EPA 600/R-11/142 | November 2011 | www.epa.gov/ord
United States
Environmental Protection
Agency
Ozone Gas Decontamination
of Materials Contaminated
with Bacillus anthracis Spores
TECHNOLOGY EVALUATION REPORT
--
o
o a
Office of Research and Development
National Homeland Security Research Center
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EPA/600/R-11/142
November 2011
Technology Evaluation Report
Ozone Gas Decontamination of Materials
Contaminated with Bacillusanthracis
Spores
UNITED STATES ENVIRONMENTAL PROTECTION AGENCY
CINCINNATI, OHIO 45268
11
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Disclaimer
The U.S. Environmental Protection Agency (EPA), through its Office of Research and
Development's National Homeland Security Research Center, funded, directed and
managed this work through Contract Number EP-C-10-001 with Battelle Memorial
Institute. This report has been peer and administratively reviewed and has been approved
for publication as an EPA document. Mention of trade names or commercial products
does not constitute endorsement or recommendation for use of a specific product.
Questions concerning this document should be addressed to:
Joseph Wood
National Homeland Security Research Center
Office of Research and Development
U.S. Environmental Protection Agency
Mail Code E343-06
Research Triangle Park, NC 27711
(919)541-5029
wood.joe@epa.gov
in
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Foreword
Following the events of September 11, 2001, addressing the critical needs related to
homeland security became a clear requirement with respect to EPA's mission to protect
human health and the environment. Presidential Directives further emphasized EPA as
the primary federal agency responsible for the country's water supplies and for
decontamination following a chemical, biological, and/or radiological (CBR) attack. To
support EPA's mission to assist in and lead response and recovery activities associated
with CBR incidents of national significance, the National Homeland Security Research
Center (NHSRC) was established to conduct research and deliver products that improve
the capability of the Agency and other federal, state and local agencies to carry out their
homeland security responsibilities.
One goal of NHSRC's research is to provide information on decontamination methods
and technologies that can be used in the response and recovery efforts resulting from a
CBR release over a wide area. The complexity and heterogeneity of the wide-area
decontamination challenge necessitates the understanding of the effectiveness of a range
of decontamination options. In addition to effective fumigation approaches, rapidly
deployable or readily available surface decontamination approaches have also been
recognized as a tool to enhance the capabilities to respond to and recover from such an
intentional CBR dispersion.
Through working with ORD's program office partners (EPA's Office of Emergency
Management and Office of Chemical Safety and Pollution Prevention) and Regional on-
scene coordinators, NHSRC is attempting to understand and develop useful
decontamination procedures for wide-area remediation. This report documents the results
of a laboratory study designed to better understand the effectiveness of ozone fumigation
to decontaminate materials contaminated with Bacillus anthracis spores; data are also
presented on the decontamination efficacy for materials contaminated with Bacillus
subtilis spores.
These results, coupled with additional information in separate NHSRC publications
(available at www.epa.gov/nhsrc) can be used to determine whether a particular
decontamination technology can be effective in a given scenario. NHSRC has made this
publication available to the response community to prepare for and recover from disasters
involving chemical and/or biological contamination. This research is intended to move
EPA one step closer to achieving its homeland security goals and its overall mission of
protecting human health and the environment while providing sustainable solutions to our
environmental problems.
Jonathan Herrmann, Director
National Homeland Security Research Center
IV
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Acknowledgments
Contributions of the following organization and individuals (reviewers of this report) are
gratefully acknowledged:
United States Environmental Protection Agency (EPA)
Office of Research and Development, National Homeland Security Research
Center
Joan Bursey
Sang Don Lee
Eletha Brady-Roberts
EPA Office of Pesticide Programs, Antimicrobials Division
Carl ton J. Kempter
United State Department of Energy
Paula Krauter (Sandia National Laboratory)
Battelle
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Executive Summary
In this study, ozone fumigation was evaluated with regard to its ability to decontaminate six
materials (glass, wood, carpet, laminate, metal ductwork, and painted wallboard paper)
inoculated with Bacillus anthracis and Bacillus subtilis spores (approximately 1 x 108 CPU).
