EPA 600/R-11/11Q | October 2011 | www.epa.gov/ord
United States
Environmental Protection
Agency
Decontamination of
Materials with Ozone Gas
in the Presence of
Vaporous Organic Compounds
TECHNOLOGY EVALUATION
REPORT

Office of Research and Development
National Homeland Security Research Center

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EPA 600/R-11/110
October 2011
Technology Evaluation Report
Decontamination of Materials with Ozone Gas in
the Presence of Vaporous Organic Compounds
UNITED STATES ENVIRONMENTAL PROTECTION AGENCY
CINCINNATI, OHIO 45268
li

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Table of Contents
Disclaimer	iv
Foreword	v
List of Figures	vi
List of Tables	vii
Abbreviations/Acronyms	viii
Acknowledgments	ix
Executive Summary	x
1.0 Introduction	1
2.0 Summary of Test Procedures	3
2.1	Preparation of Test Coupons	3
2.2	Ozone Generation and Monitoring	5
2.3	Reactive Organic Compound Introduction and Monitoring	5
2.4	Decontaminant Testing	7
2.5	Decontamination Efficacy	8
3.0 Quality Assurance/Quality Control	10
3.1	Equipment Calibration	10
3.2	QC Results	10
3.3	Audits	11
3.3.1	Performance Evaluation Audit	11
3.3.2	Technical Systems Audit	11
3.3.3	Data Quality Audit	12
3.4	Test/QA Plan Amendments and Deviations	12
3.5	QA/QC Reporting	12
3.6	Data Review	12
4.0 Results	13
4.1	Test Conditions	13
4.2	Test Results	13
4.3	Decontamination Efficacy	13
5.0 Summary and Conclusions	48
6.0 References	49
in

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Disclaimer
The U.S. Environmental Protection Agency through its Office of Research and Development
funded and managed the research described here under Work Assignment 1-11 of EPA Contract
Number EP-C-10-001 to Battelle Memorial Institute. It 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.
Questions concerning this document or its application should be addressed to:
Sang Don Lee
U.S. Environmental Protection Agency
Office of Research and Development
National Homeland Security Research Center
109 T.W. Alexander Dr. (MD-E343-06)
Research Triangle Park, NC 27711
Phone: (919) 541-4531
Fax (919) 541-0496
lee. sangdon@epa. gov
iv

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Foreword
The U.S. Environmental Protection Agency (EPA) is charged by Congress with protecting the
nation's air, water, and land resources. Under a mandate of national environmental laws, the
Agency strives to formulate and implement actions leading to a compatible balance between
human activities and the ability of natural systems to support and nurture life. To meet this
mandate, the EPA's Office of Research and Development (ORD) provides data and science
support that can be used to solve environmental problems and to build the scientific knowledge
base needed to manage our ecological resources wisely, to understand how pollutants affect our
health, and to prevent or reduce environmental risks.
In September 2002, EPA announced the formation of the National Homeland Security Research
Center (NHSRC). The NHSRC is part of the ORD; it manages, coordinates, supports, and
conducts a variety of research and technical assistance efforts. These efforts are designed to
provide appropriate, affordable, effective, and validated technologies and methods for addressing
risks posed by intentional releases of chemical, biological, and radiological agents. Research
focuses on enhancing our ability to detect, contain, and decontaminate in the event of such
releases.
The NHSRC conducts decontamination testing in an effort to provide reliable information
regarding the performance of decontamination approaches. Such testing provides independent,
quality assured performance information that is useful to decision makers in purchasing or
applying the tested approaches. Information on the variety of homeland security technologies
and topics that NHSRC research has evaluated can be found at
http://www.epa.gov/nhsrc/pubs.html.
Jonathan G. Herrmann, Director
National Homeland Security Research Center
v

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List of Figures
Figure E-l. Comparison of Efficacy Results in Duplicate Tests	xii
Figure 1. Efficacy for B. anthracis at each test condition, by coupon material. Tests shown
in chronological order	42
Figure 2. Efficacy for B. subtilisat each test condition, by coupon material. Tests shown in
chronological order	43
Figure 4-3. Comparison of Efficacy Results in Duplicate Tests	47
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List of Tables
Table E-l. Summary of Efficacy Results	xi
Table 2-1. Test Materials	4
Table 3-1. Performance Evaluation Audits	11
Table 4-1. Summary of Test Conditions	14
Table 4-2a. Inactivation of Bacillus anthracis (Ames) Spores" - Ozone Only	15
Table 4-2b. Inactivation of Bacillus subtilis Spores3 - Ozone Only	16
Table 4-3a. Inactivation of Bacillus anthracis (Ames) Spores" - 1,000 ppmv TME	17
Table 4-3b. Inactivation of Bacillus subtilis Spores" - 1,000 ppmv TME	18
Table 4-4a. Inactivation of Bacillus anthracis (Ames) Spores" - 1,000 ppmv TME (Repeat)
	19
Table 4-4b. Inactivation of Bacillus subtilis Spores" - 1,000 ppmv TME (Repeat)	20
Table 4-5a. Inactivation of Bacillus anthracis (Ames) Spores" - 2,000 ppmv TME	21
Table 4-5b. Inactivation of Bacillus subtilis Spores" - 2,000 ppmv TME	22
Table 4-6a. Inactivation of Bacillus anthracis (Ames) Spores" - 1,000 ppmv 1-Hexene	23
Table 4-6b. Inactivation of Bacillus subtilis Spores" - 1,000 ppmv 1-Hexene	24
Table 4-7a. Inactivation of Bacillus anthracis (Ames) Spores" - 1,000 ppmv 1-Hexene
(Repeat)	25
Table 4-7b. Inactivation of Bacillus subtilis Spores" - 1,000 ppmv 1-Hexene (Repeat)	26
Table 4-8a. Inactivation of Bacillus anthracis (Ames) Spores" - 1,000 ppmv 1-Hexene
(Hydrocarbon Introduced by Multiple Injections)	27
Table 4-8b. Inactivation of Bacillus subtilis Spores" - 1,000 ppmv 1-Hexene (Hydrocarbon
Introduced by Multiple Injections)	28
Table 4-9a. Inactivation of Bacillus anthracis (Ames) Spores" - 1,000 ppmv TME
(Hydrocarbon Introduced by Multiple Injections)	29
Table 4-9b. Inactivation of Bacillus subtilis Spores" - 1,000 ppmv TME (Hydrocarbon
Introduced by Multiple Injections)	30
Table 4-10a. Inactivation of Bacillus anthracis (Ames) Spores" - Ozone Only, 70% RH... 31
Table 4-10b. Inactivation of Bacillus subtilis Spores" - Ozone Only, 70% RH	32
Table 4-lla. Inactivation of Bacillus anthracis (Ames) Spores" - Ozone Only, 80% RH. .. 33
Table 4-1 lb. Inactivation of Bacillus subtilis Spores" - Ozone Only, 80% RH	34
Table 4-12a. Inactivation of Bacillus anthracis (Ames) Spores" - Ozone Only, 70% RH
(Repeat)	35
Table 4-12b. Inactivation of Bacillus subtilis Spores" - Ozone Only, 70% RH (Repeat).... 36
Table 4-13a. Inactivation of Bacillus anthracis (Ames) Spores" - Ozone Only, 80% RH
(Repeat)	37
Table 4-13b. Inactivation of Bacillus subtilis Spores" - Ozone Only, 80% RH (Repeat).... 38
Table 4-14a. Inactivation of Bacillus anthracis (Ames) Spores" - 1-Hexene, 80% RH	39
Table 4-14b. Inactivation of Bacillus subtilis Spores" - 1-Hexene, 80% RH	40
Table 4-15. Summary of Efficacy Results	41
Table 4-16. Comparison of Efficacy Results to Pooled Control Results	46
vii

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Abbreviations/Acronyms
ACS
American Chemical Society
ATCC
American Type Culture Collection
B. anthracis
Bacillus anthracis (Ames strain)
B. subtilis
Bacillus subtilis
BBRC
Battelle Biomedical Research Center
BSC
biosafety cabinet
°C
degrees Celsius
CAS
Chemical Abstracts Service
CFU
colony-forming unit(s)
CI
confidence interval
cm
centimeter
COR
Contracting Officer's Representative
EPA
U.S. Environmental Protection Agency
FID
flame ionization detector
HC1
hydrochloric acid
hr
hour
L
liter
m3
cubic meter
mbar
millibar
min
minute
mL
milliliter
|iL
microliter
NHSRC
National Homeland Security Research Center
NIST
National Institute of Standards and Technology
03
ozone
OH-
hydroxyl radical
ORD
EPA Office of Research and Development
PBS
phosphate-buffered saline
PE
performance evaluation
ppmv
parts per million by volume in air
psi
pounds per square inch
QA
quality assurance
QC
quality control
QMP
quality management plan
RH
relative humidity
rpm
revolutions per minute
SD
standard deviation
SE
standard error
SFW
sterile filtered water (cell-culture grade)
THC
total hydrocarbon
TME
tetramethyl ethyl ene
TSA
technical systems audit
viii

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Acknowledgments
Contributions of the following individuals and organizations to the development of
this document are gratefully acknowledged.
United States Environmental Protection Agency (EPA)
Joseph Wood
Worth Calfee
Leroy Mickelsen
Timothy Dean
Battelle Memorial Institute
IX

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Executive Summary
The U.S. Environmental Protection Agency's (EPA's) 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. In previous
testing for NHSRC, ozone gas (O3) was used for inactivation of spores of Bacillus anthracis and
other organisms. Unsaturated organic compounds are known to react rapidly with O3 to produce
highly reactive species (e.g., hydroxyl radicals, OH«) and reaction products (e.g., formaldehyde),
both of which may be effective sporicides. Consequently, mixtures of O3 and reactive organic
compounds may be more effective sporicides than O3 by itself. This study investigated the
effectiveness of O3 combined with a reactive gas phase organic compound for inactivating spores
of B. anthracis (Ames) and the surrogate organism Bacillus subtilis on three representative test
materials.
Experimental Procedures. Coupons of glass, bare pine wood, and galvanized metal ductwork
were inoculated with spores of B. anthracis or B. subtilis and then decontaminated by exposure
to O3 or mixtures of O3 and a reactive organic compound. Two reactive organic compounds
were used in combination with O3 (Chemical Abstracts Service [CAS] registry numbers as
indicated):
•	2,3-dimethyl-2-butene (CAS No. 563-79-1) (also known as tetramethylethylene [TME])
•	1-hexene (CAS No. 592-41-6).
In all tests, an O3 concentration of 9,000 parts per million by volume (ppmv) was maintained
over a 4-hour contact time in a 0.57 cubic meter (m3) test chamber, at room temperature
(approximately 25 degrees Celsius (°C)) and a target controlled relative humidity (RH) between
70% and 80%. Inoculated test coupons were exposed to O3 or the O3 + reactive organic
compound reaction mixture for 4 hours, and spores (Bacillus anthracis and Bacillus subtilis)
were then extracted from the coupons for determination of decontamination efficacy by
comparison to control coupons similarly inoculated but not exposed.
Results. Table E-l summarizes the efficacy results obtained with the two test organisms on the
three coupon materials.
Table E-l shows (via bold type) that 29 of the 36 efficacy results with B. anthracis in Tests 2 to
13 were significantly greater than the corresponding results in the control test (Test 1), as
determined by a comparison of 95% confidence interval (CI) values. Although Table E-l shows
many efficacy results for the test organisms that exceeded the efficacy in the control test (Test 1),
most of those efficacy results (especially for B. anthracis) are less than a 4 log reduction.
Moreover, except for the relatively high efficacy results for B. anthracis in Tests 5 and 13, the
efficacy results are not consistently dependent on the identity, concentration, number of
vaporizations, or even presence of the reactive organic compounds. The relatively high efficacy
results for B. anthracis from Tests 5 and 13 are consistent with an effect from the added 1-
hexene and/or from humidity dependence of the susceptibility of B. anthracis spores to
x

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inactivation by O3. These results indicate that use of an O3 + 1-hexene reaction mixture and RH
of at least 80% may significantly enhance decontamination efficacy for B. anthracis, relative to
the use of O3 alone. For B. subtilis a dependence of efficacy on organic compound identity,
organic compound concentration, or RH is not apparent.
Table E-l shows that only 6 of the 36 efficacy results with B. subtilis were significantly greater
than the corresponding results in the control test. Table E-l also shows that efficacy was usually
higher for B. subtilis than for B. anthracis on glass and metal coupons, but usually lower for B.
subtilis than for B. anthracis on wood coupons. Table E-l also shows (via underlined type) that
of the 39 efficacy results for B. subtilis, 22 were significantly different (by comparison of 95%
CI values) from the efficacy result for B. anthracis in the same test with the same coupon
material. This observation indicates that B. subtilis is not a suitable surrogate organism for B.
anthracis in testing with O3 + reactive organic compound reaction mixtures. The comparison of
B. subtilis and B. anthracis efficacy differed markedly with test material.
Table E-l. Summary of Efficacy Results
Test Number
Efficacy (Lo
5 Reduction)b
and Organic
B. anthracis
B. subtilis
Compound3
Glass
Wood
Metal
Glass
Wood
Metal
1. Ozone
Onlyc
1.10
1.32
0.82
2.77
0.25
2.77
2. TMEd
(1,000 ppmv)
1.78
2.36
2.05
4.39
0.81
2.83
3. TME
(1,000 ppmv)e
1.35
1.79
1.75
3.11
0.66
2.14
4. TME
(2,000 ppmv)
2.53
2.15
3.95
2.88
0.96
1.98
5. 1-Hexene
(1,000 ppmv)
6.49
4.16
4.51
4.82
0.82
4.02
6. 1-Hexene
(1,000 ppmv)6
2.36
1.83
1.65
5.15
1.66
3.03
7. 1-Hexene
(1,000 ppmv)f
1.36
1.54
1.40
4.56
0.36
1.59
8. TME
(1,000 ppmv)f
1.50
2.37
1.73
5.80
1.30
2.47
9. Ozone
Onlyc'8
1.81
2.78
1.65
4.64
1.82
2.86
10. Ozone
Onlych
1.80
2.92
2.16
5.67
1.23
2.73
11. Ozone
Onlyc'e'8
1.39
2.35
2.59
3.04
0.82
1.68
12. Ozone
Onlyceh
2.07
2.94
2.38
6.67
3.58
3.05
13. 1-Hexene
(1,000 ppmv)h
5.32
5.02
3.13
6.84
0.95
4.10
a Tests listed in chronological order in which they were performed.
XI

