using Hexane Extract!on as Quench 18
Table 15. Demonstration of Hexane as Quench for Enzyme Reaction Prior to LC/MS Analysis
forEA2192 20
Table 16. Recovery of EA 2192 from Aqueous Phase after Hexane Extraction 21
Table 17. Test Matrix for LC/MS EA 2192 22
Table 18. Liquid Chromatography/Mass Spectrometry Parameters for EA2192 Analysis 24
Table 19. Data Quality Objectives and Results for Test Measurements 25
Table 20. Performance Parameters to be Audited 27
Table 21. HD Recovery from Spike Control Coupons 28
Table 22. Monthly VX Purity Data Showing Gradual Degradation 29
Table 23. VX Recovery from Spike Control Coupons 30
Table 24. Extraction Efficiencies for CWAs from Vinyl Coupons 30
Table 25. MDL Values for VX, GD, and HD Extracted from Vinyl Using Hexane 30
Table 26. Peracetic Acid Measurements for Activated DEFENZ™ B-HD Enzyme 32
xii
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Table 27. VX Decontamination Results Using DEFENZ™ VX-G 34
Table 28. VX Decontamination Results with Concentrated Enzyme Solutions 35
Table 29. Effect of Storage of Activated Enzyme Solution on VX Decontamination Results 35
Table 30. TGD Spike Recovery from Spike Control Coupons 36
Table 31. TGD Decontamination Results Using DEFENZ™ VX-G 38
Table 32. TGD Decontamination Results with Concentrated Enzyme Solutions 39
Table 33. Effect of Storage of Activated DEFENZ™ VX-G Enzyme Solution on TGD
Decontamination Efficacy 40
Table 34. HD Decontamination Results Using DEFENZ™ B-HD 41
Table 35. Effect of Storage of Activated DEFENZ™ B-HD Enzyme Solution on HD
Decontamination Efficacy 42
Table 36. Results from Analysis for EA 2192 (Toxic VX By-product) 43
LIST OF FIGURES
Figure 1. Approach used to ensure homogeneity of DEFENZ™ 120 and DEFENZ™ 130
enzymes in 500 mL enzyme test solutions 7
Figure 2. Coupons before application of CWA (top), during DEFENZ™ VX-G
decontamination (center) and after enzymatic decontamination (bottom) with
residual plastic thickener from TGD visible, e.g., as shown by arrow 44
Figure 3. Coupons during application of VX (top) and during DEFENZ™ VX-G
decontamination (center, bottom) 45
Figure 4. Coupons before application of HD (top), during DEFENZ™ B-HD
decontamination (center) and 48 hours after decontamination (bottom) 46
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ABBREVIATIONS/ACRONYMS
AMU
BBRC
°C
ccv
CI
cm
CWA
EPA
FPD
g
gal
GC
GC/MS
GD
HD
HMRC
HPLC
IS
kg
kHz
kPa
L
LC/MS
m
MDL
MSD
min
mL
mm
atomic mass unit
Battelle Biomedical Research Center
degrees Celsius
continuing calibration verification
confidence interval
centimeter(s)
chemical warfare agent
U.S. Environmental Protection Agency
flame photometric detection/detector
gram(s)
gallon(s)
gas chromatography
gas chromatography/mass spectrometry
soman
sulfur mustard
Hazardous Materials Research Center
high performance liquid chromatography
internal standard
kilogram(s)
kilohertz
kilopascal(s)
liter(s)
liquid chromatography/mass spectrometry
meter(s)
method detection limit
mass selective detector
minute(s)
milliliters(s)
millimeter(s)
xiv
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US
HL
ng
NHSRC
NIST
OPAA
OPH
PAA
PE
psi
QA
QMP
QC
RDECOM
RDS
RH
SD
SIM
SRC
TBP
TGD
ISA
micrometer(s)
microgram(s)
microliter(s)
nanogram(s)
National Homeland Security Research Center
National Institute of Standards and Technology
organophosphorus acid anhydrolase
organophosphorus hydrolase
peracetic acid
performance evaluation
pounds per square inch
quality assurance
quality management plan
quality control
Research, Development and Engineering Command
research dilute solution(s)
relative humidity
standard deviation
selected ion monitoring
surrogate recovery compound
tributyl phosphate
thickened soman
technical systems audit
xv
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1.0 Introduction
1.1 Purpose
Protecting human health and the environment is the mission of the U.S. Environmental
Protection Agency (EPA). The imminent threat of a chemical warfare agent (CWA) release in a
building or transportation hub is driving the EPA to develop a research program that
systematically evaluates potential decontaminants of CWAs. The EPA may be tasked to clean up
these agents after a release. Most of the more efficacious decontamination technologies that have
been identified are not compatible with all surface materials due to, e.g., their corrosive or
bleaching characteristics, while some of the decontaminants may produce toxic by-products
when they react with the CWA. A need therefore exists to identify decontamination methods that
are nonreactive to building materials and that avoid toxic by-product formation. Enzymatic
decontamination technologies are benign. However, effectiveness of available enzyme
technologies against CWAs on many surfaces is unknown. In addition, the degree to which
environmental conditions such as temperature and relative humidity (RH) affect decontamination
is not known. The optimal decontaminant concentration and contact time have been determined
primarily by vendors of decontaminants and are based predominantly on stirred reactor data.
This report describes a systematic investigation to evaluate the efficacy of two enzyme-based
technologies produced by Genencor® (a Danisco Division; Palo Alto, CA): DEFENZ™ VX-G
(for decontamination of VX and G-type nerve agents) and DEFENZ™ B-HD (for
decontamination of sulfur mustard [FID]). (In May 2011, DuPont acquired a majority stake in
Danisco A/S and the Genencor® enzymes are now within DuPont Industrial Biosciences.) The
effect of the decontaminant on the building material was assessed qualitatively.
Potential benefits for DEFENZ™ VX-G include lack of toxicity, high efficiency, high
specificity and ease of use. DEFENZ™ B-HD provides an enzymatic method for on-site
production of peracetic acid for HD decontamination, thereby avoiding safety issues associated
with transportation and storage of this hazardous material.
1.2 Test Facility Description
All testing was performed at Battelle's Hazardous Materials Research Center (HMRC) or
at the Battelle Biomedical Research Center (BBRC). Both facilities are located on the same
Battelle site in West Jefferson, Ohio. Battelle is certified to work with chemical surety material
at the HMRC through its Bailment Agreement W911SR-05-H-0001 with the U.S. Army
Research, Development & Engineering Command (RDECOM). Battelle is certified to work with
chemical surety material at the BBRC through its contract with the Defense Threat Reduction
Agency (Contract Number: W81XWH-05-D-0001/DO 0001).
1.3 Project Objectives
The objective of this evaluation was to determine the decontamination efficacy of
enzymatic decontamination technologies (DEFENZ™ VX-G against VX and thickened soman
(TGD) and DEFENZ™ B-HD against HD) applied to coupons. The enzymes were initially
prepared per manufacturer's label instructions and stored and used in accordance with the label
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instructions. Efficacy of the enzymes when appropriately applied against VX, TGD, and HD was
evaluated on each of five building materials (galvanized metal, decorative laminate, industrial
carpet, wood flooring, and vinyl flooring) at one contact time (15 min as specified in the
DEFENZ™ VX-G and DEFENZ™ B-HD instructions for use). Higher concentrations of
DEFENZ™ VX-G and longer contact times were also evaluated. Specifically, a 2:1 and 3:1 mix
of recommended enzyme to water was tested with a 15 min contact time of DEFENZ™ VX-G
against VX and TGD. The recommended enzyme to water mix was also tested with a 30 and 45
min contact time for DEFENZ™ VX-G against VX and TGD while for DEFENZ™ B-HD, 30
and 60 min contact times were tested against HD.
The stability of the efficacy of prepared solutions ("pot life") with proper storage was also
evaluated. Because some decontaminants react with CWAs to produce toxic by-products, a
qualitative assessment of decontamination by-products was performed. In addition to the
chemical analyses, a qualitative visual assessment for obvious damage was made by comparing
blank coupons exposed to enzyme solution to blank coupons not exposed to enzyme solution.
Testing was performed in accordance with Test/Quality Assurance (QA) Plan for
Enzymatic Decontamination of Chemical Warfare Agents, Version 2 (July 2010) \
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2.0 Procedures
2.1 Technology Descriptions
DEFENZ™ VX-G and DEFENZ™ B-HD are enzyme-based technologies produced by
Genencor® (a Danisco Division, Palo Alto, CA). Details of the technologies are proprietary.
The instructions on how to create the default enzyme solutions are per vendor's directions.
DEFENZ™ VX-G contains granulated organophosphorus acid anhydrolase (OPAA) and
granulated organophosphorus hydrolase (OPH) enzymes which are present in a 1:10 mass ratio.
The DEFENZ™ VX-G product consists of a pouch containing two packets: (1) an
enzyme packet (110 grams (g) of granulated powder and (2) a buffer packet (250 g of powder)
containing predominantly sodium hydrogen carbonate (NaHCCb). The enzyme packet contains
two pre-mixed constituent powders: 10 g of "organophosphorous [sic] acid anhydrolase" enzyme
(DEFENZ™ 120G) and 100 g of "organophosphorous [sic] hydrolase" enzyme (DEFENZ™
130G). The enzyme and buffer dissolve in 10 liters (L) of water. According to the vendor,
DEFENZ™ VX-G has a shelf life of 3 years when stored properly in an unopened and sealed
container and a pot life (defined as time that the enzyme is active in aqueous solution) of 8 hours.
DEFENZ™ B-HD is a perhydrolase-based enzymatic system for generating peracetic
acid (PAA) as the active ingredient in the presence of water, propylene glycol diacetate, and
sodium percarbonate. It is delivered as one kilogram (kg) of slurry to which 37.85 L (10 gallons
[gal]) of water is added to activate the technology. After mixing in water, the solution is allowed
to sit for 20 min (for oxidant generation) before use. The solution must then be used within 8
hours of mixing with water.
2.2 Chemical Warfare Agents
The CWAs used to evaluate the efficacy of decontamination were VX, TGD, and HD
(Table 1). The target purity of neat agent was at least 85% and was verified for the specific agent
lot using gas chromatography (GC)-flame photometric detection (FPD) prior to beginning testing
and monthly during testing. TGD was prepared by addition of approximately 5% acrylic polymer
to neat GD at least one week prior to use in decontamination testing. This thickener was added to
reduce the volatility of GD so that sufficient GD could be recovered from a positive control
coupon.
Table 1. Chemical Warfare Agents Used
. , Manufacturer/Supplier _ ,. .,.,.,-,
Agent „ Preparation Applied to Coupons
VX US Army from EPA stocks* Neat agent (as supplied)
Neat agent (as supplied) with 5% acrylic polymer
TGD US Army from EPA stocks* (weight: volume; Paraloid K125, Rohm and Haas Company,
Philadelphia, PA)
HD US Army from EPA stocks* Neat agent (as supplied)
*EPA-owned stocks of CWAs are stored at Battelle's facilities in West Jefferson, OH.
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2.3 Building Material Coupons
This bench-scale investigation utilized small coupons of interior building materials
(presented in Table 2) contaminated with CWAs.
Table 2. Test Materials
Coupon
Manufacturer/ Surface Material
Supplier Name Size L x W Preparation
(cm)
Material
Description
Galvanized metal
ductwork
Decorative
laminate
Industry heating, ventilation, and
air conditioning standard; 24
gauge galvanized steel;
thickness 0.7 mm (Adept
Manufacturing)
Pionite® or Formica®
laminate/white matte finish; grade
10; thickness -1.2 mm
Adept Products,
Inc., West 3.5x1.5
Jefferson, OH
A' Jack Inc., , - , -
Columbus, OH
Clean with
acetone
None
Industrial grade
carpet
Shaw Industries, Inc. EcoWorx
thickness -0.7 cm
Carpet
Corporation of
• „ 3.5x1.5
America, Rome.
GA
None
Flooring material
Vinyl flooring
material
Fir plywood (bare);
thickness 0.9 cm
Armstrong Excel on
Lowe's,
Columbus, OH
Lowe's,
Columbus, OH
3.5x1
3.5x1
.5
.5
Clean with dry
air to remove
loose dust
None
2.4 Coupon Spiking
For each CWA, enzyme-based decontamination technology, contact time, and material
combination:
• Five replicate test coupons were spiked with CWA with subsequent decontamination
• Five replicate positive controls were spiked with CWA without subsequent
decontamination
• Two procedural blanks were not spiked with CWA but were decontaminated
• Two laboratory blanks were not spiked with CWA and were not decontaminated
• For DEFENZ™ VX-G 15-min contact times only, five replicate solution controls
were spiked with CWA treated with the enzyme-free buffer solution to assess whether
observed decontamination was due solely to enzymatic action or through an effect of
the buffered solution without enzyme present
• For DEFENZ™ BH-D 15-min contact times only, five replicate solution controls
were spiked with CWA treated with deionized water to assess whether observed
decontamination was solely due to enzymatic action or through an effect of water.
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Because this product is a premixed slurry, an enzyme-free product could not be
evaluated for efficacy.
All test and positive control coupons were nominally spiked with 1 microliter (jiL) of
neat or thickened CWA. This spiking volume delivered approximately 0.9 milligram (mg) of
VX, TGD, or HD. The contamination level was approximately 2 g/square meter (m2) (0.9 mg/
[3.5 centimeters (cm) x 1.5 cm] = 0.17 mg/cm2 =1.7 g/m2). VX and HD were dispensed using a
Hamilton syringe (P/N 80565 [50 |iL] equipped with a 22-gauge needle [P/N 91022] and
repeating dispenser [P/N 83700], Hamilton Co., Reno, NV).
