EPA/600/R-010/080 | September 2010 | www.epa.gov/ord
United States
Environmental Protection
Agency
Evaluation of Liquid and Foam
Technologies for the
Inactivation of Bacillus
anthracis Spores on Topsoil
INVESTIGATION REPORT
Office of Research and Development
National Homeland Security Research Center
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Evaluation of Liquid and
Foam Technologies for the
Decontamination of Bacillus
anthracis Spores on Topsoil
INVESTIGATION REPORT
U.S. ENVIRONMENTAL PROTECTION AGENCY
OFFICE OF RESEARCH AND DEVELOPMENT
NATIONAL HOMELAND SECURITY
RESEARCH CENTER
RESEARCH TRIANGLE PARK, NC 27711
Office of Research and Development
National Homeland Security Research Center
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Disclaimer
The U.S. Environmental Protection Agency (EPA), through its Office of Research and Development,
National Homeland Security Research Center, funded and directed this investigation through
a Blanket Purchase Agreement under General Services Administration contract number
GS23F0011L-3 with 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.
Questions concerning this document or its application should be addressed to:
Joseph P. Wood
National Homeland Security Research Center
Office of Research and Development (E-343-06)
U.S. Environmental Protection Agency
109 T.W.Alexander Dr.
Research Triangle Park, NC 27711
(919) 541-5029
wood.ioe(@,epa. gov
If you have difficulty accessing this PDF document, please contact Kathy Nickel (Nickel.Kathy@,
epa.gov) or Amelia McCall (McCall. Amelia@,epa. gov) for assistance.
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Acknowledgments
Contributions of the following individuals and organizations to the development of this document
are acknowledged.
United States Environmental Protection Agency (EPA)
Michael Ottlinger
Timothy Curry
Leroy Mickelsen
Battelle
Peer Reviewers
Peter Setlow, University of Connecticut
Michele Burgess, EPA Office of Emergency Management
Frank Schaefer, EPA National Homeland Security Research Center
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Contents
Disclaimer iii
Acknowledgments iv
Abbreviations/Acronyms viii
Executive Summary ix
1.0 Introduction 1
2.0 Technology Description 3
3.0 Summary of Test Procedures 5
3.1 Preparation and Analysis of Test Coupons 5
3.2 Decontamination Efficacy 7
4.0 Quality Assurance/Quality Control 9
4.1 Equipment Calibration 9
4.2 QC Results 9
4.3 Audits 9
4.3.1 Performance Evaluation Audit 9
4.3.2 Technical Systems Audit 9
4.3.3 Data Quality Audit 9
4.4 Test/QA Plan Amendments and Deviations 9
4.5 QA/QC Reporting 9
4.6 Data Review 9
5.0 pH-Amended Ultra Clorox Germicidal Bleach Test Results 11
5.1 QC Results 11
5.2 Decontamination Efficacy 11
5.3 Other Factors 11
5.3.1 Operator Control 11
5.3.2 Technology Spray Deposition 12
5.3.3 Neutralization Methodology 12
6.0 CASCAD SDF (Allen-Vanguard) Test Results 15
6.1 QC Results 15
6.2 Decontamination Efficacy 15
6.3 Other Factors 15
6.3.1 Operator Control 15
6.3.2 Technology Spray Deposition 16
6.3.3 Neutralization Methodology 16
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7.0 Oxonia Active (Ecolab) Test Results 19
7.1 QC Results 19
7.2 Decontamination Efficacy 19
7.3 Other Factors 19
7.3.1 Operator Control 19
7.3.2 Technology Spray Deposition 20
7.3.3 Neutralization Methodology 20
8.0 Klozur (FMC Corporation) Test Results 23
8.1 QC Results 23
8.2 Decontamination Efficacy 23
8.3 Other Factors 24
8.3.1 Operator Control 24
8.3.2 Technology Deposition 24
8.3.3 Neutralization Methodology 24
9.0 Performance Summary 27
9.1 pH-Amended Ultra Clorox Germicidal Bleach Results 27
9.2 CASCAD SDF Results 27
9.3 Oxonia Active Results 27
9.4 Klozur Results 27
10.0 References 29
Appendices - Technology Descriptions and Application Procedures for the Evaluated
Decontaminants
A pH-Amended Bleach Description and Application Procedure 31
B CASCAD SDF Description and Application Procedure 33
C Oxonia Active Description and Application Procedure 35
D Klozur Description and Application Procedure 37
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List of Tables
Table E-1. Summary of Quantitative Efficacy of Each Decontaminant on Topsoil Coupons x
Table 2-1. Technology Information 3
Table 5-1. Inactivation of Bacillus anthracis Ames Spores on Topsoil - pH-Amended
Ultra Clorox Germicidal Bleach 11
Table 5-2. Neutralization Testing with Bacillus anthracis (Ames) Spores on Topsoil for pH-
Amended Ultra Clorox Germicidal Bleach (60 Minute Contact Time,
Volume Equal to 4 Applications) 12
Table 5-3. Neutralization Testing with Bacillus anthracis (Ames) Spores on Topsoil for pH-
Amended Ultra Clorox Germicidal Bleach (120-Minute Contact Time,
Volume Equal to 8 Applications) 13
Table 6-1. Inactivation of Bacillus anthracis Ames Spores on Topsoil - CASCAD SDF 15
Table 6-2. Neutralization Testing with Bacillus anthracis (Ames) Spores on Topsoil for
CASCAD SDF (60 Minute Contact Time, Volume Equal to 2 Applications) 16
Table 6-3. Neutralization Testing with Bacillus anthracis (Ames) Spores on Topsoil for
CASCAD SDF (120 Minute Contact Time, Volume Equal to 4 Applications) 17
Table 7-1. Inactivation of Bacillus anthracis Ames Spores on Topsoil - Oxonia Active 19
Table 7-2. Neutralization Testing with Bacillus anthracis (Ames) Spores on Topsoil for
Oxonia Active (60 Minute Contact Time, Volume Equal to 6 Applications) 20
Table 7-3. Neutralization Testing with Bacillus anthracis (Ames) Spores on Topsoil for
Oxonia Active (120 Minute Contact Time, Volume Equal to 12 Applications) 21
Table 8-1. Inactivation of Bacillus anthracis Ames Spores on Topsoil - Klozur 23
Table 8-2. Neutralization Testing with Bacillus anthracis (Ames) Spores on Topsoil for
Klozur (24 Hour Contact Time, Volume Equal to 6 Applications) 24
Table 8-3. Neutralization Testing with Bacillus anthracis (Ames) Spores on Topsoil for
Klozur (48 Hour Contact Time, Volume Equal to 6 Applications) 25
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List of Abbreviations/Acronyms
ASTM American Society for Testing and Materials
B. anthracis Bacillus anthracis (Ames strain)
BBRC Battelle Biomedical Research Center
BSC biosafety cabinet
C Celsius
CPU colony-forming unit(s)
CI confidence interval
cm centimeter
EPA U.S. Environmental Protection Agency
g gram
hr hour
L liter
M molar
min minute
mL milliliter
NHSRC National Homeland Security Research Center
NIST National Institute of Standards and Technology
OPP EPA Office of Pesticide Programs
PBS phosphate-buffered saline
ppm parts per million
psi pounds per square inch
QA quality assurance
QC quality control
QMP quality management plan
RH relative humidity
SD standard deviation
SDF surface decontamination foam
SE standard error
SFW sterile filtered water (cell-culture grade)
STS sodium thiosulfate
TOPO Task Order Project Officer
TSA technical systems audit
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Executive Summary
The U.S. Environmental Protection Agency's (EPA)
National Homeland Security Research Center (NHSRC)
helps to protect human health and the environment
from adverse impacts of terrorist acts by carrying out
performance tests on homeland security technologies.
This report describes a study in which liquid and foam
technologies were evaluated for decontaminating test
coupons of topsoil containing Bacillus anthracis spores.
Experimental Procedures. For some tests, topsoil
coupons were prepared by filling a Parafilm®-lined 3.5
cm diameter x l cm high Petri dish with uncompacted
commercially available topsoil. For most tests, which
involved several applications of a decontaminant, the
same amount of uncompacted topsoil was placed into
a 3.4 cm high Teflon beaker of the same diameter as
the Petri dish, in order to effectively capture all of
the applied decontaminant. The topsoil used was a
commercial garden topsoil, determined by analysis to be
approximately 29% moisture by weight, with an organic
content of approximately 6% on a dry weight basis.
For testing, topsoil coupons were "contaminated" by
inoculation with the biological agent Bacillus anthracis
(Ames). The technologies evaluated for their ability to
inactivate B. anthracis (Ames) on topsoil coupons were:
• pH-Amended Ultra Clorox® Germicidal bleach
(diluted with commercial cell-culture grade sterile
filtered water (SFW) and 5% acetic acid to obtain
pH-amended solution)
• Allen-Vanguard's CASCAD™ Surface
Decontamination Foam (SDF)
• Ecolab Inc. 's Oxonia Active®
• FMC Corporation's Klozur™ (activated with
hydrogen peroxide (H2O2))
This evaluation was designed to determine the
quantitative decontamination efficacy of each
technology, in terms of the reduction in the number of
viable organisms in the topsoil coupons (log reduction).
Each decontaminant technology was tested with two
different contact times. CASCAD SDF was applied to
topsoil coupons according to the vendor's instructions,
using a dual-compartment spray bottle provided by
the vendor. Oxonia Active was applied according to
the vendor's instructions, using a conventional hand-
pumped household garden sprayer. The pH-amended
Ultra Clorox Germicidal bleach was applied according
to an application procedure developed by EPA and
Battelle, using the same type of conventional hand-
pumped household garden sprayer. For these three
decontaminants, the two contact times were 60 and 120
minutes. Spray distance, humidity, and temperature
were the same for all applications of these three
decontaminants. Klozur is designed for use as a soil
remediation treatment, and consequently a different
treatment approach was used. Klozur solution was
mixed with the topsoil in a Teflon beaker, and an equal
volume of an activating solution of hydrogen peroxide
was then immediately mixed in. An initial test of Klozur
was conducted with a 24-hour contact time, and the
second contact time of 48 hours was then chosen based
on the results of that test. Technical descriptions and
preparation and application procedures (including the
spray device, contact time, and reapplication rate) for all
the decontaminants tested are included as appendices to
this report.
Results: Application procedures and results for the
four decontaminants are summarized in the following
paragraphs.
pH-Amended Ultra Clorox Germicidal bleach - In the
first test of this decontaminant it was applied to the test
coupons until they were fully wetted, and then reapplied
at 15-minute intervals for a total of four applications
in a 60-minute contact time. In the second test of
this decontaminant it was applied to the test coupons
until they were fully wetted, and then reapplied at
15-minute intervals for a total of eight applications in
a 120-minute contact time. In addition, after each of
the eight applications in the second test, the topsoil
and the applied pH-amended Ultra Clorox Germicidal
bleach were mixed together using a glass stirring rod.
Quantitative efficacy for B. anthracis (Ames) was 0.03
log reduction with the 60-minute contact time and 0.94
log reduction with the 120-minute contact time.
CASCAD SDF - In the first test of this decontaminant
it was applied to the test coupons until they were fully
covered with foam, and then reapplied 30 minutes later
for a total of two applications in a 60-minute contact
time. In the second test of this decontaminant it was
applied to the test coupons until they were fully covered
with foam, and then reapplied at 30-minute intervals for
a total of four applications in a 120-minute contact time.
Quantitative efficacy for B. anthracis (Ames) was 0.59
log reduction with the 60-minute contact time and 1.13
log reduction with the 120-minute contact time.
Oxonia Active - In the first test of this decontaminant
it was applied to the test coupons until they were fully
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wetted, and then reapplied at 10-minute intervals for
a total of six applications in a 60-minute contact time.
In the second test of this decontaminant it was applied
to the test coupons until they were fully wetted, and
then reapplied at 10-minute intervals for a total of 12
applications in a 120-minute contact time. In addition,
after each of the 12 applications in the second test,
the topsoil and the applied Oxonia Active were mixed
together using a glass stirring rod. Quantitative efficacy
for B. anthracis (Ames) was 0.58 log reduction with the
60-minute contact time and 1.04 log reduction with the
120-minute contact time.