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b Bold type indicates result significantly greater than corresponding control (Test 1) result. Underlined type
indicates efficacy result for B. subtilis that is significantly different from the corresponding efficacy for B.
anthracis.
0 Test with 9,000 ppmv 03 only, no added reactive hydrocarbon.
d TME = tetramethylethylene.
e Repeat test.
f Reactive hydrocarbon introduced in 16 equal injections at 15-ininute intervals over 4-hour contact time, rather
than as a single injection at the start of the contact time.
g Target RH 70%.
h Target RH 80%.
The results in Table E-l raise the issue of reproducibility of the efficacy results from the testing.
Figure E-l addresses this issue by showing a comparison of efficacy results for both B. anthracis
and B. subtilis in duplicate tests. The horizontal axis of Figure E-l shows the efficacy value
found for each organism on each coupon type in the first of two duplicate tests, and the vertical
axis shows the corresponding efficacy value found in the second of two duplicate tests. The 1-
to-1 line is shown, along with parallel lines indicating a range of ±1 log reduction relative to the
1-to-l line. The duplicate test pairs from which data were drawn for Figure E-l were Tests 2 and
3, 5 and 13, 9 and 11, and 10 and 12.
Efficacy (Log Reduction), FirstTest
~ Ba Glass
¦ Ba Wood
Ba Metal
BsGlass
~ Bs Wood
A Bs Metal
Figure E-l. Comparison of efficacy results in duplicate tests.
Figure E-l shows that for B. anthracis, duplicate efficacy results agree within ±1 log reduction in
10 of the 12 cases. The duplicate results for B. subtilis agree within ±1 log reduction in 7 of the
Xll

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12 cases. Based on the limited data set it is difficult to conclude whether the material type
affects the degree of duplication of test results. Figure E-l indicates that duplication of efficacy
results can typically be achieved within approximately 1 log reduction in tests with variables
such as RH control and organic compound introduction as performed in this study.
Summary. The primary conclusion from the series of tests reported here is that the efficacy of
9,000 ppm of O3 for inactivating B. anthracis and B. subtilis spores over a 4-hour exposure was
not consistently increased by the addition of either TME or 1-hexene. A possible exception for
B. anthracis is the addition of 1,000 ppm of 1-hexene in the presence of approximately 80% RH,
which produced the only efficacy values above 4 log reduction for that organism. Those efficacy
values may result from the impact of 1-hexene reaction products and/or an effect of RH on O3
decontamination efficacy for B. anthracis. The addition of reactive organic compounds to O3 at
relatively high RH may be a valuable topic for further study.
Across the range of tests conducted, efficacy was usually higher for B. subtilis than for B.
anthracis on glass and metal coupons, but usually lower for B. subtilis than for B. anthracis on
wood coupons. These differences were statistically significant, based on a comparison of 95%
CI values. Most efficacy results were less than a 4 log reduction (especially for B. anthracis).
For B. subtilis, efficacy values above 4 log reduction were seen in nine of the 13 tests with the
glass coupons, but a dependence on organic compound identity, organic compound
concentration, or RH was not apparent.
Of the 39 efficacy results for B. subtilis, 22 were significantly different from the efficacy result
for B. anthracis in the same test with the same coupon material. This observation indicates that
B. subtilis might not be a suitable surrogate organism for B. anthracis in testing with ozone +
reactive organic compound reaction mixtures.
Efficacy results from duplicate tests agreed within ±1 log reduction in 10 of the 12 cases for B.
anthracis, and in 7 of the 12 cases for B. subtilis. Based on the limited data set it is not possible
to conclude whether the coupon material type affects the degree of duplication of test results.
These results indicate that efficacy results can typically be duplicated within approximately 1 log
reduction in tests with variables such as RH control and organic compound introduction as
performed in this study.

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1.0 Introduction
The U.S. Environmental Protection
Agency's (EPA's) 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. In response to the
needs of stakeholders, NHSRC conducts
research and 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-supported research 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 previous NHSRC testing (EPA report by
J.P. Wood, Ozone Gas Decontamination of
Materials Contaminated with Bacillus
anthracis Spores), ozone gas (O3) was used
as a fumigant for inactivation of spores of
Bacillus anthracis and other organisms
without the addition of other reactants or
oxidants. However, some unsaturated
organic compounds are known to react
rapidly with O3 to produce highly reactive
species (e.g., hydroxyl radicals, OH«) and
reaction products (e.g., formaldehyde), both
of which may be effective sporicides.
Consequently, mixtures of O3 and reactive
organic compounds may be more effective
sporicides than O3 by itself. The primary
purpose of this study was to investigate the
effectiveness of O3 combined with a reactive
gas phase organic compound for inactivating
spores of B. anthracis and the surrogate
organism Bacillus subtilis on three
representative test materials.
Two reactive organic compounds were used
in combination with O3 (Chemical Abstracts
Service [CAS] registry numbers as
indicated):
•	2,3-dimethyl-2-butene (CAS No.
563-79-1) (also known as
tetram ethyl ethylene [TME])
•	1-hexene (CAS No. 592-41-6).
In all tests, an O3 concentration of 9,000
parts per million by volume (ppmv) was
maintained over a 4-hour contact time in a
"3
0.57 cubic meter (m ) test chamber, at room
temperature (approximately 25 degrees
Celsius (°C)) and a pre-selected target
controlled humidity of 70, 75, or 80%
relative humidity (RH). Coupons of glass,
bare pine wood, and galvanized metal
ductwork were inoculated with B. anthracis
or B. subtilis spores and exposed to the O3 or
O3 + reactive organic compound reaction
mixture for 4 hours, and then spores were
extracted from the coupons for
determination of decontamination efficacy.
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The test procedures were specified in the
peer reviewed and EPA approved Test/QA
Plan. Data analysis in this study focused on
assessing whether the addition of a reactive
organic compound improved efficacy
relative to decontamination with O3 alone.
In addition, a comparison was made of
efficacy results from duplicate tests, as an
indication of the reproducibility of the entire
test procedure. The subsequent sections of
this report describe the test procedures,
document the QA/quality control (QC)
results from this study, and present and
summarize the efficacy results from the tests
performed.
2

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2.0 Summary of Test Procedures
A sequence of 13 tests was conducted using
different reactive organic compounds,
organic compound concentrations, and
organic compound introduction schedules,
with target test conditions of 9,000 ppmv
O3, room temperature (approximately 25
°C), a preselected target RH of 70, 75, or
80%, and 4-hour contact time. Section 5.1
documents the actual test conditions
achieved. In chronological order the tests
conducted were:
organic compound was introduced in a
series of 16 small vaporizations at 15-minute
intervals over the 4-hour contact time.
Repeat tests were performed in four cases as
indicated above (i.e., Tests 2 and 3; 5 and 6;
9 and 11; and 10 and 12) to assess the
reproducibility of results from duplicate
tests. In addition, Test 13 was conducted to
be a repeat of Test 5, in which the actual test
RH was nearly 80% rather than the target
RH of 75%.
1.	Control test with O3 only (no added
hydrocarbon)
2.	O3 + 1,000 ppmv TME
3.	O3 + 1,000 ppmv TME (repeat test)
4.	O3 + 2,000 ppmv TME
5.	O3 + 1,000 ppmv 1-hexene
6.	O3 + 1,000 ppmv 1-hexene (repeat
test)
7.	O3 + 1,000 ppmv 1-hexene (via
multiple vaporizations)
8.	O3 + 1,000 ppmv TME (via multiple
vaporizations)
9.	03 only, 70%RH
10.	03 only, 80% RH
11.	O3 only, 70%) RH (repeat test)
12.	O3 only, 80%) RH (repeat test)
13.	O3 + 1,000 ppmv 1-hexene, 80%>
RH.
The target RH in Tests 1 through 8 was 75
(±3)%; Tests 9 through 12 were conducted
with a target RH of either 70 (±3)% or 80
(±3)%>, as shown above. In Tests 2 through
6 and 13 the reactive organic compound was
introduced into the chamber by vaporization
of a single aliquot of the pure compound.
However, in Tests 7 and 8, the reactive
2.1 Preparation of Test Coupons
The B. anthracis (Ames) spores used for this
testing were grown from existing stock in
the Battelle Biomedical Research Center
(BBRC) and subjected to a stringent
characterization and qualification process,
required by the BBRC's standard operating
procedure for spore production, which
included the following:
•	Multi-locus variable-tandem repeat
analysis on the B. anthracis spore
suspension by the Centers for
Disease Control and Prevention.
This analysis targeted eight loci (six
chromosomally-located and one on
each of the two virulence plasmids)
and was used to discriminate B.
anthracis isolates.
•	pXOl and pX02 gene expression
testing via percent encapsulation
•	Phenotypic characterization via
microscopic morphology and colony
morphology to confirm purity and
quality of the spores
3

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•	Viability characterization via viable
spore count
•	Measurement of endotoxin content
•	Enrichment confirmation for purity.
In addition, testing was conducted by
Battelle personnel to confirm the robustness
of the spores via hydrochloric acid (HC1)
exposure. The stock spore suspension was
prepared in sterile filtered water (SFW) at an
approximate concentration of 1 x 109
spores/milliliter (mL) and stored by
refrigeration at 4 °C.
B. anthracis (Ames) and B. subtilis
(American Type Culture Collection [ATCC]
19659, grown from existing stock; identity
and purity previously verified by Battelle)
spores were inoculated onto test coupons in
an appropriate biosafety cabinet (BSC-III)
according to established BBRC procedures.
Inoculated coupons were prepared prior to
each day of experimental work. Coupons
were placed flat in the BSC-III and
o
inoculated at approximately 1 x 10 viable
spores per coupon. This inoculation was
accomplished by dispensing a 100-microliter
(|iL) aliquot of a spore stock suspension
(approximately 1 x 109 spores/mL) using a
micropipette as 10 droplets (each of 10 |iL
volume) across the surface of the test
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 test coupons remained
undisturbed overnight in the BSC to dry and
were used for testing within 24 hours after
inoculation.
The origin and specifications of the
materials used for test coupons are shown in
Table 2-1. Representative products of the
three material types were selected based on
consultation with materials suppliers. All
test coupons were 1.9 x 7.5 centimeters (cm)
in size and made from new materials.
Coupons were sterilized before use by the
means provided in Table 2-1. Autoclaving
of glass and metal coupons was done by
Battelle; gamma irradiation of wood
coupons was done by STERIS Isomedix
Services, Libertyville, IL. Sterility was
confirmed by laboratory and procedural
blanks of all three coupon materials in the
testing process.
Table 2-1. Test Materials
Material
Lot, Batch, or
Observation
Manufacturer/
Supplier Name
Material
Sterilization
Glass
C1036
Brooks Brothers
Glass; Columbus, OH
Autoclave
Wood
(untreated pine)
Generic molding
WJ Hardware;
West Jefferson, OH
Gamma
irradiation
Galvanized Metal
Not Applicable
Adept Products;
West Jefferson, OH
Autoclave
4

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2.2 Ozone Generation and Monitoring
Ozone (O3) was produced and delivered into
the 0.57 m3 BSC-III test chamber (Model
50635, The Baker Company, Sanford,
Maine) using a Model AC-2045 Ozone
Generator (IN USA, Inc., Norwood, MA),
using oxygen with 1% nitrogen content as
the feed gas to the generator. The chamber
was equipped with a continuously operating
fan that mixed injected ozone and/or reacted
hydrocarbons throughout the chamber
volume. The Model AC-2045 generator
allowed control of the electrical power used
for O3 production and the oxygen supply
pressure and flow rate, resulting in rapid
delivery and close control of the rate of O3
delivery. A flow of 10 standard liters (L)
per minute (min) of oxygen at a supply
pressure of 25 pounds per square inch (psi)
(1,724 millibars), and 100% electrical
power, raised the O3 concentration in the test
chamber from zero to 9,000 ppmv in
approximately 20 to 25 minutes. Once that
concentration was attained, the flow,
pressure, and power setting were adjusted to
0.5 standard L/min, 20 psi, and 30% power,
respectively, and introduction of O3 was
conducted intermittently to maintain the O3
concentration at 9,000 ppmv.
Ozone was monitored continuously
throughout all testing using a Model
IN2000-L2-LC Low Concentration Ozone
Analyzer (IN USA, Inc.). That analyzer
determined O3 by ultraviolet absorption,
with response based on an absolute
calibration traceable to the National Institute
of Standards and Technology (NIST). Once
the 9,000 ppmv O3 concentration was
established in the test chamber, test
operators observed the readings of the
Model IN2000-L2-LC O3 analyzer and
manually turned the Model AC-2045 O3
generator on for brief periods to maintain
the O3 concentration during the 4-hour test
periods. The exhaust gas flow of the Model
IN2000-L2-LC O3 analyzer was routed back
into the test chamber, so that O3 monitoring
did not result in any withdrawal of air from
the chamber. The readings of the O3
monitor were recorded electronically in all
testing.
In Tests 1 and 2, the decay of O3 in the test
chamber was measured before the
introduction of the reactive hydrocarbon. In
those tests, once the chamber O3
concentration reached 9,000 ppmv the O3
introduction was stopped and the O3
concentration in the closed chamber was
monitored for 60 minutes using the Model
IN2000-L2-LC O3 analyzer. Then the O3
concentration was increased back to 9,000
ppmv and the test proceeded. The O3 decay
rates determined in Tests 1 and 2 were
30%/hour (hr) and 22%/hr, respectively.
The results in Section 5 show that the
manual control of the O3 concentration
easily maintained the target 9,000 ppmv O3
in all testing, despite this natural loss of O3
in the chamber.
2.3 Reactive Organic Compound
Introduction and Monitoring
The liquid reactive organic compounds were
obtained in small quantities as American
Chemical Society (ACS) Reagent Grade
chemicals (>99% purity) (Sigma Aldrich, St.
Louis, MO). Each compound was vaporized
into the test chamber using a venturi nozzle
attached to the test chamber. The nozzle
had a small conical reservoir to hold the
liquid reactive organic compound and was
operated using pressurized clean air
(approximately 25 psi pressure). Once the
9,000 ppmv O3 concentration was
established in the test chamber, the
appropriate volume of the reactive organic
compound being used was placed into the
reservoir, and the pressurized air was
5

