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EPA-600-R-13-141
Enzymatic Decontamination of
Chemical Warfare Agent Cyclosarin (GF)
UNITED STATES ENVIRONMENTAL PROTECTION AGENCY
Washington, DC
11
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DISCLAIMER
The United States Environmental Protection Agency, through its Office of Research and
Development's National Homeland Security Research Center, funded and managed the
research described here through EPA Contract Number EP-C-10-001, Work Assignment Number
2-04 with Battelle. This document has been peer and administratively reviewed and has been
approved for publication as an Environmental Protection Agency report. It does not necessarily
reflect the views of the Environmental Protection Agency. No official endorsement should be
inferred. The Environmental Protection Agency does not endorse the purchase or sale of any
commercial products or services.
Questions concerning this document or its application should be addressed to the principal
investigator on this effort:
Lukas Oudejans, Ph.D.
Decontamination and Consequence Management Division
National Homeland Security Research Center
Office of Research and Development
U.S. Environmental Protection Agency (E343-06)
109 T.W. Alexander Dr
Research Triangle Park, NC 27711
Phone:(919)541-2973
Fax: (919) 541-0496
E-mail: oudejans.lukas@epa.gov
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
Larry Kaelin
Office of Solid Waste and Emergency Response, Office of Superfund Remediation &
Technology Innovation
Dave Mickunas
Office of Research and Development, National Homeland Security Research Center
Stuart Willison
Eletha Roberts (QA review)
Contributions of the following organization are acknowledged:
Battelle
IV
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TABLE OF CONTENS
DISCLAIMER iii
ACKNOWLEDGMENTS iv
TABLE OF CONTENTS v
LIST OF TABLES vii
LIST OF FIGURES vii
ACRONYMS viii
EXECUTIVE SUMMARY ix
1.0 Introduction 1
1.1 Background 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 Preparation of Enzyme-Based Decontamination Technologies 3
2.3.1 Preparation Procedure for DEFENZ™ VX-G 3
2.4 Building Material Coupons 5
2.5 Coupon Spiking 6
2.6 Test Matrices 7
2.6.1 Enzyme Application Rate 7
2.6.2 Simulated Enzyme Reactor Efficacy Testing 7
2.6.3 Test Matrices for DEFENZ™ VX-G against GF on Various Building Materials 8
2.7 Observation of Surface Damage 9
2.8 Extraction and Analysis 10
2.9 Method Demonstration 11
2.9.1 Recovery of GF from Test Coupons 11
2.9.2 MDL for GF Extracted from Coupon Materials 12
2.9.3 Quench of Decontamination Reaction 13
2.10 Efficacy Determination 13
2.11 Statistical Analysis 14
2.12 Analysis of By-products 14
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3.0 Quality Assurance/Quality Control 15
3.1 Control of Monitoring and Measuring Devices 15
3.2 Chemical Analysis Equipment Calibration 16
3.3 Technical Systems Audit 16
3.4 Performance Evaluation Audits 16
3.5 Data Quality Audit 17
3.6 Amendments 17
3.7 Deviations 17
4.0 Results/Discussion 18
4.1 Method Demonstration Results 18
4.2 Simulated Enzyme Reactor Results 18
4.3 By-Product Analysis 19
4.4 Coupon Decontamination Results 19
4.5 Observations of Damage to Coupons 22
5.0 Conclusions 23
6.0 References 24
VI
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LIST OF TABLES
Table ES-1. Summary of Decontamination Efficacy Results for DEFENZ™ VX-G against GF ix
Table ES-2. Simulated Enzyme Reactor Results for DEFENZ™ VX-G Enzyme and GF x
Table 1. Purity of Chemical Warfare Agents Used in Testing 3
Table 2. Formulae for Preparing DEFENZ™ VX-G Solutions 4
TableS. Test Materials 6
Table 4. Enzyme Application Amounts for Bench-Scale Coupon Testing 7
Table 5. Test Matrix for Simulated Enzyme Reactor Testing 8
Table 6. Test Matrix for Decontamination of GF with DEFENZ™ VX-G Prepared per
Manufacturer's Recommendations and 15-Min Contact Time 8
Table 7. Test Matrix for Systematic Evaluation of DEFENZ™ VX-G against GF 9
Table 8. Gas Chromatographic/Mass Spectrometric Parameters for GF Analysis 10
Table 9. Pertinent Parameters forTarget Chemical 11
Table 10. Recovery of GF Using Hexane Extraction as Quench 13
Table 11. Data Quality Objectives and Results forTest Measurements 15
Table 12. Performance Parameters Audited 17
Table 13. Extraction Efficiencies for Neat GF from Various Types of Coupons 18
Table 14. MDL Values for GF Extracted from Various Types of Coupons Using Hexane 18
Table 15. Simulated Enzyme Reactor Results for DEFENZ™ VX-G Enzyme and GF 19
Table 16. GF Decontamination Results Using DEFENZ™ VX-G (IX) 19
Table 17. GF Decontamination on Wood Flooring Using Alternative Contact Times, Repeated
Application, and/or 3X Enzyme Concentration 21
LIST OF FIGURES
Figure 1. Approach used to ensure homogeneity of DEFENZ™ 120 and DEFENZ™ 130 enzymes in
500 ml enzyme solutions 5
Figure 2. Recovered amounts of GF from materials and associated decontamination efficacy
following DEFENZ™ VX-G application (15 minute contact time) 20
Figure 3. Decontamination efficacy for 15-min, 30-min, and repeated 15-min contact time of
DEFENZ™ VX-G against GF 21
vn
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ACRONYMS
AMU
BBRC
°C
CAS
CCV
cm
CWA
EPA
FPD
g
GC
GC/MS
GD
GF
HD
IS
kHz
L
m
MDL
min
mg
ml
mm
u.m
NIST
PE
QA
QC
RH
SD
SIM
SRC
TBP
TGD
ISA
atomic mass unit
Battelle Biomedical Research Center
Degree(s) Celsius
Chemical Abstracts Service
continuing calibration verification
centimeter(s)
chemical warfare agent
U.S. Environmental Protection Agency
flame photometric detector
gram(s)
gas chromatography
gas chromatography/mass spectrometry
soman
cyclosarin
sulfur mustard
internal standard
kilohertz
liter(s)
meter(s)
method detection limit
minute(s)
milligram(s)
milliliter(s)
millimeter(s)
microgram(s)
microliter(s)
micrometer(s)
National Institute of Standards and Technology
performance evaluation
quality assurance
quality control
relative humidity
standard deviation
selected ion monitoring
surrogate recovery compound
tributyl phosphate
thickened soman
technical systems audit
Vlll
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EXECUTIVE SUMMARY
The U.S. Environmental Protection Agency (EPA) is the primary federal agency
responsible for remediation of public areas in the aftermath of a terrorist release of a chemical
warfare agent (CWA). The threat of a release in a building or transportation hub drove the EPA
to evaluate the effectiveness of DEFENZ™ VX-G, an enzyme-based technology, for
decontamination of G-type nerve agents and VX. In previous testing, thickened soman (TGD)
was the G-type agent.1 A thickened agent was used because of the high evaporation rate of
soman (GD). However, high variability that was attributed to the inherent difficulty of precise
application of small amounts of TGD (1 ul applications) onto coupons was observed with the
TGD decontamination study. Here, the efficacy of DEFENZ™ VX-G against the G-type agent
cyclosarin (GF) was systematically evaluated. Because of an evaporation rate lower than soman,
GF (without thickener) was expected to persist on building materials sufficiently to enable
decontamination efficacy testing. Application of small, precise volumes was easier to achieve
without the presence of thickener.