Klozur - In the first test of this decontaminant 1 mL of a
Klozur solution and 1 mL of a H2O2 activating solution
were added to each topsoil coupon every 60 minutes, and
mixed with the topsoil using a glass stirring rod. This
process was repeated until six total applications were
made. The decontaminant then remained in contact with
the topsoil coupons until 24 hours had elapsed since the
first application. In the second test, the same schedule
and application and mixing procedures were followed
for the six applications, but the total contact time was
48 hours. Quantitative efficacy for B. anthracis (Ames)
was 1.65 log reduction with the 24-hour contact time and
3.50 log reduction with the 48-hour contact time.
Table E-l summarizes the application procedures and
quantitative efficacy results (with 95% confidence
intervals) for all decontaminants at both contact times.
Efficacy was slightly better with the 120-minute contact
time than with the 60-minute contact time for pH-
amended Ultra Clorox Germicidal bleach, CASCAD
SDF, and Oxonia Active. However, efficacy with
these three decontaminants never exceeded about 1
log reduction, even with several applications onto the
topsoil coupons. With Klozur, efficacy was substantially
greater with the 48-hour contact time than with the 24-
hour contact time, but both Klozur efficacy results were
significantly higher than any efficacy result with the
other three decontaminants.
Table E-l. Summary of Quantitative Efficacy of Each Decontaminant on Topsoil Coupons
Decontaminant
pH-Amended
Ultra Clorox
Germicidal
Bleach
CASCAD SDF
Oxonia Active
Klozur
(activated with
fW
Total Contact
Time (minutes)
60 minutes
120 minutes
60 minutes
120 minutes
60 minutes
120 minutes
24 hours
48 hours
Number of
Applications
4
8
2
4
6
12
6
6
Mass of
Decontaminant
Applied (g)
2.6
3.6
2.9
6.5
4.0
7.1
12
12
Stirring
(Y/N)a
N
Y
N
N
N
Y
Y
Y
Quantitative
Efficacy as Log
Reduction
(± 95% CI)b
0.03 (±0.15)
0.94 (±0.10)
0.59 (±0.04)
1.13 (±0.12)
0.58 (±0.19)
1.04 (±0.10)
1.65 (±0.14)
3. 50 (±0.40)
a Stirring means mixing together of topsoil and applied decontaminant after each application.
b CI = confidence interval.
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1.0
Introduction
NHSRC works in partnership with recognized testing
organizations; with stakeholder groups consisting of
buyers, vendor organizations, scientists, engineers,
and permitters; and with participation of individual
technology developers in carrying out performance
tests on homeland security technologies. In response
to the needs of stakeholders, NHSRC evaluates
the performance of innovative homeland security
technologies by developing test plans, conducting
evaluations, collecting and analyzing data, and preparing
peer-reviewed reports. All evaluations are conducted
in accordance with rigorous quality assurance (QA)
protocols to ensure the generation of high quality data
and defensible results. NHSRC provides unbiased,
third-party information supplementary to vendor-
provided information that is useful to decision makers
in purchasing or applying the evaluated technologies.
Stakeholder involvement ensures that user needs and
perspectives are incorporated into the evaluation design
to produce useful performance information for each
evaluated technology.
In this current effort, NHSRC evaluated the performance
of four liquid or foam decontamination technologies
for their ability to inactivate Bacillus anthracis (Ames)
spores on topsoil representative of outdoor soil surfaces.
These decontaminants were selected for testing based
on existing information or data indicating potential
sporicidal efficacy on soil surfaces. Such information
or data could include EPA registration as a sterilant on
other types of surfaces, data showing sporicidal efficacy
on different materials or under different test conditions,
or known oxidizing capacity in other applications such
as soil remediation. The decontaminants tested, which
were each evaluated at two different contact times on
test coupons of commercially available topsoil, included
the following:
• pH-Amended Ultra Clorox® Germicidal bleach
(diluted with certified cell-culture grade sterile
filtered water (SFW) and 5% acetic acid to obtain
pH-amended solution)
• Allen-Vanguard's CASCAD™ Surface
Decontamination Foam (SDF)
• Ecolab Inc. 's Oxonia Active®
• FMC Corporation's Klozur™ activated with
hydrogen peroxide (H2O2).
For CASCAD SDF and Oxonia Active, testing was
performed using spray application procedures specified
by the respective vendor. For pH-amended Ultra Clorox
Germicidal bleach, testing was performed using spray
application procedures developed by EPA and Battelle
based on likely use of these decontaminants. For Klozur,
the application procedure was based on approaches
typically used in soil remediation with this product,
and involved mixing a solution of Klozur and an equal
volume of a H2O2 activating solution with the topsoil
in the coupon container. The application procedures
for all decontaminants are included as appendices to
this report. The performance of the decontamination
technologies was evaluated in terms of their quantitative
decontamination efficacy, i.e., the log reduction in viable
spores that resulted from use of the decontaminant.
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2.0
Technology Description
Table 2-1 describes the four decontamination
technologies tested, and lists the contact times and
number of applications used in testing. The information
on product composition in Table 2-1 is based on vendor-
provided information and was not confirmed in this
evaluation. Detailed technology descriptions and the
application procedures used are included as Appendices
A to D. Note that Ultra Clorox Germicidal bleach is
registered as a disinfectant, but the pH-amended solution
is not.
Table 2-1. Technology Information
Product
Vendor
General
Description/
Active
Ingredients
Components
EPA
Registration"
Contact Time"
(No. of
Applications)
Ultra Clorox
Clorox Professional
Germicidal Products
Bleach Co.
Sodium Sodium hypochlorite 6.15%, sodium
hypochlorite, hydroxide <1% (diluted with SFW, and
hypochlorous pH-amended by Battelle by adding acetic
acid acid 5%)c
67619-8
(disinfectant)
60 min (4)
120min(8)
CASCAD
SDF
Allen-
Vanguard
(Reagents prior to dissolution.) GCE-
2000 Surfactant: Sodium myristyl sulfate
10-30%, sodium (C14_16) olefin sulfonate
10-30%, ethanol denatured 3-9%, alcohols
(C1(M6) 5-10%, sodium sulfate 3-7%,
Hypochlorite, sodium xylene sulfonate 1-5%, proprietary
hypochlorous mixture of sodium and ammonium salts
acid along with co-solvent >9%; GPA-2100
Decontaminant: dichloro-isocyanuric
acid, sodium salt 70-100%; GPB-2100
Buffer: sodium tetraborate 10-30%,
sodium hydroxide 1-5%, sodium carbonate
40-65%.
None
60 min (2)
120 min (4)
Oxonia
Active
Ecolab Inc.
FMC
Corporation
peroxy acetic
Persulfate
(activated
with hydrogen
peroxide)
Hydrogen peroxide 27.5%, peroxyacetic
acid 5.8% in aqueous solution.
Sodium persulfate (Na2S2O8) >99% purity
(used as a 12% (0.5 molar) aqueous
solution, activated with hydrogen peroxide
8% solution)11
1677-129
(sterilant,
disinfectant,
sanitizer)
60 min (6)
120 min (12)
24 hr (6)
48hr(6)
Registered with the EPA Office of Pesticide Programs (OPP). Registration indicates EPA/OPP has evaluated the antimicrobial pesticide to
show its effectiveness and that the pesticide will not have unreasonable adverse effects on humans, the environment, and non-target species,
and EPA/OPP has issued a registration or license for use in the United States. Note: None of
these products is registered for use against B. anthracis.
Min = minutes, hr = hours; number of applications used in the indicated contact time shown in parentheses.
Using procedure recommended by stakeholders, 5% acetic acid was added to the bleach to obtain a pH-amended bleach solution. The
solution was prepared using 9.4 parts SFW, 1 part Ultra Clorox Germicidal bleach, and 1 part 5% glacial acetic acid to yield a solution
having a pH of 6.36 and a total chlorine content of approximately 6,200 parts per million
Equal volumes of 12% by weight (i.e., 0.5 molar) sodium persulfate and 8% by weight hydrogen peroxide solutions were applied to test
coupons, resulting in a molar ratio of 5 to 1 (hydrogen peroxide to persulfate), typical of use in soil remediation.
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Below are brief descriptions of the form and preparation
instructions of the decontamination technologies.
Greater detail on product composition, preparation, and
application procedures is provided in Appendices A to D.
• pH-Amended Ultra Clorox Germicidal bleach -
Ultra Clorox Germicidal bleach was purchased in a
one gallon container from a local retail store. The
pH-amended solution was prepared by mixing 9.4
parts SFW, 1 part Ultra Clorox Germicidal bleach,
and 1 part 5% glacial acetic acid. The diluted, pH-
adjusted final solution was applied using a hand-
pressurized portable garden sprayer.
• CASCAD SDF - One CASCAD solution
was prepared by diluting 31.2 g of GPA-2100
(decontaminant) to 300 mL with SFW, and the other
solution was made by diluting 7.2 g of GPB-2100
(buffer) and 18 mL of GCE-2000 (surfactant) to
300 mL with SFW. The application process used a
dual spray bottle designed by the vendor to deliver
equal portions of the two solutions through a single
spray nozzle, and equipped with a diffuser mesh to
produce the foam.
• Oxonia Active - A decontaminant solution
containing 5,000 parts per million (ppm)
peroxyacetic acid was prepared fresh daily by
diluting 76 mL of Oxonia Active to 1 L with SFW.
The diluted solution was applied using a hand-
pressurized portable garden sprayer.
• Klozur - Klozur is a white crystalline solid
consisting of >99% pure sodium persulfate. A
solution of 12% sodium persulfate was prepared by
dissolving 12 grams of Klozur in SFW and diluting
to 100 mL; this solution was 0.5 molar (M) in
sodium persulfate. Each application of the Klozur
solution to a topsoil coupon consisted of mixing
1 mL of 0.5M persulfate solution with the topsoil,
followed immediately by addition and mixing of
1 ml of 8% H2O2 solution. The vendor of Klozur
indicates that the H2O2 activates formation of highly
reactive sulfate radicals (SO4"«). Equal volumes
of the persulfate and H2O2 solutions resulted in
a persulfate/H2O2 molar ratio of 5 to 1, a typical
ratio recommended for the use of Klozur in soil
remediation.
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3.0
Summary of Test Procedures
Test procedures were performed in accordance with the
peer-reviewed test/QA plan and are briefly summarized
here.
3.1 Preparation and Analysis of Test
Coupons
The test soil was Gardenscape® topsoil, batch number
PY1A0597, purchased from a national chain home-
and-garden store. This topsoil had a moisture content
of approximately 29%, and an organic content of
approximately 6% on a dry weight basis, as determined
by analysis in a commercial laboratory using a standard
American Society for Testing and Materials (ASTM)
method1 Most test coupons consisted of approximately
7 grams of uncompacted topsoil in a Parafilm®-lined
3.5 cm diameter x l cm deep circular glass Petri dish.
For tests that involved more than two applications
of the decontaminant, the same mass of topsoil was
placed in a Parafilm-lined 3.5 cm diameter x 3.4 cm
deep circular Teflon beaker. That larger container was
needed to capture the decontaminant from the multiple
applications. An amendment to the test/QA plan was
prepared, reviewed, and approved for this change in the
form of the coupon container.
The Bacillus anihracis (Ames) spores used for this
testing were prepared from a qualified stock of the
Ames strain at the Battelle Biomedical Research
Center (BBRC). All spore lots were subject to a
stringent characterization and qualification process,
required by Battelle's standard operating procedure
for spore production. Specifically, all spore lots were
characterized prior to use by observation of colony
morphology, direct microscopic observation of spore
morphology and size, and determination of percent
refractivity and percent encapsulation. In addition, the
number of viable spores were determined by colony
count and expressed as colony forming units per
milliliter (CFU/mL). (Theoretically, once plated onto
bacterial growth media, each viable spore germinates
and yields one CPU.) Variations in the expected colony
phenotypes were recorded. Endotoxin concentration of
each spore preparation was determined by the Limulus
Ameocyte Lysate assay. Genomic DNA was extracted
from the spores and DNA fingerprinting was done to
confirm the genotype. The virulence of the spore lot
was measured by challenging guinea pigs intradermally
with a dilution series of spore suspensions, and virulence
was expressed as the intradermal median lethal dose. In
addition, testing was conducted for robustness of the
spores via HC1 resistance. The stock spore suspension
was prepared in SFW at an approximate concentration of
1 x 109 spores/mL and stored by refrigeration at 4 °C.