TGD was dispensed using a positive displacement pipette (P/N F148504 [5-10 jiL] and
C-10 [10 jiL] tip, Rainin Instrument LLC, Oakland CA). The pipette was initially set to dispense
1.4 jiL to account for losses along the pipette wall and tip, nominally yielding 1 jiL applied to the
coupon. Adjustments were made to the pipette setting based on ongoing experience to improve
accuracy of the volume applied. For the initial lot of TGD, the volume was increased to 1.6 jiL;
for a new lot, the volume was decreased to 1.2 jiL.
Polytetrafluoroethylene (Teflon®) spike control coupons (P/N 5Y43BYD, Thomas
Scientific, Swedesboro, NJ) were evaluated, one at the beginning, one at the middle and one at
end of each trial (total of three spike control coupons per trial). A day of decontamination and
subsequent extraction and analysis is referred to as a "trial". Each spike control coupon was
spiked with three droplets of neat or thickened CWA, using the same pipette and pipette settings
as were used for spiking the test and positive control coupons, then immediately placed in 20
milliliters (mL) of extraction solution, shaken for 15 seconds and passively extracted for one
hour. The first spike control coupon was prepared at the beginning of the evaluation. The second
spike control coupon was prepared midway through application of agent to test coupons and
positive controls. The final spike control coupon was prepared after the last test coupon was
contaminated. The mass of CWA per spiked droplet applied to test and positive control coupons
is assumed to be equal to the mean of the CWA per droplet recovered from the spike control
coupons calculated as follows:
_ 3% CWA/ . .
9 droplets ^ '
where:
a = Mean mass of CWA per spiked droplet
CWA, = Mass of CWA recovered from the ith spike control coupon
2.5 Preparation of Enzyme-Based Decontamination Technologies
2.5.1 Preparation Procedure for DEFENZ™ VX-G
The DEFENZ™ VX-G enzyme pouch contained two types of enzymes appropriate for
G-type agents (DEFENZ™ 120) and VX (DEFENZ™ 130). Because the enzymes were together
in a single pouch but may not be thoroughly mixed, the following method was used to ensure
homogeneity among enzyme solutions prepared using only a portion of the enzyme mixture to
make less than 10 L of enzyme solution.
The enzyme packet and buffer packet were opened and the contents were separately
weighed. The weight ratio between the enzyme and the buffer (110:250) was the ratio used to
5
-------
create smaller quantities. Laboratory batches of the buffer (sufficient to produce 500 mL of
enzyme solution) were prepared by dividing the contents of the buffer packet (nominally 250 g)
into 20 equal portions (12.5 g ± 0.1 g each) in separate appropriately labeled scintillation vials
(03-337-14/vial; 02-912-068/cap, Fisher Scientific, Pittsburgh, PA). The vials of buffer powder
were stored at ambient temperature in a desiccator until needed.
Laboratory batches of enzymes (each sufficient to produce 500 mL of enzyme solution)
were prepared, as shown in Figure 1, to ensure product uniformity as much as practical. The
enzyme packet contents (DEFENZ™ VX-G enzyme; nominally 110 g) were divided into five
equal portions (samples 22 g ± 0.1 g each) using an analytical balance (Model AX-205 ID #
C21236, Mettler-Toledo, Toledo, OH). Each sample was retained in a weighing pan (08-732-
103, Fisher Scientific, Pittsburgh, PA). Five mixed samples (22 g ± 0.1 g each) were then
produced by transferring an equal amount (4.4 g ± 0.1 g) from each sample into each of five new
weighing pans (08-732-103, Fisher Scientific, Pittsburgh, PA). Twenty batches (5.5 g± 0.25 g
each) were then produced by transferring an equal amount (1.1 g ± 0.05 g) from each mixed
prepared sample into each of 20 scintillation vials (03-337-14/vial; 02-912-068/cap, Fisher
Scientific, Pittsburgh, PA). Each vial, sufficient to prepare 500 mL of enzyme solution, was
marked to indicate that the vial contained DEFENZ™ VX-G enzyme (5.5 g) and stored at
ambient temperature in a desiccator until needed.
Enzyme solutions were prepared fresh each day of testing in accordance with
manufacturer's instructions, but with smaller, proportionate amounts of enzyme (5.5 g) and
buffer (12.5 g). The preparation of DEFENZ™ VX-G is shown in Table 3. Deionized water was
used to prepare the solutions.
Table 3. Enzyme-Based Decontamination Technology Concentrations
Formulae for Preparing DEFENZ™ VX-G Solutions
\Vater
Enzyme (g) Buffer (g)
Weight (g) in packet/10,000 mL x Weight (g) in packet/10,000 mL x 4,000 mL
4,000 mL (nominally 44 g) (nominally 100 g)
1 vial containing DEFENZ™ VX-G 1 vial containing DEFENZ™ VX-G buffer (sodium t
enzyme, 5.5 g ± 0.25 g hydrogen carbonate), 12.5 g ± 0.1 g
* This solution was prepared and used in method development to establish the mass of DEFENZ™ VX-G that
remains on coupons of various types when applied with a sprayer (see Section 2.6.1).
trThese solutions were prepared for use in decontamination testing.
The pH of the enzyme solution was measured and documented prior to each day of testing using
a pH meter (pFt/Ion Analyzer Model 350, Corning, Lowell, MA). The enzyme solutions used
were verified as being within a specified range of pH (8.3 ± 0.5).
The time that the enzyme solution was prepared was documented along with the time at
which the enzyme solution was used (applied to test coupons); elapsed time from preparation to
use was documented.
-------
Packet containing 110 g mix of DEFENZ™ 120 and DEFENZ™ 130 enzymes
22 g mixed
sample (x 5)
Packet contents were equally divided
to create 5 x 22-g samples
1
r
22 g
sample
i
r
22 g
sample
^
r
22 g
sample
4
22 g
sample
I
22 g
sample
From each 22 g sample, 4.4 g were
used to create a mixed sample (22 g
total); this procedure was repeated
five times
From each mixed sample, 1.1 g was
transferred to a vial to create a batch of
enzyme of sufficient mass (5.5 g) for
preparing 500 mL of enzyme solution;
this procedure was repeated 20 times
Figure 1. Approach used to ensure homogeneity of DEFENZ™ 120 and DEFENZ™ 130
enzymes in 500 mL enzyme test solutions.
2.5.2 Preparation Procedure for DEFENZ™ B-HD
Genencor® instructions for use of DEFENZ™ B-HD enzyme state: Mix the entire
contents (1.0 kg) into 10 gallons [37.85 L] of water. Agitate until dissolved and allow 20 min
before use. Use within 8 hours.
Because DEFENZ™ B-HD is a solution containing insoluble matter, a method was
required to prepare user-ready solutions of less than 37.85 L. Genencor® recommended the use
of good agitation followed by a fast transfer of the aliquots to make them as representative as
possible. According to Genencor®, the key for successful operation of this enzyme is the
-------
generated peracetic acid concentration. The amount of peracetic acid in each batch was measured
before use.
The following method was employed to prepare and ensure the quality of bench-scale
batches of the enzyme:
1. The mass (1.001 kg) and volume (730 mL) of the parent product were measured.
2. The ratio of the solid phase (435 mL) to the total volume (730 mL) was determined to be
0.6.
3. Twelve aliquots of the stock enzyme, sufficient for about 650 mL of activated enzyme
after the addition of water, were prepared. Solid material was transferred to graduated
conical tubes to the 7.5 mL level (the level was measured after about 5 min of settling);
the liquid phase was added to 12.5 mL. This procedure maintained a solid phase to liquid
phase ratio of 0.6, the same as the parent stock solution. The stability of the ratio was
verified after 1 hour; no increased settling of the solid phase was observed.
4. The net weight of each enzyme aliquot was calculated as the difference in pre-weight and
post-weight of the conical tube. The mean and standard deviation of the samples are
tabulated in Table 4.
Table 4. Weight of Enzyme Aliquots and Volume of Make-up Water Required
Aliquot
#
1
2
3
4
5
6
7
8
9
10
11
12
Pre-Weight Post-Weight
(g)
12.79
12.93
12.80
12.86
12.94
12.84
12.69
12.74
12.77
12.81
12.72
12.66
Standard
(g)
29.15
30.40
29.83
29.99
30.76
29.70
30.69
30.38
30.31
29.94
30.27
29.92
Average:
Deviation (SD):
%Relative SD:
Net Weight of Enzyme Aliquot
(g)
16.36
17.47
17.03
17.13
17.82
16.86
18.00
17.64
17.54
17.13
17.55
17.26
17.32
0.45
2.60%
Make-up Water
(mL)
619
662
644
649
674
638
681
668
664
648
664
653
The following method was employed to activate and use the enzyme on each day of
testing:
-------
1. Deionized water ("make-up water") was added to an aliquot of the stock enzyme solution
in a conical tube. The amount of water was based on the mass of that specific aliquot of
enzyme so that the label instruction ratio of 1 kg/10 gal (1000 g/37,850 mL; 1 g/37.9 mL)
was maintained.
2. The concentration of peracetic acid in the solution was determined 25 min after an aliquot
of the DEFENZ™ B-HD was activated by diluting 1 mL of the prepared enzyme sample
with 9 mL of deionized water. The pH was measured and adjusted to a range of two to
five with acetic acid. A sample of the activated DEFENZ™ B-HD enzyme solution was
diluted 1:10 (to get within the range of the test strips) and EM Quant® Peracetic Acid
Test Strips (Number 100011, EMD Chemicals, Gibbstown, NJ) were then used to
measure the peracetic acid concentration. The activated enzyme solution was considered
acceptable for use if the peracetic acid concentration was >2500 ppm.
3. The pH of the enzyme solution was measured prior to each day of use using a pH meter
(pH/Ion Analyzer Model 350, Corning, Lowell, MA) and documented.
4. Except for the delayed testing ("pot life test"), the enzyme solution was applied to the test
coupons no sooner than 25 min after mixing the enzyme with water and no later than 60
min after mixing the enzyme with water.
5. The time that the enzyme solution was prepared each day was documented along with the
time at which the enzyme solution was used (applied to test coupons); elapsed time from
preparation to use was documented.
2.6 Test Matrices
2.6.1 Spray Application Demonstration to Select Enzyme Application Rate
In field application of the enzyme product, use of a sprayer would be likely. In laboratory
tests, the enzyme-based decontamination technologies were delivered to coupon surfaces as
measured amounts from syringes or pipettes in order to reduce variability in amounts applied
when compared to a spray application. A demonstration was used to determine the mass of
enzyme solution (DEFENZ™ VX-G) that would be applied to a surface in a typical spray
application. These data provided material-specific target values for the amount of enzyme
solution to be applied to coupons to evaluate decontamination efficacy. The amount of enzyme-
based decontamination technology that carried over into neutralization or extraction was
determined by: (1) weighing the coupon before application of the enzyme-based
decontamination technology, (2) spraying the enzyme solution onto the coupons, (3) waiting for
the shortest contact time, and (4) weighing the coupon. The residual enzyme solution was
calculated as the difference in the mass of the coupon with residual enzyme-based
decontamination technology after the contact time less the mass of the coupon before application
of the enzyme-based decontamination technology.
DEFENZ™ VX-G solution (4,000 mL) was prepared as described in Section 2.5.1. The
solution was held at ambient laboratory conditions for one hour before use in the sprayer.
Four 1.5 x 3.5 cm coupons of each test material (galvanized metal, decorative laminate,
industrial carpet, wood flooring, and vinyl flooring) were weighed on a calibrated balance. The
20 coupons were placed on a horizontal surface and arranged side by side to form a row with the
long sides next to each other and approximately 5 - 8 cm between the coupons.
-------
The enzyme solution was applied to the coupons in controlled tests using a full-scale
pressurized tank sprayer (Solo® Model 425 DLX, Solo, Newport News, VA). The sprayer was
selected as representative of garden-type sprayers that would be commercially available to
decontamination response teams in local stores across the nation.
The coupons were sprayed with a sweeping motion after establishing uniform flow of
liquid decontaminant from the sprayer at 207 kPa (30 psi). The tip of the sprayer nozzle was held
0.5 - 0.6 meters above the coupons and at an angle of 90° to the substrate surface. Spraying was
continued by sweeping side to side until a continuous film of liquid covered the surface of the
material. The rate of the sweeping motion was approximately one linear foot per second.
After spraying was completed, the coupons were covered loosely with a Petri dish to
hinder evaporation until the final weight of each coupon was determined. Each sprayed coupon
was weighed on a calibrated balance (Mettler Toledo PG 5002-SDR, Zurich Switzerland) to
obtain its final weight. The mass of the enzyme-based decontamination technology applied to the
coupon was determined by subtracting the initial coupon weight from the coupon weight post-
spraying. For each type of material, the average amount of each enzyme-based decontamination
technology retained on a coupon was calculated.
The test was repeated with three additional sets of coupons in order to characterize
average results for sprayer performance with DEFENZ™ VX-G enzyme-based decontamination
technology. The density of the enzyme-based decontamination technology was used, along with
the average mass found for spraying the liquid on each of the materials, to calculate the average
volume of enzyme-based decontamination technology retained on the coupons. The results of the
spray-and-weigh demonstration were the basis for selecting the amount of enzyme solution to be
applied to the CWA on the coupons in subsequent bench-scale testing. The same amounts, by
volume, of DEFENZ™ B-HD were used as DEFENZ™ VX-G; spray tests were not repeated.