Efficacy results, i.e., GF recovered from test coupons after decontamination with the
enzyme product relative to GF recovered from positive control coupons, are summarized in
Table ES-1. Application of DEFENZ™ VX-G prepared at the manufacturer's recommended
concentration ("IX") reduced the amount of GF on the coupon with a 15 minute (min) contact
time. A >90% efficacy was observed against GF on non-porous galvanized metal and decorative
laminate. Lower efficacy (77%, 80%) was observed against GF on vinyl flooring and industrial
carpet, respectively, while the observed efficacy against GF on wood flooring was the lowest
(36%).
Table ES-1. Summary of Decontamination Efficacy Results for DEFENZ™ VX-G against GF
Material
Galvanized metal
Wood flooring
Industrial Carpet
Vinyl flooring
Decorative laminate
Wood flooring
Wood flooring
Wood flooring
Wood flooring
Wood flooring
Contact
Time, min*
15
15
15
15
15
30
15+15
15
30
15+15
Concentration
IX
IX
IX
IX
IX
IX
IX
3X
3X
3X
Mean Test Coupons
Efficacy
92%
36%
80%
77%
94%
47%
61%
53%
44%
79%
* Manufacturer recommends 15-min contact time; 15+15 indicates that after an initial application with a 15-min
contact time, the enzyme was reapplied for an additional 15-min contact time; ibid for 30+30.
f IX is enzyme diluted with deionized water per manufacturer's recommendation; 3X is enzyme diluted with one-
third of the recommended water.
ix
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The standard DEFENZ™ VX-G enzyme preparation (IX) was tested on wood flooring at a longer
contact time (30 min). The efficacy did not increase significantly with the 30-min contact time
compared to a 15-min contact time (Student's t-test p = 0.29). This result was obtained in spite
of the potential for evaporative loss during the additional contact period. Reapplication of the
IX enzyme for a second 15-min period resulted in a significant increase in efficacy (p = 0.014),
presumably due to the replenishment with fresh enzymes. Given the results from the single 30-
min application of the enzyme, the increased efficacy with a second application of the enzyme
is unlikely to be explained by increased evaporation.
Concentrations of DEFENZ™ VX-G enzymes higher than the manufacturer's
recommendation ("3X") were tested with 15-min and 30-min contact times, and with repeated
15-min applications. Results are shown in Table ES-1. Increasing the concentration to 3X did not
significantly increase efficacy at 15-min (p = 0.17) or 30-min (no improvement) contact times
compared to decontamination for 15-min using the IX concentration. With repeated 15-min
application of the 3X enzyme, efficacy was significantly higher (p = 0.009) compared to
decontamination for 15-min using the IX concentration. However, the higher (3X)
concentration with repeated 15-min application did not result in significantly greater efficacy
than the repeated 15-min application using the IX concentration (p = 0.17). In summary,
reapplication of the standard enzyme preparation was demonstrated to increase efficacy.
Higher enzyme concentrations and/or longer contact times did not significantly increase
efficacy against GF.
A simulated enzyme reactor test was performed for GF in which a neat CWA
(here, GF) is added to the enzyme solution in a vial (no coupon surface present) and sonicated
for a contact time of 15 min as a simulation of the stirring process during a normal enzyme
reactor test. This test simulates conditions generally used by a vendor to claim a product's
efficacy against a CWA. The result of the simulated enzyme reactor test is shown in Table ES-2.
DEFENZ™ VX-G exhibited higher efficacy against GF when compared to the coupon testing.
Ninety-nine percent of GF was decomposed with the 15 min contact time. This more dynamic
interaction is apparently important in reaching a higher efficacy against GF. Lower efficacy
results obtained during coupon testing can be explained by the more static interaction of the
enzyme solution with GF on the test coupon.
Table ES-2. Simulated Enzyme Reactor Results for DEFENZ™ VX-G Enzyme and GF
Blank Mean Positive Control Mean Test Mean
CWA Enzyme Used _ . . ... ,,.^,
Solution, u.g Total Mass, ug (SD) Total Mass, u.g (SD) Efficacy
GF DEFENZ™ VX-G ND* 704(66) 10(13) 99%
*ND indicates no GF was detected.
No obvious visual damage resulted from the application of the enzyme solution. Caution should
be used in extrapolating from the bench testing to field application of the enzymes. However,
given the observed efficacies of the DEFENZ™ enzymes against GF and the lack of visible
damage to a range of indoor building materials, the enzymes appear to be useful for
decontaminating this CWA on indoor building materials after a terrorist release.
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1.0 Introduction
1.1 Background
Protecting human health and the environment is the mission of the U.S. Environmental
Protection Agency (EPA). The 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 for CWAs. The EPA may be tasked to clean-up these agents
after a release in a public setting. Information about suitable decontamination technologies is
limited and optimal decontaminant concentration and contact times have been determined
primarily by vendors with limited third party verification. Effectiveness of available enzymatic
decontamination technologies against CWAs on surfaces is generally unknown.