B. anthracis fAmes) spores were inoculated onto test
coupons in an appropriate biosafety cabinet (BSC-II
or -III) according to established Battelle procedures.
Inoculated coupons were prepared fresh for each day of
experimental work, by placing coupons in the BSC and
inoculating at approximately 1 x 10s spores per coupon.
This inoculation was accomplished by dispensing a 100-
uL aliquot of the spore stock suspension (approximately
1 x 109 spores/mL) using a micropipette as 10 droplets
(each of 10 uL volume) across the surface of the coupon.
This approach provided more uniform distribution
of spores across the coupon surface than would be
obtained through a single drop of the suspension. After
inoculation, the coupons remained undisturbed overnight
in the BSC to dry. Blank (uninoculated) topsoil coupons
were held in a separate cabinet from the spiked coupons,
to avoid contamination of the blanks with spores during
the drying period.
On the day following spore inoculation, coupons
intended for decontamination (including blanks) were
transferred into a glove box (test chamber) where
the decontamination technology was applied using
the apparatus and application conditions specified in
Appendices A to D of this report. For pH-amended
Ultra Clorox bleach, CASCAD SDF, and Oxonia
Active, application was done by spraying the
decontaminant onto the test coupons multiple times.
The decontamination spray distance of 30 cm (12
inches), humidity (< 70% relative humidity (RH)), and
temperature (20 to 25 °C) were the same for all spray
applications. For CASCAD SDF and Oxonia Active the
amount of decontaminant, contact time, spray pressure,
application and reapplication procedures, etc., were as
specified by the respective vendor. For pH-amended
Ultra Clorox Germicidal bleach, these parameters were
chosen by EPA based on common use of these products
and reasonable application procedures for small-scale
evaluation. Oxonia Active and pH-amended Ultra
Clorox Germicidal bleach were both applied using
the same type of commercial hand-pressurized garden
sprayer (1.5 L (51 oz.) GardenPlus, Part No. 0036943,
LG Sourcing, Inc., Wilkesboro, NC) equipped with a
pressure gauge (Ashcroft 9795T117, General Purpose
0-15 pounds per square inch (psi), Grade B (Certified
accuracy ± 2% at mid-scale)). CASCAD SDF was
applied using a vendor-supplied dual-compartment
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spray bottle with a mesh diffuser to create the foam. In
testing of both pH-amended Ultra Clorox Germicidal
bleach and Oxonia Active with a 120-minute contact
time, the additional step was taken of mixing the applied
decontaminant with the topsoil after each application
using a glass stirring rod. An amendment to the test/
QA plan was prepared, reviewed, and approved for this
addition to the test procedure for this decontaminant.
For testing of Klozur, the 0.5 M persulfate solution and
8% H2O2 activating solution were poured as liquids
(not sprayed) in 1 mL amounts directly onto each
topsoil coupon, and mixed with the topsoil after each
application using a 10 cm long glass stirring rod. This
approach, and the concentrations and applied volumes
of the two solutions, were chosen by EPA to follow the
vendor's recommendations for use of the product in soil
remediation
Deposition tests were conducted before any testing with
pH-amended Ultra Clorox Germicidal bleach, CASCAD
SDF, and Oxonia Active, using topsoil coupons that
had not been inoculated with spores. In these tests
the decontaminant was applied to three pre-weighed
test coupons exactly as specified (i.e., according to
the respective Appendix A, B, or C), and the mass of
decontaminant applied on each coupon was determined.
The average deposited mass of decontaminant was then
used in neutralization trials, to determine the amount
of neutralizing agent needed to stop the action of the
decontaminant after the prescribed contact time. No
deposition test was conducted for Klozur, because that
decontaminant was added volumetrically to the topsoil
rather than spray-applied.
The neutralization trials were designed to determine the
required concentration of a neutralizing chemical chosen
(or recommended by the vendor) to stop the action of the
decontaminant being tested after the prescribed contact
time. The required concentration of neutralizer was
determined in trial runs for each contact time for each
decontaminant tested. In each of those trial runs a range
of neutralizer concentrations was tried, to determine the
concentration that most effectively stopped the action
of the decontaminant (as indicated by the maximum
recovery of viable spores in simulated coupon extracts).
An amount of topsoil equivalent to a test coupon was
included in each control sample and each test sample
containing neutralizer in each trial run. The results of
those neutralization trials are shown in the respective
results chapters (Chapters 5 to 8).
Following decontamination, the topsoil from
each coupon (along with any associated pooled
decontaminant) was transferred aseptically to a sterile
50mL conical vial containing 10 mL of extraction
solution. All extraction solutions consisted primarily
of sterile phosphate-buffered saline (PBS) solution
with Triton X-100 surfactant (i.e., 99.9% PBS solution,
0.1% Triton X-100), and the appropriate concentration
of the neutralizer. The topsoil from each coupon was
then extracted by agitation of the 50 mL vial on an
orbital shaker for 15 minutes at approximately 200
revolutions per minute (rpm) at room temperature.
Each glass stirring rod used to mix topsoil and applied
decontaminant was extracted in the 50 mL vial along
with the topsoil with which it was used. Following
extraction 1 mL of the topsoil extract was removed,
and a series of dilutions through 10~7 were prepared in
SFW. An aliquot (0.1 mL) of the undiluted extract and
each serial dilution was then spread plated onto tryptic
soy agar plates and incubated overnight at 35 to 37 °C.
Plates were enumerated within 18 to 24 hours of plating.
Theoretically, once plated onto the growth medium,
each recovered viable spore germinates and yields one
colony forming unit (CPU). The number of CFU/mL
was determined by multiplying the average number of
colonies per plate by the reciprocal of the dilution, and
accounting for the 0.1 mL volume of extract or dilution
that was plated.
The use of topsoil as a test coupon required development
of techniques to assure adequate recovery of inoculated
B. anihracis spores, and the absence of interference from
native soil microorganisms in counting of recovered
spores. The topsoil used in testing was not sterilized
before use. A heat shock procedure was found to
minimize interference by native microorganisms,
and was used on all topsoil coupons. Specifically,
inoculated or blank topsoil was extracted in the PBS/
Triton X-100 solution, and the recovered supernatant
was heat shocked in a water bath at 65 °C for one
hour before being serially diluted and plated. Topsoil
samples inoculated with B. anthracis spores all showed
the presence of a single homogeneous species, with all
colonies of uniform size and morphologically distinctive
for B. anthracis. Blank topsoil samples showed growth
of colonies of other, native, Bacillus species, which
were not seen with the inoculated topsoil samples.
Consequently, although topsoil blanks showed some
growth, that growth did not occur with extracts of
inoculated topsoil, so no interference existed in terms of
counting recovered spores. The mechanism by which
growth of native Bacillus is suppressed in the extracts of
inoculated topsoil was not investigated, but may involve
monopolization of nutrients by the large numbers of
inoculated spores. By this procedure, the recovery of
B. anthracis (Ames) spores inoculated onto topsoil
was found to be approximately 50%. The heat shock
procedure for use of topsoil differed from the procedures
used previously with other materials and stated in the
test/QA plan; an appropriate amendment to the plan was
prepared and approved before any testing with topsoil
coupons was conducted.
-------
Before further decontamination tests, the test chamber
was cleaned using the vendor-supplied method for
neutralizing the decontamination reagent (see the
appendices to this report). If no instructions for
neutralization were provided, the test chamber was
cleaned following procedures established under the
BBRC Facility Safety Plan.
Laboratory blanks controlled for sterility, and procedural
blanks controlled for viable spores inadvertently
introduced to test coupons. The procedural blanks were
inoculated with an equivalent amount of 0.1 mL of
"stock suspension" that did not contain the biological
agent. To be considered acceptable for quantitative
efficacy determination, extracts of laboratory or
procedural blanks were to contain no CPU. The mean
percent spore recovery was calculated using results from
positive control coupons (inoculated, not decontaminated
(sprayed with SFW instead of the decontaminant)), by
means of the following equation:
Mean % Recovery = [Mean CPU /CPU ., ] x 100 (1)
J l pc spike-1 v '
where Mean CPU c is the mean number of CPU
recovered from five replicate positive control coupons,
and CFUspike is the number of CPU inoculated onto
each of those coupons. The value of CFUspike is known
from enumeration of the stock spore suspension. Spore
recovery from positive control coupons was calculated
for B. anthracis in each decontaminant test, and the
results are included in Chapters 5 through 8.
3.2 Decontamination Efficacy
The quantitative performance or efficacy of the
decontamination technology was assessed by
determining the number of viable organisms remaining
on each test coupon, and in any decontaminant captured
with the coupon, after decontamination. These data were
compared with the number of viable organisms extracted
from the positive control coupons sprayed with SFW,
which was the matrix for the spore suspension used to
inoculate the test coupons.
The number of colony-forming units (CPU) of B.
anthracis m extracts of test and positive control
coupons was determined to calculate efficacy of the
decontaminant. Efficacy is defined as the extent (as
Iog10 reduction) to which viable spores extracted from
test coupons after decontamination were less numerous
than the viable spores extracted from positive control
coupons subjected only to an inert SFW spray, at the
same temperature and contact time as the decontaminant
application. First, the logarithm of the CPU count value
from each coupon extract was determined, and then the
mean of those logarithm values was determined for each
set of control and associated test coupons, respectively.
Efficacy of a decontaminant for a test organism on
topsoil was calculated as the difference between those
mean log values, i.e.:
Efficacy = (loglo CFUc,) - (k>g,0 CFUtj) (2)
where Iog10CFUc.refers to they individual logarithm
values obtained from the positive control coupons
and Iog10CFUt.refers to they individual logarithm
values obtained from the corresponding test coupons,
and the overbar designates a mean value. In tests
conducted under this plan, there were five control and
five corresponding test coupons (i.e.,7 = 5). In the case
where no CPUs were found in a coupon extract, a CPU
count of 1 was assigned, resulting in a log CPU of zero
for that coupon.
The variances (i.e., the square of the standard deviation
(SD)) of the log10CFt/c and log10CFW values were also
calculated for both the control and test coupons (i.e.,
SD2c and SLPt.), and were used to calculate the pooled
standard error (SE) for the efficacy value calculated in
Equation 2, as follows:
SD2t,.
(3)
where the number 5 again represents the number y of
coupons in both the control and test data sets. Thus each
efficacy result is reported as a log reduction value with
an associated SE value.
The significance of differences in efficacy was assessed
based on the 95% confidence interval of each efficacy
result. The 95% confidence interval (CI) is:
95% CI = Efficacy ± (1.96 x SE) (4)
Differences in efficacy were judged to be significant if
the 95% CIs of the two efficacy results did not overlap.
The efficacy results are presented in a series of tables in
Chapters 5 through 8 for each decontaminant technology.
-------
-------
4.0
Quality Assurance/Quality Control
Quality assurance/quality control (QC) procedures were
performed in accordance with the test/QA plan for this
evaluation, except as noted below. QA/QC procedures
are summarized below.
4.1 Equipment Calibration
All equipment (e.g., pipettes, incubators, biological
safety cabinets) and monitoring devices (e.g.,
temperature, RH) used at the time of evaluation were
verified as being certified, calibrated, or validated.
4.2 QC Results
Quality control efforts conducted during decontaminant
testing included positive control coupons (inoculated,
not decontaminated), procedural blanks (not inoculated,
decontaminated), laboratory blanks (not inoculated, not
decontaminated), and spike control samples (analysis of
the stock spore suspension). The results for these QC
samples in each decontaminant evaluation are included
in the results chapter for each respective decontaminant
(see Chapters 5 through 8). One QC issue regarding
spore inoculation was addressed during testing, and is
noted in Section 4.4.
4.3 Audits
4.3.7 Performance Evaluation Audit
No performance evaluation audit was performed for B.
cmthracis (Ames) because a quantitative standard for
such a biological material does not exist.
4.3.2 Technical Systems Audit
Battelle QA staff conducted a technical systems audit
(TSA) at the BBRC on November 10, 2009 during
testing of Oxonia Active to ensure that the evaluation
was being conducted in accordance with the test/QA
plan. In the TSA, test procedures were compared to
those specified in the test/QA plan, and data acquisition
and handling procedures were reviewed. Observations
and findings from the TSA were documented and
submitted to the Battelle Task Order Leader for
response. No adverse findings resulted from the TSA.