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provided to the venturi. Vaporization of the
liquid organic compound into the chamber
was completed within approximately 20
seconds. In most tests the entire amount of
organic compound needed to produce the
target concentration was vaporized into the
chamber in a single injection. However, in
Test 7 with 1-hexene and Test 8 with TME,
the total amount of the compound was
divided and introduced in 16 equal
injections spaced at 15-minute intervals
throughout the 4-hour contact time.
The volume of each liquid reactive organic
compound needed to produce the target
vapor phase concentration was first
estimated based on the chamber volume, and
trial runs were then conducted to confirm
that the vaporization procedure produced the
correct organic compound concentration.
Each organic compound was monitored
using a Model 20 Heated Total Hydrocarbon
(THC) Analyzer (VIG Industries, Inc.,
Anaheim, CA) based on flame ionization
detection (FID). The continuous THC
analyzer and its supplies of hydrogen (Zero
Grade [<0.5 ppmv THC] or better) and
combustion air (Ultra Zero Grade [<0.1
ppmv THC or better]) (Air Li qui de, Troy,
MI) were placed near the test chamber. The
continuous THC analyzer drew its 3 L/min
sample flow from a three-way valve that
allowed sampling of air from either the test
chamber or the surrounding laboratory. The
THC analyzer was calibrated with a NIST-
traceable propane standard certified to better
than 5% accuracy (EPA Protocol Mixture;
Air Liquide, Troy, MI), and the monitoring
results were corrected to account for the
number of carbons in the target molecule.
For example, THC analyzer response to
TME (a six-carbon molecule) was twice the
response to an equimolar concentration of
propane (a three-carbon molecule).
Because of the high reactivity of the organic
compounds with O3, the trial runs duplicated
all aspects of the test procedure except the
introduction of O3. The organic compound
delivery was activated in the same way as in
a decontamination test, and the organic
compound concentration in the chamber was
monitored using the THC analyzer until the
concentration was stable. The three-way
valve on the inlet of the THC analyzer was
switched to allow the analyzer to sample
laboratory air instead of chamber air
whenever chamber air monitoring was not
being conducted. Based on trial runs, liquid
volumes of 2.8 and 5.6 mL of neat TME
were used to produce the 1,000 ppmv and
2,000 ppmv concentrations, respectively. A
liquid volume of 2.9 mL of neat 1-hexene
produced the 1,000 ppmv concentration of
that compound. In the two tests in which
multiple injections of the organic compound
were made, neat TME was introduced by 16
injections of 175 |iL each, and neat 1-
hexene was introduced by 16 injections of
181 |iL each. To assure complete delivery
of these small volumes of the organic
compounds, the syringe used to introduce
the organic compound into the conical
reservoir was pre-flushed with the organic
compound, and the organic compound was
injected directly into the very bottom of the
conical reservoir (i.e., without contact with
the sides of the reservoir).
No monitoring of organic compound
concentrations was conducted during actual
tests with O3 present. The organic
compound was not monitored partially
because of the high reactivity of the organic
compounds with O3, which presumably
destroyed the compounds immediately upon
their introduction to the chamber. Also, the
organic compound was not monitored
because of the necessity to minimize
exposure of the THC analyzer to the highly
corrosive O3 concentrations.
6

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2.4 Decontaminant Testing
In all tests a wet/dry bulb hygrometer
(comprised of two National Institute of
Standards and Technology [NIST]-traceable
thermometers [Fisher 13-990-270])
incorporating two NIST-traceable
thermometers was used to monitor the
chamber temperature every 2 to 15 minutes.
The wet/dry bulb temperatures were also
used to determine the chamber RH by means
of an online calculator
(www.ringbell.co.uk/info/humid.htm). The
inoculated coupons were placed into the test
chamber in closed containers (HPL838P,
Lock & Lock, Farmers Branch, TX), one
holding control coupons and one holding
test coupons. Prior to introduction of O3
into the chamber the containers were opened
and the coupons were allowed to equilibrate
with the RH in the chamber. Then the
containers were closed and O3 delivery to
the chamber began. When the O3
concentration reached 9,000 ppmv, the
container of test coupons was opened, and
the reactive organic compound was
vaporized into the test chamber where it
reacted with the O3. The test coupons were
left in contact with the O3 + organic reactant
mixture for 4 hours, and the container was
then closed again and the chamber was
purged with air to exhaust the O3 to an
appropriate vent. During the 4-hour contact
time the test operator adjusted the O3
delivery as described in Section 2.2 to
maintain the O3 concentration at the 9,000
ppmv target level.
For each combination of coupon material
and organism (B. anthracis or B. sub/His),
five replicate test coupons (inoculated with
spores and exposed to the O3 + reactive
organic compound), five replicate positive
control coupons (inoculated and not
exposed), one procedural blank (not
inoculated, exposed), and one laboratory
blank (not inoculated, not exposed) of each
coupon material were used in each test.
Following the 4-hour exposure, each test
coupon was transferred aseptically to a
sterile 50-mL conical vial containing 10 mL
of sterile phosphate-buffered saline (PBS)
solution with 0.1% Triton X-100 surfactant
(i.e., 99.9% PBS, 0.1% Triton X-100).
Coupons were then extracted by agitation on
an orbital shaker for 15 minutes at
approximately 200 revolutions per minute
(rpm) at room temperature. Following
extraction, 1 mL of the coupon extract was
removed, and a series of dilutions through
10"7 was prepared in SFW. An aliquot (0.1
mL) of the undiluted extract and/or of each
serial dilution was then spread-plated onto
tryptic soy agar plates (in triplicate) and
incubated overnight at 35 to 37 °C.
Resulting colonies were enumerated within
18 to 24 hours of plating. This incubation
period was determined to be ideal for
Bacillus species to grow colonies of
adequate size for enumeration and for
inspection of physical morphology to ensure
that a homogeneous culture grew on the
nutrient agar and that the target organism
was recovered. The inoculated and
recovered spore values are reported as
colony-forming units (CFU), as determined
by the enumeration process. Theoretically,
once plated onto bacterial growth media,
each recovered viable spore germinates and
yields one CFU. 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 the extract or dilution
that was plated.
Laboratory blank coupons controlled for
sterility and procedural blanks controlled for
viable spores inadvertently introduced to test
coupons. The procedural blanks were
spiked with an equivalent amount of 0.1 mL
7

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of "stock suspension" that did not contain
the biological agent. The target acceptance
criterion was that extracts of laboratory or
procedural blanks were to contain no CFU.
where Mean CFUpc is the mean number of
CFU recovered from five replicate positive
control coupons of a single material, and
CFUSpike is the number of CFU spiked onto
each of those coupons. The value of
CFUSpike is known from enumeration of the
stock spore suspension. Spore recovery was
calculated for B. anthracis and B. subtilis on
each coupon material in each test, and the
results are included in Section 4.
2.5 Decontamination Efficacy
The performance or efficacy of the O3 or O3
+ reactive organic compound reaction
mixture was assessed by determining the
number of viable organisms remaining on
each test coupon after the 4-hour contact
time. Those numbers were compared to the
number of viable organisms extracted from
the positive control coupons.
where logio CFUcy refers to the j individual
logarithm values obtained from the positive
control coupons and logio CFUty refers to
the j individual logarithm values obtained
from the corresponding test coupons, and
the overbar designates a mean value. In the
tests reported here, there were five control
and five corresponding test coupons (i.e. J =
5) for each coupon material. In the case
where no viable spores were found in any of
the five test coupon extracts after the 4-hour
contact time, a CFU abundance of 1 was
assigned, resulting in a logio CFU of zero
The mean percent spore recovery from each
coupon material was calculated using results
from positive control coupons (inoculated,
not exposed) by means of the following
equation:
(1)
The number of viable spores of B. anthracis
or B. subtilis in extracts of test and positive
control coupons was determined to calculate
efficacy of the O3 or O3 + reactive organic
compound reaction mixture. Efficacy is
defined as the extent (as logio reduction) by
which viable spores extracted from test
coupons after the contact time were less
numerous than the viable spores extracted
from unexposed positive control coupons, at
the same temperature, RH, and contact time
(i.e., higher efficacy indicates greater
effectiveness at inactivating spores). First,
the logarithm of the CFU abundance from
each coupon extract was determined, and
then the mean of those logarithm values was
determined for each set of positive control
and associated test coupons, respectively.
Efficacy of a decontaminant for a test
organism on the ith coupon material was
calculated as the difference between those
mean log values, i.e.:
(2)
for that material. In such a case, the final
efficacy on that material was reported as
greater than or equal to (>) the value
calculated by Equation 2. Efficacy values in
all instances where complete kill was
observed should be regarded as equal, as
numerical differences are due to variation in
inoculum titer and recovery efficiencies
from the various surfaces.
The variances (i.e., the square of the
standard deviation (SD)) of the logio CFUcy
and logio CFUty values were also calculated
Mean % Recovery = [Mean CFUpc/CFUSpike] x 100
Efficacy = (log10 CFUct]) - (log10 CFUt1})
8

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for both the control and test coupons (i.e.,
S2Cij and S2/.,/), and were used to calculate the
pooled standard error (SE) for the efficacy
value calculated in Equation 2, as follows:
S2c, S2i
SE = J	^ + —
V 5 5
where the number 5 again represents the
number j 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.
Differences in efficacy were judged as
significant if the 95% CIs of the two
efficacy results did not overlap. The
(3)
The significance of differences in efficacy
across different coupon materials and spore
types was assessed based on the 95%
confidence interval (CI) of each efficacy
result. For a set of five results (4 degrees of
freedom) the 95% CI is:
(4)
efficacy results are presented in Section 4
for each reactive organic compound by
coupon material.
95% CI = Efficacy ± (2.78 x SE)
9

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3.0 Quality Assurance/Quality Control
QA/ QC procedures were performed in
accordance with the applicable QMP and the
test/QA plan for this evaluation, as
amended, except as noted below. QA/QC
procedures and associated results are
summarized below.
3.1	Equipment Calibration
All equipment (e.g., pipettes, incubators,
biological safety cabinets), monitoring
devices (e.g., O3, temperature) and THC
analyzer used at the time of evaluation were
verified as being certified, calibrated, or
validated.
3.2	QC Results
QC samples generated during decontaminant
testing included positive control coupons
(inoculated, not exposed), procedural blanks
(not inoculated, exposed), laboratory blanks
(not inoculated, not exposed), and spike
control samples (analysis of the stock spore
suspension). The results for these QC
samples in each decontaminant evaluation
are included in Section 4 along with the
results from all tests.
Most positive control spore recovery results
were within the target range of 5 to 120% of
the spiked spores. Positive control recovery
values on glass and galvanized metal ranged
from approximately 26 to 94% for B.
anthracis and from approximately 23 to
83% for B. subtilis. However, positive
control recovery values on bare wood were
lower, ranging from 4.1 to 11.6% for B.
anthracis (with three recovery values less
than 5%) and from 1.4 to 13.3% for B.
subtilis (with 10 recovery values less than
5%). These relatively low spore recoveries
from wood coupons, especially for B.
subtilis, are consistent with recoveries of
these organisms found from this material in
previous testing.(1'2) All positive control
spore recoveries were more than sufficient
for determining the efficacy of O3 + reactive
organic compound decontaminant mixtures.
For example, the lowest positive control
spore recovery of 1.4% was sufficient to
determine an efficacy of up to 6.2 log
reduction. A memorandum documenting
thedeviation was prepared, approved, and
retained in the test files noting the
acceptance of the low spore recovery values.
All procedural and laboratory blanks met the
target criterion of no observed CFU in
testing with both B. anthracis and B.
subtilis.
Spike control samples were taken from the
B. anthracis and B. subtilis spore
suspensions on each day of testing, and
serially 10-fold 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
was known after completion of each day's
testing. The target criterion was to maintain
a spore suspension density of 1 x 109/mL (±
1 log),(1'2) leading to a spike of 1 x 107 to 1 x
109 spores on each test coupon. All of the
actual B. anthracis and B. subtilis spike
values for the 13 days of testing were well
within that range, with B. anthracis spike
n
values ranging from 5.7x10 to 1.54 x
10