All testing was conducted under ambient laboratory conditions (maximum and minimum
range during testing were 17 degrees Celsius [°C] - 20 °C and RH 51% - 58%). The same
amounts of enzyme solutions were applied to each spot of CWA contamination on the 3.5 cm by
1.5 cm coupons for both the DEFENZ™ VX-G and DEFENZ™ B-HD decontamination testing
and are shown in Table 5. The amount of enzyme solution applied to each spot of CWA in this
study was the same as the amount of cleaner applied to CWA in a prior study2 for the following
materials: galvanized metal - 0.06 mL, decorative laminate - 0.06 mL, wood flooring - 0.09 mL,
and carpet -0.12 mL. Vinyl flooring was not used in the prior study. Note that, in field use,
mechanical removal of CWA by splash and runoff may occur in addition to the enzymatic
degradation. In this investigation, an effort was made to differentiate the chemical degradation
derived from the functioning of the enzyme solution from mechanical removal by gently
applying the enzyme solution directly to the CWA spots. The amount of enzyme applied directly
to the CWA spots was approximately equal to the mass of enzyme retained on the entire surface
of the nonporous coupons. Both wood and carpet retain a high mass of enzyme solution. The
higher mass retained on industrial carpet and wood flooring is assumed to have soaked into the
coupon and may not reflect the mass of enzyme available to interact with the CWA.
(Note: The spray demonstration method used in this study to determine the amount of
enzyme solution retained on coupons was the same method used in a prior study2 to determine
the amount of cleaning solutions that were retained on building materials. The mass of enzyme
10
-------
solution retained on the coupons after a four-second spray [exclusive of splash and runoff] is
shown in Table 5. The measured density of DEFENZ™ VX-G was 1.022 g/mL.)
Table 5. Enzyme Application Amounts for Bench-Scale Testing
Measured Mass of Enzyme Solution
1VT o t p 1* j o 1
Enzyme Solution, g (SD) Application, mL
Galvanized metal
Decorative laminate
Wood flooring
Industrial carpet
Vinyl flooring
0.053 (0.018)
0.075 (0.020)
0.220(0.119)
0.166(0.070)
0.084 (0.022)
0.06
0.06
0.09
0.12
0.06
2.6.2 DEFENZ™ VX-G Test Matrices
The DEFENZ™ VX-G enzyme-based decontamination technology was evaluated against
VX and TGD using a 15-min contact time and manufacturer-specified enzyme concentration for
the material combinations shown in Table 6. The test coupons were spiked with VX or TGD and
allowed to weather for 30 minutes; then the DEFENZ VX-G enzyme was added for the specified
contact time for the decontamination test. The positive control coupons were spiked with VX or
TGD and allowed to weather for 30 minutes plus the 15 minutes contact time used for the test
coupons.
The only potentially toxic by-product from VX decontamination is EA 2192. EA 2192
cannot be determined using GC/MS. No potentially toxic by-products from TGD
decontamination were expected. Therefore, no GC/MS analysis was performed to quantify toxic
by-products from VX or TGD decontamination. Instead, LC/MS was used for qualitative
analysis of EA 2192 in VX/decontaminant solutions as described in Section 2.11.
Table 6. Test Matrix for Decontamination of CWA with DEFENZ™ VX-G Prepared per
Manufacturer's Recommendations and 15-Min Contact Time
. , i»/r * • i Test Positive Solution Procedural Laboratory
Agent Matenal „ * ^ . , -t /-,..,# n, , t ™ i s
Coupons Controls Controls Blanks Blanks
VX
VX
VX
VX
VX
TGD
TGD
TGD
TGD
TGD
Galvanized metal
Decorative Laminate
Industrial Carpet
Wood Flooring
Vinyl Flooring
Galvanized metal
Decorative Laminate
Industrial Carpet
Wood Flooring
Vinyl Flooring
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
Test coupons are spiked with CWA and undergo decontamination.
t Positive controls are spiked with CWA but do not undergo decontamination.
11
-------
* Solution controls were controls where the coupon with CWA applied to surface was able to interact with enzyme-
free buffer solution.
* Procedural blanks are not spiked with CWA but undergo decontamination; one additional procedural blank was
held for 48 hours (or longer if over a weekend) and examined for visually-obvious changes.
§ Laboratory blanks were not spiked with CWA and did not undergo decontamination.
An adaptive management approach was used in which testing results were used to modify
subsequent testing. Because of low efficacies against VX on all types of materials tested here, no
useful data would be generated by using shorter contact times as anticipated in the test/QA plan1.
Therefore, DEFENZ™ VX-G efficacy was not evaluated at shorter contact times. Instead,
efficacies at two higher enzyme concentrations (namely, 2X and 3X) were evaluated for VX on
galvanized metal and TGD on laminate. Galvanized metal and laminate were selected because
these materials exhibited the least efficacy observed with a 15-min contact time of DEFENZ™
VX-G against VX (shown in Section 4.2.3) and TGD (shown in Section 4.2.4), respectively. The
question being answered was whether a higher enzyme concentration would increase efficacy for
decontaminating materials on which the lower (vendor recommended) enzyme concentration had
the least efficacy. The test matrix for higher concentration enzyme solutions is shown in Table 7.
Table 7. Test Matrix for Increased DEFENZ™ VX-G Enzyme Concentration
. -. , . , _,. Enzyme Test Positive Procedural Laboratory
Agent Matenal Time, „ , ,. „ „ , . _. . _. .
. Concentration Coupons Controls Blanks Blanks
mm r
VX
VX
TGD
TGD
Galvanized
metal
Galvanized
metal
Decorative
Laminate
Decorative
Laminate
15
15
15
15
2X
3X
2X
3X
5
5
5
5
5
5
5
5
2
2
2
2
2
2
2
2
The manufacturer's instructions call for the contents of the enzyme packets to be mixed
into 10 L of water. For the 2X concentration (i.e., two times the concentration recommended by
the manufacturer), the contents would be mixed with 5 L of water and for the 3X concentration
the enzyme would be mixed in 3.3 L of water. Actual mixtures were based on this proportion
applied to the amounts of enzyme in the "batch packets" as follows:
• 2X mix: Add the contents intended for 500 mL to 250 mL of deionized water
• 3X mix: Add the contents intended for 500 mL to 167 mL of deionized water.
The granulated enzyme and buffer dissolve completely in deionized water at the 2X and 3X
concentration levels. A 5X concentration was considered for testing; however, the enzyme or
buffer products did not dissolve completely at this concentration and, therefore, a 5X
concentration was not tested.
Efficacy as a function of pot life time, defined as the time between preparation of the
enzyme solution and actual application onto the coupon surface, was evaluated using the test
12
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matrix in Table 8. Enzyme solutions (3X DEFENZ™ VX-G) were prepared and placed in sealed
containers in a refrigerator (approximately 4 °C) in accordance with the storage conditions for
prepared enzyme that are recommended by Genencor®: "below 15 °C (59 °F) and out of
sunlight." One hour before use (5, 14, and 23 hours, respectively), the 3X enzyme solutions were
removed from the refrigerator and allowed to sit at room temperature. The 3X enzyme solution
was then applied to decontaminate carpet spiked with VX or TGD. A 15-min contact time was
used for the pot life test. Carpet was selected for the pot life test because carpet demonstrated
high efficacy with a contact time of 15 min for DEFENZ™ VX-G against VX (shown in Section
4.2.3) and TGD (shown in Section 4.2.4), respectively. The extraction and analysis procedures
were identical to the procedures used for the other efficacy testing.
Table 8. Test Matrix for Effect of Storage of Activated 3X DEFENZ™ VX-G Enzyme
Solution on VX Decontamination Results
. , n..., Pot Life, Test Positive Procedural Laboratory
Agent Matenal , „ „ , . _. . _. .
hours Coupons Controls Blanks Blanks
VX
VX
VX
TGD
TGD
TGD
Carpet
Carpet
Carpet
Carpet
Carpet
Carpet
6
15
24
6
15
24
5
5
5
5
5
5
5
5
5
5
5
5
2
2
2
2
2
2
2
2
2
2
2
2
2.6.3 DEFENZ™ B-HD Test Matrices
DEFENZ™ B-HD was evaluated against FID following the test matrix in
Table 9. The amounts of DEFENZ™ B-HD applied for decontamination of each type of material
was the same as for DEFENZ™ VX-G and is shown in Table 5. The test coupons were spiked
with FID and allowed to weather for 30 minutes; then the DEFENZ B-HD enzyme was added for
the specified contact time for the decontamination test. The positive control coupons were spiked
with HD and allowed to weather for 30 minutes plus the specified contact time used for the test
coupons.
Table 9. Test Matrix for Decontamination of CWA with DEFENZ™ B-HD
Agent
HD
HD
HD
HD
HD
Material
Galvanized Metal
Decorative Laminate
Industrial Carpet
Wood Flooring
Vinyl Flooring
Test
Coupons
5*
5
5*
5
5
Positive
Controls
5*
5
5*
5
5
Solution
Controls
5*
5
5*
5
5
Procedural
Blanks
2*
2
2*
2
2
Laboratory
Blanks
2*
2
2*
2
2
Qualitative assessment of decontamination by-products was performed using full scan GC/MS.
13
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Because efficacies against HD were not high on any material after a 15-min contact time,
DEFENZ™ B-HD efficacy was not evaluated at shorter contact times. Instead, efficacies at two
higher contact times, 30 min and 60 min, were evaluated for HD on two materials (carpet and
vinyl). Carpet and vinyl were selected for testing at the longer contact times because DEFENZ™
B-HD exhibited low efficacies against FID on these materials at a 15-min contact time (shown in
Section 4.2.5). No solution controls were included and the test matrix is shown in Table 10.
Table 10. Test Matrix for Longer Contact Times with DEFENZ™ B-HD
Agent
HD
HD
HD
HD
Material
Carpet
Carpet
Vinyl
Vinyl
Contact
Time,
min
30*
60*
30
60
Test
Coupons
5
5
5
5
Positive
Controls
5
5
5
5
Solution
Controls
0
0
0
0
Procedural
Blanks
2
2
2
2
Laboratory
Blanks
2
2
2
2
Qualitative assessment of decontamination by-products was performed using full scan GC/MS.
Efficacy of DEFENZ™ B-HD solution as a function of pot life time was evaluated using
the test matrix shown in Table 11. The DEFENZ™ B-HD solution was mixed as described in
Section 2.5.2. The prepared enzyme solution was allowed to sit at room temperature for 2, 4, and
6 hours before use. The enzyme solution was then applied to decontaminate wood spiked with
HD. The contact time was 15 min. Wood was selected for the pot life test because DEFENZ™
B-HD demonstrated high efficacy against HD with a contact time of 15 min (shown in Section
4.2.5).
Table 11. Test Matrix for Delayed Application of DEFENZ™ B-HD Enzyme Solution
. , T./T x • i P°t Life, Test Positive Procedural Laboratory
Agent Material , „ „ x . _. . ™ ,
hours Coupons Controls Blanks Blanks
HD
HD
HD
Wood
Wood
Wood
2
4
6
5
5
5
5
5
5
2
2
2
2
2
2
2.7 Observation of Surface Damage
Representative digital photographs were taken of coupons before and after they were
exposed to the enzyme solution decontamination for 15 min to document observed impacts or
absence thereof. One of the decontaminated procedural blanks of each material type was rinsed
with deionized water and allowed to dry. Additional photographs were taken of this procedural
blank that was rinsed but not extracted two days after testing (or more if 'two days' fell on a
weekend) to document any visually obvious changes that occurred to a procedural blank. They
were visually inspected and compared to coupons not exposed to the decontamination treatment
to look for obvious changes in appearance of the procedural blanks (for example, in the color,
reflectivity, or apparent roughness of the coupon surfaces). Differences were recorded in the
evaluation records. In addition, representative photographs were taken two days after testing to
14
-------
document any visually obvious changes that may have occurred. Observations and photographs
of pre- and post-decontaminant coupons are included in Section 4.4.
2.8 Extraction and A nalysis
After the appropriate contact time the test, positive control, solution control, procedural
blank, and laboratory blank coupons were transferred to individual extraction bottles (P/N
89044-462, VWR International, West Chester, PA) containing 10 mL of hexane with an internal
standard (IS). The hexane extraction solvent contained naphthalene as an IS. The extraction
bottles were sealed, shaken by hand for about 5-10 seconds, and placed into a sonicator. After all
bottles containing coupons to be extracted for a given time point were placed in the sonicator,
they were sonicated at 40 - 60 kilohertz (kHz) for 10 min. Within 30 min after the completion of
sonication, a 1.0 mL aliquot was transferred to a GC vial (P/N 06-718-476 and 03-391-6, Fisher
Scientific [Restek Corp], Hanover Park, IL 60133) and sealed. This process was repeated for all
samples until each test, positive control, solution control, procedural blank, and laboratory blank
coupon had been shaken, sonicated, and aliquoted for analysis.
All test, positive control, solution control, procedural blank, and laboratory blank
coupons were individually extracted and the amount of CWA in the extraction solution was
determined using a GC/mass spectrometer (MS, Model 6890, Agilent Technologies, Santa Clara,
CA). Samples were analyzed using an Agilent (Palo Alto, CA) 6890N Series GC interfaced to a
5973 network quadrupole mass selective detector (MSD). Chromatographic separation of the
analytes was conducted using an RTX-5 (cross-linked methyl silicone) fused silica capillary
column, 30.0 m (or 29.5 m) length x 0.25 millimeter (mm) diameter x 0.5 micrometer (|im)
coating thickness. The GC/MS parameters for VX, GD and HD analysis are shown in Table 12.