This report describes a systematic investigation to evaluate the efficacy of an enzyme-
based technology produced by Genencor , (a Danisco Division, Palo Alto, CA): DEFENZ™ VX-G
(for decontamination of VX and G-type nerve agents). (In May 2011, DuPont acquired a
majority stake in Danisco A/S and the Genencor enzymes are now marketed within DuPont
Industrial Biosciences.) In previous testing, the focus was on thickened soman (TGD) as the G-
type agent.1 Thickened agent was used because of the high evaporation rate of soman (GD).
However, the high variability that was observed with the TGD decontamination study was
attributed to the inherent difficulty of precise application of small amounts of TGD (1 microliter
[ul] applications) onto coupons. Here, the efficacy of DEFENZ™ VX-G against the G-type agent
cyclosarin (GF) is evaluated systematically. Because GF has a lower evaporation rate than
soman, GF (without thickener) was expected to persist sufficiently on building materials to
enable decontamination efficacy testing. In comparison, the vapor pressure of GF is ~9 times
lower than the value for GD.
1.2 Test Facility Description
All testing was performed at the Battelle Biomedical Research Center (BBRC) site in
West Jefferson, Ohio. 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 main objective of this evaluation was to determine the decontamination efficacy of
DEFENZ™ VX-G enzyme decontamination technology against GF applied to coupons made from
materials consistent with items found in indoor environments. The efficacy was evaluated as a
function of material type, time, repeated application, and concentration. The enzyme was
initially prepared per manufacturer's directions, stored and used in accordance with the label
instructions. Efficacy of the enzyme when appropriately applied against GF was evaluated on
each of five different 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 instructions for use). Higher concentrations of DEFENZ™ VX-G, longer contact
times, and repeated applications were also evaluated. Specifically, the enzyme solution
1
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prepared according to the manufacturer's recommendations was tested with a 30-min contact
time and a 15-min contact time with a reapplication and additional 15-min contact time to
decontaminate GF on wood. Wood was selected here as the building material associated with
the lowest efficacy against GF in the first round of experiments. In addition, a 3 times higher
concentration of recommended enzyme to water was tested with a 15-min contact time, 30-
min contact time, and a 15-min contact time with a reapplication and an additional 15-min
contact time to decontaminate GF on wood.
As a secondary objective, the effects of the enzyme-based decontamination
technologies on the building materials were qualitatively evaluated by visual inspection,
identifying changes in color, reflectivity or roughness. Such assessment would indicate whether
material incompatibility was observed.
Simulated enzyme reactor testing was performed to determine the decontamination
efficacy of enzyme decontamination technologies (DEFENZ™ VX-G against GF). This test
simulates conditions generally used by a vendor to claim a product's efficacy against a CWA.
Results of this simulation would indicate whether this more dynamic environment is important
in reaching a higher efficacy against a CWA. Such test is also without potential confounds
arising from application to and extraction from material coupons.
Testing was performed in accord with Test/Quality Assurance (QA) Plan for Enzymatic
Decontamination of Chemical Warfare Agents, Version 2 (July 2010) (available upon request).2
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2.0 Procedures
2.1 Tech no lo gy Descrip tions
DEFENZ™ VX-G is an enzyme-based technology produced by Genencor (a Danisco
Division, Palo Alto, CA). The details of the technologies are proprietary. Instructions for creating
default enzyme solutions are listed and were followed as per vendor's directions.
The DEFENZ™ VX-G product consists of a pouch containing two packets:
• Enzyme packet (110 grams [g]) of granulated powder
• Buffer packet (250 g of powder) containing predominantly sodium hydrogen carbonate
(NaHCOs).
The enzyme packet contains two pre-mixed constituent powders: 10 g of "organophosphorous
[sic] acid anhydrolase" enzyme (DEFENZ™ 120) and 100 g of "organophosphorous [sic]
hydrolase" enzyme (DEFENZ™ 130). The enzyme and buffer dissolve in 10 liters (L) of water.
2.2 Chemical Warfare Agents
The CWA used to evaluate the efficacy of decontamination in this report was GF
(cyclohexyl methylphosphonofluoridate, CAS Registry Number 329-99-7), (Table 1). The target
purity of the neat agent was expected to be at least 85%. Purity for each ampoule of GF was
determined using gas chromatography (GC)-flame photometric detection (FPD) prior to
beginning testing. Observed purity was more than the required 85%.
Table 1. Purity of Chemical Warfare Agents Used in Testing
.„ .. .. Observed Neat
Agent Manufacturer/Supplier Name run A D •
GF US Army from EPA stocks* 99%
*EPA-owned stocks of CWAs are stored at Battelle's facilities in West Jefferson, OH.
2.3 Preparation of Enzyme-Based Decontamination Technologies
2.3.1 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. This method enabled the same proportions as
recommended by the vendor to be used to prepare batches smaller than 10 L.
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
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)
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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 Table 2 and 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 (22.0 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 (22.0 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 (5.50 g ± 0.25 g each) were
then produced by transferring an equal amount (1.1 g ± 0.05 g) from each mixed sample
prepared in 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 contains DEFENZ™ VX-G enzyme (5.5 g) and stored at ambient
temperature in a desiccator until needed.
The manufacturer's instructions call for the contents of the enzyme packets to be mixed
into 10 L of water. Enzyme solutions were prepared fresh each day of testing in accordance
with manufacturer's instructions (IX), but with smaller proportionate amounts of enzyme (5.5
g) and buffer (12.5 g). Deionized water was used to prepare the solutions. The pH of the
enzyme solution was measured and documented prior to each day of use using a pH meter (pH
meter Model SevenMuIti, Mettler Toledo, Columbus, OH). The enzyme solutions used were
verified as being pH 8.3 ± 0.3.
For the 3X concentration, the full packet of enzyme would be mixed in 3.3 L of water.
Actual 3X mixtures were based on this proportion applied to the amounts of enzyme in the
"batch packets" as follows: add the contents intended for 500 ml to 167 ml of deionized water.
The preparation of DEFENZ™ VX-G per manufacturer's recommended concentration (IX) and
3X concentration is shown in Table 2.