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o
10 /coupon and B. subtilis spike values
ranging from 6.17 x 107 to 1.29 x
o
10 /coupon.
3.3 Audits
3.3.1 Performance Evaluation Audit
A performance evaluation (PE) audit was
performed on the THC monitor used to
determine the chamber concentrations of the
added reactive hydrocarbons, by analysis of
an independent hydrocarbon standard. A PE
audit was also conducted on the two
thermometers used to determine chamber
temperature and RH by means of wet/dry
bulb measurements. Those audits consisted
of comparisons to an independent NIST-
traceable thermometer. Also, the laboratory
digital clock used in testing was subjected to
a PE audit by comparison to an independent
time measurement. All of these PE audits
were conducted prior to the start of testing.
Table 3-1 shows the results of the PE audits,
and indicates that all PE audits met the
target tolerances set out in the amended
test/QA plan. Footnotes to Table 3-1
identify the PE audit standards used for
comparison. No PE audit was performed
involving O3, as appropriate certified
standards do not exist. Similarly, no PE
audit was conducted addressing the
concentration or purity of the B. anthracis
(Ames) or B. subtilis organisms because
quantitative standards for these biological
materials do not exist.
Table 3-1. Performance Evaluation Audits

Audit
Allowable
Actual
Measurement
Procedure
Tolerance
Tolerance
Total hydrocarbons
Analysis of independent
propane standard3
± 10%
1.8%
Temperature
Dry bulb thermometer
Wet bulb thermometer
Compared to NIST-traceable
thermometer13
± 2 °C
All <0.2 °C for 13
instances
Time
Compared time to independent time
measurement0
± 2 sec/hr
0 seconds in 2 hr
a: PE audit standard was 300 ppm Certified Master Class propane standard, cylinder number ALM058033,
expiration November, 2013 (AirLiquide, Houston, TX).
b: PE audit standard was a Type 13-990-270 NIST-traceable thermometer (Fisher Scientific, Hampton, NH).
c: PE audit standard was www.time.gov. the official U.S. time service of NIST and the U.S. Naval Observatory.
3.3.2 Technical Systems Audit
EPA QA staff conducted a technical systems
audit (TSA) of the test procedures at the
BBRC between March 8 and 11, 2011, to
ensure that the evaluation was being
conducted in accordance with the amended
test/QA plan and the QMP. As part of the
TSA, test procedures were compared to
those specified in the test/QA plan, and data
acquisition and handling procedures were
reviewed. Battelle QA staff also conducted
a separate TSA at the BBRC on March 31,
2011. As with the EPA audit, test
procedures were compared to those
specified in the test/QA plan. Observations
and findings from these TSAs were
documented and submitted to the Battelle
Work Assignment Leader for response. No
adverse findings resulted from either of
these TSAs. TSA records were permanently
stored with the Battelle QA Manager.
11

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3.3.3 Data Quality Audit
3.5 QA/QC Reporting
At least 10% of the 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.
3.4 Test/QA Plan Amendments and
Deviations
The test/QA plan for this evaluation was
adapted by amendment of a peer-reviewed,
fully approved plan established for a
previous evaluation. A memorandum noting
a deviation was prepared, approved, and
retained in the test files for this evaluation,
related to acceptance of several control
sample spore recoveries outside the target
range of 5 to 120% recovery. Those
recoveries are noted in Section 5.1. None of
those deviations had any significant effect
on the reported efficacy determinations.
Each audit was documented in accordance
with the QMP. The results of the audits
were submitted to the EPA (i.e., to the
NHSRC Quality Assurance Manager and the
EPA Contracting Officer's Representative
[COR]).
3.6 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.
12

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4.0 Results
This section summarizes the test conditions
maintained in the testing and presents the
detailed spore recovery and efficacy results
from each of the 13 tests.
4.1	Test Conditions
Table 4-1 summarizes the temperature and
RH conditions and O3 concentrations
established in the test chamber for each test.
Shown are the mean, SD, and range of the
temperature, RH, and O3 concentration
during the 4-hour contact time in each test.
Table 4-1 shows that the test conditions
were tightly controlled in all tests and
closely similar from one test to another. The
target test conditions, and the tests
conducted as repeats (i.e., duplicates), are
indicated by footnotes to Table 4-1.
4.2	Test Results
4.3 Decontamination Efficacy
Table 4-15 summarizes the efficacy results
found for each of the two organisms on each
of the three test materials in the 13 tests.
This table shows the efficacy values listed in
Tables 4-2 through 4-14 by organism and
test material, but does not repeat the 95% CI
values shown in those tables. Instead,
efficacy values in Table 4-15 are shown in
bold type when they are significantly greater
than the corresponding efficacy result in the
control test (Test 1). Also, the B. subtilis
efficacy results are underlined when they are
significantly different from the
corresponding efficacy results for B.
anthracis. As noted in Section 2.5, efficacy
results were judged to be significantly
different if their 95% CI values did not
overlap.
Tables 4-2a through 4-14b show the results
of all 13 tests. Each table lists the four types
of coupons used for each of three coupon
materials and shows the spore inoculum on
each test and positive control coupon, the
mean log of the observed CFU recovered
from each coupon type, the mean percent
spore recovery on each coupon type, and the
calculated efficacy on each material (with
95% CI). Within each of Tables 4-2 through
4-14, the "a" table shows results for B.
anthracis, and the "b" table shows results
for B. subtilis.
13

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Table 4-1. Summary of Test Conditions
Test Number
and Organic
Compoundb
Test Condition3
T Avg.
(±SD)
(°C)
T Range
(°C)
RH Avg.
(±SD)
(%)
RH Range
(%)
03 Avg.
(±SD)
(ppmv)
03 Range
(ppmv)
1. Ozone
Onlyc
24.9 ±0.7
23.3 -26.0
75.3 ±2.3
71.7-79.1
9,001 ±43
8,885 -
9,096
2. TMEd
(1,000 ppmv)
25.1 ±0.6
24.0-25.8
76.4 ±2.7
71.0-79.1
9,022 ± 23
8,408 -
9,299
3. TME
(1,000 ppmv)6
24.6 ±0.4
24.0-25.5
74.2 ± 1.9
71.1-78.3
9,035 ± 78
8,734-
9,269
4. TME
(2,000 ppmv)
24.3 ±0.5
23.8 -25.3
75.0 ± 1.6
72.8- 78.2
9,046 ± 127
8,020 -
9,207
5. 1-Hexene
(1,000 ppmv)
24.1 ±0.6
23.0-25.0
78.7 ± 1.0
75.9- 80.0
9,002 ± 127
8,092 -
9,215
6. 1-Hexene
(1,000 ppmv)6
23.6 ±0.8
21.8-24.8
74.4 ±2.0
70.5-76.8
9,001 ±227
7,173 -
9,250
7. 1-Hexene
(1,000 ppmv)f
24.1 ±0.7
22.5 -25.0
73.3 ± 1.6
71.3-76.4
9,007 ±91
8,632-
9,287
8. TME
(1,000 ppmv)f
23.8 ±0.7
22.5 -24.8
76.3 ± 1.9
70.3-79.5
9,059 ±119
8,482-
9,379
9. Ozone
Onlyc'8
23.6 ±0.5
22.5 -24.5
71.7 ± 1.0
68.8- 72.8
9,025 ± 139
8,152 -
9,283
10. Ozone
Onlych
23.2 ±0.7
21.8-24.0
79.5 ± 1.9
77.2- 82.0
8,995 ±111
8,681 -
9,344
11. Ozone
Onlyc'6'8
23.2 ±0.6
22.5 -24.3
70.7 ±2.1
66.6-74.0
9,050 ± 142
8,666-
9,347
12. Ozone
Onlyceh
23.9 ±0.5
22.8-24.8
81.4 ±2.7
78.1 - 84.3
9,025 ± 124
8,699-
9,251
13. 1-Hexene
(1,000 ppmv)h
24.2 ±0.6
22.5 -25.0
81.4 ± 1.5
77.6- 82.5
8,980 ±205
7,945 -
9,420
a T = temperature, RH = relative humidity, 03 = ozone, SD = standard deviation. Target conditions were 25 °C and
9,000 ppmv 03; target RH was 75%, unless otherwise denoted by footnotes g and h, below.
b Tests listed in chronological order in which they were performed.
0 Test with 9,000 ppmv 03 only, no added reactive hydrocarbon.
d TME = tetramethylethylene.
e Repeat test.
f Reactive hydrocarbon introduced in 16 equal injections at 15-minute intervals over 4-hour contact time, rather
than as a single injection at the start of the contact time.
8 Target RH 70%.
h Target RH 80%.
14

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Table 4-2a. Inactivation of Bacillus anthracis (Ames) Spores" - Test 1, Ozone Only	
T .	Mean of Logs A
Test Material	"rrF"! h™ °f Observed R ean °	Efficacy ± CI
CFU	Kecovery
Glass
Positive Controlsb	8.23 x 107	7.63 ± 0.06	51.7 ±6.8
Test Coupons0	8.23 x 107	6.53 ± 0.06	4.1 ±0.54 1.10 ± 0.10
Laboratory Blankd 0 0	0
Procedural Blank6 0 0	0
Bare Wood
Positive Controls	8.23 x 107	6.86 ± 0.08	8.9 ±1.7
TestCoupons	8.23 x 107	5.54 ±0.11	0.43±0.13 1.32 ± 0.18
Laboratory Blank 0 0	0
Procedural Blank 0 0	0
Galvanized Metal
Positive Controls	8.23 x 107	7.75 ± 0.09	69.0 ±13.3
TestCoupons	8.23 x 107	6.93 ± 0.48	16.7 ± 16.7 0.82 ±0.61
Laboratory Blank 0 0	0
Procedural Blank 0 0	0
a Data are expressed as the mean (± SD) of the logs of the number of spores (CFU) observed on five individual
coupons, the mean percent recovery on those five coupons, and decontamination efficacy (log reduction).
CI = confidence interval (± 2.78 x SE).
b Positive Controls = inoculated, not decontaminated coupons (sprayed with SFW).
0 Test Coupons = inoculated, decontaminated coupons.
d Laboratory Blank = not inoculated, not decontaminated coupon.
e Procedural Blank = not inoculated, decontaminated coupon.
Not Applicable.
15

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Table 4-2b. Inactivation of Bacillus subtilis Spores3 - Test 1, Ozone Only	
T .	Mean of Logs A
Test Material	"rrF"! h™ °f Observed R ean °	Efficacy ± CI
CFU	Kecovery
Glass
Positive Controlsb	7.77 x 107	7.59 ±0.14	51.7 ±17.2
Test Coupons'	7.77 x 107	4.82 ± 1.46	0.94 ± 1.6 2.77 ±1.81
Laboratory Blankd 0 0	0
Procedural Blank6 0 0	0
Bare Wood
Positive Controls	7.77 x 107	6.18 ±0.26	2.2 ±1.3
Test Coupons	7.77 x 107	5.93 ±0.04	1.1 ±0.1 0.25 ± 0.32
Laboratory Blank 0 0	0
Procedural Blank 0 0	0
Galvanized Metal
Positive Controls	7.77 x 107	7.66 ±0.16	61.9 ±23.3
Test Coupons	7.77 x 107	4.88 ± 0.75	0.22 ±0.22 2.77 ±0.96
Laboratory Blank 0 0	0
Procedural Blank 0 0	0
a Data are expressed as the mean (± SD) of the logs of the number of spores (CFU) observed on five individual
coupons, the mean percent recovery on those five coupons, and decontamination efficacy (log reduction).
CI = confidence interval (± 2.78 x SE).
b Positive Controls = inoculated, not decontaminated coupons (sprayed with SFW).
0 Test Coupons = inoculated, decontaminated coupons.
d Laboratory Blank = not inoculated, not decontaminated coupon.
e Procedural Blank = not inoculated, decontaminated coupon.
Not Applicable.
16

-------
Table 4-3a. Inactivation of Bacillus anthracis (Ames) Spores" - Test 2, 1,000 ppmv TME
T .	Mean of Logs A
Test Material	"rrF"! h™ °f Observed R ean °	Efficacy ± CI
CFU	Kecovery
Glass
Positive Controlsb	9.40 x 107	7.54 ± 0.04	37.0 ±3.8
Test Coupons0	9.40 x 107	5.76±0.12	0.62±0.17 1.78 ±0.16
Laboratory Blankd 0 0	0
Procedural Blank6 0 0	0
Bare Wood
Positive Controls	9.40 x 107	6.86 ± 0.39	11.3 ±12.7
Test Coupons	9.40 x 107	4.50 ± 0.34	0.04 ±0.03 2.36 ±0.65
Laboratory Blank 0 0	0
Procedural Blank 0 0	0
Galvanized Metal
Positive Controls	9.40 x 107	7.69 ± 0.09	52.9 ±10.4
TestCoupons	9.40 x 107	5.64 ±0.11	0.48±0.12 2.05±0.18
Laboratory Blank 0 0	0
Procedural Blank 0 0	0
a Data are expressed as the mean (± SD) of the logs of the number of spores (CFU) observed on five individual
coupons, the mean percent recovery on those five coupons, and decontamination efficacy (log reduction).
CI = confidence interval (± 2.78 x SE).
b Positive Controls = inoculated, not decontaminated coupons (sprayed with SFW).
0 Test Coupons = inoculated, decontaminated coupons.
d Laboratory Blank = not inoculated, not decontaminated coupon.
e Procedural Blank = not inoculated, decontaminated coupon.
Not Applicable.
17

-------
Table 4-3b. Inactivation of Bacillus subtilis Spores3 - Test 2,1,000 ppmv TME
Mean of Logs
Test Material	'"rrs"! h™ °f Observed	Mean /o	Efficacy ± CI
CFU	Kecovery
Glass
Positive Controlsb
Test Coupons0
Laboratory Blankd
Procedural Blank6
1.07
1.07
106
10s
0
0
7.50 ±0.03
3.12 ± 1.25
0
0
29.9 ±2.3
0.01 ±0.02
0
0
4.39 ± 1.55
Bare Wood
Positive Controls
Test Coupons
Laboratory Blank
Procedural Blank
1.07
1.07
10s
10s
0
0
6.82 ±0.53
6.01 ±0.13
0
0
13.3 ± 21.3
1.0 ± 0.3
0
0
0.81 ±0.67
Galvanized Metal
Positive Controls
Test Coupons
Laboratory Blank
Procedural Blank
1.07
1.07
10s
10s
0
0
7.65 ±0.08
4.82 ±0.08
0
0
42.5 ±7.9
0.06 ±0.01
0
0
2.83 ±0.14
a Data are expressed as the mean (± SD) of the logs of the number of spores (CFU) observed on five individual
coupons, the mean percent recovery on those five coupons, and decontamination efficacy (log reduction).
CI = confidence interval (± 2.78 x SE).
b Positive Controls = inoculated, not decontaminated coupons (sprayed with SFW).
0 Test Coupons = inoculated, decontaminated coupons.
d Laboratory Blank = not inoculated, not decontaminated coupon.
e Procedural Blank = not inoculated, decontaminated coupon.
Not Applicable.
18