Decontamination testing was conducted at concentrations of approximately 7,000, 9,000,
9,800, 11,000 and 12,000 ppmv ozone for various contact times (4, 6, 8, 9, or 12 hours) at
target levels of 22 ± 5 °C and 75% or 85% relative humidity (%RH) ± 5% RH.
Decontamination results are summarized in Tables ES-1 and ES-2. Five replicates of
each test material were evaluated at each condition.
Table ES-1. Inactivation (Log Reduction) of B. anthracis spores
Target Test
Conditions,
Ozone/%RH+
7,000 ppmv,
85%RH±5%
7,000 ppmv,
75% RH ± 5%
9,000 ppmv,1
85%RH±5%
9,000 ppmv,1
85%RH±5%
9,000 ppmv,
75% RH ± 5%
9,800 ppmv,
85%RH±5%
9,800 ppmv1,
85% RH ± 5%
ll,000ppmv,f
85%RH±5%
12,000 ppmv,f
85%RH±5%
Contact
Time, hr
4
6
8
4
6
8
4
6
8
6
9
12
4
6
8
6
9
12
6
9
12
6
9
12
6
9
12
Glass
4.39
2.71
6.25
1.86
2.33
2.33
2.64
3.26
3.92
4.81
5.68
7.11
1.72
1.70
1.88
6.61
7.33
6.95
5.13
5.11
6.13
4.04
4.16
6.05
6.52
6.69
7.66
Wood
4.13
5.04
6.70a
2.53
3.35
2.95
6.01
6.31
6.68a
6.34
6.71a
6.71a
2.31
2.21
2.56
6.90a
6.90a
6.60
6.54a
6.54a
6.54a
5.50
6.43a
6.43a
7.04a
6.74
7.04a
Material
Carpet
4.10
6.13
7.82a
2.06
3.03
4.38
4.31
7.93a
7.93a
7.90a
7.90a
7.90a
2.33
3.16
4.29
7.92a
7.92a
7.92a
7.76a
7.76a
7.76a
7.51
7.82a
7.82a
6.21
7.97a
7.97a
Type*
Laminate
3.11
2.79
4.99
1.51
1.65
1.58
2.21
3.76
3.10
2.75
3.78
2.74
1.27
1.27
1.49
3.52
3.35
5.39
3.10
3.70
4.05
2.27
4.43
4.92
5.05
6.93
5.82
Metal
Ductwork
2.33
1.98
2.95
1.48
1.44
1.70
1.79
2.17
2.28
2.81
3.90
2.55
1.55
1.40
1.40
3.34
4.00
4.97
2.58
3.07
3.85
2.50
3.70
4.72
5.88
5.79
6.50
Wallboard
Paper
7.60a
6.87
7.60a
5.21
6.97
6.34
7.17
7.66a
7.66a
7.74a
7.74a
7.74a
3.65
5.32
5.42
7.84a
7.84a
7.84a
7.48a
7.48a
7.48a
7.53a
7.53a
7.53a
7.49a
7.19
7.49a
Immediately following inoculation, all materials were kept at 85% ± 5% RH for approximately 24 hours prior
to introduction of ozone.
* Data are expressed as mean log reduction.
+ Target temperature was 22 °C ± 5 °C.
1 Measurements were taken using a different, high level ozone monitor as described in Section 4.2.
1 Conditions tested twice in order to evaluate longer contact times (i.e., 9 and 12 hours).
a Material was completely decontaminated, i.e., no spores were detected on any of the five replicate coupons.