TSA records were permanently stored with the NHSRC
Q A Manager.
4.3.3 Data Quality Audit
At least 10% of all test data acquired during the
evaluation were audited. A Battelle QA auditor traced
the data from the initial acquisition, through reduction
and statistical analysis, to final reporting to ensure
the integrity of the reported results. All calculations
performed on the data undergoing the audit were
checked.
4.4 Test/QA Plan Amendments and
Deviations
One amendment to the test/QA plan was prepared,
reviewed, approved, and distributed to all parties
involved in this evaluation. That amendment
implemented both the use of the taller Teflon container
for tests with multiple applications of decontaminant,
and the use of a glass stirring rod to mix the topsoil and
applied decontaminant during testing. The TSA cited in
Section 4.3.2 showed that all test procedures followed
the test/QA plan, i.e., no deviations were recorded as a
result of that TSA. A deviation was prepared, approved,
and placed in the evaluation file regarding acceptance
of spore inoculation counts outside the target range of
1 x 108/coupon (± 25%) in the testing of Ultra Clorox
Germicidal bleach (60 minute test) and Klozur (24 hour
test). These occurrences are noted in Sections 5.1 and
8.1, respectively.
4.5 QA/QC Reporting
Each audit was documented, and the results of the audits
were submitted to the EPA (i.e., to the NHSRC QA
Manager and the Task Order Project Officer (TOPO)).
4.6 Data Review
Records and data generated in the evaluation received
a QC/technical review before they were utilized
in calculating or evaluating results and prior to
incorporation in reports. All data were recorded by
Battelle staff. The person performing the QC/technical
review was involved in the experiments and added his/
her initials and the date to a hard copy of the record
being reviewed. This hard copy was returned to the
Battelle staff member who stored the record.
-------
-------
5.0
pH-Amended Ultra Clorox Germicidal
Bleach Test Results
5.1 QC Results
In testing of pH-amended Ultra Clorox Germicidal
bleach with B. anthracis, percent recoveries of
inoculated spores from the positive control coupons were
88.6% and 108% in the 60-minute and 120-minute tests,
respectively. These percent recovery values were well
within the acceptable range of 1 to 150% stated in the
test/QAplan.
All procedural and laboratory blanks met the criterion
of no observed CPUs morphologically characteristic of
B. anthracis (Ames), although other (native) organisms
were found on some blank coupons.
Spike control samples were taken from the spore
suspension on each day of testing and serially diluted,
nutrient plated, and counted to establish the spore
density used to inoculate the coupons. This process
takes approximately 24 hours, so spore density is known
after completion of each day's testing. The target
criterion is a spore suspension density of 1 x lOVmL
(± 25%), leading to an inoculation of 1 x 10s spores
(± 25%) on each test coupon. The actual inoculation
values for B. anthracis testing were 6.97 x 107 and 8.13
x 107 CPU/coupon, respectively, on the two days of
testing. Thus on the first test day the coupons received
a spore inoculation that was slightly low relative to the
target criterion. In light of the high spore recoveries
and low efficacy observed, testing from that day was
not repeated. However, a deviation from the test/QA
plan documenting the acceptance of this low inoculation
value was prepared, approved, and retained in the project
files.
5.2 Decontamination Efficacy
The quantitative efficacy results for pH-amended Ultra
Clorox Germicidal bleach at the two contact times of
60 and 120 minutes are presented in Table 5-1. The
decontamination efficacy of pH-amended Ultra Clorox
Germicidal bleach on topsoil was 0.03 log reduction
with the 60-minute contact time (four applications)
and 0.94 log reduction with the 120-minute contact
time (eight applications, with mixing of the topsoil
and applied decontaminant). The latter efficacy result
is statistically significantly greater than the former,
indicating that the additional applications, longer
contact time, and/or mixing with topsoil improved
the effectiveness of inactivation. However, even with
eight applications and mixing of the decontaminant and
topsoil, the efficacy of Ultra Clorox Germicidal bleach
was less than 1 log reduction.
Table 5-1. Inactivation of Bacillus anthracis Ames Spores
on Topsoila- pH-Amended Ultra Clorox Germicidal Bleach
Contact Time
(Number of
Applications)
60 Minutes (4)
Positive Controls'
Test Coupons0
Laboratory Blank4'
Procedural Blank'''
120 Minutes
(8, stirred)
Positive Controls'
Test Coupons0
Laboratory Blank4'
Procedural Blank6-'
Mean of
Inoculum Logs of
(CFU) Observed
CFU±SD
6.97 xlO7 7.77±0.13
6.97 xlO7 7. 74 ±0.12
0 0
0 0
8. 13 xlO7 7.94 ±0.08
8. 13 xlO7 6.99 ±0.09
0 0
0 0
Mean% .
Decontamination
"SID17 Efficacy ± CI
88.6 ±23.0
81.6 ±26.3 0.03 ±0.15
0
0
108 ±18
12.3 ±2.3 0.94±0.10
0
0
a Data are expressed as mean (± SD) of the logs of total number of spores (CFU) observed
on individual coupons, mean percent recovery, and decontamination efficacy (log
reduction). CI = confidence interval (± 1.96 x SE)
b Inoculated, not decontaminated coupon (sprayed with SFW).
c Inoculated, decontaminated coupon.
d Laboratory Blank = not inoculated, not decontaminated coupon.
e Procedural Blank = not inoculated, decontaminated coupon.
f Endogenous organisms were found in topsoil blanks; no organisms other than B. anthracis
Ames were found on inoculated coupons.
"-" Not Applicable.
5.3 Other Factors
5.3.7 Operator Control
The pH-amended Ultra Clorox Germicidal bleach
was prepared according to the procedure described in
Appendix A, by mixing 9.4 parts SFW, 1 part Ultra
Clorox Germicidal bleach, and 1 part 5% acetic acid.
The bleach used was obtained through a retail purchase,
and the bottle was unopened until the first day of use.
The actual resulting solution used for testing with B.
anthracis had a pH of 6.36 and a total chlorine content
of approximately 6,200 ppm. The pH-amended Ultra
Clorox Germicidal bleach was freshly prepared prior to
each testing day (i.e., the preparation was assigned a one
day shelf-life and excess was discarded at the end of the
day).
All trials were conducted at ambient conditions inside
a climate-controlled laboratory. The temperature inside
the testing chamber was equilibrated to the ambient
laboratory temperature of approximately 22 (± 1) °C.
-------
The RH was monitored with a hygrometer traceable
to the National Institute of Standards and Technology
(NIST). The RH in the test chamber was controlled as
necessary by an automatic dehumidifier set to maintain
RH below 70%. That system never activated during
decontamination with pH-amended Ultra Clorox
Germicidal bleach, and therefore the testing chamber RH
was always less than 70% during testing.
5.3.2 Technology Spray Deposition
The pH-amended Ultra Clorox Germicidal bleach was
applied according to the procedure in Appendix A.
The pH-amended Ultra Clorox Germicidal bleach was
applied 12 inches from the topsoil coupons until the
topsoil appeared saturated with liquid. The same type of
handheld garden sprayer was used to apply the control
application (SFW) and the pH-amended Ultra Clorox
Germicidal bleach. Each sprayer was slightly modified
to accommodate a pressure gauge to ensure that the
spray was applied using 4 to 6 psi pressure. After the
prescribed contact time, each topsoil coupon and the
collected decontaminant were placed in a 50 mL conical
vial for extraction.
To assess pH-amended Ultra Clorox Germicidal bleach
deposition, triplicate topsoil coupons were weighed, and
these values were recorded. Then the triplicate coupons
were sprayed with pH-amended Ultra Clorox Germicidal
bleach according to the test procedure in Appendix A.
The pre-application weights were then subtracted from
the post-application weights, which included the weight
of decontaminant captured with each coupon. This
process was conducted separately for the 60-minute and
120-minute application procedures (Appendix A) which
required four and eight applications of pH-amended
Ultra Clorox Germicidal bleach, respectively. The mass
of Ultra Clorox Germicidal bleach deposited on topsoil
coupons in the 60-minute test (4 applications) averaged
2.60 g, and in the 120-minute test (8 applications)
averaged 3.55 g. The density of the diluted bleach
deposited on the test coupons was not measured directly,
but was estimated to be approximately 1.0 g/mL. Based
on that density, volumes of 2.60 and 3.55 mL were
then used in trials to determine the amount of sodium
thiosulfate (STS) needed to neutralize the Ultra Clorox
Germicidal bleach (Section 5.3.3).
5.3.3 Neutralization Methodology
Neutralization of the pH-amended Ultra Clorox
Germicidal bleach was achieved with STS. The results
of the neutralization trials for B. cmthracis with the
60-minute and 120-minute applied volumes of pH-
amended Ultra Clorox Germicidal bleach are shown
in Tables 5-2 and 5-3, respectively. Based on these
neutralization results, a concentration of 1.0% STS in
the extraction solution was chosen for neutralization of
the pH-amended Ultra Clorox Germicidal bleach in the
60-minute test, and a concentration of 1.5% STS was
chosen for neutralization in the 120-minute test.
Table 5-2. Neutralization Testing with Bacillus anthracis
(Ames) Spores on Topsoil for pH-Amended Ultra Clorox
Germicidal Bleach (60 Minute Contact Time, Volume
Equal to 4 Applications)
Treatment
pH-amended Ultra
Clorox Germicidal
bleach + Spores1
pH-amended Ultra
Clorox Germicidal
bleach + PBS +
Triton X- 100 +
Spores3-11
PBS + Triton
X- 100 + Spores
(Control)b
pH-amended Ultra
Clorox Germicidal
bleach + PBS
+ Triton X- 100
+ 0.5% STS +
Spores3-1'
pH-amended Ultra
Clorox Germicidal
bleach + PBS
+ Triton X- 100
+ 1.0% STS +
Sporesa-b
pH-amended Ultra
Clorox Germicidal
bleach + PBS
+ Triton X- 100
+ 1.5% STS +
Spores8-1'
Inoculum
(CFU)
9.00 x 107
9.00 x 107
9.00 x 107
9.00 x 107
9.00 x 107
9.00 x 107
Total
Observed
CFU
0
0
9.00 x 107
7.93 x 107
9.10x 107
7.63 x 107
%of
Control
0
0
"
88.2
101.1
84.7
a pH-amended Ultra Clorox Germicidal bleach volume of 2.60 mL corresponds
to mean gravimetric deposition on topsoil coupons.
b lOmL volume of PBS includes 0.1% of Triton X-100 surfactant and indicated
% of STS; total volume for all samples with pH-amended Ultra Clorox
Germicidal bleach = 12.60 mL (10 mL PBS/Tnton/STS + 2.60 mL pH-
amended Ultra Clorox Germicidal bleach).
"-" Not Applicable.
-------
Table 5-3. Neutralization Testing with Bacillus a nth rat is
(Ames) Spores on Topsoil for pH-Amended Ultra Clorox
Germicidal Bleach (120-Minute Contact Time, Volume
Equal to 8 Applications)
Treatment
pH-amended Ultra
Clorox Germicidal
bleach + Spores1
pH-amended Ultra
Clorox Germicidal
bleach + PBS +
Triton X- 100 +
Spores1'"
pH-amended Ultra
Clorox Germicidal
bleach + Triton
X- 100 + Spores
(Control)b
pH-amended Ultra
Clorox Germicidal
bleach + PBS
+ Triton X- 100
+ 1.0%STS +
Spores1'"
pH-amended Ultra
Clorox Germicidal
bleach + PBS
+ Triton X- 100
+ 1.5%STS +
Spores"*
pH-amended Ultra
Clorox Germicidal
bleach + PBS
+ Triton X- 100
+ 2.0% STS +
Spores1-"
Inoculum
(CFU)
8.57 x 107
8.57 x 107
8.57 x 107
8.57 x 107
8.57 x 107
8.57 x 107
Total
Observed
CFU
0
0
7.91 x 107
7.65 x 107
8.73 x 107
7.93 x 107
%of
Control
0
0
_
96.7
110.4
100.3
a pH-amended Ultra Clorox Germicidal bleach volume of 3.55 mL corresponds
to mean gravimetric deposition on topsoil coupons.
b lOmL volume of PBS includes 0.1% of Triton X-100 surfactant and
indicated % of STS; total volume for all samples with pH-amended Ultra
Clorox Germicidal bleach = 13.55 mL (10 mL PBS/Triton/ STS + 3.55 mL
pH-amended Ultra Clorox Germicidal bleach).