-------
Table 4-4a. Inactivation of Bacillus anthracis (Ames) Spores" - Test 3, 1,000 ppmv TME
(Repeat)	
Test Material
Inoculum
(CFU)
Mean of Logs
of Observed
CFU
Mean %
Recovery
Efficacy ± CI
Glass
Positive Controls'3
Test Coupons0
Laboratory Blankd
Procedural Blank6
1.44
1.44
10s
10s
0
0
7.68 ±0.08
6.33 ±0.13
0
0
33.8 ±5.9
1.6 ±0.4
0
0
1.35 ±0.19
Bare Wood
Positive Controls
Test Coupons
Laboratory Blank
Procedural Blank
1.44
1.44
10s
10s
0
0
6.86 ±0.16
5.07 ±0.24
0
0
5.3 ±2.0
0.09 ±0.05
0
0
1.79 ±0.36
Galvanized Metal
Positive Controls
Test Coupons
Laboratory Blank
Procedural Blank
1.44
1.44
106
108
0
0
7.91 ±0.09
6.16 ± 0.14
0
0
57.6 ± 10.7
1.1 ±0.4
0
0
1.75 ±0.21
a Data are expressed as the mean (± SD) of the logs of the number of spores (CFU) observed on five individual
coupons, the mean percent recovery on those five coupons, and decontamination efficacy (log reduction).
CI = confidence interval (± 2.78 x SE).
b Positive Controls = inoculated, not decontaminated coupons (sprayed with SFW).
0 Test Coupons = inoculated, decontaminated coupons.
d Laboratory Blank = not inoculated, not decontaminated coupon.
e Procedural Blank = not inoculated, decontaminated coupon.
Not Applicable.
19

-------
Table 4-4b. Inactivation of Bacillus subtilis Spores3 - Test 3,1,000 ppmv TME (Repeat)
	 Mean of Logs Mean o/o
Test Material	of Observed „	Efficacy ± CI
Cfu	Recovery
Inoculum
(CFU)
Glass
Positive Controlsb
Test Coupons0
Laboratory Blankd
Procedural Blank6
1.29
1.29
106
10s
0
0
7.48 ±0.11
4.37 ± 1.21
0
0
23.8 ±5.3
0.34 ±0.73
0
0
3.11 ± 1.50
Bare Wood
Positive Controls
Test Coupons
Laboratory Blank
Procedural Blank
1.29
1.29
10s
10s
0
0
6.68 ±0.13
6.03 ±0.24
0
0
3.9 ± 1.2
0.9 ±0.4
0
0
0.66 ±0.35
Galvanized Metal
Positive Controls
Test Coupons
Laboratory Blank
Procedural Blank
1.29
1.29
10s
10s
0
0
7.77 ±0.05
5.63 ±0.16
0
0
46.2 ±5.6
0.35 ±0.13
0
0
2.14 ±0.22
a Data are expressed as the mean (± SD) of the logs of the number of spores (CFU) observed on five individual
coupons, the mean percent recovery on those five coupons, and decontamination efficacy (log reduction).
CI = confidence interval (± 2.78 x SE).
b Positive Controls = inoculated, not decontaminated coupons (sprayed with SFW).
0 Test Coupons = inoculated, decontaminated coupons.
d Laboratory Blank = not inoculated, not decontaminated coupon.
e Procedural Blank = not inoculated, decontaminated coupon.
Not Applicable.
20

-------
Table 4-5a. Inactivation of Bacillus anthracis (Ames) Spores"
Mean of Logs
Test Material	of Observed
CFU
- Test 4, 2,000 ppmv TME
Mean %
Inoculum
(CFU)
Recovery
Efficacy ± CI
Glass
Positive Controlsb
Test Coupons0
Laboratory Blankd
Procedural Blank6
1.54
1.54
106
10s
0
0
7.73 ±0.06
5.20 ±0.36
0
0
35.1 ±5.1
0.13 ±0.10
0
0
2.53 ±0.46
Bare Wood
Positive Controls
Test Coupons
Laboratory Blank
Procedural Blank
1.54
1.54
10s
10s
0
0
6.96 ±0.10
4.81 ±0.62
0
0
6.1 ± 1.4
0.08 ±0.10
0
0
2.15 ±0.78
Galvanized Metal
Positive Controls
Test Coupons
Laboratory Blank
Procedural Blank
1.54
1.54
10s
10s
0
0
7.84 ±0.05
3.89 ±0.33
0
0
45.2 ±5.2
0.006 ±0.005
0
0
3.95 ±0.42
a Data are expressed as the mean (± SD) of the logs of the number of spores (CFU) observed on five individual
coupons, the mean percent recovery on those five coupons, and decontamination efficacy (log reduction).
CI = confidence interval (± 2.78 x SE).
b Positive Controls = inoculated, not decontaminated coupons (sprayed with SFW).
0 Test Coupons = inoculated, decontaminated coupons.
d Laboratory Blank = not inoculated, not decontaminated coupon.
e Procedural Blank = not inoculated, decontaminated coupon.
Not Applicable.
21

-------
Table 4-5b. Inactivation of Bacillus subtilis Spores3 - Test 4, 2,000 ppmv TME
Mean of Logs
Test Material	'"rrs"! h™ °f Observed	Mean /o	Efficacy ± CI
CFU	Kecovery
Glass
Positive Controlsb
Test Coupons0
Laboratory Blankd
Procedural Blank6
1.04
1.04
106
10s
0
0
7.61 ±0.09
4.73 ± 1.24
0
0
39.7 ±8.6
0.46 ±0.78
0
0
2.88 ± 1.55
Bare Wood
Positive Controls
Test Coupons
Laboratory Blank
Procedural Blank
1.04
1.04
10s
10s
0
0
5.95 ±0.64
5.00 ±0.36
0
0
2.0 ±2.5
0.12 ± 0.10
0
0
0.96 ±0.91
Galvanized Metal
Positive Controls
Test Coupons
Laboratory Blank
Procedural Blank
1.04
1.04
10s
10s
0
0
7.74	±0.04
5.75	±0.21
0
0
52.8 ±4.7
0.6 ±0.3
0
0
1.98 ±0.26
a Data are expressed as the mean (± SD) of the logs of the number of spores (CFU) observed on five individual
coupons, the mean percent recovery on those five coupons, and decontamination efficacy (log reduction).
CI = confidence interval (± 2.78 x SE).
b Positive Controls = inoculated, not decontaminated coupons (sprayed with SFW).
0 Test Coupons = inoculated, decontaminated coupons.
d Laboratory Blank = not inoculated, not decontaminated coupon.
e Procedural Blank = not inoculated, decontaminated coupon.
Not Applicable.
22

-------
Table 4-6a. Inactivation of Bacillus anthracis (Ames) Spores" - Test 5, 1,000 ppmv
1-Hexene	
T t M t l	Inoculum Mf nh°^ L°!f Mean %	ff	„
Test Material	,„„.n	ot Observed „	Elbcacy ± CI
(CFU)	CFU	Recovery	J
Glass
Positive Controls'3	5.70 x 107	7.60 ± 0.02	70.6 ±3.4
Test Coupons0	5.70 x 107	1.11 ± 1.54	0.001 ±0.001 6.49±1.92
Laboratory Blankd 0 0	0
Procedural Blank6 0 0	0
Bare Wood
Positive Controls	5.70 x 107	6.75 ± 0.07	10.0 ±1.6
Test Coupons	5.70 x 107	2.60 ± 1.80	0.01 ±0.02 4.16 ±2.24
Laboratory Blank 0 0	0
Procedural Blank 0 0	0
Galvanized Metal
Positive Controls	5.70 x 107	7.61 ±0.09	72.2 ±14.7
Test Coupons	5.70 x 107	3.10 ±0.45	0.003 ± 0.002 4.51 ±0.56
Laboratory Blank 0 0	0
Procedural Blank 0 0	0
a Data are expressed as the mean (± SD) of the logs of the number of spores (CFU) observed on five individual
coupons, the mean percent recovery on those five coupons, and decontamination efficacy (log reduction).
CI = confidence interval (± 2.78 x SE).
b Positive Controls = inoculated, not decontaminated coupons (sprayed with SFW).
0 Test Coupons = inoculated, decontaminated coupons.
d Laboratory Blank = not inoculated, not decontaminated coupon.
e Procedural Blank = not inoculated, decontaminated coupon.
Not Applicable.
23

-------
Table 4-6b. Inactivation of Bacillus subtilis Spores3 - Test 5,1,000 ppmv 1-Hexene	
	 Mean of Logs Mean o/o
Test Material	of Observed „	Efficacy ± CI
Cfu	Recovery
Inoculum
(CFU)
Glass
Positive Controlsb
Test Coupons0
Laboratory Blankd
Procedural Blank6
1.07
1.07
106
10s
0
0
7.69 ±0.08
2.87 ±0.78
0
0
45.9 ±8.2
0.001 ±0.001
0
0
4.82 ±0.98
Bare Wood
Positive Controls
Test Coupons
Laboratory Blank
Procedural Blank
1.07
1.07
10s
10s
0
0
6.54 ±0.30
5.72 ±0.55
0
0
3.8 ±2.1
0.9 ± 1.2
0
0
0.82 ±0.77
Galvanized Metal
Positive Controls
Test Coupons
Laboratory Blank
Procedural Blank
1.07
1.07
10s
10s
0
0
7.77 ±0.11
3.75 ±0.37
0
0
56.3 ± 13.4
0.007 ±0.005
0
0
4.02 ±0.47
a Data are expressed as the mean (± SD) of the logs of the number of spores (CFU) observed on five individual
coupons, the mean percent recovery on those five coupons, and decontamination efficacy (log reduction).
CI = confidence interval (± 2.78 x SE).
b Positive Controls = inoculated, not decontaminated coupons (sprayed with SFW).
0 Test Coupons = inoculated, decontaminated coupons.
d Laboratory Blank = not inoculated, not decontaminated coupon.
e Procedural Blank = not inoculated, decontaminated coupon.
Not Applicable.
24

-------
Table 4-7a. Inactivation of Bacillus anthracis (Ames) Spores" - Test 6, 1,000 ppmv
1-Hexene (Repeat)	
Test Material
Inoculum
(CFU)
Mean of Logs
of Observed
CFU
Mean %
Recovery
Efficacy ± CI
Glass
Positive Controls'3
Test Coupons0
Laboratory Blankd
Procedural Blank6
1.36
1.36
10s
10s
0
0
7.55 ±0.03
5.19 ±0.22
0
0
26.3 ± 1.8
0.13 ±0.08
0
0
2.36 ±0.28
Bare Wood
Positive Controls
Test Coupons
Laboratory Blank
Procedural Blank
1.36
1.36
10s
10s
0
0
6.78 ±0.13
4.95 ±0.40
0
0
4.6 ± 1.5
0.09 ±0.08
0
0
1.83 ±0.52
Galvanized Metal
Positive Controls
Test Coupons
Laboratory Blank
Procedural Blank
1.36
1.36
106
108
0
0
7.84 ±0.06
6.20 ±0.06
0
0
51.8 ± 7.7
1.2 ±0.2
0
0
1.65 ±0.11
a Data are expressed as the mean (± SD) of the logs of the number of spores (CFU) observed on five individual
coupons, the mean percent recovery on those five coupons, and decontamination efficacy (log reduction).
CI = confidence interval (± 2.78 x SE).
b Positive Controls = inoculated, not decontaminated coupons (sprayed with SFW).
0 Test Coupons = inoculated, decontaminated coupons.
d Laboratory Blank = not inoculated, not decontaminated coupon.
e Procedural Blank = not inoculated, decontaminated coupon.
Not Applicable.
25

-------
Table 4-7b. Inactivation of Bacillus subtilis Spores" - Test 6,1,000 ppmv 1-Hexene
(Repeat)	
Test Material
Inoculum
(CFU)
Mean of Logs
of Observed
CFU
Mean %
Recovery
Efficacy ± CI
Glass
Positive Controls'3
Test Coupons0
Laboratory Blankd
Procedural Blank6
1.16
1.16
10s
10s
0
0
7.55 ±0.09
2.40 ±0.82
0
0
31.1 ± 6.2
0.001 ±0.003
0
0
5.15 ± 1.03
Bare Wood
Positive Controls
Test Coupons
Laboratory Blank
Procedural Blank
1.16
1.16
10s
10s
0
0
6.72 ± 0.29
5.06 ±0.17
0
0
5.3 ±2.7
0.11 ±0.04
0
0
1.66 ±0.42
Galvanized Metal
Positive Controls
Test Coupons
Laboratory Blank
Procedural Blank
1.16
1.16
106
108
0
0
7.79 ±0.04
4.76 ±0.23
0
0
53.6 ±4.4
0.06 ±0.03
0
0
3.03 ±0.29
a Data are expressed as the mean (± SD) of the logs of the number of spores (CFU) observed on five individual
coupons, the mean percent recovery on those five coupons, and decontamination efficacy (log reduction).
CI = confidence interval (± 2.78 x SE).
b Positive Controls = inoculated, not decontaminated coupons (sprayed with SFW).
0 Test Coupons = inoculated, decontaminated coupons.
d Laboratory Blank = not inoculated, not decontaminated coupon.
e Procedural Blank = not inoculated, decontaminated coupon.
Not Applicable.
26