Table 12. Gas Chromatography/Mass Spectrometry Parameters for VX and GD Analysis
Parameters Description
VXandGD
HD
Analysis Method:
Model & SN:
Data System:
Liner Type:
GC/MS (Scan)
GC/MS (Scan)
HP6890N GC (CN10331014) &
5973N MSD (US30985853)
MSD ChemStation
4 mm Split/Splitless
RTX-5MS, 30.0 m or 29.5 m length,
0.25 mm diameter, 0.5 (im film coating
HP6890N GC (US00042609) &
5973N MSD (US 1046065 8)
MSD ChemStation
4 mm Split/Splitless
RTX-5MS, 30.0 m length, 0.25 mm
diameter, 0.25 (im film coating
Column:
Mode:
Inlet (Injector)
Temperature:
Detector Temperature:
Sample Size:
thickness
Constant flow (1.5 mL/min)
250 °C
230 °C
1 pL
thickness
Constant flow (1
250 °C
230 °C
1 pL
.3 mL/min)
15
-------
50 °C (hold 2.0 mm) increase at 20 4Q oc (hdd IQ ^ mcrease at
Oven Program for VX °C/minto 250 °C (holdO.Omin),
and GD Analysis: increase at 35 °C/min to 300 °C (hold 8 °C/iran to 30° °C^\d 0.0 mm),
0 5 min) increase to 325 °C (hold 0.0 mm)
The mass selective detector was operated in the full-scan mode for compounds ranging
from 28 to 500 atomic mass units (AMUs). Where concentrations were too low to quantitate in
the full-scan mode, the samples were scanned again in the selected ion monitoring (SIM) mode.
The GC/MS measurements were used to compare and evaluate co-extractive sample components
and CWA response. Table 13 outlines the ion masses which were used to quantitate the CWAs.
CWA decontamination by-products were qualitatively assessed by examining the GC
chromatogram in full scan mode.
Table 13. Pertinent Parameters for Target Chemicals
Analyte SIM Ions
VX
TGD
HD
114, 127,72
99, 126, 69, 82
158, 109, 160
2.9 Method Demonstration
2.9.1 Recovery of CWA from Test Coupons
Method demonstration was conducted and consistent with previous testing, to establish
that extraction efficiencies (recoveries) from test coupons were sufficiently high and to establish
method detection limits (MDL[s]) for CWAs and material combinations for which such
information had not previously been demonstrated in the laboratory. The extraction efficiency
was determined as a percent of the agent recovered from the spiked coupon relative to the
amount spiked. The extraction method was acceptable if the extraction efficiency was 40% -
120% with a coefficient of variance between samples not exceeding 30%.
Hexane was selected to extract VX, TGD, and HD from the aqueous phase based on prior
experience.2'3 Vinyl was the only material for which no recovery had been demonstrated.
Therefore, eight vinyl coupons were each spiked with 10 uL of 875 micrograms (ug)/mL VX or
1,000 ug/mL GD research dilute solutions (RDS; CWA diluted with hexane [GC Resolv grade,
Fisher Scientific, Pittsburg, PA]). The surrogate recovery compound (SRC) was also applied to
the coupon surface. Sufficient hexane to cover the coupons (10 mL) was used for each
extraction. The coupons were transferred into hexane within 0.5 min of spiking with dilute CWA
solution. Immediately after transfer, the vial was capped and shaken by hand for 5-10 seconds
and placed into a sonicator. After all vials containing coupons to be extracted were placed into
the sonicator, the samples were sonicated at 50-60 kHz for approximately 10 min. Within 30 min
after the completion of sonication, an aliquot of extract was transferred to a GC vial (P/N HP-
5181-880, VWR [Agilent Technologies], West Chester, PA) and sealed. The amount of spiked
CWA was confirmed using control samples where the same dilute solution was spiked directly
into hexane and analyzed.
16
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Because GD RDS recoveries were low, extraction efficiency demonstration was
subsequently repeated by spiking each of two vinyl coupons with 1 jiL of neat GD and 1 |iL of
neat SRC using a Hamilton syringe (P/N 80565 [50 |iL]). The coupons were placed in 10 mL of
hexane within 0.5 min of spiking with neat agent and SRC. The process and analysis, described
in the preceding paragraph, was repeated for the extracts of vinyl spiked with neat GD.
HD recovery from vinyl was determined by spiking each of three vinyl coupons with
10 |igHD (10 |iL of a 1 mg/mL RDS solution).
The aliquots of hexane extracts of coupons spiked with CWA (1 uL) and aliquots of
hexane containing the same spike amount as applied to the coupons were analyzed for CWAs as
described in Section 2.8.
Extraction efficiency was calculated using a series of equations. The CWA concentration
in a coupon extract or spiked hexane sample was determined by Equation 2:
A C
where:
As = Area of the target analyte peak in the sample
A;s = Area of the internal standard peak
Cs = Concentration of the target analyte in the sample (|j,g/mL)
C;s = Concentration of the internal standard (|j,g/mL)
M = Slope of the GC calibration line
W = Y intercept of the GC calibration line
GC concentration results (|j,g/mL) were converted to total mass by multiplying by extract
volume:
Mm=CxEv (3)
where:
Mm= Measured mass of CWA (|j,g)
C = GC concentration (|j,g/mL)
EV = Volume of extract (mL)
Extraction efficiency was then defined as:
„ . rr,, . (Mm of CWA on Test Coupon^}
Extraction Efficiency = — ^— ^ - - — x 1 00% (4)
[ Mm of CWA in Hexane )
2.9.2 MDL for VX, GD, and HD Extracted from Vinyl
The MDLs for VX, GD, and HD extracted from vinyl were determined following the
EPA procedure.4 The MDLs were calculated as follows:
MDL = t(n-l,l-a= 0.99) x SD (5)
17
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where:
t(n-l,l-a = 0.99) = the Student's t-value for a 99% confidence level and
standard deviation estimate with n-1 degrees of freedom.
SD = standard deviation of the replicate analyses
2.9.3 Quench of Decontamination Reaction
Hexane extraction was expected to remove the CWA (reactant) from the aqueous phase
in which the enzyme is active thereby halting (quenching) the decontamination reaction.
Enzymes are not expected to be functional in the nonpolar phase so other additives are not
expected to be needed to quench the reaction. The neutralization method was assumed not to be
impacted by the coupon material. Quench methods were therefore evaluated using solution tests.
The use of hexane extraction as a quench method was assessed as follows:
1. Fifty jiL of enzyme was added (using a positive displacement pipette (P/N M-250 [250
|iL] and D-200 [2-200 |iL] tip, Gilson Inc., Middleton, WI) to a vial containing 10 mL of
hexane and IS (naphthalene-dg) and 1 uL of CWA, shaken for 15 seconds, and allowed to
stand for 10 min.
2. Distilled water, equivalent to the amount of enzyme solution in Step 1, was added (using
a positive displacement pipette) to a vial containing 10 mL of hexane and IS
(naphthalene-dg) and 1 uL of CWA, shaken for 15 seconds, and allowed to stand for 10
min.
3. The extracts from Steps 1 and 2 were analyzed using GC/MS. Extraction alone, without
additional neutralization, was acceptable for GC/MS analysis if the amount of CWA
recovered in Step 1 (enzyme present) was at least 70% of the amount of CWA recovered
in Step 2 (no enzyme present). All Agent recoveries with hexane exceeded the required
70% (Table 14).
Table 14. Recovery of Agent Using Hexane Extraction as Quench
Recovery with Water, Recovery with Enzyme, Mean % Hexane Recovery
Agent ug/mL ug/mL "with Quenched Enzyme"
(SD) n = 3 (SD) n = 3 Compared to "with Water"
vx
GD
HD
20.1 (0.20)
21.8(0.18)
21.0. (0.35)
18.1 (0.24)
22.3 (0.33)
19.0(1.53)
90
102
90
2.9.4 EA 2192 LC/MS Analysis
Liquid chromatography/mass spectrometry (LC/MS) analysis for the EA 2192 by-product
in the aqueous phase required that VX degradation by the enzyme be quenched. Hexane would
be considered effective as a quench if (1) VX recovery from hexane was not reduced by enzyme
in the aqueous phase (VX recovery >70%) and (2) the amount of EA 2192 recovered from the
aqueous phase was >70% of an EA 2192 spike. This recovery was demonstrated by spiking 1 uL
of neat VX containing 648 ug of VX (900 ug/uL for pure VX x 72% purity), using a Hamilton
syringe (P/N 80565 [50 |iL], into each of six 60-mL vials (GLC-04869, Qorpak, Bridgeville, PA)
18
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containing 5 mL hexane with 5 ug/mL naphthalene-d8 (IS) and capping the vial. A solution
containing deionized water (2 mL) and decontaminant (50 uL) was then added to each of three
vials of VX in hexane with IS. Similarly, 2.05 mL of deionized water was added to each of the
three remaining vials containing VX in hexane with IS. All vials were closed and sonicated for
10 min (50-60 kHz). The mixtures were allowed to sit for 15 min to enable the polar and
nonpolar phases to separate. The hexane phase from each vial was analyzed for VX using
GC/MS. The aqueous phase was frozen at -20 °C and subsequently analyzed for EA 2192 using
LC/MS as a positive control (containing EA 2192 as a naturally-occurring by-product) to
compare to EA 2192 present after VX was decontaminated with DEFENZ™ VX-G.
The recovery of EA 2192 in water or aqueous enzyme (when sonicated with hexane) was
evaluated. Ten microliters of an EA 2192 solution (1.4 ug EA 2192/mL of deionized water) was
spiked into each of six 60-mL vials (GLC-04869, Qorpak, Bridgeville, PA) containing 5 mL
hexane IS. A solution of deionized water (2 mL) and decontaminant (50 uL) was added to three
vials containing hexane and IS. Deionized water (2.050 mL) was added to each of the remaining
three vials containing hexane and IS. The vials were closed and sonicated for 10 min (50-60
kHz), then allowed to sit for 15 min to enable the polar and nonpolar phases to separate. The
aqueous phase was analyzed for EA 2192 using LC/MS.
The results of the hexane extraction of VX to quench the enzyme reaction are shown in
Table 15. High percentages of VX were recovered from the hexane layer in the presence of water
(90% recovered) or the aqueous solution of DEFENZ™ VX-G enzymes (103%). Further, there
was no significant difference between the amount of VX recovered from hexane with water
present or with enzyme present (p = 0.35). Because the recoveries of VX were >70%, hexane
extraction of the VX reaction was considered sufficient to quench the enzyme reaction in
subsequent decontamination efficacy testing.
EA 2192 is a degradation by-product that was present in the VX spike as observed in the
aqueous phase of both the water/hexane (with VX) extract and the DEFENZ™ VX-G/hexane
(with VX) extract. There was significantly less EA 2192 extracted from the neat VX (in hexane)
with water present than from the neat VX (in hexane) with enzyme present (p = 0.005).
However, these samples were transported between laboratories and stored at less than -20 °C for
several months between the time of the VX quench test and the analysis of the EA 2192 quench
test. Transportation and the passage of time may account for this anomaly. As noted below, when
analyzed immediately, no significant differences were noted in EA 2192m water or in
DEFENZ™ VX-G solution.
The results of the EA 2192 stability demonstration are shown in Table 16. From
water without enzyme mean recovery was 99%. From water with enzyme mean recovery was
79%. The recoveries of the EA 2192 spike from the aqueous phase of the DEFENZ™ VX-
G/hexane extraction met the test/Q A criterion of exceeding 70% of the amount of EA 2192
recovered from the aqueous phase of the water/hexane extract receiving comparable treatment,
so hexane extraction of VX was considered sufficient to quench the enzyme reaction. There was
no significant difference in the amount of EA 2192 extracted by hexane with water present or
with enzyme present (p = 0.15).
To test for potential ion suppression by the enzyme solution that could result in
artificially low EA 2192 values, a known mass of EA 2192 (2.55 ng) was added to both
decontamination samples and positive control samples and quantified. A mean of 95% (SD 35%)
19
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of the added EA 2192 was recovered from the positive control samples and a mean of 82% (SD
7%) of the added EA 2192 was recovered from the test samples. Based on the results from this
test, there was no ion suppression.