"-" Not Applicable.
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6.0
CASCAD SDF (Allen-Vanguard) Test Results
6.1 QC Results
In testing of CASCAD SDF with B. anthracis, percent
recoveries of inoculated spores from the positive control
coupons were 80.1% and 106% in the 60-minute and
120-minute tests, respectively. These percent recovery
values were well within the acceptable range of 1 to
150% stated in the test/QA plan.
All procedural and laboratory blanks met the criterion
of no observed CPUs morphologically characteristic of
B. anthracis (Ames), although other (native) organisms
were found on some blank coupons.
Spike control samples were taken from the spore
suspension on each day of testing, and serially diluted,
nutrient plated, and counted to establish the spore
density used to inoculate the coupons. This process
takes approximately 24 hours, so the spore density is
known after completion of each day's testing. The
target criterion is to maintain a spore suspension density
of 1 x lOVmL (± 25%), leading to an inoculation of 1
x 10s spores (± 25%) on each test coupon. The actual
inoculation values for two days of B. anthracis testing
were 7.80 x 107 CPU/coupon and 8.40 x 107 CFU/oupon,
within the ±25% tolerance of the 1 x 108/coupon target.
6.2 Decontamination Efficacy
The quantitative efficacy results for CASCAD SDF at
the two contact times of 60 minutes (two applications)
and 120 minutes (four applications) are presented in
Table 6-1. The decontamination efficacy of CASCAD
SDF on topsoil was 0.59 log reduction with the
60-minute contact time (two applications) and 1.13
log reduction with the 120-minute contact time (four
applications). The 120-minute efficacy result is
statistically significantly greater than the 60-minute
result, indicating that the additional applications of
CASCAD SDF and/or the longer contact time improved
the effectiveness of inactivation. However, even with
four applications and the 120-minute contact time the
efficacy of CASCAD SDF was only slightly greater than
1 log reduction.
6.3 Other Factors
6.3.7 Operator Control
On each day of testing, the two components of Allen-
Vanguard's CASCAD SDF were prepared according
to the vendor's instructions in Appendix B. The spray
nozzle was then placed onto the dual-chamber bottle,
in which each chamber of the bottle contained one of
the two CASCAD SDF reagent solutions. Prior to
each application, the CASCAD SDF spray nozzle was
primed by repeatedly spraying into an absorbent cloth
to clear any air bubbles that may have formed between
applications. After each application, the spray nozzle
was removed from the bottle and any residual CASCAD
SDF was removed by repeated pulls on the trigger of the
Table 6-1. Inactivation of Bacillus anthracis Ames Spores on Topsoila- CASCAD SDF
Contact Time
(Number of
Applications)
60 Minutes (2)
Positive Controls'1
Test Coupons0
Laboratory Blank45
Procedural Blanke'f
120 Minutes (4)
Positive Controls'1
Test Coupons0
Laboratory Blank45
Procedural Blanke'f
Inoculum
(CFU)
7.80 x 107
7.80 x 107
0
0
8.40 x 107
8.40 x 107
0
0
Mean of Logs of
Observed CFU
±SD
7.79 ± 0.04
7.21 ±0.02
0
0
7.95 ±0.06
6.81 ±0.12
0
0
Mean % Recovery
±SD
80.1 ±6.8
20.6 ±0.9
0
0
106. 3 ±14.7
8.0 ±2.2
0
0
Decontamination
Efficacy ± CI
0.59 ±0.04
-
1.13±0.12
a Data are expressed as mean (± SD) of the logs of total number of spores (CFU) observed on individual coupons, mean percent
recovery, and decontamination efficacy (log reduction).
CI = confidence interval (± 1.96 x SE).
b Inoculated, not decontaminated coupon (sprayed with SFW).
c Inoculated, decontaminated coupon.
d Laboratory Blank = not inoculated, not decontaminated coupon.
e Procedural Blank = not inoculated, decontaminated coupon.
f Endogenous organisms were found in topsoil blanks; no organisms other than B. anthracis Ames were found on inoculated coupons.
"-" Not Applicable.
-------
spray nozzle. The spray nozzle was then placed onto a
dual chamber bottle that contained only SFW so as to
completely clean out the spray nozzle until its next use.
All tests were conducted under ambient conditions
inside a climate-controlled laboratory. The temperature
inside the test chamber was equilibrated to the ambient
laboratory temperature of approximately 22 (± 1) °C.
The RH inside the test chamber was monitored with a
NIST-traceable hygrometer. Whenever the RH reached
70%, an automatic dehumidification system attached to
the testing chamber was actuated until the RH dropped
below 70%. The dehumidifier did not actuate during
the prescribed contact times with the CASCAD SDK
Therefore, the testing chamber RH was always less than
70% during decontamination with CASCAD SDK
6.3.2 Technology Spray Deposition
Allen-Vanguard's CASCAD SDF was applied according
to the procedure included as Appendix B of this report.
CASCAD SDF was applied from a distance of 12 inches
from the topsoil coupons, with the aim of fully covering
the topsoil coupon with approximately a 3/8 inch layer
of foam. After the prescribed contact time, each topsoil
coupon and the collected decontaminant were placed in a
50 mL conical vial for extraction.
To assess CASCAD SDF deposition, triplicate topsoil
coupons were weighed, and these values were recorded.
Then the triplicate coupons were sprayed with CASCAD
SDF according to the test procedure in Appendix B.
The preapplication weights were then subtracted from
the post-application weights, which included the weight
of decontaminant captured with each coupon. The
resulting mass was used to estimate the amount of STS
needed to effectively neutralize the CASCAD SDF. This
process was conducted separately for the 60-minute and
120-minute application procedures (Appendix B) which
required two and four applications of CASCAD SDF,
respectively.
The mass of CASCAD SDF deposited on topsoil
coupons in the 60-minute test (2 applications) averaged
2.93 g, and in the 120-minute test (4 applications)
averaged 6.46 g. The density of the CASCAD SDF
deposited on the test coupons was not measured directly,
but was estimated to be approximately 1.0 g/mL, based
on the compositions of the two component solutions that
produce the delivered foam (see Appendix B). Based
on that density, volumes of 2.93 and 6.46 mL were then
used in trials to determine the amount of STS needed to
neutralize the CASCAD SDF (Section 6.3.3).
6.3.3 Neutralization Methodology
Neutralization of CASCAD SDF was achieved with
STS. The results of the neutralization trials for B.
cmthracis with the 60-minute and 120-minute application
procedures are shown in Tables 6-2 and 6-3, respectively.
Based on these neutralization results, a concentration
of 1.5% STS in the extraction solution was chosen
for neutralization of CASCAD SDF in the 60-minute
test, and a concentration of 2.0% STS was chosen for
neutralization in the 120-minute test.
Table 6-2. Neutralization Testing with Bacillus anthmcis (Ames) Spores on Topsoil for CASCAD SDF (60 Minute Contact
Time, Volume Equal to 2 Applications)
Treatment
CASCAD SDF + Spores1
CASCAD SDF + PBS + Triton X-100 + Spores1-"
PBS + Triton X-00 + Spores (Control)
CASCAD SDF + PBS + Triton X-100 + 0.5% STS + Spores"-"
CASCAD SDF + PBS + Triton X-100 + 1 .0% STS + Spores1-"
CASCAD SDF + PBS + Triton X-100 + 1 .5% STS + Spores"-"
Inoculum Total Observed
(CFU) (CFU)
8.69 x
8.69 x
8.69 x
8.69 x
8.69 x
8.69 x
107 0
107 0
107 1.06xl08
107 3.45 x 107
107 8.87 x 107
107 l.OOxlO8
% of Control
0
0
-
32.4
83.3
94.4
a CASCAD SDF volume of 2.93 mL corresponds to mean gravimetric deposition on topsoil coupons.
b 10 mL volume of PBS includes 0.1 % of Triton X-l 00 surfactant and indicated % of STS; total volume for all samples with CASCAD SDF = 12.93 mL (10 mL
PBS+Tnton +STS + 2.93 mL CASCAD SDF).
"-" Not Applicable.
-------
Table 6-3. Neutralization Testing with Bacillus anthracis (Ames) Spores on Topsoil for CASCAD SDF (120 Minute Contact
Time, Volume Equal to 4 Applications)
Treatment
Inoculum
(CFU)
Total
„, ,
Observed
(CFU)
...
% of
CASCAD SDF + Spores1
8.00 x 107
0
CASCAD SDF + PBS + Triton X-100 + Spores1'
8.00 x 107
PBS + Triton X-100 + Spores (Control)"
8.00 x 107
7.81 x 107
CASCAD SDF + PBS + Triton X-100 + 1 .5% STS + Spores1'"
8.00 x 107
6.87 x 107
88.0
CASCAD SDF + PBS + Triton X-100 + 2.0% STS + Spores1-
8.00 x 107
8.49 x 107
108.7
CASCAD SDF + PBS + Triton X-100 + 2.5% STS + Spores1'
8.00 x 107
7.88 x 107
101.0
CASCAD SDF + PBS + Triton X-100 + 3.0% STS + Spores1-
8.00 x 107
8.47 x 107
108.4
a CASCAD SDF volume of 6.46 mL corresponds to mean gravimetric deposition on topsoil coupons.
b 10 mL volume of PBS includes 0.1% of Triton X-100 surfactant and indicated % of STS; total volume for all samples with CASCAD SDF = 16.46 mL (10 mL
PBS+Tnton +STS + 6.46 mL CASCAD SDF).
"-" Not Applicable.
-------
-------
7.0
Oxonia Active (Ecolab) Test Results
7.1 QC Results
In testing of Oxonia Active with B. anthracis, percent
recoveries of inoculated spores from the positive control
coupons were 79.5% and 102% in the 60-minute and
120-minute tests, respectively. These percent recovery
values were well within the acceptable range of 1 to
150% stated in the test/QA plan.
All procedural and laboratory blanks met the criterion
of no observed CPUs morphologically characteristic of
B. anthracis (Ames), although other (native) organisms
were found on some blank coupons.
Spike control samples were taken from the spore
suspension on each day of testing, and serially diluted,
nutrient plated, and counted to establish the spore
density used to spike the coupons. This process takes
approximately 24 hours, so the spore density is known
after completion of each day's testing. The target
criterion is to maintain a spore suspension density of 1 x
lOVmL (± 25%), leading to a spike of 1 x 108 spores (±
25%) on each test coupon. The actual spike values for
two days of B. anthracis testing were 8.23 x 107 CPU/
coupon and 9.07 x 107 CPU/coupon, well within the
±25% tolerance of the 1 x 108/coupon target.
7.2 Decontamination Efficacy
The quantitative efficacy results for Oxonia Active
at the two contact times of 60 and 120 minutes are
presented in Table 7-1. The decontamination efficacy
of Oxonia Active on topsoil was 0.58 log reduction
with the 60-minute contact time (six applications) and
1.04 log reduction with the 120-minute contact time
(12 applications, with mixing of the topsoil and applied
decontaminant). The latter efficacy result is statistically
significantly greater than the former, indicating that
the additional applications, longer contact time, and/
or mixing with topsoil improved the effectiveness of
inactivation. However, even with 12 applications and
mixing of the decontaminant and topsoil, the efficacy
of Oxonia Active was only slightly greater than 1 log
reduction.
7.3 Other Factors
7.3.7 Operator Control
On each day of testing, Oxonia Active was prepared
Table 7-1. Inactivation of Bacillus anthracis Ames Spores on Topsoil11 - Oxonia Active
Contact Time
(Number of Applications)
60 Minutes (6)
Positive Controls'1
Test Coupons0
Laboratory Blank45
Procedural Blanke'f
120 Minutes
(12, stirred)
Positive Controlsb
Test Coupons0
Laboratory Blank45
Procedural Blanke'f
Inoculum
(CFU)
8.23 x 107
8.23 x 107
0
0
9.07 x 107
9.07 x 107
0
0
Mean of Logs of
Observed CFU
±SD
7.81 ±0.05
7.23 ±0.21
0
0
7.96 ±0.05
6.93±0.11
0
0
Mean % Recovery
±SD
79.51 ±8.6
23.0 ±14.1
0
0
102.1 ±11. 5
9.6 ±2.4
0
0
Decontamination
Efficacy ± CI
-
0.58±0.19
-
-
1.04 ±0.10
.
a Data are expressed as mean (± SD) of the logs of total number of spores (CFU) observed on individual coupons, mean percent recovery, and decontamination efficacy
(log reduction).