-------
Table 4-8a. Inactivation of Bacillus anthracis (Ames) Spores" - Test 7, 1,000 ppmv
1-Hexene (Hydrocarbon Introduced by Multiple Injections)	
T t M t l	Inoculum Mf nh°^ L°!f Mean %	ff	„
Test Material	,„„.n	ot Observed „	Emcacy ± CI
(CFU)	CFU	Recovery	J
Glass
Positive Controls'3	1.31 x 10s	7.77 ± 0.06	45.5 ±6.0
Test Coupons0	1.31 x 10s	6.41±0.05	2.0±0.2 1.36 ±0.09
Laboratory Blankd 0 0	0
Procedural Blank6 0 0	0
Bare Wood
Positive Controls	1.31 x 10s	7.10 ±0.30	11.6 ±7.9
TestCoupons	1.31 x 108	5.56 ±0.41	0.4±0.3	1.54±0.63
Laboratory Blank	0	0	0
Procedural Blank	0	0	0
Galvanized Metal
Positive Controls	1.31 x 108	7.86 ± 0.05	55.0 ±6.8
TestCoupons	1.31 x 108	6.46 ± 0.02	2.2±0.1 1.40±0.07
Laboratory Blank 0 0	0
Procedural Blank 0 0	0
a Data are expressed as the mean (± SD) of the logs of the number of spores (CFU) observed on five individual
coupons, the mean percent recovery on those five coupons, and decontamination efficacy (log reduction).
CI = confidence interval (± 2.78 x SE).
b Positive Controls = inoculated, not decontaminated coupons (sprayed with SFW).
0 Test Coupons = inoculated, decontaminated coupons.
d Laboratory Blank = not inoculated, not decontaminated coupon.
e Procedural Blank = not inoculated, decontaminated coupon.
Not Applicable.
27

-------
Table 4-8b. Inactivation of Bacillus subtilis Spores3
(Hydrocarbon Introduced by Multiple Injections)
Test 7,1,000 ppmv 1-Hexene
Test Material
Inoculum
(CFU)
Mean of Logs
of Observed
CFU
Mean %
Recovery
Efficacy ± CI
Glass
Positive Controls'3
Test Coupons0
Laboratory Blankd
Procedural Blank6
1.11
1.11
10s
10s
0
0
7.56 ± 0.11
3.00 ±0.31
0
0
33.5 ±8.6
0.001 ±0.001
0
0
4.56 ±0.41
Bare Wood
Positive Controls
Test Coupons
Laboratory Blank
Procedural Blank
1.11
1.11
10s
10s
0
0
6.55 ±0.16
6.19 ±0.43
0
0
3.3 ± 1.1
1.8 ± 1.1
0
0
0.36 ±0.57
Galvanized Metal
Positive Controls
Test Coupons
Laboratory Blank
Procedural Blank
1.11
1.11
106
108
0
0
7.86 ±0.12
6.27 ±0.45
0
0
67.1 ± 17.0
2.6 ±2.6
0
0
1.59 ±0.57
a Data are expressed as the mean (± SD) of the logs of the number of spores (CFU) observed on five individual
coupons, the mean percent recovery on those five coupons, and decontamination efficacy (log reduction).
CI = confidence interval (± 2.78 x SE).
b Positive Controls = inoculated, not decontaminated coupons (sprayed with SFW).
0 Test Coupons = inoculated, decontaminated coupons.
d Laboratory Blank = not inoculated, not decontaminated coupon.
e Procedural Blank = not inoculated, decontaminated coupon.
Not Applicable.
28

-------
Table 4-9a. Inactivation of Bacillus anthracis (Ames) Spores"
(Hydrocarbon Introduced by Multiple Injections)	
Test 8, 1,000 ppmv TME
Test Material
Inoculum
(CFU)
Mean of Logs
of Observed
CFU
Mean %
Recovery
Efficacy ± CI
Glass
Positive Controls'3
Test Coupons0
Laboratory Blankd
Procedural Blank6
1.50
1.50
10s
10s
0
0
7.81 ±0.04
6.31 ±0.09
0
0
42.8 ±4.0
1.4 ±0.3
0
0
1.50 ± 0.13
Bare Wood
Positive Controls
Test Coupons
Laboratory Blank
Procedural Blank
1.50
1.50
10s
10s
0
0
6.78 ±0.11
4.41 ±0.32
0
0
4.1 ± 1.0
0.02 ±0.01
0
0
2.37 ±0.42
Galvanized Metal
Positive Controls
Test Coupons
Laboratory Blank
Procedural Blank
1.50
1.50
106
108
0
0
7.75 ±0.06
6.02 ±0.11
0
0
37.5 ±5.4
0.7 ±0.2
0
0
1.73 ±0.15
a Data are expressed as the mean (± SD) of the logs of the number of spores (CFU) observed on five individual
coupons, the mean percent recovery on those five coupons, and decontamination efficacy (log reduction).
CI = confidence interval (± 2.78 x SE).
b Positive Controls = inoculated, not decontaminated coupons (sprayed with SFW).
0 Test Coupons = inoculated, decontaminated coupons.
d Laboratory Blank = not inoculated, not decontaminated coupon.
e Procedural Blank = not inoculated, decontaminated coupon.
Not Applicable.
29

-------
Table 4-9b. Inactivation of Bacillus subtilis Spores3
(Hydrocarbon Introduced by Multiple Injections)
Test 8,1,000 ppmv TME
Test Material
Inoculum
(CFU)
Mean of Logs
of Observed
CFU
Mean %
Recovery
Efficacy ± CI
Glass
Positive Controls'3
Test Coupons0
Laboratory Blankd
Procedural Blank6
1.08
1.08
10s
10s
0
0
7.38 ±0.16
1.58 ± 1.54
0
0
23.4 ±8.1
0.001 ±0.001
0
0
5.80 ± 1.93
Bare Wood
Positive Controls
Test Coupons
Laboratory Blank
Procedural Blank
1.08
1.08
10s
10s
0
0
6.10 ±0.29
4.80 ±0.62
0
0
1.4 ± 1.2
0.14 ±0.22
0
0
1.30 ±0.86
Galvanized Metal
Positive Controls
Test Coupons
Laboratory Blank
Procedural Blank
1.08
1.08
106
108
0
0
7.70 ±0.05
5.23 ±0.68
0
0
47.1 ±5.7
0.4 ±0.4
0
0
2.47 ±0.84
a Data are expressed as the mean (± SD) of the logs of the number of spores (CFU) observed on five individual
coupons, the mean percent recovery on those five coupons, and decontamination efficacy (log reduction).
CI = confidence interval (± 2.78 x SE).
b Positive Controls = inoculated, not decontaminated coupons (sprayed with SFW).
0 Test Coupons = inoculated, decontaminated coupons.
d Laboratory Blank = not inoculated, not decontaminated coupon.
e Procedural Blank = not inoculated, decontaminated coupon.
Not Applicable.
30

-------
Table 4-10a. Inactivation of Bacillus anthracis (Ames) Spores" - Test 9, Ozone Only, 70%
RH	
T t M t l	Inoculum Mf nh°^ L°!f Mean %	ff	„
Test Material	,„„.n	ot Observed „	Elbcacy ± CI
(CFU)	CFU	Recovery	J
Glass
Positive Controls'3	1.21 x 10s	7.87 ± 0.09	61.9 ±12.3
Test Coupons'	1.21 x 10s	6.06 ± 0.46	1.3 ±0.9 1.81 ±0.58
Laboratory Blankd 0 0	0
Procedural Blank6 0 0	0
Bare Wood
Positive Controls	1.21 x 10s 6.75 ±0.10	4.8 ±1.2
Test Coupons	1.21 x 10s 3.97 ±0.44	0.01 ±0.01	2.78 ± 0.56
Laboratory Blank	0 0	0
Procedural Blank	0 0	0
Galvanized Metal
Positive Controls	1.21 x	10s 7.98 ± 0.03	79.1 ±6.2
TestCoupons	1.21 x	10s 6.33 ±0.11	1.8 ±0.5 1.65 ±0.15
Laboratory Blank	0 0	0
Procedural Blank	0 0	0
a Data are expressed as the mean (± SD) of the logs of the number of spores (CFU) observed on five individual
coupons, the mean percent recovery on those five coupons, and decontamination efficacy (log reduction).
CI = confidence interval (± 2.78 x SE).
b Positive Controls = inoculated, not decontaminated coupons (sprayed with SFW).
0 Test Coupons = inoculated, decontaminated coupons.
d Laboratory Blank = not inoculated, not decontaminated coupon.
e Procedural Blank = not inoculated, decontaminated coupon.
Not Applicable.
31

-------
Table 4-10b. Inactivation of Bacillus subtilis Spores" - Test 9, Ozone Only, 70% RH	
	 Mean of Logs Mean o/o
Test Material	of Observed „	Efficacy ± CI
Cfu	Recovery
Inoculum
(CFU)
Glass
Positive Controlsb
Test Coupons0
Laboratory Blankd
Procedural Blank6
1.09
1.09
106
10s
0
0
7.61 ±0.08
2.97 ± 1.13
0
0
37.9 ±6.5
0.02 ±0.03
0
0
4.64 ± 1.41
Bare Wood
Positive Controls
Test Coupons
Laboratory Blank
Procedural Blank
1.09
1.09
10s
10s
0
0
6.67 ±0.29
4.85 ±0.25
0
0
5.4 ±4.7
0.07 ±0.04
0
0
1.82 ±0.48
Galvanized Metal
Positive Controls
Test Coupons
Laboratory Blank
Procedural Blank
1.09
1.09
10s
10s
0
0
7.84 ±0.09
4.98 ±0.67
0
0
64.5 ± 12.3
0.2 ±0.3
0
0
2.86 ±0.84
a Data are expressed as the mean (± SD) of the logs of the number of spores (CFU) observed on five individual
coupons, the mean percent recovery on those five coupons, and decontamination efficacy (log reduction).
CI = confidence interval (± 2.78 x SE).
b Positive Controls = inoculated, not decontaminated coupons (sprayed with SFW).
0 Test Coupons = inoculated, decontaminated coupons.
d Laboratory Blank = not inoculated, not decontaminated coupon.
e Procedural Blank = not inoculated, decontaminated coupon.
Not Applicable.
32

-------
Table 4-11 a. Inactivation of Bacillus anthracis (Ames) Spores" - Test 10, Ozone Only, 80%
RH	
T t M t l	Inoculum Mf nh°^ L°!f Mean %	ff	„
Test Material	,„„.n	ot Observed „	Elbcacy ± CI
(CFU)	CFU	Recovery	J
Glass
Positive Controls'3	1.08 x10s	7.98 ± 0.04	88.3 ± 7.5
Test Coupons'	1.08 x 10s	6.18 ±0.05	1.4 ±0.2 1.80 ±0.08
Laboratory Blankd 0 0	0
Procedural Blank6 0 0	0
Bare Wood
Positive Controls	1.08 x10s	6.85 ±0.12	6.8 ±1.9
Test Coupons	1.08 x 10s	3.93 ±0.56	0.02 ±0.02 2.92 ±0.71
Laboratory Blank 0 0	0
Procedural Blank 0 0	0
Galvanized Metal
Positive Controls	1.08 x10s	8.01 ±0.04	94.4 ±8.9
TestCoupons	1.08 x 10s	5.84±0.23	0.7±0.4 2.16±0.28
Laboratory Blank 0 0	0
Procedural Blank 0 0	0
a Data are expressed as the mean (± SD) of the logs of the number of spores (CFU) observed on five individual
coupons, the mean percent recovery on those five coupons, and decontamination efficacy (log reduction).
CI = confidence interval (± 2.78 x SE).
b Positive Controls = inoculated, not decontaminated coupons (sprayed with SFW).
0 Test Coupons = inoculated, decontaminated coupons.
d Laboratory Blank = not inoculated, not decontaminated coupon.
e Procedural Blank = not inoculated, decontaminated coupon.
Not Applicable.
33

-------
Table 4-llb. Inactivation of Bacillus subtilis Spores" - Test 10, Ozone Only, 80% RH
	 Mean of Logs Mean o/o
Test Material	of Observed „	Efficacy ± CI
Cfu	Recovery
Inoculum
(CFU)
Glass
Positive Controlsb
Test Coupons0
Laboratory Blankd
Procedural Blank6
1.03
1.03
106
10s
0
0
7.58 ±0.23
1.92 ±2.03
0
0
40.6 ± 15.3
0.01 ±0.02
0
0
5.67 ±2.54
Bare Wood
Positive Controls
Test Coupons
Laboratory Blank
Procedural Blank
1.03
1.03
10s
10s
0
0
6.19 ± 0.17
4.96 ±0.26
0
0
1.6 ± 0.8
0.1 ±0.1
0
0
1.23 ±0.38
Galvanized Metal
Positive Controls
Test Coupons
Laboratory Blank
Procedural Blank
1.03
1.03
10s
10s
0
0
7.87 ±0.05
5.14 ± 1.09
0
0
72.2 ±7.8
0.4 ±0.4
0
0
2.73 ± 1.35
a Data are expressed as the mean (± SD) of the logs of the number of spores (CFU) observed on five individual
coupons, the mean percent recovery on those five coupons, and decontamination efficacy (log reduction).
CI = confidence interval (± 2.78 x SE).
b Positive Controls = inoculated, not decontaminated coupons (sprayed with SFW).
0 Test Coupons = inoculated, decontaminated coupons.
d Laboratory Blank = not inoculated, not decontaminated coupon.
e Procedural Blank = not inoculated, decontaminated coupon.
Not Applicable.
34