Table 15. Demonstration of Hexane as Quench for Enzyme Reaction Prior to LC/MS
Analysis for EA 2192
Sample Type
Water in
Hexane
Water in
Hexane
Water in
Hexane
DEFENZ™
VX-G in
Hexane
DEFENZ™
VX-G in
Hexane
DEFENZ™
VX-G in
Hexane
VX
Spike,
US
648
648
648
648
648
648
VX
Recovered,
US
572.5
650
532
589
794.5
612
MeanVX
Recovery,
ug (SD)
584.8
(60.0)
665.2
(112.6)
Recovery
88%
100%
82%
91%
123%
94%
MeanVX EA2192
% Recovered,
Recovery ug
0.763
90% 0.744
1.084
1.365
103% 2.128
1.712
MeanEA
2192
Recovery
0.864
(0.191)
1.735
(0.382)
20
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Table 16. Recovery of EA 2192 from
Sample Type
EA 2192 (Water/ Hexane)
EA 2192 (Water/ Hexane)
EA 2192 (Water/ Hexane)
EA 2192 DEFENZ™
VX-G in Hexane
EA 2192 DEFENZ™
VX-G in Hexane
EA 2192 DEFENZ™
VX-G in Hexane
EA 2192
Spike, ng
14
14
14
14
14
14
Aqueous Phase
EA 2192
Recovered, ng
11.77
16.11
13.55
12.44
9.76
11.05
after Hexane
EA 2192
Recovery
84%
115%
97%
89%
70%
79%
Extraction
MeanEA
2192
Recovery,
ng (SD)
6.74
(1.07)
5.41
(0.66)
MeanEA
2192 %
Recovery
99%
79%
2.10 Efficacy Determination
Decontamination efficacy was determined by measuring the amount of residual CWA on
test coupons and comparing them with positive controls (spiked with CWA, not decontaminated
and analyzed after the same "contact time" as the test coupons). Aliquots of extracts from blanks,
positive controls, and decontaminated coupons were analyzed for CWAs according to methods
described in Section 2.8. Decontamination efficacy was calculated as follows:
1) Calculation of CWA (or SRC) concentration in a coupon extract sample is determined by
Equation 5:
(5)
where:
2)
As = Area of target analyte peak in sample
A;s = Area of internal standard peak
Cs = Concentration of target analyte in sample (ug/mL)
Cis = Concentration of the internal standard (ug/mL)
M = slope of the GC calibration line
W = Y intercept of GC calibration line.
GC concentration results (|j,g/mL) are converted to total mass by multiplying by the
extract volume:
(6)
where:
Mm= Measured mass of target analyte (CWA or SRC) (|j,g)
Cs = GC concentration (|j,g/mL) of target analyte
Ev = Volume of extract (mL).
21
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3) Decontamination efficacy (percent removal achieved during decontamination) is then
defined as:
™ °f _ x100% (7)
^ M- of CWA on Positive Control Coupon
where:
Mm = Measured mass of CWA (|j,g) on an individual test coupon (or solution control
coupon)
M- = Mean measured mass of CWA (|j,g) from five positive control coupons.
The mean efficacy is the average efficacy from five test coupons included in a given
decontamination test (i.e., enzyme type, enzyme concentration, and contact time). A statistically
significant efficacy is defined when the average measured amount of agent recovered from the
test coupons after decontamination is statistically significantly (Student's t-test p < 0.05) lower
than the average amount recovered from the positive control coupons (i.e., those without
decontamination application).
2.11 Analysis of By-Products
Based on previous non-enzymatic decontamination testing,1 a variety of by-products may
be produced during decontamination of HD, such as: o-mustard; 2-thiophene acetonitrile; 3-
chloro-2-methylthiopropene; thiocyanic acid, 2-(2-butoxyethoxy) ethyl ester; l,3-bis(ethylthio)
propane; di vinyl sulfone; bis(beta-chloroethyl) sulfone (mustard sulfone); and bis(beta-
chloroethyl) sulfoxide (mustard sulfoxide). The GC/MS instrumentation was operated in the full
scan mode to detect such toxic CWA by-products in coupon extracts. A National Institute of
Standards and Technology (NIST) 2002 mass spectral library was used to tentatively identify
compounds in the mass spectra. Reports were generated using ChemStation software (Version
D.01.02.16 [15 June 2004], Agilent, Santa Clara, CA).
LC/MS was used for qualitative analysis for EA 2192 in VX/decontaminant solutions.
EA 2192 is a highly toxic VX degradation by-product. LC/MS was used because EA 2192
cannot be resolved using GC analytical methods. The test matrix for the LC/MS by-product
analysis is shown in Table 17.
Table 17. Test Matrix for LC/MS EA 2192 _
Agent Sample Type # Replicates
, ,v DEFENZ™ VX-G concentrated (3X) enzyme solution with CWA (test .
VA i 4- \ *
solutions)
VX Water (rather than enzyme) with CWA (positive control) 3
None Neutralized enzyme solution (solution blank) 3
For each "test solution" sample, 50 uL of the liquid decontaminant was added using a
Hamilton syringe (P/N 80565 [50 uL]) to a 20-mL vial (66022-060, VWR) containing
1 uL of neat VX and mixed. The contact time between the concentrated (3X) enzyme and the
VX solution was 15 min. The "3X" decontaminant is the enzyme solution prepared with only
one-third the label-specified dilution with water. As positive control solutions, deionized water
(50 uL) was added to each of three 20-mL vials containing 1 uL of neat VX.
22
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All vials containing test and positive control solution were closed and sonicated for 1 min
(50-60 kHz) after decontaminant or deionized water, respectively, was added. The mixture was
allowed to sit for 15 min. The reaction was halted at the end of the contact time by extraction
with hexane to remove the VX substrate from the aqueous solution containing the active enzyme.
Specifically, 50 uL of deionized water and 5 mL hexane were added to each of the test and
positive control solutions. After the hexane was added to the vials, they were closed and
sonicated for 10 min (50-60 kHz), then allowed to sit for 15 min to enable the polar and nonpolar
phases to separate. The aqueous phase of each positive control and test solution was serially
diluted with deionized water (1:10 of 1:25 of 1:50).
The EA 2192 was analyzed using an LC/MS system that consisted of a Shimadzu
20xR solvent delivery system, a Prodigy ODS-3, 2.1 x 150 mm, 5 jim analytical column
(Phenomenex, Torrance, CA) for chromatographic separation, and an Applied Biosystems 5500
mass spectrometer operated using positive electrospray. LC/MS parameters are shown in Table
18. Data were acquired for ion transitions 240>139 and 240>128. Samples that were not
analyzed the same day were stored at -20 °C or lower.
During method demonstration, a standard curve of EA 2192 in water was analyzed at 0.2,
0.5, 1.0, 2.5, 5.0, 9.8, and 25.2 ng/mL. The response to EA 2192 was found to be quadratic over
this range of concentrations. Quench efficacy using hexane to remove VX (thereby halting
decontamination by the DEFENZ™ VX-G solution) was verified in method demonstration.
Effectively recovery of EA 2192 from the aqueous phase after extraction with hexane was also
verified.
Because the analysis was qualitative, calibration with EA 2192 standards was not
included in the analysis of EA 2192 as a by-product of enzymatic decomposition. Rather, the
relative proportion of EA 2192 extracted from a VX solution, with or without enzymatic
decontamination (DEFENZ™ VX-G "3X" solution), was determined.
23
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Table 18. Liquid Chromatography/Mass Spectrometry Parameters for EA 2192 Analysis
Equipment and Parameters
Description
High Performance Liquid Chromatography
(HPLC)
Mass Spectrometer
Mass Spectrometer Source
Mass Spectrometer Software
HPLC Column
HPLC Column Temperature
Mobile Phase Components
Gradient Profile
Injection Volume
Run Time
Shimadzu 20xR Series
AB SCEX Triple Quad™ 5500
TurboIonSpray® probe (Electrospray), positive ion
mode
Analyst 1.5.1
Phenomenex Prodigy ODS-3, 2.1 x 150 mm, 5 um
Ambient
A = water: acetonitrile, 98:2 (v:v)
B = 0.2% formic acid in acetonitrile: isopropanol 80:20
(v:v)
Time, min
0
1
8
8.5
8.6
20
%B
0
0
25
25
0
0
Flow rate,
mL/min
0.2
0.2
0.2
0.2
0.3
0.3
lOuL
20 min
24
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3.0 Quality Assurance/Quality Control
3.1 Control of Monitoring and Measuring Devices
QC requirements and results are shown in Table 19.
Table 19. Data Quality Objectives and Results for Test Measurements
Parameter
Time
Mass
pH
Background
Contaminants
Mass of CWA (in
extraction solvent)
Mass of CWA (in
neutralized enzyme
solution)
Mass of SRC (test
and positive control
coupons and
laboratory and
procedural blanks)
Mass of CWA (on
positive controls)
Mass of CWA (on
spike controls)
Mass of CWA (on
laboratory blank)
Measurement
Method
Timer/data logger
Balance with daily
calibration check using
standard weights
pH meter
Analyze blank solvent
using GC- or LC/MS
Extract in solvent and
analyze using GC- or
LC/MS
Extract in solvent and
analyze using GC- or
LC/MS
Extract in solvent and
analyze using GC/MS
Extraction/
chromato graphic
quantitation
Extraction/
chromato graphic
quantitation
Extraction/
chromato graphic
quantitation
QC Requirement
Two seconds/hour; check once
before beginning testing
Balance precision at least O.lx
lowest measured value
Calibrate with two standard buffer
solutions spanning range of interest
70% of CWA spike is recovered;
determine once during method
demonstration
>70% of CWA, spike is recovered;
determine once during method
demonstration
>70% recovery of SRC (which
provides a check for matrix effects)
Results were considered an outlier
if the recovery value for analyte
from a coupon fell outside three
standard deviations of the mean.
Criterion applies only if
concentration of analyte is >5 times
the MDL
>85% of CWA spike target
-------
3.2 Chemical Analysis Equipment Calibrations
A six-point calibration for CWA and tributyl phosphate (TBP) as an SRC was generally
used with a lower calibration level of 0.5 |ig/mL and an upper range of approximately 50 |ig/mL.
Due to saturation, some analytes had only a five-point curve with an upper range of 25 jig/mL.
Naphthalene-dg was used as an IS for quantitation of CWAs and TBP. An average response
(relative standard deviation <15%) or linear regression (or, in a few cases, quadratic regression)
curve fit was applied to the calibration data. Samples exceeding the upper calibration limit were
diluted to a concentration within the calibration range and reanalyzed.
Continuing calibration verification (CCV) standards were included prior to sample
analysis, following every fifth sample and at the end of each batch of samples. Two or more
CCV concentrations were used, one of which was equal to the low calibration standard (0.5
|ig/mL and 10.0 |ig/mL for VX and TGD; 0.5 |ig/mL and 12.0 |ig/mL for HD) and CCV
response within 25% of nominal concentration was acceptable. Samples analyzed prior to or
following CCVs that were outside acceptance limits were re-analyzed.
For GC/MS, the neat CWA was diluted with hexane to prepare standard solutions that
were analyzed to construct a standard curve within an appropriate range. The standard solutions
were included each day that an analysis was performed. The GC/MS or LC/MS calibration
curves met the following performance requirements:
• r2 greater than 0.98
• % bias for the lowest standard less than 25%
• % bias for the remaining standards less than 15%
• % bias for the lowest calibration check standard less than 35%
• % bias for the remaining calibration check standard less than 20%
• Difference between replicate samples less than 20%.
3.3 Technical Systems Audit (TSA)
The QA Manager performed a TSA during the performance of the decontamination
testing. The purpose of the TSA was to ensure that testing was performed in accordance with the
test/QA plan1. In the audit, a QA Officer reviewed the sampling and analysis methods used,
compared actual test procedures to those specified in the test/QA plan1, and reviewed data
acquisition and handling procedures. The QA Manager prepared a report, the findings of which
were addressed either by modifications to the test procedures or by documentation in the test
records.
The TSA report noted that efforts to compensate for TGD losses caused by adhesion to
pipette wall and tip were documented as a deviation from the test/QA plan.l The impact of the
inconsistent TGD applications to coupons was to have high variability that made it less likely
that significant efficacy would be detected.
The TSA report also noted that the samples not used on the same day were stored at
-20° C rather than 4° C specified in the test/QA plan.l The lower temperature was a more
stringent condition than that specified in the test/QA plan1 and was not expected to have any
adverse impacts on the results.
26
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The ISA report also noted that the sonication at 50-60 kHz corresponds to the factory
specification and was not verified. The test/QA plan1 did not require verification of the
frequency of soni cation and precise sonication frequency was not considered to be a critical
parameter.
3.4 Performance Evaluation A udits
A performance evaluation (PE) audit was conducted for each performance parameter
shown in Table 20 to assess the quality of the measurements made during testing. The audits for
temperature, RH, concentration, and time were performed once during testing by analyzing a
standard that is independent of standards used during the testing. Table 20 summarizes the
acceptance criteria and results for the PE audit.
Table 20. Performance Parameters to be Audited
Parameter
Audit Procedure Expected Tolerance
PE Audit Results
Time
Compare time to
independent clock or
watch value
±2 seconds/hour
Both timers used during testing
were compared and found to be
within 2-second requirement
Chemical Mass
Use GC/MS to measure
SRC from secondary
source and compare to
primary source
Determine mass of agent
delivered to Teflon®
spike control coupons
and compare to target
application level
±10%
>85% of spike target
Primary and secondary sources
were found to be within +/-10%
tolerance requirement
For determining mass of agent
delivered to Teflon, see Section
3.6 below
Mass
Use balance to determine
the mass of a reference
weight
±0.1 g
Balance used was within annual
calibration and calibration
checks performed regularly to
±0.1 g criterion
3.5 Data Quality Audit
The Battelle QA Manager audited at least 10% of the investigation data and traced the
data from initial acquisition through reduction and statistical comparisons to final reporting. All
data analysis calculations were checked.
3.6 Spike Control Data
HD recoveries from spike control coupons are shown in Table 21 for each day of testing.
Examination of the mass spectra revealed that no HD was detected on any laboratory blank or
procedural blank coupons (except one procedural blank that was inadvertently spiked).
27
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Table 21. HD Recovery from Spike Control Coupons
Recovery, ug/mL Percent of Expected
(SD, n = 3) Concentration
8/2/2011 33 92
8/18/2011 45 125
Because of deviations from the test/QA plan,l the spike control data for VX and TGD are shown
in Section 3.8.