CI = confidence interval (± 1.96 x SE).
b Inoculated, not decontaminated coupon (sprayed with SFW).
c Inoculated, decontaminated coupon.
d Laboratory Blank = not inoculated, not decontaminated coupon.
e Procedural Blank = not inoculated, decontaminated coupon.
f Endogenous organisms were found in topsoil blanks; no organisms other than B. anthracis Ames were found on inoculated coupons.
"-" Not Applicable.
-------
according to the vendor's explicit instructions as stated
in Appendix C. After the Oxonia Active was diluted in
SFW, the product was tested to ensure that the active
component (peroxyacetic acid) was within the range
specified by the vendor. This check was done using a
test kit also provided by the vendor (High Oxonia Active
Test Kit 322). All such checks showed the prepared
solution to be in the correct range. The diluted Oxonia
Active was then transferred to a handheld garden sprayer
modified with a pressure gauge to ensure that the spray
was applied using 4 to 6 psi pressure.
All tests were conducted at ambient conditions inside
a climate-controlled laboratory. The temperature
inside the test chamber was equilibrated to the ambient
laboratory temperature of approximately 22 (± 1) °C.
The RH inside the test chamber was monitored with a
NIST-traceable hygrometer. Whenever the RH reached
70%, as it did during reapplications of the Oxonia
Active, the dehumidification system attached to the
testing chamber was actuated until the RH dropped
below 70%. Therefore, the testing chamber RH was
always < 70% during decontamination with Oxonia
Active.
7.3.2 Technology Spray Deposition
Oxonia Active was applied according to the procedure
included as Appendix C of this report. Oxonia Active
was applied from a distance of 12 inches from the topsoil
coupons. Reapplication of the Oxonia Active was made
every 10 minutes. The same type of handheld garden
sprayer was used to apply the control application (SFW)
and the Oxonia Active. Each sprayer was slightly
modified to accommodate a pressure gauge to ensure
that the spray was applied using 4 to 6 psi pressure.
After the required contact time, each topsoil coupon and
the collected decontaminant were placed in a 50 mL
collection vial for extraction.
To assess Oxonia Active deposition, triplicate topsoil
coupons were weighed, and these values were recorded.
Then the triplicate coupons were sprayed with Oxonia
Active according to the test procedure in Appendix C.
The pre-application weights were then subtracted from
the post-application weights, which included the weight
of decontaminant captured with each coupon. This
process was conducted separately for the 60-minute
and 120-minute application procedures (Appendix C)
which required six and 12 applications of Oxonia Active,
respectively. The mass of Oxonia Active deposited on
topsoil coupons in the 60-minute test (6 applications)
averaged 4.00 g, and in the 120-minute test (12
applications) averaged 7.07 g. The density of the Oxonia
Active deposited on the test coupons was not measured
directly, but was estimated to be approximately 1.0
g/mL, based on the preparation instructions for the
decontaminant (see Appendix C). Based on that density,
volumes of 4.00 and 7.07 mL were then used in trials to
determine the amount of STS needed to neutralize the
Oxonia Active (Section 7.3.3).
7.3.3 Neutralization Methodology
Neutralization of the Oxonia Active was achieved with
STS. The results of the neutralization trials for B.
cmthracis with the 60-minute and 120-minute application
procedures are shown in Tables 7-2 and 7-3, respectively.
Based on these neutralization results, a concentration
of 3% STS in the extraction solution was chosen for
neutralization of Oxonia Active in the 60-minute
test, and a concentration of 5% STS was chosen for
neutralization in the 120-minute test.
Table 7-2. Neutralization Testing with Bacillus anthracis (Ames) Spores on Topsoil for Oxonia Active (60 Minute Contact
Time, Volume Equal to 6 Applications)
Treatment
Oxonia Active + Spores"
Oxonia Active + PBS + Triton X-100 + Spores1-"
PBS + Triton X-100 + Spores (Control)"
Oxonia Active + PBS + Triton X-100 + 2.0% STS + Spores1'"
Oxonia Active + PBS + Triton X-100 + 2.5% STS + Spores1-"
Oxonia Active + PBS + Triton X-100 + 3.0% STS + Spores1-"
Oxonia Active + PBS + Triton X-100 + 3.5% STS + Spores1-"
Inoculum
(CFU)
8.60 x 107
8.60 x 10'
8.60 x 107
8.60 x 107
8.60 x 107
8.60 x 107
8.60 x 107
Total Observed
(CFU)
0
0
8.95 x 107
7.61 x 107
8.69 x 107
9.35 x 107
9.61 x 107
% of Control
0
0
-
85.0
97.2
104.5
107.5
a Oxonia Active volume of 4.00 mL corresponds to mean gravimetric deposition on soil coupons.
b 10 mL volume of PBS includes 0.1% of Triton X-100 surfactant and indicated % of STS; total volume for all samples with OxomaX-lOO/STS + 4.00 mL Oxonia Active).
"-" Not Applicable.
-------
Table 7-3. Neutralization Testing with Bacillus anthracis (Ames) Spores on Topsoil for Oxonia Active (120 Minute Contact
Time, Volume Equal to 12 Applications)
Treatment
Oxonia Active + Spores1
Oxonia Active + PBS + Triton X-100 + Spores"
PBS + Triton X-100 + Spores (Control)1
Oxonia Active + PBS + Triton X-100 + 3.0% STS + Spores"
Oxonia Active + PBS + Triton X-100 + 3.5% STS + Spores"
Oxonia Active + PBS + Triton X-100 + 4.0% STS + Spores"
Oxonia Active + PBS + Triton X-100 + 4.5% STS + Spores"
Oxonia Active + PBS + Triton X-100 + 5.0% STS + Spores"
Total
Inoculum fr, .
^TTTT\ Observed % of Control
(CFU) (CFU)
8.87>
8.87 >
8.87 >
8.87>
8.87>
8.87 >
8.87 >
8.87 >
< 107 0 0
< 107 0 0
< 107 8.69 x 107
< 107 0 0
< 107 0 0
< 107 0 0
< 107 6.39 x 107 73.5
< 107 8.31 x 107 95.6
a Oxonia Active volume of 7.07 mL corresponds to mean gravimetric deposition on soil coupons.
b 10 mL volume of PBS includes 0.1% of Triton X-100 surfactant and indicated % of STS; total volume for all samples with Oxonia Active = 17.07 mL (10 mL of PBS/
Triton X-100/STS + 7.07 mL Oxonia Active).
"-" Not Applicable.
-------
-------
8.0
Klozur (FMC Corporation) Test Results
8.1 QC Results
In testing of Klozur with B. anthracis, percent recoveries
of inoculated spores from the positive control coupons
were 41% and 95% in the 24-hour and 48-hour tests,
respectively. These percent recovery values were well
within the acceptable range of 1 to 150% stated in the
test/QAplan.
The procedural and laboratory blanks met the criterion
of no observed CPUs in quantitative efficacy testing with
B. anthracis (Ames).
Spike control samples were taken from the spore
suspension on each day of testing, and serially diluted,
nutrient plated, and counted to establish the spore
density used to spike the coupons. This process takes
approximately 24 hours, so the spore density is known
after completion of each day's testing. The target
criterion is to maintain a spore suspension density of 1
x 109/mL (± 25%), leading to a spike of 1 x 10s spores
(± 25%) on each test coupon. The actual spike values
for the two B. anthracis tests were 6.93 x 107 CPU/
coupon and 7.53 x 107 CPU/coupon for the 24 hour and
48 hour tests, respectively. Thus in the 24 hour test the
coupons received a spore inoculation that was slightly
low relative to the target criterion. In light of the high
spore recoveries and low efficacy observed, testing from
that day was not repeated. However, a deviation from
the test/QAplan documenting the acceptance of this low
inoculation value was prepared, approved, and retained
in the project files.
8.2 Decontamination Efficacy
The quantitative efficacy results for Klozur at the two
contact times of 24 hours and 48 hours are presented
in Table 8-1. The decontamination efficacy of Klozur
on topsoil was 1.65 log reduction with the 24-hour
contact time (six applications) and 3.50 log reduction
with the 48-hour contact time (also six applications).
These efficacy results are both statistically significantly
higher than any efficacy value obtained with the other
decontaminants, and are also significantly different from
one another. The relatively high log reduction of 3.5
logs (compared to other decontaminants) achieved with
Klozur with the 48-hour contact time may be due to the
relatively large volume of combined oxidants (Klozur
persulfate and H2O2) used and the extended contact time,
as well as a more effective chemistry.
Table 8-1. Inactivation of Bacillus anthracis Ames Spores on Topsoil11 - Klozur
Contact Time
(Number of Applications)
Inoculum (CFU)
Mean of Logs of
Observed CFU
±SD
Mean % Recovery
±SD
Decontamination
Efficacy ± CI
24 Hours (6, stirred)
Positive Controls'1
Test Coupons0
Laboratory Blank45
Procedural Blanke'f
6.93xl07 7.48 ±0.16
6.93xl07 5.83 ±0.04
0 0
0 0
45.9 ±19.6
0.97 ±0.09
0
0
-
1.65 ±0.14
-
-
48 Hours (6, stirred)
Positive Controls'1
Test Coupons0
Laboratory Blank45
Procedural Blanke-f
7.53 x 10'
7.53 x 10'
0
0
7.85 ±0.08
4.35 ±0.45
0
0
95.0 ±15.7
0.039 ±0.022
0
0
3.50 ±0.40
a Data are expressed as mean (± SD) of the logs of total number of spores (CFU) observed on individual coupons, percent recovery, and decontamination efficacy (log
reduction).
CI = confidence interval (± 1.96 * SE).
b Inoculated, not decontaminated coupon (sprayed with SFW).
c Inoculated, decontaminated coupon.
d Laboratory Blank = not inoculated, not decontaminated coupon.
e Procedural Blank = not inoculated, decontaminated coupon.
f Endogenous organisms were found in topsoil blanks; no organisms other than B. anthracis Ames were found on inoculated coupons.
"-" Not Applicable.
-------
8.3 Other Factors
8.3.1 Operator Control
On each day of testing, a 0.5 M Klozur solution was
prepared according to the vendor's instructions as stated
in Appendix D. The activating solution of 8% H2O2 was
obtained from a commercial supplier. Unlike the other
decontaminants tested, Klozur and the H2O2 activating
solution were not sprayed onto the soil coupons, but
were added to each coupon directly in 1 mL aliquots.
All tests were conducted at ambient conditions inside a
climate-controlled laboratory. The temperature inside the
test chamber was equilibrated to the ambient laboratory
temperature of 22 (± 1) °C. The RH in the test chamber
was monitored with a NIST-traceable hygrometer. A
dehumidification system on the test chamber was
designed to actuate whenever the RH reached 70%. That
system never activated during decontamination with
Klozur, and therefore the chamber RH was always less
than 70% during testing.
8.3.2 Technology Deposition
Klozur was applied according to the procedure included
as Appendix D of this report. After the required
contact time, each topsoil coupon and the collected
decontaminant were placed in a 50 mL collection vial for
extraction.
Deposition experiments were not needed for Klozur,
since the Klozur and H2O2 solutions were applied to the
topsoil coupons volumetrically. The total amount of
the applied solutions was 12 mL for each of the 24-hour
and 48-hour contact times (6 applications, each of 1 mL
of Klozur solution and 1 mL of H2O2 solution). This
volume was used to estimate the amount of STS needed
to effectively neutralize the Klozur/H2O2 mixture for
both contact times.
8.3.3 Neutralization Methodology
Neutralization of Klozur/H2O2was achieved with STS.