Table 2. Formulae for Preparing DEFENZ™ VX-G Solutions
Enzyme (g)
Buffer (g)
Water (ml)
Manufacturer's
Recommended
Concentration (Ix)
1 vial containing DEFENZ™ VX-G
enzyme, 5.5 g ±0.25g
1 vial containing DEFENZ™ VX-G buffer
(sodium hydrogen carbonate),
12.5 g± 0.1 g
500
3X Preparation
1 vial containing DEFENZ™ VX-G
enzyme, 5.5 g ± 0.25 g
1 vial containing DEFENZ™ VX-G buffer
(sodium hydrogen carbonate),
12.5 g± 0.1 g
167
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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
22 g
sample
1
22 g
sample
i
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 was repeated five times
From each mixed sample, 1.1 g were
transferred to a vial to create a batch of
enzyme of sufficient mass (5.5 g) for
preparing 500 mL of enzyme solution;
this was repeated 20 times
Figure 1. Approach used to ensure homogeneity of DEFENZ7
in 500 ml enzyme solutions.
2.4 Building Material Coupons
120 and DEFENZ™ 130 enzymes
This bench-scale investigation utilized small coupons of interior building materials
(presented in Table 3) contaminated with GF.
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Table 3. Test Materials
Material
Galvanized metal
ductwork
Decorative
laminate
Industrial grade
carpet
Wood Flooring
material
Vinyl flooring
material
Description
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
Shaw Industries Inc. EcoWorx
thickness ~0.7 cm
Fir plywood (bare); thickness 0.9 cm
Armstrong Excelon
Manufacturer/
Supplier Name
Adept Products,
Inc., West
Jefferson, OH
A' Jack Inc.,
Columbus, OH
Carpet
Corporation of
America, Rome,
GA
Lowe's,
Columbus, OH
Lowe's,
Columbus, OH
Coupon .
, . Material
Surface Size
LxW(cm) PreParat'°"
3.5 x 1.5 C'ean Wlth
acetone
Clean with dry
3.5x1.5 air to remove
loose dust
Clean with dry
3.5x1.5 air to remove
loose dust
Clean with dry
3.5x1.5 air to remove
loose dust
Clean with dry
3.5x1.5 air to remove
loose dust
2.5 Coupon Spiking
For each contact time and material combination:
• Five replicate test coupons were spiked with GF with subsequent decontamination;
• Five replicate positive controls were spiked with GF without subsequent
decontamination;
• Two procedural blank coupons were not spiked with GF but were decontaminated;
• Two laboratory blank coupons that were not spiked with GF and were not
decontaminated.
All test and positive control coupons were spiked with a nominal 1 ul of neat GF,
delivering approximately 1.1 milligram (mg) of GF. The contamination level was approximately
2 g/square meter (m2) (1.1 mg/ [3.5 centimeters (cm) x 1.5 cm] = 0.21 mg/cm2 = 2.1 g/m2). GF
was dispensed using a calibrated Hamilton syringe (P/N CAL80975 [50 ul] equipped with a 22-
gauge needle [P/N 91022] and repeating dispenser [P/N 83700], Hamilton Co., Reno NV).
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 1 ul droplets of neat GF, using the same syringe and repeating dispenser
settings as 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
6
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hour. The first spike control coupon was prepared at the beginning of the evaluation. The
second spike control coupon was prepared midway though 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 shown in Equation 1:
a= (1)
9 droplets
where:
(X = Mean mass of CWA per spiked droplet
CWAj = Mass of CWA recovered from the Ith spike control coupon.
2.6 Test Matrices
2.6.1 Enzyme Application Rate
A backpack type sprayer would be the most likely method for application of an enzyme
solution in the field setting. However, for this laboratory study, in order to reduce variability in
amounts of enzyme solution applied to the small coupons, the enzyme solution was delivered
to coupon surfaces as measured amounts from pipettes. In a previous study, a spray application
was used to determine the mass of enzyme solution (DEFENZ™ VX-G) that would be applied to a
surface in a typical spray application.1 These data provided material-specific target values for
the amount of enzyme solution to be applied to coupons to evaluate decontamination efficacy.
The applied enzyme solution amounts are shown in Table 4. Enzyme solutions were applied to
coupons using a positive displacement pipette ((P/N M-250 [250 ul] and D-200 [2-200 ul] tip,
Gilson Inc., Middleton, Wl).
Table 4. Enzyme Application Amounts for Bench-Scale Coupon Testing _
Enzyme Solution
Material
Application (ml)
Galvanized metal
Decorative laminate
Wood flooring
Industrial carpet
Vinyl flooring
0.06
0.06
0.09
0.12
0.06
2.6.2 Simulated Enzyme Reactor Efficacy Testing
A simulated enzyme reactor test was performed for GF utilizing DEFENZ™ VX-G. The
simulated enzyme reactor test involves combining neat GF with the enzyme solution in a vial
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(no coupon surface present) with sonication of the vial at 50-60 kiloHertz (kHz) during a contact
time of 15 min as a simulation of stirring during an enzyme reactor test. The test matrix is
shown in Table 5.
Neat agent (1 ul) was delivered using a calibrated Hamilton syringe (P/N CAL80975 [50
ul] equipped with a 22-gauge needle [P/N 91022] and repeating dispenser [P/N 83700],
Hamilton Co., Reno NV) into each vial designated as a test sample or positive control. The
enzyme decontaminant (60 ul) was added to each test sample. This amount was selected
because it is consistent with the application to nonporous surfaces in coupon testing. The GF
and enzyme solution were always in contact during sonication. Positive control samples for the
simulated enzyme reactor testing were vials spiked with GF to which 60 uL of Dl water was
added (i.e., no enzymatic decontamination). Procedural blanks are defined as vials with only the
60 ul enzyme solution and no GF.
GF 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/internal standard (IS), (naphthalene-d8), then sonicating at
50-60 kHz for 10 min. The GF amount present in the vials was determined by the gas
chromatography/mass spectrometry (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 GF decontamination.
Table 5. Test Matrix for Simulated Enzyme Reactor Testing
Number of Test Number of Positive Number of
Agent Enzyme Product „ „ „. .
Samples Controls Blanks
GF DEFENZ™VX-G 331
2.6.3 Test Matrices for DEFENZ™ VX-G against GF on Various Building Materials
The DEFENZ™ VX-G enzyme-based decontamination technology was evaluated against
GF using a 15-min contact time and manufacturer-specified enzyme concentration for the
material combinations as shown in Table 6. The test coupons were spiked with GF and allowed
to weather for 30 minutes; then the (60 uL) DEFENZ™ VX-G enzyme was added for the specified
contact time for the decontamination test. The positive control coupons were spiked with GF
and allowed to weather for 30 minutes plus the contact time for the corresponding
decontamination test. When the appropriate time had been reached (equivalent to contact
time), all coupons were spiked with surrogate recovery compound (SRC), tributyl phosphate
(TBP), and placed into separate vials containing 10 ml of hexane (GC Resolv grade, Fisher
Scientific, Pittsburg, PA) containing the IS (naphthalene-d8), and the coupons were extracted,
and analyzed as described in Section 2.8. This SRC was added as a check for possible matrix
effects.