3.7 Amendments
Seven amendments were incorporated into the test/QA plan. l A brief summary of the
amendments follows:
• Amendment 1: Additional details were added to decrease losses during the spray-and-
weigh demonstration; language was added detailing the LC/MS evaluation of EA 2192.
• Amendment 2: A required deliverable of the work assignment, the amendment described
test/QA changes to perform additional efficacy testing at shorter or longer times for high
and low efficacy materials, respectively, and evaluate loss of efficacy as a function of
time after the enzyme solution is prepared.
• Amendment 3: Added language to perform neat GD extractions to determine extraction
efficiency; added language to describe method demonstration for the use of hexane to
quench the aqueous solution for LC/MS analysis.
• Amendment 4: Provided details for preparing uniform aliquots of DEFENZ™ VX-G
from the bulk materials received.
• Amendment 5: Provided flexibility to modify tests specified for "high efficacy" materials
when high efficacy was not observed for any materials.
• Amendment 6: A required deliverable of Amendment 1 to contract EP-C-10-001 Work
Assignment 1-04, provided test/QA details necessary to apply the plan to testing of
DEFENZ™ B-HD.
• Amendment 7: A revised LC/MS analysis to compare EA 2192 extracted from VX with
and without enzyme present.
3.8 Deviations
Deviation 1: On March 4, after testing had begun on March 2, the monthly purity tests
showed that actual delivery of neat agent was 80.7% VX (Table 22) rather than the 85% required
by Section B 1.1 of the test/QA plan.l VX is known to become unstable and degrade
unpredictably and rapidly during storage. Because of this phenomenon, U.S. Department of
Defense research typically requires 70% purity as the acceptance criterion. Monthly quantitative
analysis of VX stock solutions is performed to identify stocks that are degrading. Normal, but
unpredictable, degradation of VX occurred between the monthly test of purity and the use of the
VX in testing.
28
-------
Table 22. Monthly VX Purity Data Showing Gradual Degradation
Date Agent Purity %
4-Mar-ll VX 80.7
3-Feb-ll VX 89.3
7-Jan-ll VX 91.3
The deviation is not believed to have impacted test results. The testing results indicated
little or no decontamination efficacy at the lower contamination level generated by the less pure
(80.7%) VX challenge. If the decontamination is ineffective at 80.7%, ineffectiveness at the full
(85%) challenge is expected. Therefore, all samples were included in the analysis.
Deviation 2: The spike controls from the TGD trials showed actual delivery of CWA
was sometimes outside the range required by the test/QA plan.l Thickened agent was
particularly difficult to deliver with accuracy and precision. Microliter levels of application of
thickened agent are imprecise due to the high viscosity of the material and drag on the pipette tip.
Excess TGD from the reservoir sticks to the outside of the pipette tip. Wiping off this excess
TGD from the tip can pull the TGD aliquot out of the pipette. As specified in the test/QA plan,l
based on prior experience the pipette was set to dispense 1.4 uL of TGD in order to deliver 1 uL
(expected measurement of 100 ug/mL). However, the TGD recovered from controls
corresponded to a volume higher than target being delivered to the coupons. Specifically, rather
than observing recoveries equivalent to 100 ug/mL, deliveries to the coupon ranged from a mean
of 134 ug/mL for galvanized metal to 288 ug/mL for carpet.
Based on the accuracy of the first tests, professional judgment was used to adjust the
pipette setting in subsequent testing to get closer to the test/QA plan1 targets. The TGD was also
replaced with fresh material before sub sequent testing.
Deviation 3: Teflon® spike control coupons are used by Battelle as a standard to detect
unexpected problems, e.g., low VX purity. VX recoveries from spike control coupons are shown
in Table 23 for each day of testing. VX recoveries from spike control coupons were 72% or
greater. However, recoveries were generally lower than the 85% recovery specified in the
test/QA plan.*
Because the testing was repeated with longer decontamination times, the deviation was
not believed to impact conclusions drawn from the testing.
29
-------
Table 23. VX Recovery from Spike Control Coupons
Mean Recovery, ug/mL % of Target
(SD) n = 3
107.7(6.4)
128.4(0.9)
126.9(3.8)
114.8(1.6)
118.2(1.6)
115.8(2.1)
72
86
85
77
79
77
4.0 Results/Discussion
4.1 Method Demonstration Resu Its
The extraction methods accepted for use met the acceptance criterion (see Section 2.9.1)
of being in the range of 40% -120% recovery with a coefficient of variance between samples not
exceeding 30%. Use of RDS of VX, GD, and HD for the extraction efficiency demonstration was
a deviation from the test/QA plan. * VX and HD recoveries were above 70% and therefore
acceptable (Table 24). GD RDS recoveries were below 70% and therefore repeated with neat
GD. Extraction efficiencies with neat GD were above 70%.
Table 24. Extraction Efficiencies for CWAs from Vinyl Coupons
CWA Mean Extraction Efficiency Coefficient of Variance
VX(RDS) 114% 7.0%
GD(Neat) 107% 11%
HD(RDS) 94% 3.0%
The MDLs for VX, GD, and HD extracted from vinyl using 10 mL of hexane are shown
in Table 25.
Table 25. MDL Values for VX, GD, and HD Extracted from Vinyl Using Hexane
MDL, ug (10 mL Extract)
Material VX GD HD
Vinyl 1.81 0.96 1.52
4.2 Decontamination Results
30
-------
4.2.1 Measurement of pH of Enzyme Solution on Coupons
The pH of the enzyme solutions on the various coupons was measured using broad range
(0-14) pH indicator strips (#9590, EMD, Gibbstown, NJ). The DEFENZ™ VX-G solution, ready
for use, had a pH between 8 and 9 as measured with the pH indicator strips. The pH indicator
strips were selected because a pH meter could not measure small (100 uL) droplets on the
various material surfaces. A broad range pH indicator was selected because the potential pH
range from the various surfaces was not known. The DEFENZ™ VX-G on the surface of
laminate, wood, carpet, and vinyl, measured after a 15 min contact time, exhibited a pH of 9,
except for galvanized metal which exhibited a pH of 10. The DEFENZ™ B-HD enzyme
preparation exhibited a pH of about 8. No change in pH (pH remained about 8) was observed for
the enzyme after 15 min contact time with the surface of the galvanized metal, laminate, wood,
carpet, and vinyl coupons.
4.2.2 Measurement of Peracetic Acid in DEFENZ™ B-HD Enzyme Solution
The results of the measurement of the peracetic acid concentration in the activated
DEFENZ™ B-HD enzyme solution are shown in Table 26. Each activated enzyme preparation
was greater than the minimum recommended 2500 ppm level. The strips measure in 100 mg/mL
increments and the test solution was diluted 1:10 before the test strips were used. A reading of
100 mg/mL measured in the dilute solution was therefore reported as 1000 mg/mL in the
activated solutions.
31
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Table 26. Peracetic Acid Measurements for Activated DEFENZ™ B-HD Enzyme
Test
Date
7/22/1 1
7/22/1 1
8/2/1 1
8/2/1 1
8/2/1 1
8/18/11
8/18/11
8/18/11
8/18/11
Use
Method
development
(quench)
Method
development
(quench)
15 min
contact time
15 min
contact time
15 min
contact time
6 hr pot life
test
4 hr pot life
test
2 hr pot life
test
30 and 60
min contact
time
Time
Prepared
1018
0926
0950
1053
1148
0710
0717
0930
0838
Time Peracetic Acid
Tested
1056
0959
1014
1116
1215
0752
1315
0801
1117
0814
0930
0907
1143
Peracetic Acid,
mg/mL
4000
4000-5000
4000
4000-5000*
4000
4000
3000-4000*
4000
3000-4000*
4000
4000
4000
3000-4000*
* Test strip key is in 100 mg/mL increments and was used to test a 1:10 dilution of the enzyme. A 400 mg/mL
measurement in the dilute solution was therefore reported as 4000 mg/mL in the full strength enzyme solution. The
readings that were intermediate between color values were reported as a range, e.g., 4000-5000 indicating a
measurement above 4000, but below 5000.
4.2.3 VX Decontamination
No VX was found on any laboratory blank coupon. No VX was found on any procedural
blank coupon.
Decontamination efficacy results (mean and SD) using DEFENZ™ VX-G enzymes
prepared in accordance with manufacturer's instructions are shown in Table 27. The p-value is
the result of comparison of the mean of the positive control coupon and the test coupon
recoveries using the two-tailed Student's t-test. Differences were considered statistically
significant if p < 0.05. This means that the average measured amount of agent recovered from
32
-------
the test coupons after decontamination was statistically significantly lower than the average
amount recovered from the positive control coupons (i.e., those without decontamination
application).
A statistically significant efficacy was observed for DEFENZ™ VX-G enzymes with a
15 min contact time for VX on all materials except vinyl. Efficacy was observed for VX on
vinyl, but, because of variability in the results, the efficacy was not statistically significant.
Results from the solution controls indicate some efficacy against VX on wood or carpet that may
be attributable to ingredients in the DEFENZ™ VX-G product other than the enzyme.
The standard DEFENZ™ VX-G enzyme preparation was tested at longer contact times
(30 and 45 min) to evaluate whether efficacy would increase. Galvanized metal was used
because of the low efficacy observed for VX on galvanized metal at 15 min. Results for
decontamination of VX using longer contact times are shown in Table 27. Efficacy was observed
to increase with increasing contact time.
33
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Table 27. VX Decontamination Results Using DEFENZ™ VX-G
Material
Laminate
Wood
Carpet
Vinyl
Galvanized
metal
Galvanized
metal
Galvanized
metal
Contact
Time,
niin
15
15
15
15
15
30
45
Mean Positive Controls, ug
(SD; 95% Confidence
Interval [CI])
700
(21; 681-719)
410
(134; 293-5 27)
718
(46; 678-759)
646
(74; 581-712)
716
(34; 686-747)
918
(54; 871-965)
874
(30; 848-900)
Mean Solution Control
Coupons, ug
(SD; 95% CI)
683
(53; 637-730 )
299
(115; 199-400)
458
(108; 363-553)
703
(34; 673-733)
747
(43; 709-785)
891
(9; 883-899)
859
(17; 844-874)
Mean Test Coupons, ug
(SD; 95% CI)
618
(34; 589-648)
207
(143; 82-332)
580
(57; 530-630)
524
(11 8; 421 -627)
635
(62; 582-689 )
711
(29; 686-736)
645
(42; 608-681)
Mean Solution
Control
Efficacy
2%
27%
36%
-9%
-4%
3%
2%
Mean Test
Coupons Efficacy
12%
p<0.01
50%
p = 0.05
19%
p<0.01
19%
p = 0.09
11%
p = 0.04
23%
p<0.01
26%
p<0.01
34
-------
Higher concentrations of DEFENZ™ VX-G enzymes were tested with a 15 min contact
time to evaluate whether efficacy would increase. Results for decontamination of VX using
higher concentrations than the manufacturer's recommendation (2X and 3X) DEFENZ™ VX-G
solutions with a 15 min contact time are shown in Table 28. Efficacy was observed to increase
with increasing enzyme concentration. A 5X concentration resulted in incomplete dissolution of
the enzyme or buffer products and was not tested.
Table 28. VX Decontamination Results with Concentrated Enzyme Solutions
Material
Galvanized
metal
Galvanized
metal
Enzyme
Concentration
2X
3X
Contact
Time,
min
15
15
Mean Positive
Controls, ug
(SD; 95% CI)
854
(23; 834-875)
841*
(79; 772-910)
Mean Test
Coupons, ug
(SD; 95% CI)
608
(33; 579-637)
509*
(45; 458-561)
Mean Test
Coupons
Efficacy
29%
p<0.01
39%*
p<0.01
Only three values used due to sample processing error.
Results for decontamination of VX using concentrated (3X) DEFENZ™ VX-G solutions
stored at 4 °C after preparation for use are shown in Table 29. The prepared enzyme solution was
stored in a refrigerator for the time shown in Table 29 minus one hour, and then exposed to
ambient conditions for one hour. Contact time was 15 min. The p-value resulting from
comparison of the mean of the 6-hour delay test coupons to the 24-hour delay coupons (p = 0.35)
indicates that no significant difference in efficacy was observed after the longer delay period.
The p-value resulting from comparison of the mean of the 15-hour delay test coupons to the 24-
hour delay coupons (p = 0.37) indicates that no significant difference in efficacy was observed
after the longer delay period. No VX was found on any laboratory blank or procedural blank
coupons. Refrigerated storage for up to 24 hours appears to maintain the activity of the
DEFENZ™ VX-G enzyme solution when used for decontamination of VX.
Table 29. Effect of Storage of Activated Enzyme Solution on VX Decontamination Results
Material
Carpet
Carpet
Carpet
Enzyme
Concentration
3X
3X
3X
Pot Life,
hours
6
15
24
Contact
Time, min
15
15
15
Positive
Controls, ug
(SD; 95% CI)
810*
(44; 788-833)
1Y1CO11 1C81
Coupons, ug
528
(130; 415-642)
520
(104; 429-611)
429
(183; 269-588)
iviean lest
Coupons
Efficacy
35%
p<0.01
36%
p<0.01
47%
p<0.01
*Mean of 15 positive control coupons.
35
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4.2.4 TGD Decontamination
Application of small, precise amounts of thickened agent was difficult. Pipette settings
were manipulated based on historical applications and trial-to-trial observations. As shown in
Table 30, within trial and between trial variability in the mass recovered from spike control
coupons was high.