The results of the neutralization trials for B. anthmcis
with the 24-hour and 48-hour contact times are shown
in Tables 8-2 and 8-3, respectively. The neutralization
of Klozur with STS was not as effective as the
neutralization of other decontaminants, as the maximum
spore recoveries relative to the control were less than
20%, even with STS comprising 20% of the PBS/Triton
X-100/STS extraction solution. This result may be due
to the large volume of Klozur + H2O2 solution used (i.e.,
12 mL). In any case, at the maximum spore recovery
conditions there was less than a 1 log loss in viable
spores, leaving ample spores for determination of the
efficacy of Klozur. Based on these neutralization results,
a concentration of 5% STS in the extraction solution
was chosen for neutralization of Klozur in the 24-hour
test, and a concentration of 20% STS was chosen for
neutralization in the 48-hour test.
Table 8-2. Neutralization Testing with Bacillus anthracis (Ames) Spores on Topsoil for Klozur
(24 Hour Contact Time, Volume Equal to 6 Applications)
Treatment
Inoculum
(CFU)
Total
Observed % of Control
(CFU)
Klozur + Spores1
7.87 x 107
0
0
Klozur + PBS + Triton X-100 + Spores1-"
7.87 x 107
PBS + Triton X-100 + Spores (Control)"
7.87 x 107 5.60 x 107
Klozur + PBS + Triton X-100 + 2.5% STS + Spores1-1
7.87 x 107 1.09 x 106
2.0
Klozur + PBS + Triton X-100 + 5% STS + Spores1-
7.87 x 107
1.01 x 107
18.1
Klozur + PBS + Triton X-100 + 7.5% STS + Spores1-1
7.87 x 107
3.84 x 106
a Decontaminant volume of 12.0 mL consists of 6.0 mL 0.5 M Klozur solution and 6.0 mL of 8% HO solution.
b lOmL volume of PBS includes 0.1% of Triton X-100 surfactant and indicated % of STS; otal volume for all samples with Klozur = 22.0 mL
(10 mL of PBS/Triton X-100/STS + 12.0 mL Klozur/H2O2).
"-" Not Applicable.
-------
Table 8-3. Neutralization Testing with Bacillus anthracis (Ames) Spores on Topsoil for Klozur
(48 Hour Contact Time, Volume Equal to 6 Applications)
Treatment
Klozur +
Klozur +
Spores1
PBS
+ Triton X- 100 +
Spores8-1'
PBS + Triton X-100 + Spores (Control)"
Klozur +
Klozur +
Klozur +
Klozur +
Klozur +
PBS
PBS
PBS
PBS
PBS
+ Triton X- 100 +
+ Triton X- 100 +
+ Triton X- 100 +
+ Triton X- 100 +
+ Triton X- 100 +
10,
12,
15,
17,
20,
.0% STS
.5% STS
.0% STS
.5% STS
.0% STS
+ Spores1-"
+ Spores1-"
+ Spores1-"
+ Spores1-"
+ Spores1-"
Inoculum
(CFU)
7.53 >
7.53 >
7.53 >
7.53 >
7.53 >
7.53 >
7.53 >
7.53 >
<107
<107
<107
<107
<107
<107
<107
<107
Total Observed
(CFU)
0
0
5.17x
7.33 x
6.85 x
6.45 x
8.00 x
9.70 x
107
106
106
106
106
106
% of Control
0
0
-
14,
13,
12,
15,
18,
.2
.3
.5
.5
.8
a Decontaminant volume of 12 mL consists of 6.0 mL 0.5 M Klozur solution and 6.0 mL of 8% H2O2 solution.
b lOmL volume of PBS includes 0.1% of Triton X-100 surfactant and indicated % of STS; total volume for all samples with Klozur = 22.0mL(10mL
of PBS/Triton X-100/STS + 12.0 mL Klozur/H2O2).
"-" Not Applicable.
-------
-------
9.0
Performance Summary
9.1 pH-Amended Ultra Clorox
Germicidal Bleach Results
• The quantitative efficacy of pH-amended Ultra
Clorox Germicidal bleach for inactivating B.
anthracis (Ames) spores ontopsoil was 0.03 (±
0.15) log reduction with four applications and
a 60-minute contact time, and 0.94 (± 0.10) log
reduction with eight applications and a 120-minute
contact time that included mixing the topsoil and
decontaminant after each application.
• The efficacy result with the 120-minute contact time
is statistically significantly greater than the efficacy
at the 60-minute contact time.
9.2 CASCAD SDF Results
• The quantitative efficacy of CASCAD SDF
for inactivating B. anthracis (Ames) spores on
topsoil was 0.59 (± 0.04) log reduction with two
applications and a 60-minute contact time, and 1.13
(±0.12) log reduction with four applications and a
120-minute contact time.
• The efficacy result with the 120-minute contact time
is statistically significantly greater than the efficacy
at the 60-minute contact time.
9.3 Oxonia Active Results
• The quantitative efficacy of Oxonia Active for
inactivating B. anthracis (Ames) spores on topsoil
was 0.58 (± 0.19) log reduction with six applications
and a 60-minute contact time, and 1.04 (± 0.10) log
reduction with 12 applications and a 120-minute
contact time that included mixing the topsoil and
decontaminant after each application.
• The efficacy result with the 120-minute contact time
is statistically significantly greater than the efficacy
at the 60-minute contact time.
9.4 Klozur Results
• The quantitative efficacy of Klozur for inactivating
B. anthracis (Ames) spores ontopsoil was 1.65 (±
0.14) log reduction with six applications and a 24-
hour contact time, and 3.50 (± 0.40) log reduction
with six applications and a 48-hour contact time.
• The efficacy results at the 24 and 48-hour contact
times are statistically significantly different from
one another, and both efficacy results with Klozur
are statistically significantly higher than any efficacy
result with any other decontaminant tested.
-------
-------
10.0
References
1. American Society for Testing and Materials Method
D2974-07a, Moisture, Ash and Organic Matter of Peat
and Other Organic Soils, ASTM International, West
Conshohocken, PA, 2007, DOI: 10.1520/D2974-07A,
www.astm.org.
-------
-------
Appendix A:
pH-Amended Bleach Description
and Application Procedure
General Description
For testing of efficacy against B. anthracis on soil, pH-
amended bleach consisted of bleach diluted in water and
its pH adjusted by addition of acetic acid. Specifically,
Ultra Clorox® Germicidal Bleach was used, which
contains 6.15% by weight sodium hypochlorite (NaOCl)
and < 1.0% sodium hydroxide (NaOH) in aqueous
solution. This product is strongly basic, having a pH
between 11-12, and has a density of 1.08 to 1.11 g/mL.
The pH adjustment is achieved by addition of 5% acetic
acid. The recipe for preparation of pH-amended bleach
for use as a decontaminant is as follows:
• Prepare 5% acetic acid solution by diluting 50
mL of glacial acetic acid up to 1 L with SFW in a
volumetric flask.
• Mix 9.4 parts SFW, 1 part Ultra Clorox® Germicidal
Bleach, and 1 part 5% acetic acid. The resulting
solution will have a pH of about 6.8 and a mean
total chlorine content, estimated based on dilution,
of about 6,200 ppm.
The active decontaminating agents in this final solution
are hypochlorite (OC1") and hypochlorous acid. The
effectiveness of bleach as a biological decontaminant is
widely known.
In previous testing of pH-amended bleach as a
decontaminant, neutralization of the bleach solution was
achieved using STS. The bleach formulation used in this
evaluation differed from that used in previous testing,
so the determination of the neutralization procedure
was repeated to establish neutralization conditions
appropriate for this evaluation.
Application Procedure for Testing
Based on previous test results with pH-amended bleach,
and considering the topsoil surface material to be used
in this testing, an application procedure for use in testing
was developed. The intent of this procedure was to
employ conventional and readily available equipment in
a relatively simple application process. Trial runs were
conducted to establish the appropriate concentration of
STS for neutralization of the pH-amended bleach.
The pH-amended bleach was prepared fresh shortly
before use on each day of testing, as described above.
The pH of the solution was measured and recorded as
part of the test data. A new non-corroding garden pump
sprayer was used to apply the solution of pH-amended
bleach to the test coupon surfaces. An identical sprayer
was used to apply SFW to positive control test coupons.
Each sprayer was fitted with a pressure gauge to indicate
the internal delivery pressure of the sprayer. The internal
pressure of each sprayer was maintained in a normal
range for use (i.e., 4 to 6 psi) throughout all applications.
Based on laboratory tests, such a range of pressures
produces a stable spray suitable for application on the
scale of coupon testing.
Testing of pH-amended bleach was conducted with two
different contact times, i.e., 60 and 120 minutes. The
step-by-step application procedure for the 60 minute
contact time was:
• Apply the pH-amended bleach solution to the test
coupons (or SFW to the positive control coupons)
from a distance of about one foot (12 inches)
using the sprayer at a delivery pressure within the
specified range, until the test coupon surfaces are
fully wetted by the solution.
• Re-apply the solution three times, i.e., at 15
minutes after the first application, 30 minutes after
the first application, and 45 minutes after the first
application.
• If necessary, pump up the pressure in the sprayer
before application to maintain pressure within the
specified range.
• When 60 minutes have elapsed since the start of
the first application, place the coupons into the
extraction solution (containing the neutralization
agent) along with any collected runoff of pH-
amended bleach.
The step-by-step application procedure for the 120
minute contact time was:
• Apply the pH-amended bleach solution to the test
coupons (or SFW to the positive control coupons)
from a distance of about one foot (12 inches)
using the sprayer at a delivery pressure within the
specified range, until the test coupon surfaces are
fully wetted by the solution.
• Re-apply the solution seven times, i.e., at 15,
30, 45, 60, 75, 90, and 105 minutes after the first
application.
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After each application, thoroughly mix the applied
bleach solution with the soil in the test coupon,
using a glass stirring rod.
If necessary, pump up the pressure in the sprayer
before application to maintain pressure within the
specified range.
When 120 minutes have elapsed since the start of
the first application, place the coupons and each
associated glass stirring rod into the extraction
solution (containing the neutralization agent) along
with any collected runoff of pH-amended bleach.
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Appendix B:
CASCAD Description and
Application Procedure
General Description
CASCAD Surface Decontamination Foam (SDF)
uses two liquid solutions (A and B) which react to
form a foam as they are mixed upon release from the
application device. These two solutions are made from
three separate reagents, having chemical composition as
follows:
• GPA-2100 (decontaminant) - solid reagent in
powder form consisting of dichloroisocyanuric acid
sodium salt, 70 to 100% by weight;
• GPB-2100 (buffer) - solid reagent in powder form
consisting of sodium tetraborate 10 to 30%, sodium
hydroxide 1 to 5 %, and sodium carbonate 40 to
65% by weight;
• GCE-2000 (surfactant) - liquid reagent consisting of
sodium myristyl sulfate 10 to 30%, sodium (C14-
16) olefin sulfonate 10 to 30%, ethanol denatured
3 to 9%, alcohols (C10-16) 5-10%, sodium sulfate
3 to 7%, sodium xylene sulfonate 1 to 5%, and a
proprietary mixture of sodium and ammonium salts
along with water and co-solvent >9% by weight.
The A and B solutions are prepared from these reagents
by the following procedure:
l.Make solutionAby adding 31.2 g (four7.8 g
packets) of GPA-2100 to 250 mL of SFW in a
graduated cylinder, and then dilute with SFW to 300
mL.
2. Mix with a micro stir bar until dissolved
3. Make solution B by adding 7.2 g (four 1.8 g
packets) of GPB-2100 to 250 mL of SFW in a
graduated cylinder.
4. Mix with a micro stir bar until dissolved.
5. Add 18 mL (four 4.5 mL packets) of GCE-2000 to
the solution from Step 4, mix, and then dilute with
SFW to 300 mL
For use on the small scale needed for testing, a manual
spray application bottle (the 600 mL Hand Held
Decontamination System) has been developed by Allen-
Vanguard that draws solutions A and B from separate
compartments and delivers them as a foam through a
single spray head. To fill and operate the Hand Held
System, follow these steps:
1. Pull the Locking Lever on the front of the
bottle housing forward and lift. This will
open the housing and expose the solution
bottles, which are labeled "A" and "B".
2. With the housing opened remove the caps
(turn counter clockwise) and pull out the
solution suction lines from the solution
bottles.
3. With the caps and suction lines removed
from both the "A" and "B" solution bottles:
a. Pour solution A into the bottle labeled
"A", and pour solution B into the
bottle labeled "B".
b. Assure that both bottles are seated in
the housing with the "B" bottle at the
front.
c. Place the suction lines back into the
"A" and "B" bottles and tighten both
"A" and "B" caps by turning them in a
clockwise direction.