Table 6. Test Matrix for Decontamination of GF with DEFENZ™ VX-G Prepared per
Manufacturer's Recommendations and 15-Min Contact Time
-------
Agent
GF
GF
GF
GF
GF
Material
Galvanized Metal
Decorative Laminate
Industrial Carpet
Wood Flooring
Vinyl Flooring
Test
Coupons
5
5
5
5
5
Positive
Controlsf
5
5
5
5
5
Procedural
Blanks*
2
2
2
2
2
Laboratory
Blanks5
2
2
2
2
2
Test coupons are spiked with GF and undergo decontamination.
Positive controls are spiked with GF but do not undergo decontamination.
* Procedural blanks are not spiked with GF but undergo decontamination; one of the three procedural blanks was
extracted and analyzed, the second procedural blank was not extracted but was held for 48 hours (or longer if over
a weekend) and examined for visually-obvious changes. See Section 2.7.
§ Laboratory blanks were not spiked with GF and did not undergo decontamination.
DEFENZ™ VX-G efficacy against GF on wood was evaluated with a repeated application
(a total of two applications of 15 min contact time each), a longer contact time (30 min), and at
a higher enzyme concentrations (3X) at 15 min, two applications of 15 min each, and a 30 min
application. Wood flooring was selected because this material exhibited the least efficacy
observed with a 15-min contact time against GF. The question being answered was whether a
longer contact time, reapplication, or higher enzyme concentrations would increase efficacy for
decontaminating materials on which the vendor-recommended enzyme concentrations and
contact time had the least efficacy. The test matrix for the systematic evaluation of enzyme
efficacy against GF is shown in Table 7.
Table 7. Test Matrix for Systematic Evaluation of DEFENZ™ VX-G against GF
Material
Wood
flooring
Contact
Time
15+15
30
15
15+15
30
Concentration
IX
IX
3X
3X
3X
Test
Coupons
5
5
5
5
5
Positive
Controls
5
5
5
Procedural
Blanks
2
2
2
Laboratory
Blanks
2
2
2
2.7 Observation of Surface Damage
Procedural blanks 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).
Observations were recorded in the evaluation records.
-------
2.8 Extraction and Analysis
After the appropriate contact time the test, positive control, procedural blank, and
laboratory blank coupons were transferred to individual extraction bottles (S236-0040, Fisher
Scientific, Pittsburgh, PA) containing 10 ml of hexane with naphthalene-d8 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 were placed in
the sonicator, they were sonicated at 50 - 60 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 06-718-439 and 06-
719-003, Fisher Scientific [Restek Corp], Hanover Park, IL) 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 GF in the extraction solution was
determined using a GC/MS, (Model 6890, Agilent Technologies, Santa Clara, CA) interfaced with
a 5973 network quadrupole mass-selective detector. Chromatographic separation of the
analytes was conducted using an RTX-5MS (cross-linked methyl silicone) fused silica capillary
column, 30.0 meter (m) length x 0.25 millimeter (mm) diameter x 0.25 micrometer (u.m) coating
thickness. The GC/MS parameters for GF analysis are shown in Table 8.
Table 8. Gas Chromatographic/Mass Spectrometric Parameters for GF Analysis
Parameters
Analysis Method GC/MS (Scan)
Model & SN HP6890N GC (CN10331014) & 5973N MSD (US30985853)
Data System MSD ChemStation
Liner Type 4 mm Split/Splitless
Column RTX-5MS, 30 m length, 0.25 mm diameter, 0.25 urn film coating thickness
Mode Constant Pressure
Inlet (Injector) Temperature 250 °C
Transfer Line Temperature 280 °C
Sample Size 1 ul
40 °C (1.0 min hold) to 100 °C @ 30 °C/min
Oven Program for Analysis to 150 °C @ 5 °C/min
to 325 °C (1.0 min hold) @ 15 °C/min*
* The final temperature (325 °C) is used to ensure all compounds eluted and carryover between runs was avoided.
The mass selective detector was operated in the full-scan mode for compounds ranging
from 40 to 400 atomic mass units (AMUs). The GC/MS measurements were used to compare
and evaluate co-extractive sample components and GF response. Table 9 outlines the selected
ion monitoring (SIM) masses that were used to quantify GF.
10
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Table 9. Pertinent Parameters for Target Chemical
Analyte SIM Ions
GF 99, 67, 54, 81
2.9 Method Demonstration
2.9.1 Recovery of GF from Test Coupons
Method demonstration was conducted, consistent with previous testing1, to establish
that extraction efficiencies (recoveries) from test coupons were sufficiently high and to
establish method detection limits (MDL[s]) for GF from the five materials included in the
testing. The extraction efficiency was determined as a percent of the GF 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%.
Recovery efficiencies were determined by spiking each of three coupons of each
material type with 1.0 ul of neat GF. Hexane [GC Resolv grade, Fisher Scientific, Pittsburg, PA]
was selected to extract GF from the aqueous (enzyme containing) phase. The SRC was also
applied to the coupon surface (1.0 ul). 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 GF. Immediately after transfer, the vial was capped and shaken by hand for 5-10 seconds
and placed into a sonicator. After all vials containing the 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 06-718-439 and 06-719-003, Fisher Scientific [Restek Corp], Hanover Park, IL) and sealed.
The amount of spiked GF was confirmed using control samples where the GF was spiked
directly into hexane and analyzed.
The aliquots of hexane extracts of coupons spiked with GF (1 ul) and aliquots of hexane
containing the same spike amount as applied to the coupons were analyzed for GF as described
in Section 2.8.