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Table 4-12a. Inactivation of Bacillus anthracis (Ames) Spores" - Test 11, Ozone Only, 70%
RH (Repeat)	
Test Material
Inoculum
(CFU)
Mean of Logs
of Observed
CFU
Mean %
Recovery
Efficacy ± CI
Glass
Positive Controls'3
Test Coupons0
Laboratory Blankd
Procedural Blank6
1.24
1.24
10s
10s
0
0
7.83 ±0.16
6.44 ±0.14
0
0
56.6 ± 16.2
2.3 ±0.7
0
0
1.39 ±0.27
Bare Wood
Positive Controls
Test Coupons
Laboratory Blank
Procedural Blank
1.24
1.24
10s
10s
0
0
6.84 ±0.08
4.49 ± 0.27
0
0
5.7 ± 1.1
0.03 ± 0.02
0
0
2.35 ±0.35
Galvanized Metal
Positive Controls
Test Coupons
Laboratory Blank
Procedural Blank
1.24
1.24
106
108
0
0
7.98 ±0.13
5.39 ±0.23
0
0
79.8 ±24.5
0.2 ±0.1
0
0
2.59 ±0.33
a Data are expressed as the mean (± SD) of the logs of the number of spores (CFU) observed on five individual
coupons, the mean percent recovery on those five coupons, and decontamination efficacy (log reduction).
CI = confidence interval (± 2.78 x SE).
b Positive Controls = inoculated, not decontaminated coupons (sprayed with SFW).
0 Test Coupons = inoculated, decontaminated coupons.
d Laboratory Blank = not inoculated, not decontaminated coupon.
e Procedural Blank = not inoculated, decontaminated coupon.
Not Applicable.
35

-------
Table 4-12b. Inactivation of Bacillus subtilis Spores" - Test 11, Ozone Only, 70% RH
(Repeat)	
Test Material
Inoculum
(CFU)
Mean of Logs
of Observed
CFU
Mean %
Recovery
Efficacy ± CI
Glass
Positive Controls'3
Test Coupons0
Laboratory Blankd
Procedural Blank6
1.03
1.03
10s
10s
0
0
7.77 ±0.12
4.72 ± 0.25
0
0
58.3 ± 16.5
0.06 ±0.03
0
0
3.04 ±0.34
Bare Wood
Positive Controls
Test Coupons
Laboratory Blank
Procedural Blank
1.03
1.03
10s
10s
0
0
6.21 ±0.10
5.39 ±0.39
0
0
1.6 ±0.3
0.3 ±0.3
0
0
0.82 ±0.50
Galvanized Metal
Positive Controls
Test Coupons
Laboratory Blank
Procedural Blank
1.03
1.03
106
108
0
0
7.76 ±0.09
6.08 ±0.57
0
0
57.0 ± 11.6
2.6 ±4.0
0
0
1.68 ±0.71
a Data are expressed as the mean (± SD) of the logs of the number of spores (CFU) observed on five individual
coupons, the mean percent recovery on those five coupons, and decontamination efficacy (log reduction).
CI = confidence interval (± 2.78 x SE).
b Positive Controls = inoculated, not decontaminated coupons (sprayed with SFW).
0 Test Coupons = inoculated, decontaminated coupons.
d Laboratory Blank = not inoculated, not decontaminated coupon.
e Procedural Blank = not inoculated, decontaminated coupon.
Not Applicable.
36

-------
Table 4-13a. Inactivation of Bacillus anthracis (Ames) Spores" - Test 12, Ozone Only, 80%
RH (Repeat)	
T t M t l	Inoculum Mf nh°^ L°!f Mean %	ff	„
Test Material	,„„.n	ot Observed „	Elbcacy ± CI
(CFU)	CFU	Recovery	J
Glass
Positive Controls'3	9.87 x 107	7.50 ±0.01	32.0 ±1.0
Test Coupons'	9.87 x 107	5.43 ±0.25	0.3 ± 0.2 2.07 ±0.31
Laboratory Blankd 0 0	0
Procedural Blank6 0 0	0
Bare Wood
Positive Controls	9.87 x 107	6.84 ±0.17	7.5 ±3.1
Test Coupons	9.87 x 107	3.90 ±0.82	0.03 ± 0.05 2.94 ±1.04
Laboratory Blank 0 0	0
Procedural Blank 0 0	0
Galvanized Metal
Positive Controls	9.87 x 107	7.63 ± 0.08	44.4 ± 8.6
TestCoupons	9.87 x 107	5.25 ±0.40	0.2±0.2 2.38 ± 0.51
Laboratory Blank 0 0	0
Procedural Blank 0 0	0
a Data are expressed as the mean (± SD) of the logs of the number of spores (CFU) observed on five individual
coupons, the mean percent recovery on those five coupons, and decontamination efficacy (log reduction).
CI = confidence interval (± 2.78 x SE).
b Positive Controls = inoculated, not decontaminated coupons (sprayed with SFW).
0 Test Coupons = inoculated, decontaminated coupons.
d Laboratory Blank = not inoculated, not decontaminated coupon.
e Procedural Blank = not inoculated, decontaminated coupon.
Not Applicable.
37

-------
Table 4-13b. Inactivation of Bacillus subtilis Spores" - Test 12, Ozone Only, 80% RH
(Repeat)	
T t M t l	Inoculum Mf nh°^ L°!f Mean %	ff	„
Test Material	,„„.n	ot Observed „	Enicacy ± CI
(CFU)	CFU	Recovery	J
Glass
Positive Controls'3	6.33 x 107	7.52 ± 0.06	52.1 ±7.2
Test Coupons'	6.33 x 107	0.84 ± 1.23	0.0002 ± 0.0004 6.67 ±1.53
Laboratory Blankd 0 0	0
Procedural Blank6 0 0	0
Bare Wood
Positive Controls	6.33 x 107	6.34 ±0.38	4.6 ±3.9
Test Coupons	6.33 x 107	2.76 ± 0.40	0.001 ±0.001 3.58 ±0.68
Laboratory Blank 0 0	0
Procedural Blank 0 0	0
Galvanized Metal
Positive Controls	6.33 x 107	7.66 ±0.17	77.8 ±32.7
TestCoupons	6.33 x 107	4.61 ±0.90	0.2±0.1 3.05 ±1.13
Laboratory Blank 0 0	0
Procedural Blank 0 0	0
a Data are expressed as the mean (± SD) of the logs of the number of spores (CFU) observed on five individual
coupons, the mean percent recovery on those five coupons, and decontamination efficacy (log reduction).
CI = confidence interval (± 2.78 x SE).
b Positive Controls = inoculated, not decontaminated coupons (sprayed with SFW).
0 Test Coupons = inoculated, decontaminated coupons.
d Laboratory Blank = not inoculated, not decontaminated coupon.
e Procedural Blank = not inoculated, decontaminated coupon.
Not Applicable.
38

-------
Table 4-14a. Inactivation of Bacillus anthracis (Ames) Spores" - Test 13,1-Hexene,
80% RH	
T t M t l	Inoculum Mf nh°^ L°!f Mean %	ff	„
Test Material	,„„.n	ot Observed „	Elbcacy ± CI
(CFU)	CFU	Recovery	J
Glass
Positive Controls'3	1.27 x 10s	7.81 ±0.02	50.8 ±2.8
Test Coupons'	1.27 x 10s	2.49 ±2.28	0.01 ±0.01 5.32 ±2.82
Laboratory Blankd 0 0	0
Procedural Blank6 0 0	0
Bare Wood
Positive Controls	1.27 x 10s	6.99 ±0.17	8.2 ±3.6
Test Coupons	1.27 x 10s	1.97 ± 1.33	0.0005 ± 0.0007 5.02 ±1.66
Laboratory Blank 0	0	0
Procedural Blank 0	0	0
Galvanized Metal
Positive Controls	1.27 x 10s	7.79 ± 0.04	48.8 ±4.6
Test Coupons	1.27 x 10s	4.66 ± 0.40	0.05 ± 0.03 3.13 ±0.50
Laboratory Blank 0 0	0
Procedural Blank 0 0	0
a Data are expressed as the mean (± SD) of the logs of the number of spores (CFU) observed on five individual
coupons, the mean percent recovery on those five coupons, and decontamination efficacy (log reduction).
CI = confidence interval (± 2.78 x SE).
b Positive Controls = inoculated, not decontaminated coupons (sprayed with SFW).
0 Test Coupons = inoculated, decontaminated coupons.
d Laboratory Blank = not inoculated, not decontaminated coupon.
e Procedural Blank = not inoculated, decontaminated coupon.
Not Applicable.
39

-------
Table 4-14b. Inactivation of Bacillus subtilis Spores" - Test 13,1-Hexene, 80% RH	
T .	Mean of Logs A
Test Material	"rrF"! h™ °f Observed R ean °	Efficacy ± CI
CFU	Kecovery
Glass
Positive Controlsb	6.17 x 107	7.51 ±0.06	52.4 ±8.1
Test Coupons'	6.17 x 107	0.67 ± 0.92	^OOO^ 6.84 ±1.15
Laboratory Blankd 0 0	0
Procedural Blank6 0 0	0
Bare Wood
Positive Controls	6.17 x 107	5.94 ±0.08	1.4 ±0.2
Test Coupons	6.17 x 107	4.99 ± 0.68	0.3 ± 0.3 0.95 ± 0.85
Laboratory Blank 0 0	0
Procedural Blank 0 0	0
Galvanized Metal
Positive Controls	6.17 x 107	7.70 ±0.12	83.4 ±26.8
TestCoupons	6.17 x 107	3.60±0.23	0.007±0.004 4.10±0.32
Laboratory Blank 0 0	0
Procedural Blank 0 0	0
a Data are expressed as the mean (± SD) of the logs of the number of spores (CFU) observed on five individual
coupons, the mean percent recovery on those five coupons, and decontamination efficacy (log reduction).
CI = confidence interval (± 2.78 x SE).
b Positive Controls = inoculated, not decontaminated coupons (sprayed with SFW).
0 Test Coupons = inoculated, decontaminated coupons.
d Laboratory Blank = not inoculated, not decontaminated coupon.
e Procedural Blank = not inoculated, decontaminated coupon.
Not Applicable.
40

-------
Table 4-15. Summary of Efficacy Results
Test Number
and Organic
Compound3
Efficacy (Lo
5 Reduction)b
B. anthracis
B. subtilis
Glass
Wood
Metal
Glass
Wood
Metal
1. Ozone
Onlyc
1.10
1.32
0.82
2.77
0.25
2.77
2. TMEd
(1,000 ppmv)
1.78
2.36
2.05
4.39
0.81
2.83
3. TME
(1,000 ppmv)6
1.35
1.79
1.75
3.11
0.66
2.14
4. TME
(2,000 ppmv)
2.53
2.15
3.95
2.88
0.96
1.98
5. 1-Hexene
(1,000 ppmv)
6.49
4.16
4.51
4.82
0.82
4.02
6. 1-Hexene
(1,000 ppmv)6
2.36
1.83
1.65
5.15
1.66
3.03
7. 1-Hexene
(1,000 ppmv)f
1.36
1.54
1.40
4.56
0.36
1.59
8. TME
(1,000 ppmv)f
1.50
2.37
1.73
5.80
1.30
2.47
9. Ozone
Onlyc'8
1.81
2.78
1.65
4.64
1.82
2.86
10. Ozone
Onlych
1.80
2.92
2.16
5.67
1.23
2.73
11. Ozone
Onlyc'6'8
1.39
2.35
2.59
3.04
0.82
1.68
12. Ozone
Onlyceh
2.07
2.94
2.38
6.67
3.58
3.05
13. 1-Hexene
(1,000 ppmv)h
5.32
5.02
3.13
6.84
0.95
4.10
a Tests listed in chronological order in which they were performed.
b Bold type indicates result significantly greater than corresponding control (Test 1) result. Underlined type
indicates efficacy result for B. subtilis that is significantly different from the corresponding efficacy for B.
anthracis.
0 Test with 9,000 ppmv 03 only, no added reactive hydrocarbon.
d TME = tetramethylethylene.
e Repeat test.
f Reactive hydrocarbon introduced in 16 equal injections at 15-minute intervals over 4-hour contact time, rather
than as a single injection at the start of the contact time.
8 Target RH 70%.
h Target RH 80%.
41

-------
Table 4-15 shows (via bold type) that 29 of
the 36 efficacy results with B. cmthracis in
Tests 2 to 13 were significantly higher than
the corresponding results in the control test
(Test 1), as determined by a comparison of
95% CI values. In contrast, only 6 of the 36
efficacy results with B. subtilis were
significantly higher than the corresponding
results in the control test. The relatively
high frequency of significant efficacy results
for B. cmthracis is in part due to the low
efficacy and relatively narrow 95% CI
values observed in the control test with that
organism (see Table 4-2a). Moreover,
except for the relatively high efficacy results
for B. anthracis in Tests 5 and 13, the
efficacy results are not clearly dependent on
the identity, concentration, number of
vaporizations, or even presence of the
reactive organic compounds. This
observation is illustrated in Figures 4-1 and
4-2, which display the efficacy values found
in the 13 tests for B. anthracis and B.
subtilis, respectively. The relatively high
efficacy results for B. anthracis from Tests 5
and 13 (Figure 4-1) are consistent with an
effect from the added 1-hexene and/or a
humidity dependence of the susceptibility of
B. anthracis spores to inactivation by O3.
These results indicate that use of an O3 + 1-
hexene reaction mixture and RH of at least
80% may significantly enhance
decontamination efficacy for B. anthracis,
relative to the use of O3 alone. However,
higher humidity in the absence of an added
organic compound did not have a clear
effect on efficacy for B. anthracis. A
correspondingly elevated efficacy is also not
consistently apparent in Tests 5 and 13 with
B. subtilis (Figure 4-2). For B. subtilis,
efficacy values above 4 log reduction were
seen in nine of the 13 tests with the glass
coupons, but a dependence on organic
compound identity, organic compound
concentration, number of vaporizations, or
RH is not obvious.
B. anthracis
1 Tir
Glass
Wood	Galv. Metal
Test Material
11.	Control
12.	1000 ppm TME
3. 1000 ppm TME
14.	2000 ppm TME
15.	1000 ppm 1-Hexene
6. 1000 ppm 1-Hexene
I 7. 1000 ppm 1-Hexene 15 min
18. 1000 ppm TME 15 mm
9. No added HC, 70% RH
HO. No added HC, 80% RH
11.	No added HC, 70% RH
12.	No added HC, 80% RH
13.	1000 ppm 1-Hexene, 80% RH
Figure 4-1. Efficacy for B. anthracis at each test condition, by coupon material. Tests
shown in chronological order.
42