4. Hold the suction line up with one hand
while closing the top of the housing with
the other hand. Make certain that the
Locking Lever snaps into its recess when
the housing top closes. The suction line
may be pinched closed if this procedure is
not followed correctly; the condition of the
line can be checked by looking through the
housing at the suction line.
5. To use the 600 mL Hand Held
Decontamination System, grasp the neck
of the housing with your dominant hand
and place the finger of this hand on the
trigger of the foam nozzle. Aim the tip of
the foam nozzle in the direction of the area
to be decontaminated and pump the trigger.
The trigger may have to be squeezed three
or four times to evacuate the air in the
suction line before foam is discharged.
Application Procedure for Testing
CASCAD SDF was applied to soil coupons using the
vendor-developed dual spray applicator. In previous
testing, neutralization of the CASCAD SDF was
achieved by addition of 0.5% STS to the extraction
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solution. Trial runs were conducted before testing
to establish the appropriate STS concentration for
neutralization of the applied CAS CAD SDK
Testing of CASCAD SDF was conducted with two
different contact times, 60 minutes and 120 minutes. In
testing with a 60-minute contact time, each soil coupon
was prepared by filling a 1 cm-deep glass Petri dish
with soil; in testing with a 120-minute contact time, the
same mass of soil was placed in a 3.5 cm-deep Teflon
beaker having approximately the same internal diameter
as the Petri dish. This difference was implemented to
assure capture of the added depth of foam resulting from
additional applications of the CASCAD SDF. The step-
by-step application procedures for testing of CASCAD
SDF with soil coupons was as indicated below.
• Follow the instructions provided above for
preparation of the reagent solutions and loading of
the manual spray applicator.
• Squeeze the trigger of the applicator head a few
times while pointing the applicator into a laboratory
sink or other waste container, until any air is cleared
from the applicator and CASCAD SDF is delivered
from the applicator as a foam.
For the 60-minute contact time:
• Apply the CASCAD SDF to the soil coupons using
the manual applicator from a distance of about one
foot (12 inches) while moving the nozzle, until the
soil is entirely covered with no less than one (1)
centimeter (3/8") deep foam.
• Allow the foam to remain on the coupons for 30
minutes.
• Reapply more CASCAD SDF and allow the foam to
remain on the coupons for an additional 30 minutes.
• When a total of 60 minutes has elapsed since
the first application, place each coupon into
the extraction solution (containing the STS
neutralization agent) along with any CASCAD SDF
accumulated on the coupon.
For the 120-minute contact time:
• Apply the CASCAD SDF to the soil coupons using
the manual applicator from a distance of about one
foot (12 inches) while moving the nozzle, until the
soil is entirely covered with no less than one (1)
centimeter (3/8") deep foam.
• Allow the foam to remain on the coupons for 30
minutes.
• Reapply more CASCAD SDF at 30 minutes, 60
minutes, and 90 minutes after the first application
(for a total of four applications).
• When a total of 120 minutes has elapsed since
the first application, place each coupon into
the extraction solution (containing the STS
neutralization agent) along with any CASCAD SDF
accumulated on the coupon.
After use, empty and clean the manual spray applicator
according to the instructions below.
Cleaning the Hand Held Decontamination
System
Clean the CASCAD SDF system after use by the
following procedure.
1. Dump any remaining decontamination
solution from both the "A" and "B" bottles
and dispose of the solutions following
appropriate waste procedures.
2. Thoroughly rinse both bottles with SFW,
then fill each bottle with SFW.
3. Place the filled bottles back into the
housing, insert the suction lines, and close
the housing.
4. Pump the trigger until the suction lines
and foam nozzle are free from the
decontamination solution.
5. Flush the interior and the exterior of the
housing, and the caps used while mixing
the solution, thoroughly with SFW.
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Appendix C:
Oxonia Actve Description and
Application Procedure
General Description
Oxonia Active®, a liquid sanitizer made by Ecolab
Inc., consists of 27.5% hydrogen peroxide (H2O2) and
5.8% peroxyacetic acid (CH3CO(O2)H) by weight in
water (density =1.13 g/mL). Oxonia Active is used for
sterilizing a variety of surfaces and containers in food,
packaging, and other industries, and can be applied as
a liquid or foam, or as droplets by fogging the target
area. A temporary approval (crisis exemption) of
Oxonia Active was granted by the U.S. Environmental
Protection Agency for decontamination of Bacillus
anihracis spores on non-porous surfaces, at defined
temperatures, contact times, and dilution of the product.
Application Procedure for Testing
This application procedure for use of Oxonia Active
on topsoil was developed in consultation with the
vendor. The aim is to use a relatively simple application
process that is likely to be effective when carried out
with conventional and readily available equipment.
Trial runs were conducted to establish the appropriate
concentration of STS to be used in the spore extraction
solution for neutralization of Oxonia Active.
For testing, a decontaminant solution containing 5,000
ppm peroxyacetic acid was prepared fresh daily by
diluting 76 mL of Oxonia Active to 1 L with SF W.
The Ecolab Peroxyacid Test Kit was used for periodic
verification of the peroxyacetic acid concentration in the
undiluted Oxonia Active from which the decontaminant
solution was prepared.
A non-corroding garden pump sprayer was used to
apply the diluted Oxonia Active solution to the test
coupons. An identical sprayer was used to apply SFW
to positive control test coupons. Each sprayer was fitted
with a pressure gauge to indicate the internal delivery
pressure of the sprayer, which was maintained in a
normal range for use (i.e., 4 to 6 psi) in all applications.
Based on laboratory tests, such a range of pressures
produces a stable spray, suitable for application on the
scale of coupon testing. All applications were done at
normal room temperature (approximately 20 °C (68
°F)). Testing of Oxonia Active was conducted with two
different contact times, 60 minutes and 120 minutes.
The step-by-step application procedure for Oxonia
Active is shown below:
For the 60-minute contact time:
• Apply the decontaminant solution to the soil test
coupons (or SFW to the positive control coupons)
from a distance of about one foot (12 inches)
using the sprayer at a delivery pressure within
the specified range. Spray the solution onto the
coupons until the test coupons are visibly wet.
• Reapply the decontaminant solution if coupon
surfaces become visibly dry, and regardless of the
wetness of the coupons re-apply the decontaminant
solution at 10 minute intervals after the initial
application.
• If necessary, pump up the pressure in the sprayer
before application to maintain pressure within the
specified range.
• When 60 minutes have elapsed since the start of
the first application, place each soil coupon into the
extraction solution (containing the neutralization
agent) along with any decontaminant solution
accumulated on the coupon.
For the 120-minute contact time:
• Apply the decontaminant solution to the soil test
coupons (or SFW to the positive control coupons)
from a distance of about one foot (12 inches)
using the sprayer at a delivery pressure within the
specified range. Spray the solution onto the coupons
until the test coupons are visibly wet. Stir the soil
with a glass stirring rod to thoroughly mix the soil
with the applied decontaminant solution. Use a
different glass stirring rod for each of the test and
positive control coupons.
• Reapply the decontaminant solution if coupon
surfaces become visibly dry, and regardless of the
wetness of the coupons re-apply the decontaminant
solution at 10 minute intervals after the initial
application. After each application stir the soil with
a glass stirring rod to thoroughly mix the soil with
the applied decontaminant solution.
• If necessary, pump up the pressure in the sprayer
before application to maintain pressure within the
specified range.
• When 120 minutes have elapsed since the start
of the first application, rinse each glass stirring
rod with 10 mL of the extraction solution
(containing the neutralization agent) and place each
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corresponding coupon into the extraction solution
along with any collected runoff of decontaminant
solution.
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Appendix D:
Klozur Description and Application Procedure
General Description
Klozur™ is a solid reagent made by FMC Corporation
that is used for in situ and ex situ chemical oxidation
of contaminants in environmental remediation
applications. Klozur consists of sodium persulfate
(Na2S2O8) of purity > 99%, in the form of white,
odorless crystals. In remediation applications, Klozur
is injected into contaminated soil or groundwater, and
activated by mixing in appropriate proportions with
hydrogen peroxide (H2O2) solutions of up to 8% H2O2
by weight, according to instructions published by FMC
Corporation1. Activation of Klozur with H2O2 generates
sulfate radicals (SO4"«), which are capable of destroying
a wide range of organic contaminants.
Application Procedure for Testing
This application procedure for use of Klozur on
topsoil was developed by EPA and Battelle based on
information published by the vendor. The aim is to use
a relatively simple application process that is likely to be
effective when carried out with conventional and readily
available equipment. The procedure involves application
of Klozur, followed by application of the H2O2 activating
solution, consistent with the recommended approach for
soil remediation. Trial runs were conducted to establish
the appropriate concentration of STS to be used in the
spore extraction solution for neutralization of the Klozur/
H2O2 mixture after decontamination.
For testing, a 0.5 M solution of persulfate was prepared
by dissolving 12 grams of Klozur in SFW and diluting
with SFW to 100 mL final volume. This solution is
11.9% persulfate by weight. Commercially prepared 8%
H2O2 solution was purchased for use in testing. Equal
volumes of these Klozur and H2O2 solutions provide a
molar ratio of 5:1 (H2O2:persulfate), which is a typical
ratio for application in site remediation activities.
Application of Klozur to the topsoil test coupons was
done by injecting 1 mL of the 0.5 M persulfate solution
into each coupon using a laboratory pipet, and mixing
the soil and persulfate solution thoroughly using a glass
stirring rod. A 1 mL volume of the 8% H2O2 activating
solution was then applied to the coupon in the same
manner and mixed with the soil using the same glass
stirring rod. This process was repeated for multiple
applications. All applications were done at normal room
temperature (approximately 20 °C (68 °F)). Testing
of Klozur was conducted with two different total
contact times, 24 hours and 48 hours. The step-by-step
application procedure for Klozur was:
For the 24-hour contact time:
• Inject 1 mL of the 0.5 M persulfate solution into
each test coupon and associated blank coupon.
Mix the topsoil and persulfate solution thoroughly
using a glass stirring rod. Inject 1 mL of SFW into
each positive control coupon and associated blank
coupon, and thoroughly mix the topsoil and SFW
with a glass stirring rod. (Use a different stirring
rod for each topsoil coupon.)
• Immediately inject 1 mL of 8% H2O2 solution into
each test coupon and associated blank coupon, and
thoroughly mix the topsoil and injected solutions
again with the stirring rod. Inject another 1 mL
of SFW into each positive control coupon and
associated blank coupon, and thoroughly mix the
topsoil and SFW again with the stirring rod.
• Repeat both application steps above at intervals of
60 minutes after the initial application, until a total
of six applications have been made.
• Keep all coupons in the test enclosure until 24 hours
have elapsed since the first application, and then
place each soil coupon into the extraction solution
(containing the neutralization agent) along with
all the decontaminant solution accumulated on the
coupon.
For the 48-hour contact time:
• Inject 1 mL of the 0.5 M persulfate solution into
each test coupon and associated blank coupon.
Mix the topsoil and persulfate solution thoroughly
using a glass stirring rod. Inject 1 mL of SFW into
each positive control coupon and associated blank
coupon, and thoroughly mix the topsoil and SFW
with a glass stirring rod. (Use a different stirring
rod for each topsoil coupon.)
• Immediately inject 1 mL of 8% H2O2 solution into
each test coupon and associated blank coupon, and
thoroughly mix the topsoil and injected solutions
again with the stirring rod. Inject another 1 mL
of SFW into each positive control coupon and
associated blank coupon, and thoroughly mix the
topsoil and SFW again with the stirring rod.
• Repeat both application steps above at intervals of
60 minutes after the initial application, until a total
of six applications have been made.
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Keep all coupons in the test enclosure until 48 hours
have elapsed since the first application, and then
place each soil coupon into the extraction solution
(containing the neutralization agent) along with
all the decontaminant solution accumulated on the
coupon.
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Reference
Procedure for Activating Klozur™Persulfate with
an 8% Hydrogen Peroxide Solution, Document
02-01-EIT-DH, FMC Corporation, Philadelphia,
PA, Copyright 2008 FMC Corporation. All rights
reserved
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United States
Environmental Protection
Agency
PRESORTED STANDARD
POSTAGES 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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