Extraction efficiency was calculated using a series of equations. The GF concentration in
a coupon extract or spiked hexane sample was determined by Equation 2:
A C
-^=M^ + W
A,s c,s (2)
where:
As = Area of the target analyte peak in the sample
AjS = Area of the internal standard peak
Cs = Concentration of the target analyte in the sample (ug/mL)
C/s = Concentration of the internal standard (ug/mL)
M = Slope of the GC calibration line
11
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W = Y intercept of the GC calibration line
GC concentration results (u,g/mL) were converted to total mass by multiplying by extract
volume as shown in Equation 3:
Mm=CxEv (3)
where:
Mm= Measured mass of CWA (u,g)
C = GC concentration (u,g/mL)
Ev = Volume of extract (ml)
Extraction efficiency was then defined by Equation 4 as:
F , +• v-ff- • (Mm of CWA on Test Coupon^]
Extraction Efficiency = —^-^- — x 100% (4)
^ Mm of CWA in Hexane )
where:
Mm= Measured mass of CWA (u,g) recovered from an individual test coupon or
recovered from hexane spiked with CWA.
2.9.2 MDL for GF Extracted from Coupon Materials
In addition to determining extraction efficiencies, the MDL was determined for analysis
of the GF from each of the five building materials included in the testing by following the EPA
guidelines (40 Code of Federal Regulations, Part 136, Appendix B).3 To achieve the low spike
levels for this testing required the use of dilute solutions of GF. Eight replicate coupons of each
material type were laid out in the hood on a clean surface. The coupons were spiked with 10 ul
of ~1,000 microgram (u.g)/mL of GF in hexane (~10 u.g of agent). The actual spike mass was
recorded. The coupons were also spiked with 1 ul of SRC. Within 5 min of initiation of spiking,
the coupons were placed, spiked side down, into separate bottles containing 10 ml of
hexane/IS. The bottles were immediately placed into a sonicator and sonicated for 10 min at
50- 60 kHz. At the completion of sonication, the bottles were removed from the sonicator and
within 30 min an aliquot of each sample was pulled using a Pasteur pipette and placed into a GC
vial for analysis.
The MDLs were calculated as shown in Equation 5:
MDL = t(n-l,l-a = 0.99) xSD (5)
where:
t(n-l,l-a = 0.99) = the Students' t value for a 99% confidence level and
standard deviation estimate with n-1 degrees of freedom
SD = standard deviation of the replicate analyses.
12
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2.9.3 Quench of Decontamination Reaction
Hexane extraction was expected to remove the GF (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 non-polar 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. Enzyme (60 ul) was added (using a positive displacement pipette (P/N M-250 [250 ul]
and D-200 [2-200 uL] tip, Gilson Inc, Middleton, Wl) to a vial containing 10 ml of hexane
and IS (naphthalene-ds) and 1 ul of GF, 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/IS and 1 uL of GF,
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 if the amount of GF recovered in Step 1
(enzyme present) was at least 70% of the amount of GF recovered in Step 2 (no enzyme
present).
All GF recoveries with hexane exceeded the required 70% (Table 10).
Table 10. Recovery of GF Using Hexane Extraction as Quench
Recovery with Water, Recovery with Enzyme, Mean % Recovery "with
Agent ug (SD) ug (SD) Quenched Enzyme" Compared
n = 3 n = 3 to "with Water"
GF 1008(109) 826(11) 82
2.10 Efficacy Determination
The decontamination efficacy was determined by measuring the amount of residual GF
on test coupons and comparing this amount with positive controls (spiked with GF, 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 GF
according to methods described in Section 2.8. Decontamination efficacy was calculated as
follows:
1. Concentration of GF (or SRC) in a coupon extract sample is determined by Equation 2
2. GC concentration results (ug/mL) are converted to total mass by multiplying by the
extract volume shown in Equation 3.
3. Decontamination efficacy (percent removal achieved during decontamination) is then
defined in Equation 6 as:
13
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E = \l __ M of CWA on Test Coupon _ }% (g)
M m of CWA on Positive Control Coupon
where:
M = Measured mass of CWA (u,g) on an individual test coupon
Mm = Mean of measured mass of CWA (u,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).
2. 1 1 Statistical Analysis
The standard deviation is calculated as shown in Equation 7:
f=1(^-M)2 (7)
where:
o = standard deviation
u. = mean
x, = lvalue of the variable being evaluated, e.g., control coupon
N = total number of elements in the population.
A two-tailed Student's t-test is used to compare the means of the positive control
coupon and the test coupon recoveries. Unequal variance between the populations is assumed.
A p-value is the result of the comparison. Results are considered significant if p < 0.05.
A two-tailed Student's t-test is also used to compare the means of the test coupons
subjected to alternative treatment (longer contact time, repeated applications, and/or higher
enzyme concentrations) to the standard treatment (IX concentration applied once for 15 min).
Unequal variance between the populations is assumed. A p-value is the result of comparison.
Results are considered significant if p < 0.05.
2.12 Analysis of By-products
The GC/MS instrumentation was operated in the full scan mode to detect (toxic) GF
decontamination by-products in the extracts of the simulated enzyme reactor tests. 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).
14
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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 11.
Table 11. Data Quality Objectives and Results for Test Measurements
Parameter
Measurement
Method
QC Requirement
Results
Time
Timer/data logger
Two seconds/hour; check once
before beginning testing
Passed requirement
Mass
Balance with daily
calibration check using
standard weights
Balance precision at least O.lx
lowest measured value
Daily balance calibration
check passed QC
requirement
pH
Calibrate with two standard buffer
pH meter solutions spanning range of
interest
Daily calibration check
passed QC requirement
Background
Contaminants
Analyze blank solvent
using GC/MS
70% of GF spike is recovered;
determine once during method
demonstration
GF recoveries met the QC
requirement
MassofCWA(in
neutralized enzyme
solution)
Extract in solvent and
analyze using GC/MS
>70% of GF, spike is recovered;
determine once during method
demonstration
GF recoveries met the QC
requirement
Mass of SRC (test
and positive control
coupons and
laboratory and
procedural blanks)
Extract in solvent and
analyze using GC/MS
>70% recovery of SRC (which
provides a check for matrix effects)
All SRC recoveries met the
QC requirement
Mass of CWA (on
positive controls)
Extraction/
chromatographic
quantitation
Result were considered an outlier if
the recovery value for analyte from
a coupon falls outside of three
standard deviations of the mean.