-------
Figure 4-2. Efficacy for B. subtilis at each test condition, by coupon material. Tests shown
in chronological order.
B. subtilis
¦g 4
PS
01 ¦>
Gla ss
nm
I 11
l II
Wood
Test Material
Galv. Metal
11.	Control
12.	1000 ppm IME
3. 1000 ppm IME
14.	2000 ppm IME
15.	1000 ppm 1-Hexene
6. 1000 ppm 1-Hexene
i 7. 1000 ppm 1-Hexene 15 min
18. 1000 ppm IME 15 min
9. No added HC 70% RH
110. No added HC 80% RH
11.	No added HC 70% RH
12.	No added HC 80% RH
13.1000 ppm 1-Hexene 80% RH
Efficacy was usually higher for B. subtilis
than for B. anthracis on glass and metal
coupons, but usually lower for B. subtilis
than for B. cmthracis on wood coupons. For
example, with glass coupons, nine of 13
efficacy results for B. subtilis exceeded 4 log
reduction, whereas only two efficacy results
for B. cmthracis exceeded 4 log reduction.
Both of those results for B. anthracis were
in tests conducted with 1,000 ppm of 1-
hexene at an RH of approximately 80% (i.e.,
Tests 5 and 13). Those two tests also
produced the two highest efficacy results for
B. anthracis on wood and two of the three
highest results on metal coupons. A
relatively high efficacy of nearly 4 log
reduction was also observed for B. anthracis
in Test 4 with 2,000 ppm TME, but only on
galvanized metal.
Table 4-15 also shows (via underlined type)
that 22 of the 39 efficacy results for B.
subtilis were significantly different from the
corresponding efficacy result for B.
anthracis in the same test with the same
coupon material. This observation indicates
that B. subtilis is not a suitable surrogate
organism for B. anthracis in testing with
ozone + reactive organic compound reaction
mixtures. However, the comparison of B.
subtilis and B. anthracis efficacy differs
markedly with test material. With glass
coupons, nine of the 13 B. subtilis efficacy
results differ significantly from the
corresponding B. anthracis results, and in all
nine cases efficacy was higher for B.
subtilis. Similarly, with galvanized metal
coupons, six of the 13 B. subtilis efficacy
results differ significantly from the
corresponding B. anthracis results, and in
five of those six cases efficacy was higher
43

-------
for B. subtilis. In contrast, with wood
coupons, seven of the 13 B. subtilis efficacy
results differ significantly from the
corresponding B. anthracis results, and in all
seven cases the efficacy was lower for B.
subtilis.
A potential limitation of this study is the use
of a single test run as the control for
comparison to all other tests. The results
shown above do not indicate a consistent
effect of the chamber RH on the efficacy
results for either B. anthracis or B. subtilis
and consequently it is attractive to consider
Tests 9 through 12 as additional control tests
in that no reactive organic compound was
introduced during those tests. As a result, an
evaluation has been done in which the
efficacy results for each coupon material and
organism from Test 1 and Tests 9 through
12 were pooled to calculate a control
efficacy and 95% CI value for comparison
to the results of Tests 2 through 8 and Test
13. For the pooling of data, the logs of the
spore counts obtained for the five positive
control coupons and the five test coupons of
each material with each organism in Test 1
and Tests 9 though 12 were compiled. This
compilation resulted in 25 positive control
log values and 25 test log values for each
combination of coupon material and
organism. The mean and variance of the
positive control log values and of the test log
values were calculated, and used to
determine efficacy and 95% CI values for
each of the pooled data sets using the
equations in Section 2.5. The 95% CI of the
resulting efficacy values was calculated
using a multiplier in Equation 4 of 2.065,
consistent with 25 data points (24 degrees of
freedom). The results of this analysis are
summarized in Table 4-16, which shows the
mean efficacy and 95% CI values for the
pooled data sets, and the resulting
indications of significant differences in
efficacy for Tests 2 through 8 and Test 13.
Table 4-16 shows that several of the mean
efficacy and 95% CI values of the pooled
data are quite different from (i.e., higher
than) those values found in the single
control test (Test 1, Tables 4-2a and b).
Comparison of the Test 2 through 8 and Test
13 results to the pooled control results in
Table 4-16 shows that nine of the 24
efficacy results for B. anthracis differ
significantly from the pooled control results,
with eight efficacy results (entries in bold
type) significantly higher than the pooled
control results, and one efficacy result (Italic
type; Test 7, metal) significantly less than
the control result. Six of the 24 efficacy
results for B. subtilis differ significantly
from the pooled control results, with three
efficacy results significantly higher than the
pooled control results and three efficacy
results (Test 3, wood and Test 7, wood and
metal) significantly less than the control
result. Tests 5 and 13 were most effective in
producing efficacy results that were
significantly higher than the pooled control
results; this result is consistent with the
results shown in Table 4-15 and discussed
above. Overall, the results in Table 4-16 do
not alter the finding that addition of a
reactive hydrocarbon to ozone does not
necessarily enhance decontamination
efficacy. However, those results also
support the observation noted above that
addition of 1-hexene at RH of approximately
80%) may significantly enhance
decontamination efficacy for B. anthracis,
relative to the use of O3 alone.
4.4 Reproducibility of Test Results
The results in Table 4-15 and Figures 4-1
and 4-2 raise the issue of reproducibility of
the efficacy results from the testing. What
degree of agreement can be expected in
duplicate test runs, and consequently what
differences in efficacy can be considered to
be significant (beyond the comparison of
44

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95% CI values)? Figure 4-3 addresses this
issue by showing a comparison of efficacy
results for both B. anthracis and B. subtilis
in duplicate tests.

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Table 4-16. Comparison of Efficacy Results to Pooled Control Results
Test Number
and Organic
Compound3
Efficacy (Lo
5 Reduction)b
B. anthracis
B. subtilis
Glass
Wood
Metal
Glass
Wood
Metal
Pooled
Controls
(Ozone Only)c
1.63
(± 0.20)
2.46
(± 0.33)
1.92
(± 0.30)
4.56
(± 0.83)
1.54
(± 0.48)
2.62
(± 0.38)
2. TMEd
(1,000 ppmv)
1.78
2.36
2.05
4.39
0.81
2.83
3. TME
(1,000 ppmv)6
1.35
1.79
1.75
3.11
0.66
2.14
4. TME
(2,000 ppmv)
2.53
2.15
3.95
2.88
0.96
1.98
5. 1-Hexene
(1,000 ppmv)
6.49
4.16
4.51
4.82
0.82
4.02
6. 1-Hexene
(1,000 ppmv)6
2.36
1.83
1.65
5.15
1.66
3.03
7. 1-Hexene
(1,000 ppmv)f
1.36
1.54
1.40
4.56
0.36
1.59
8. TME
(1,000 ppmv)f
1.50
2.37
1.73
5.80
1.30
2.47
13. 1-Hexene
(1,000 ppmv)8
5.32
5.02
3.13
6.84
0.95
4.10
a Tests listed in chronological order in which they were performed.
b Bold type indicates result significantly greater than corresponding pooled control result. Italic type indicates
result significantly less than corresponding pooled control result.
0 Efficacy (± 95% CI) shown based on pooled results from all tests with 9,000 ppmv 03 and no added reactive
hydrocarbon (i.e., Test 1 and Tests 9 through 12).
d TME = tetramethylethylene.
e Repeat test.
f Reactive hydrocarbon introduced in 16 equal injections at 15-minute intervals over 4-hour contact time, rather
than as a single injection at the start of the contact time.
g Target RH 80%.
46

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¦O
c
o
u
OJ
CO
"c*
o
u
3
¦O
OJ
en
tXD
o
~ Ba G ass
Ba Wood
Ba Metal
O Bs Glass
~ Bs Wood
A Bs Meta
Efficacy (Log Reduction), First Test
Figure 4-3. Comparison of efficacy results in duplicate tests.
The horizontal axis of Figure 4-3 shows the
efficacy value found with each organism on
each coupon type in the first of two
duplicate tests, and the vertical axis shows
the corresponding efficacy value found in
the second of two duplicate tests. Coupon
types are distinguished by the shape of the
symbol. B. cmthracis results are shown in
Figure 4-3 by filled symbols and B. subtilis
results by open symbols. The 1-to-l line is
shown, along with parallel lines indicating a
range of ±1 log reduction relative to the 1-
to-1 line. The duplicate test pairs from
which data were drawn for Figure 4-3 were
Tests 2 and 3; 5 and 13; 9 and 11; and 10
and 12. These duplicates include tests
conducted with TME (Tests 2 and 3), with
1-hexene (Tests 5 and 13), and with no
added organic compound (Tests 9 through
12). Note that Test 13 (rather than Test 6) is
used as a duplicate for Test 5, because the
RH achieved in Test 5 (78.7%, Table 4-1)
was considerably higher than that in Test 6
(74.4%). Test 13 was conducted at 80% RH
as a duplicate of Test 5 because of concern
about potential humidity dependence of the
efficacy of O3 for inactivating B. cmthracis.
Figure 4-3 shows that for B. anthracis,
duplicate efficacy results agree within ±1
log reduction in 10 of the 12 cases. The
duplicate results for B. subtilis agree within
±1 log reduction in 7 of the 12 cases. Based
on the limited data set it is not possible to
conclude whether the material type affects
the degree of duplication of test results.
Figure 4-3 indicates that efficacy results can
typically be duplicated within approximately
1 log reduction in tests with variables such
as RH control and organic compound
introduction as performed in this study.
47

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5.0 Summary and Conclusions
The primary conclusion of the series of tests
reported here is that the efficacy of 9,000
ppm of O3 for inactivating B. anthracis and
B. subtilis spores over a 4 hour exposure
was not consistently increased by the
addition of either TME or 1-hexene, whether
that addition was a single vaporization or
multiple vaporizations. A possible
exception for B. anthracis is the addition of
1,000 ppm of 1-hexene in the presence of
80% RH, which produced the only efficacy
values above 4 log reduction for that
organism. Those efficacy values may result
from both the impact of 1-hexene reaction
products and the reported effect of RH on O3
decontamination efficacy for B. anthracis.
However, elevated humidity in the absence
of an added organic compound did not
increase O3 efficacy for B. anthracis. The
addition of reactive organic compounds to
O3 at relatively high RH may be a valuable
topic for further study.
Across the range of tests conducted, efficacy
was usually higher for B. subtilis than for B.
anthracis on glass and metal coupons, but
usually lower for B. subtilis than for B.
anthracis on wood coupons. Most efficacy
results were less than a 4 log reduction
(especially so for B. anthracis). For B.
subtilis, efficacy values above 4 log
reduction were seen in nine of the 13 tests
with the glass coupons, but a dependence on
organic compound identity, organic
compound concentration, number of
vaporizations, or RH was not apparent.
Of the 39 efficacy results for B. subtilis, 22
were significantly different from the
efficacy result for B. anthracis in the same
test with the same coupon material. This
observation indicates that B. subtilis is not a
suitable surrogate organism for B. anthracis
in testing with ozone + reactive organic
compound reaction mixtures. However, the
comparison of B. subtilis and B. anthracis
efficacy differed markedly with test
material. With glass coupons, nine of the 13
B. subtilis efficacy results differed
significantly from the corresponding B.
anthracis results, and in all nine cases
efficacy was higher for B. subtilis.
Similarly, with galvanized metal coupons,
six of the 13 B. subtilis efficacy results
differed significantly from the
corresponding B. anthracis results, and in
five of those six cases efficacy was higher
for B. subtilis. In contrast, with wood
coupons, seven of the 13 B. subtilis efficacy
results differed significantly from the
corresponding B. anthracis results, and in all
seven cases the efficacy was lower for B.
subtilis.
Efficacy results from duplicate tests agreed
within ±1 log reduction in 10 of the 12 cases
for B. anthracis, and in 7 of the 12 cases for
B. subtilis. Based on the limited data set it is
not possible to conclude whether the coupon
material type affects the degree of
duplication of test results. These results
indicate that efficacy results can typically be
duplicated within approximately 1 log
reduction in tests with variables such as RH
control and organic compound introduction
as performed in this study.
48

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6.0 References
1. Wood, J.P., Choi, Y., Rogers, J., Kelly,
T., Willenberg, Z., and Riggs, K.
"Evaluation of Efficacy for the
Inactivation of Bacillus anthracis Spores
on Building and Outdoor Materials
Using Liquid and Foam Spray Sporicidal
Technologies", J. Applied Microbiology.
110:5. 1,262-1,273 (2011).
2. Calfee, M.W., Choi, Y., Rogers, J.,
Kelly, T., Willenberg, Z., and Riggs, K.,
"Lab-Scale Assessment to Support
Remediation of Outdoor Surfaces
Contaminated with Bacillus anthracis
Spores", J. of Bioterrorism and
Biodefense. 2:110. doi: 10.4172/2157-
2526.1000110(2011).
49

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