Table 30. TGD Spike Recovery from Spike Control Coupons
Pipette Setting, uL
1.4
1.2
1.2
1.2
1.2
1.6
Recovery, ug/mL
(SD, n = 3)
420
(169)
163
(33)
187
(54)
61
(7)
71
(4)
202
(8)
%of
Target
280
108
125
41
47
135
CWA Lot
1
1
1
2
2
2
Results for decontamination of TGD using DEFENZ™ VX-G are shown in Table 31.
The p-value is the result of comparison of the mean of the positive control coupon and the test
coupon recoveries using Student's t-test. No GD was found on any laboratory blank or
procedural blank coupon.
Statistically significant efficacy was observed for DEFENZ™ VX-G against GD (applied
as TGD) on carpet after a 15 min contact time. No efficacy was observed for DEFENZ™ VX-G
against TGD on galvanized metal, wood, vinyl, or laminate after a 15 min contact time. With
longer contact times (30 min), a statistically significant efficacy was observed for DEFENZ™
VX-G against TGD on laminate. At 45 min contact time, efficacy was observed, but, due to
variability, was not significantly different from the positive controls.
Data shown in Table 31 suggest that the solution controls containing the DEFENZ™
VX-G ingredients without the enzymes have consistent higher recoveries than positive controls.
This may be due to the reduced evaporation of TGD in the presence of a buffered water droplet
covering TGD. Analysis of p-values from the comparison of the mean of the positive control
coupon and solution control recoveries using Student's t-test shows, however, that the
differences are not statistically significant (p value ranges from 0.08 to 0.67 across the five
materials).
36
-------
Based on the high variability in the use of small volumes of thickened agents, future
testing should apply larger volumes of thickened agent (to correspondingly larger coupons).
Further, longer contact times may be appropriate to increase the likelihood of observing
significant differences.
37
-------
Table 31. TGD Decontamination Results Using DEFENZ™ VX-G
Material
Galvanized
metal
Wood
Carpet
Vinyl
Laminate
Laminate
Laminate
Contact
Fime, niin
15
15
15
15
15
30
45
Mean Positive Controls,
US
(SD; 95% CI)
1347
(257; 1121-1572)
1525
(169; 1377-1673)
2877
(172; 2727-3027)
2177
(679; 1582-2773)
1381
(162; 1239-1523)
460
(92; 379-540)
634
(180; 476-792)
Mean Solution Control
Coupons, ug
(SD; 95% CI)
1867
(617; 1326-2408)
1694
(489; 1266-2123)
3290
(780; 2606-3973)
2362
(648; 1794-2930)
2016
(610; 1481-2550)
624
(110; 528-720)
820
(501; 381-1260)
Mean Test Coupons, ug
(SD; 95% CI)
1410
(545; 932-1 888)
1665
(883; 800-2530)
1659
(366; 1338-1979)
1524
(749; 868-2180)
1887
(890; 1107-2668)
238
(61; 185-292)
481
(337; 186-777)
Mean Solution __ _, ,
„ , , Mean Test
Control ^ ,,,.,.
Efficacy Coupons EffKacy
-39% -5%
p = 0.82
-9%
-U% p = 0.77
^14% 42%
p<0.01
30%
-8%
-46% "37%
p = 0.27
-36% 48%
p<0.01
^29% 24%
p = 0.41
38
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Higher concentrations of DEFENZ™ VX-G enzymes were tested with a 15-min contact
time to evaluate whether efficacy would increase. Results for decontamination of TGD using
more concentrated DEFENZ™ VX-G solutions are shown in Table 32. The p-value is the result
of comparison of the mean of the positive control coupon and the test coupon recoveries using
Student's t-test. No GD was found on any laboratory blank coupon or procedural blank coupon.
Statistically significant efficacy at 15 min was observed with both the 2X and 3X enzyme
concentrations against TGD on laminate. This contrasts with no statistically significant efficacy
measured with the standard enzyme solution (following manufacturer's recommendation) at 15
min.
Table 32. TGD Decontamination Results with Concentrated Enzyme Solutions
Material
Laminate
Laminate
Enzyme
Concentration
2X
3X
Contact
Time, min
15
15
Mean Positive
Controls, ug
(SD; 95% CI)
226
(35; 195-257)
225
(31; 198-252)
Mean Test
Coupons, ug
(SD; 95% CI)
72
(23; 52-92)
110
(53; 63-156)
Mean Test
Coupons
Efficacy
68%
p<0.01
51%
p<0.01
Results for decontamination of TGD using concentrated (3X) DEFENZ™ VX-G
solutions stored at 4 °C after preparation for use are shown in Table 33. The prepared enzyme
solution was stored in a refrigerator for the time shown in Table 33 minus one hour and then
exposed to ambient conditions for one hour. Contact time was 15 min. The p-value is the result
of comparison of the mean of the positive control coupon and the test coupon recoveries using
Student's t-test. The p-value resulting from comparison of the mean of the 6-hour delay test
coupons to the 24-hour delay coupons (p = 0.67) indicates that no significant difference in
efficacy is observed after the longer delay period. The p-value resulting from comparison of the
mean of the 15-hour delay test coupons to the 24-hour delay coupons (p = 0.43) indicates that no
significant difference in efficacy is observed after the longer delay period.
No GD was found on any laboratory blank coupon. Small amounts of GD were recovered
from carpet procedural blanks in the 6 hour pot test (10.0 jig), 15 hour pot test (9.9 jig), and 24
hour pot test (12.4 jig).
Refrigerated storage for up to 24 hours appears to maintain the efficacy of the
DEFENZ™ VX-G enzyme solution when used for decontamination of GD.
39
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Table 33. Effect of Storage of Activated DEFENZ™ VX-G Enzyme Solution on TGD
Decontamination Efficacy
-- , . , Enzyme Pot Life,
Matenal „ \ ,. , '
Concentration hours
Carpet 3X 6
Carpet 3X 15
Carpet 3X 24
*Mean of 15 positive control coupons.
4.2.5 HD Decontamination
Contact Mean Positive Mean Test
Time, Controls, ug Coupons, ug
min (SD; 95% CI) (SD; 95% CI)
1074
(797; 376-1773)
1597* 1038
(399; 1395-1799) (296; 779-1298)
15
(186; 744-1070)
Mean Test
Coupons
Efficacy
33%
(p = 0.22)
35%
(p<0.01)
43%
p< 0.001
Results for decontamination of HD using DEFENZ™ B-HD solution prepared per label
instructions are shown in Table 34. Contact time was 15, 30, or 60 min. The p-value is the result
of comparison of the mean of the positive control coupon and the test coupon recoveries using
Student's t-test. The p-values resulting from comparison of the mean of the test and positive
control coupons indicates that statistically significant efficacy (p < 0.05) of DEFENZ™ B-HD
against HD is observed for galvanized metal, laminate, wood, carpet, and vinyl coupons with a
15-min contact time. The standard DEFENZ™ B-HD enzyme preparation was tested at longer
contact times (30 and 60 min) to evaluate whether efficacy would increase. Carpet and vinyl
were used because of the low efficacy observed for HD on carpet and vinyl at 15 min. Efficacy
of DEFENZ™ B-HD against HD on vinyl and carpet was higher after a 60 min contact time than
after a 15- or 30-min contact time.
40
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Table 34. HP Decontamination Results Using DEFENZ™ B-HD
Material
Galvanized
metal
Laminate
Wood
Carpet
Vinyl
Vinyl
Vinyl
Carpet
Carpet
Contact
Time,
niin
15
15
15
15
15
30
60
30
60
Mean Positive
Controls, ug
(SD; 95% CI)
1111
(105; 1019-1203)
1222
(128; 1096-1348)^
849
(94; 767-932)
1171
(71; 1109-1233)
1094
(43; 1056-1132)
816
(64; 760-872)
947
(188; 782-1112)
1063
(146; 935-1191)
990
(64; 934-146)
Mean Solution Control
Coupons, ug
(SD; 95% CI)
1038
(25; 1016-1060)
1092
(21; 1073-1110)
754
(26; 73 1-777)
1146
(72; 1083-1209)
1062
(60; 1009-1114)
Not attempted
Not attempted
Not attempted
Not attempted
Mean Test Coupons, ug
(SD; 95% CI)
843
(125; 702-984)*
894
(89; 816-972)
601
(64; 545-657) J
983
(67; 924-1041)
826
(73; 762-890)
761
(65; 705-818)
613
(53; 566-660)
905
(105; 812-997)
690
(56; 641-739)
Mean Solution
Control Efficacy
7%
11%
11%
3%
3%
Not attempted
Not attempted
Not attempted
Not attempted
Mean Test Coupons
Efficacy
24%
p O.04
27%
p<0.01
29%
p<0.01
16%
p = <0.01
24%
p = <0.01
7%
p = 0.22
35%
p = 0.01
15%
p = 0.09
30%
p = <0.01
*Results only for three coupons because two coupons were tipped or dropped during transfer.
t-
Results for only four coupons because one coupon was tipped or dropped during transfer.
One outlier excluded from analysis.
41
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Results are shown for decontamination of HD using DEFENZ™ B-HD solution prepared
per label instructions and stored at ambient laboratory temperature (approximately 21° C) after
preparation for use. The prepared enzyme solution was stored for the time shown in Table 35
before use. Contact time was 15 min. The p-value is the result of comparison of the mean of the
positive control coupon and the test coupon recoveries using the two-tailed Student's t-test. The
p-value resulting from comparison of the mean of the 2-hour delay test coupons to the 6-hour
delay coupons (p = 0.24) indicates that no significant difference in efficacy is observed after the
longer delay period. Storage for up to 6 hours at approximately 21° C appears to maintain the
efficacy of the DEFENZ™ B-HD enzyme solution.
Table 35. Effect of Storage of Activated DEFENZ™ B-HD Enzyme Solution on HD
Decontamination Efficacy
Pot Life,
Matenal
Hours
Wood 2
Wood 4
Wood 6
_ Mean Positive
Contact controls, ug
Time, mm
(SD)
662
15
(32)
15
(109)
930
15
(167)
Mean Test
Coupons, ug
(SD)
611
(129)
728*
(160)
718
(138)
Mean Test
Coupons
Efficacy
8%
p = 0.44
4%
p = 0.79
23%
p = 0.06
Results for only four coupons; outlier excluded, possible dilution error.
4.3 By-Product Analysis
Bis (beta-chloroethyl) sulfone was the only FID by-product of interest detected in coupon
extracts or positive control samples using full scan GC/MS. Bis (beta-chloroethyl) sulfone was
detected only in the hexane extract for the 60-min decontaminated carpet samples. The peak was
very small (just above baseline).
The peak areas for EA 2192 visible in the LC/MS method with and without application of
DEFENZ™ VX-G to VX are shown in Table 36. Note that these are raw peak areas for positive
control and test solutions diluted with deionized water as follows: 1:10, 1:25, 1:50. Qualitative
analysis showed EA 2192 to be present with and without decontamination. This observation may
be expected because EA 2192 is a by-product from natural degradation of VX. Further,
significantly less EA 2192 was observed (p < 0.005) after application of the enzyme than in the
controls (without enzyme). Assuming that the calibration standards were stable since
preparation, EA 2192 in positive controls averaged 78.5 |ig/mL and EA 2192 in test solutions
averaged 27.5 |ig/mL. (Because the work was semi-quantitative, the stability of calibration
standards was not verified. However, the quadratic fit for the seven point calibration curves had
an r2 = 0.993.)
42
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DEFENZ™ VX-G in contact with VX (containing natural EA 2192 by-product) does not
appear to produce EA 2192 but rather to reduce EA 2192 that may be naturally present as a VX
contaminant.
Table 36. Results from Analysis for EA 2192 (Toxic VX By-product)
Sample ID
Positive Control Sample 1
Positive Control Sample 2
Positive Control Sample 3
Test Solution Sample 1
Test Solution Sample 2
Test Solution Sample 3
Peak Average
Area PeakArea
5549039
5289832
4803737
5516719
2773475
1452236 1964603
1668098
% Remaining
Compared to
Positive
Control
NA
37.1
% Less Than
Positive
Control
NA
62.9
4.4 Observations of Damage to Coupons
The DEFENZ™ VX-G treatment resulted in no obvious change to any coupons. The
acrylic polymer used to thicken GD was visible on coupons after decontamination. Typical
photographs before, during, and after treatment are shown in Figures 2 and 3. The DEFENZ™
VX-G treatment resulted in no obvious visible damage to any of the coupons either immediately
after decontamination or two days after the decontamination. Typical photographs taken before,
during, and after treatment are shown in Figure 4.
No detailed examination or testing for structural damage was included in this evaluation.
Damage, if any occurred, that is not readily visible would not be likely to be detected in this
evaluation.
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Figure 2. Coupons before application of CWA (top), during DEFENZ™ VX-G
decontamination (center) and after enzymatic decontamination (bottom) with residual
plastic thickener from TGD visible, e.g., as shown by arrow.
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Figure 3. Coupons during application of VX (top) and during DEFENZ™ VX-G
decontamination (center, bottom).
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Figure 4. Coupons before application of HD (top), during DEFENZ™B-HD
decontamination (center) and 48 hours after decontamination (bottom).