Criterion applies only if
concentration of analyte is
>5 times the MDL
No outliers were noted
Mass of CWA (on
spike controls)
Mass of CWA (on
laboratory blank)
Extraction/
chromatographic
quantitation
Extraction/
chromatographic
quantitation
^oco/ f^c -i * * All spike control recoveries
>85% of GF spike target "T
met the QC requirement
No GF was detected on any
-------
Quality checks on the prepared DEFENZ™ VX-G solutions were obtained through pH
measurement of the solution. All prepared solutions were pH = 8.0
3.2 Chemical Analysis Equipment Calibration
A six-point calibration for GF and the SRC was generally used with a lower calibration
level of 0.5 u.g/mL and an upper range of approximately 50 u.g/mL Naphthalene-d8 was used as
the IS for quantitation of GF and TBP. An average response (relative standard deviation <15%)
and quadratic regression curve fit were 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 CCV
concentrations were used (0.5 u.g/mL and 25 u.g/mL), one of which was equal to the low
calibration standard. A CCV response within 25% of nominal concentration was acceptable. One
CCV was low, so the analysis was repeated for that batch of test samples.
For GC/MS, the neat GF 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 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%; and
• difference between replicate samples less than 20%.
The calibration curve r2 was >0.99 and the % bias for all standards was ±7%.
3.3 Technical Systems Audit
The QA Manager performed a Technical Systems Audit (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/quality assurance (QA) plan. In the audit a QA Officer reviewed the
sampling and analysis methods used, compared actual test procedures to those specified in the
test/QA plan, 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 addressed the systematic decontamination of GF. The TSA report noted that all
work followed written procedures. No issues were noted.
3.4 Performance Evaluation Audits
A performance evaluation (PE) audit was conducted for each performance parameter
shown in Table 12 to assess the quality of the measurements made during testing. The audits
16
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for mass, chemical mass, pH, and time were performed once during testing by analyzing a
standard(s) that is independent of standards used during the testing.
Table 12. Performance Parameters 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
Spike controls were at 87% of spike
target
Use balance to determine
Mass 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
pH
Use pH meter to determine
pH of a standard solution
±0.1 pH units
pH meter was found to be within
±0.1 pH units
3.5 Data Quality Audit
The 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 Amendments
Nine amendments were incorporated into the test/QA plan. It included enzymatic
decontamination tests against TGD, HD and VX.1 A brief summary of the amendment related to
decontamination of GF follows:
• Amendment 8: A required deliverable of Amendment 1 to contract EP-C-10-001 Work
Assignment 2-04, provided test/QA details necessary to apply the plan to testing of
DEFENZ™VX-G against GF.
3.7 Deviations
No deviations from the test/QA plan were noted for the work described in this report.
17
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4.0 Results/Discussion
4.1 Method Demonstration Results
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%. GF recoveries were 85% to 103% and the coefficients of variance
for triplicate samples were 1.3% to 8.7% and, therefore, acceptable (Table 13).
Table 13. Extraction Efficiencies for Neat GF from Various Types of Coupons
Material
Teflon
Galvanized metal
Wood flooring
Industrial carpet
Vinyl flooring
Decorative laminate
Mean Extraction Efficiency
88% (n=4)
93% (n=3)
84% (n=3)
94% (n=3)
101% (n=3)
102% (n=3)
Coefficient of Variance
3.0%
6.3%
8.5%
1.3%
4.6%
8.7%
The MDLs for GF extracted from various types of test coupons using 10 ml of hexane
are shown in Table 14.
Table 14. MDL Values for GF Extracted from Various Types of Coupons Using Hexane
Material GF MDL, u,g (10 ml Extract)
Galvanized metal 0.11
Wood flooring 0.35
Industrial carpet 0.45
Vinyl flooring 0.25
Decorative laminate 0.20
4.2 Simulated Enzyme Reactor Results
The results of the simulated enzyme reactor results are summarized in Table 15.
DEFENZ™ VX-G (mixed with water at the ratio recommended by the manufacturer)
demonstrated efficacy against GF. Mean efficacy against GF after the 15-min contact time was
99%. DEFENZ™ VX-G exhibited here a higher efficacy against GF when compared to the coupon
testing. Ninety-nine percent of GF was decomposed with the 15 min contact time. This more
dynamic interaction is apparently important in reaching a higher efficacy against GF. Lower
efficacy results obtained during coupon testing can be explained by the more static interaction
of the enzyme solution with GF on the test coupon.
18
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The temperature profile for a fifteen min sonication resulted in a rise of about 7 °C from
17.4 °C when sonication began to 24.2 °C at 15 min.
Table 15. Simulated Enzyme Reactor Results for DEFENZ™ VX-G Enzyme and GF
Blank Mean Positive Control
. T * i iv/i Mean Test Total Mass, Mean
CWA Enzyme Used Solution, Total Mass,
„ ' lrn. MS SD Efficacy
ug ug (SD)
GF DEFENZ™ VX-G ND* 704(66) 10(13) 99%
*ND indicates no GF was detected, <0.5 u.g/mL (lower calibration limit).
No GF was detected in the blank solution that was part of the simulated enzyme reactor
testing.
4.3 By-Product Analysis
The simulated enzyme reactor GC/MS data were examined in full scan mode for
qualitative differences between control and test samples. No substantial differences were
observed in chromatographic peaks between the test and control samples.
4.4 Coupon Decontamination Results
Decontamination efficacy results (mean and SD) using DEFENZ™ VX-G enzymes prepared
in accordance with the manufacturer's instructions are shown in Table 16. Efficacy was
observed for all materials ranging from a low of 36% for wood to 94% for decorative laminate.
A graphical representation of the amounts recovered and associated decontamination efficacy
is provided in Figure 2. Amounts recovered from the positive control coupons were lower than
observed during extraction efficiency demonstration. Lower recoveries observed here maybe
indicative of some evaporative loss or degradation of GF during the 45-min time between
spiking and extraction of the positive control coupons.
Table 16. GF Decontamination Results Using DEFENZ™ VX-G (IX)
Material
Galvanized metal
Wood flooring
Industrial carpet
Vinyl flooring
Decorative laminate
Contact Time,
min
15
15
15
15
15
Mean Positive Control
Coupons,
ug(SD)
780 (80)
580 (56)
920 (61)
850 (77)
660 (40)
Mean Test
Coupons,
ug(SD)
60(7)
370 (25)
180 (93)
200 (27)
40(3)
Mean* Test Coupons
Efficacy, %
92
36
80
77
94
Calculation of mean efficacy based on analytical results before rounding of mean positive control and mean test
coupon
19
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1200
1000-
ZJ
» 800-
^
o
co 600-
U_
O
8
0)
200
92%
V
I I Positive Controls
I I Test Coupons
^y Efficacy
94%
V
80%
V
rfi
36%
V
77%
V
100
80
60
40
o
W
o
t
LU
20
0
Galvanized metal Wood flooring Industrial carpet Vinyl flooring Decorative laminate
Material
Figure 2. Recovered amounts of GF from materials and associated decontamination efficacy
following DEFENZ™ VX-G application (15 minute contact time).
The standard DEFENZ™ VX-G enzyme preparation (IX) was tested at a longer
contact time (30 min) and with two sequential 15-min applications to evaluate whether efficacy
would increase. Wood flooring material was used as the coupon material because of the lower
efficacy observed for GF on wood with a 15-min contact time. Results for decontamination of
GF using the longer contact time and repeated applications are shown in Table 17 and
visualized in Figure 3. Efficacy did not significantly increase with a 30-min contact time
compared to a 15-min contact time (p = 0.29). Reapplication of the IX enzyme for two
sequential 15 min periods resulted in a significant increase in efficacy (p = 0.014). Given the
results from the single 30-min application of the enzyme, the increased efficacy with a second
application of the enzyme is unlikely to be explained by increased evaporation.
20
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Table 17. GF Decontamination on Wood Flooring Using Alternative Contact Times, Repeated
Application, and/or 3X Enzyme Concentration
Mean Positive Control
Contact Time,
min
30
15 + 15
15
30
15+15
Concentration
IX
IX
3X
3X
3X
Coupons,
ug(SD)
580 (46)
700 (147)
730 (68)
Mean Test Coupons,
ug(SD)
310(51)
230 (45)
330 (64)
410 (61)
150 (83)
Mean* Test Coupons
Efficacy, %
47
61
53
44
79
""Calculation of mean efficacy based on analytical results before rounding of mean positive control and mean test
coupon
100.
.
c
I 60-\
N
LU
8
40-
20-
0-
1X concentration
3X concentration
15 30
Contact time (minutes)
Figure 3. Decontamination efficacy for 15-min, 30-min, and repeated 15-min contact time of
DEFENZ™ VX-G against GF
Concentrations of DEFENZ™ VX-G enzymes higher than the manufacturer's
recommendation (3X) were tested with 15-min and 30-min contact times, and with a repeated
15-min application of the 3X enzyme to evaluate whether efficacy against GF would increase.
Results for decontamination of GF using higher concentrations than the manufacturer's
recommendation with a 30-min contact time and sequential 15-min applications are shown in
Figure 3. Increasing the concentration to 3X did not significantly increase efficacy at 15-min (p =
0.17) or 30-min (no improvement) contact times compared to decontamination for 15-min
using the IX concentration. With a repeated 15-min application of the 3X enzyme efficacy was
21
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significantly higher (p = 0.009) compared to decontamination for 15-min using the IX
concentration. However, the higher (3X) concentration with repeated 15-min applications did
not result in significantly greater efficacy than the repeated 15-min applications using the IX
concentration (p = 0.17).
The GF recovered from wood positive control coupons varied from 580 u.g to 730 u.g
across the four sets of test coupons. The differences were not attributable to longer periods of
evaporation. Non-homogeneity of the wood, differences in wood moisture content, or other
factors associated with the material may account for these differences in recovery. However,
no testing has been done to determine the cause of this difference. The observed improved
efficacies with longer interaction times and repeated application cannot be explained solely by
the wide range in recovered amounts from positive controls.
Quality control (QC) measurements included laboratory blanks, procedural blanks, dose
confirmation, and Teflon spike control measurements. GF was not found on any laboratory
blank coupon. GF was not found on galvanized metal procedural blank coupons. GF was
detected at low levels on procedural control coupons of other material types (<0.50 u.g/mL
extract). Dose confirmation (measured concentration in direct spike of 1 ul into 10.0 ml of
solvent) was 111% of the target concentration of 110 u.g/mL Recoveries from Teflon spike
controls were 86% (standard deviation [SD] = 7%) and 91% (SD = 3%) of the expected
concentration acquired as part of the default contact time tests and longer interaction time
tests, respectively.
4.5 Observations of Damage to Coupons
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. No detailed
examination or testing for structural damage was included in this evaluation. Damage, if any
occurred that is not readily visible, would likely not be detected in this evaluation.
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5.0 Conclusions
Simulated enzyme reactor testing demonstrated significant efficacy of DEFENZ™ VX-G
against GF with a 15 min contact time. GF was reduced by 99%. The impact on the efficacy of an
observed temperature increase (about 7 °C) associated with sonication (15 minutes) as part of
the simulated enzyme reactor tests was not further evaluated. Temperature typically impacts
enzyme performance. However, data using the CWA simulant paraoxon suggests that no
appreciable effect on enzyme activity occurs in the 5 - 35 °C range.4
The application of DEFENZ™ VX-G resulted in less GF recovered from all materials
tested: galvanized metal, wood flooring, industrial carpet, vinyl flooring, and decorative
laminate with a 15-min contact time. Tests on wood flooring showed efficacy increased with
repeated 15-min applications. Increasing the single application contact time to 30 min or
increasing the enzyme concentration to 3X did not significantly increase efficacy.
A comparison of the simulated enzyme reactor testing efficacy (99% reduction in GF
amount recovered) versus the surface decontamination (36-94% reduction in GF amount
recovered) suggests that the more static interaction of the surface decontamination tests
reduces the enzymatic reactivity. Reactor based testing efficacy results may therefore only be
considered as an upper limit to the reduction of a CWA (here, GF) from a surface.
Given the observed efficacies and the lack of visible damage to a range of indoor
building materials, DEFENZ™ VX-G enzyme appears to be a technology useful for removing GF
from 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.
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6.0 References
1. U.S. EPA Report, Enzymatic Decontamination of Chemical Warfare Agents, U.S.
Environmental Protection Agency, Washington, DC, EPA/600/R-12/033, 2012
2. Test/Quality Assurance (QA) Plan for Enzymatic Decontamination of Chemical Warfare
Agents, Version 2 (July 2010). Available upon request from EPA
3. 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 (June 30,
1986)
4. Impact of Environmental Conditions on the Enzymatic Decontamination of a Material Surface
Contaminated with Chemical Warfare Agent Simulants. Lukas Oudejans, Barbara
Wyrzykowska-Ceradini, Craig Williams, Dennis Tabor, and Jeanelle Martinez. Accepted for
publication in Industrial, Chemical and Engineering Research, June 2013
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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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