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5.0 Conclusions
DEFENZ™ VX-G exhibited statistically significant efficacy (p < 0.05) against VX on
laminate, wood, carpet, and galvanized metal) with a 15 min contact time; DEFENZ™ VX-G did
not exhibit statistically significant efficacy against VX on vinyl with a 15 min contact time (p =
0.09). Tests on galvanized metal showed that efficacy increased with increasing contact time (30
and 45 min) and when higher concentrations of the enzymes (2X and 3X manufacturer's
recommendations) were employed.
DEFENZ™ VX-G exhibited statistically significant efficacy (p < 0.05) against TGD on
carpet, but not against TGD on galvanized metal (p = 0.82), wood (p = 0.77), vinyl (p = 0.19) or
laminate (p = 0.27) with a 15-min contact time. DEFENZ™ VX-G enzymes applied to TGD on
laminate showed that although no statistically significant efficacy was observed with a 15-min
contact time, there was statistically significant efficacy with a 30-min contact time. After a 45-
min contact time, less GD was recovered from laminate treated with enzyme than from positive
controls, but the difference was not significant. High variability in recoveries of GD and
substantial loss of GD from coupons due to natural attenuation challenge efforts to determine
efficacy. While the results are valid, future testing involving thickened agents should use larger
application volumes to reduce variability.
DEFENZ™ B-HD exhibited statistically significant efficacy against HD on all five
materials tested (laminate, wood, carpet, vinyl, and galvanized metal).
No toxic by-products were found to be produced by use of the DEFENZ™ VX-G or
DEFENZ™ B-HD enzymes and no damage to the test material coupons was visually observed
from the use of the enzymes.
The observed modest efficacies of the DEFENZ™ VX-G or DEFENZ™ B-HD enzymes
are similar to those obtained with e.g., diluted bleach products2 under similar test conditions.
Combined with the observation that toxic by-products are not produced and the lack of visible
damage to a range of indoor building materials, the enzymes appear to be technologies that might
be considered for use against VX, G-agents, or HD on indoor building materials after a terrorist
release.
Caution should be used in extrapolating from the bench testing to field application of the
enzymes. A full-scale test using spray equipment and larger surfaces is warranted to ensure that
the laboratory results are scalable. With the preceding caveat, based on the results of this
evaluation, the following application guidance is suggested:
1. Use a higher concentration of DEFENZ™ VX-G (dilute with only one third the amount
of water recommended by the manufacturer) and at least a 45-min contact time to
increase efficacy against VX when a variety of surface types may be contaminated.
2. Natural attenuation of GD (even when thickened) will likely be high from many surfaces,
but the use of DEFENZ™ VX-G may increase the rate of removal of the agent. Use a
higher concentration of DEFENZ™ VX-G (use only one third the amount of water
recommended by the manufacturer) and at least a 45 min contact time for surfaces
contaminated with TGD.
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3. Use a 60-min contact time for DEFENZ™ B-HD enzymes, prepared according to
manufacturer's instructions, for decontaminating HD.
4. While the DEFENZ™ VX-G and DEFENZ™ B-HD enzymes demonstrate modest
efficacy, a substantial portion of the chemical agents (VX, TGD, and HD) can be
extracted from the test materials even after the longest contact times and using the highest
enzyme concentrations evaluated. Use of longer contact times or repeated applications
may further reduce the chemical agent to acceptable levels.
The effectiveness of using longer contact times or repeated applications as approaches to
reduce chemical agents to acceptable levels is an important knowledge gap; further investigation
is recommended. Temperature effects were not tested in this evaluation and testing was
performed under ambient laboratory conditions. Temperature typically impacts enzyme
performance (rule of thumb, reaction rates increase by 50% to 100% with a 10° C rise in
temperature). Efficacy would be expected to decrease at lower temperatures and increase with
higher temperatures within an (unknown) optimal range. Higher temperatures will denature
enzymes. Further investigation is recommended to understand the impact of temperature on
efficacy.
Activity of enzymes depends strongly on the manufacturer's production process. Hence,
the results obtained in this report reflect solely on the commercially available DEFENZ™
enzyme products rather than the associated enzymes.
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6.0 References
1. Test/Quality Assurance (QA) Plan for Enzymatic Decontamination of Chemical Warfare
Agents, Version 2 (July 2010). Available upon request from EPA
2. U.S. EPA. 2011. Evaluation of Household or Industrial Cleaning Products for Remediation of
Chemical Agents. U.S. Environmental Protection Agency. EPA/600/R-11/055.
3. Rogers, J., T. Hayes, D. Kenny, I. MacGregor, K. Tracy, R. Krile, M. Nishioka, M. Taylor, K.
Riggs, H. Stone, and S. P. Ryan. 2008. Decontamination of Toxic Industrial Chemicals and
Chemical Warfare Agents on Building Materials Using Chlorine Dioxide Fumigation and
Liquid Oxidant Technologies. U.S. Environmental Protection Agency, Washington, DC,
EPA/600/R-08/125.
4. Code of Federal Regulations Title 40: Protection of Environment Part 136 - Guidelines
establishing test procedures for the analysis of pollutants. Appendix B - Definition and
Procedure for the Determination of the Method Detection Limit—Revision 1.11
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APPENDIX A
SIMULATED ENZYME REACTOR TESTS
SUMMARY:
Simulated enzyme reactor testing was performed to determine the decontamination efficacy of
enzyme decontamination technologies (DEFENZ™ VX-G against GD and VX and DEFENZ™
B-HD against FID) without potential confounds arising from application to and extraction from
material coupons. This simulated reactor test is defined here as a test where neat CWA interacts
with the enzyme solution in a vial (no coupon surface present) and includes sonication during a
contact time of 15 min as a simulation of the stirring process. The results of the simulated reactor
tests are shown in Table AS-1. DEFENZ VX-G exhibited efficacy against GD and VX. Ninety-
nine percent or more of both G agents was decomposed within the 15 min contact time. Lower
efficacy was observed against VX (23%). DEFENZ B-HD exhibited a 44% efficacy in 15 min
against HD.
Table A-l. Simulated Reactor Results for Enzymes and CWA
CWA
GD
VX
HD
Enzymes Used
DEFENZ™ VX-G
DEFENZ™ VX-G
DEFENZ™ B-HD
Blank
Solution,
Ug
ND*
ND*
ND*
Mean Positive Control
Total Mass, ug
(SD)
430(18)
480(11)
1110 (5)
Mean Test Total
Mass, ug
(SD)
ND*
370(10)
620 (270)
Mean
Efficacy
>99%
23%
44%
*ND indicates no CWA was detected
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APPROACH:
Procedures were followed as described in the main text. Deviations from the coupon testing
procedures are described here.
Chemical Warfare Agents
The purity of neat CWA (Table A-2) used was greater than 85%, except for VX which had purity
of 71%. The neat VX purity was accepted for the single simulated reactor test. This change to the
test/QA plan was documented in an amendment. VX is known to degrade once an ampoule is
opened. Thus, 85% purity is a difficult criterion to achieve except for the initial aliquot removed
from an ampoule. The U.S. Army accepts VX with a purity of 70% ± 10% for testing. Therefore,
the decision was made not to open a new ampoule of VX, but to use the VX from the opened
ampoule and to take the purity into account when making analytic standards.
Table A-2. Purity of Chemical Warfare Agents Used in Testing
. Manufacturer/Supplier Neat CWA
Agent ,T „ .,
Name Purity
VX US Army from EPA stocks* 71 %
GD US Army from EPA stocks* 91 %
HD US Army from EPA stocks* 99%
*EPA-owned stocks of CWA are stored at Battelle's facilities in West Jefferson, OH
Test Matrix
A simulated reactor test was performed for GD and VX utilizing DEFENZ™ VX-G and for HD
utilizing DEFENZ™ B-HD. The simulated reactor test is defined here as a test where a neat
CWA interacts with the enzyme solution in a vial (no coupon surface present). The test involved
sonication of the vial at 50-60 kHz during a contact time of 15 min as a simulation of the stirring
process. The test matrix is shown in Table A-3. The simulated reactor test was not performed for
thickened GD (TGD) to avoid difficulties in dispensing TGD as described in Section 4.2.4.
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One microliter (1 jiL) of neat agent was pipetted using a calibrated Hamilton syringe (P/N
CAL80975 [50 |iL] equipped with a 22-gauge needle [P/N 91022] and repeating dispenser [P/N
83700], Hamilton Co., RenoNV) into each vial designated as a test sample or positive control.
Sixty microliters (60 jiL) of the appropriate enzyme decontaminant was added to each test
sample. This amount was selected because it is consistent with the application to nonporous
surfaces in coupon testing. The CWA and enzyme solution were always in contact during
sonication. Positive control samples were vials spiked with CWA to which 60 jiL of DI water
was added (i.e., no enzymatic decontamination). Blanks are defined as vials with only the 60 jiL
enzyme solution and no CWA.
CWA was extracted individually by transferring the solution from each test, positive control, and
blank vial each into a separate 40 mL glass bottle (S236-0040, Fisher Scientific, Pittsburgh, PA)
that contained 10 mL of hexane/IS, then sonicating at 50-60 kHz for 10 min. The CWA amount
present in the vials was determined by the GC/MS analysis method in use for analysis of the
coupon extracts. Samples that were not analyzed the same day were stored at -20 °C ± 3 °C or
colder. GC/MS results were reviewed to identify by-products from CWA decontamination.
Table A-3. Test Matrix for Simulated Reactor Testing
Agent
GD
VX
HD
Enzyme Product
DEFENZ™ VX-G
DEFENZ™ VX-G
DEFENZ™ B-HD
Number of
Test Samples
3
3
3
Number of
Positive Controls
3
3
3
Number of
Blanks
1
1
1
Extraction and Analysis
The GC/MS parameters where different from the main text during analysis of the simulated
enzyme reactor testing for VX, GD, and HD analysis are shown in Table A-4.
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Table A-4. Gas Chromatographic/Mass Spectrometry Parameters for VX, GD, and HD
Analysis where different from Coupon Testing (See Table 12, main body of text)
Parameters VX and GD HD
Analysis Method GC/MS (Scan) GC/MS (Scan)
Mode Constant Pressure Constant Pressure
40 °C (1.0 min), 100 °C (0.0 min) @ 40 °C (2.0 min), 150 °C (0.0 min) @
Oven Program for 30 °C/min, 150 °C (0.0 min) @ 15 °C/min, 280 °C (0.0 min) @
Analysis 5 °C/min, 275 °C (0.0 min) @ 30 °C/min, 300 °C (0.0 min) @
15 °C/min, 325 °C (1.0 min) 30 °C/min, 325 °C (3.0 min)
Quality Assurance/Quality Control where Different from Coupon Testing
Amendments
Revised the purity criterion for accepting VX for use in testing from 85% to 71%; lower limit is
consistent with the U.S. Army purity acceptance criterion (70% ±10%).
RESULTS/DISCUSSION OF SIMULATED ENZYME REACTOR TESTS
The results of the simulated reactor results are summarized in Table A-5. DEFENZ™ VX-G
(mixed with water at the ratio recommended by the manufacturer) demonstrated efficacy against
GD and VX. No GD was detected after the 15 min contact time; mean efficacy was >99%. Mean
efficacy against VX was 23% after 15 min. DEFENZ™ B-HD (prepared per manufacturer's
guidance for small volumes and diluted with water in the recommended ratio of water to enzyme
solution) after a contact time of 15 min demonstrated 44% efficacy against FID compared to
positive controls. The temperature profile for fifteen min sonication was demonstrated to result
in a rise of about 7 °C from 17.4 °C when soni cation begins to 24.2 °C at 15 min.
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Table A-5. Simulated Reactor Results for Enzymes and CWA
CWA
GD
VX
HD
Enzymes Used
DEFENZ™ VX-G
DEFENZ™ VX-G
DEFENZ™ B-HD
Blank
Solution,
ug
ND*
ND*
ND*
Mean Positive Control
Total Mass, ug
(SD)
430(18)
480(11)
1110 (5)
Mean Test Total
Mass, ug
(SD)
ND*
370(10)
620 (270)
Mean
Efficacy
>99%
23%
44%
*ND indicates no CWA was detected
No CWA was detected in any blank solution in the simulated reactor testing.
The simulated reactor GC/MS data were examined for qualitative differences between control
and test samples. The qualitative differences in peaks in the test sample compared to the control
sample were as follows:
• GD - increased GD diester [Dipinacolyl methylphosphonate]
• VX - increased VX sulfide [Bis(2-diisopropylaminoethyl) sulfide] and butyric acid
• HD - methane sulfonamide, chloroacetic acid, and carbonic acid were found only in test
samples.
Note that certain known by-products, particularly the EA 2192 from VX, may be present, but
would not be detected using GC/MS.
CONCLUSIONS SIMULATED REACTOR TESTING
Simulated reactor testing demonstrated significant efficacy of DEFENZ VX-G against VX and
GD with a 15 min contact time. GD amount was reduced by > 99%. Mean efficacy against VX
was lower, 23%. Simulated reactor testing demonstrated significant efficacy of DEFENZ B-FfD
against FID with a 15 min contact time. Mean efficacy against FID was 44%. In most instances,
simulated reactor testing efficacies were higher than observed efficacies during coupon testing,
albeit in the same range. The largest difference was observed for DEFENZ VX-G product
against GD as the presence of the thickener to create TGD as used during coupon
decontamination may have resulted in a significantly reduced efficacy against TGD.
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United States
Environmental Protection
Agency
PRESORTED STANDARD
POSTAGE & FEES PAID
EPA
PERMIT NO. G-35
Office of Research and Development (8101R)
Washington, DC 20460
Official Business
Penalty for Private Use
$300
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