VI
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Table ES-2. Inactivation (Log Reduction) of B. subtilis
Target Test
Conditions,
Ozone/%RH+
7,000 ppmv,
85% RH ± 5%
7,000 ppmv,
75%RH±5%
9,000 ppmv,1
85% RH ± 5%
9,000 ppmv,1
85%RH±5%
9,000 ppmv,
75%RH±5%
9,800 ppmv,
85% RH ± 5%
9,800 ppmv1,
85%RH±5%
12,000 ppmv, f
85% RH ± 5%
Contact
Time, hr
4
6
8
4
6
8
4
6
8
6
9
12
4
6
8
6
9
12
6
9
12
6
9
12
Glass
4.51
6.51
7.60
1.18
1.88
2.42
2.84
3.87
4.60
6.53
6.89
7.11
2.77
3.91
6.28
5.68
6.59
7.08
7.28a
6.11
6.47
6.08
7.30
7.60a
Wood
1.48
4.00
3.30
0.38
1.71
0.42
0.74
1.04
1.53
2.62
3.41
5.87
0.69
0.78
1.14
4.54
3.64
5.70
6.32a
6.32a
6.32a
2.80
3.77
3.13
Material
Carpet
1.72
2.82
2.01
0.83
0.87
1.60
2.06
2.15
2.89
3.84
5.14
5.99
1.87
2.02
2.32
4.35
5.34
6.69
4.97
6.84
7.05
3.69
4.05
4.72
Type*
Laminate
6.49
6.57
8.02 a
1.01
1.25
1.53
2.78
2.48
2.91
7.59a
7.59a
7.29
2.70
2.50
3.84
7.51
7.82a
7.82a
3.23
5.98
7.26a
7.80a
7.80a
7.80a
Metal
Ductwork
2.42
2.51
3.45
0.39
0.44
0.55
1.23
1.32
2.15
7.17
6.86
7.47a
1.71
1.75
2.80
6.17
7.65a
6.31
5.86
6.47
6.66
4.69
6.42
6.26
Wallboard
Paper
6.84
7.44 a
7.44 a
3.07
5.81
5.17
3.17
5.30
5.52
6.75a
6.75a
6.75a
5.87
4.08
6.67
6.87a
6.87a
6.87a
5.97a
5.97a
5.97a
6.57a
6.57a
6.57a
Immediately following inoculation, all materials were kept at 85% ± 5% RH for approximately 24 hours prior
to introduction of ozone.
* Data are expressed as mean log reduction.
+ Target temperature was 22 °C ± 5 °C.
1 Measurements were taken using a different high level ozone monitor as described in Section 4.2.
1 Conditions tested twice in order to evaluate longer contact times (i.e., 9 and 12 hours).
a Material was completely decontaminated, i.e., no spores were detected on any of the five replicate coupons.
The highest log reductions achieved for B. anthracis were on wallboard paper, carpet,
and wood, with >6 log reduction on over 67% of tests on these materials. (A six log
reduction or greater has been proposed as a requirement by EPA for registration as a
sporicidal decontaminant^.) For B. subtilis, the highest efficacies were achieved on
glass, laminate and wallboard paper, with over 54% of the tests on these materials
resulting in log reductions >6. At the highest concentration (12,000 ppmv) and after 12
hours, greater than 6 log reduction was observed on all materials except laminate (which
had a log reduction of 5.82) for B. anthracis; and > 6 log reduction on all materials
except wood and carpet for B. subtilis.
No visible damage to the test materials was observed following ozone fumigation.
vn
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Contents
Disclaimer iii
Foreword iv
Acknowledgments v
Executive Summary vi
Abbreviations/Acronyms xii
1.0 Introduction 1
2.0 Procedures 2
2.1 Biological Agent 2
2.2 Test Materials 2
2.3 Coupon Inoculation 3
2.4 Coupon Extraction and Biological Agent Quantification 4
2.5 Calculations 4
2.6 Surface Damage 6
3.0 Quality Assurance/Quality Control 7
3.1 Performance Evaluation Audit 7
3.2 Technical Systems Audit 7
3.3 Data Quality Audit 7
3.4 QA/QC Reporting 8
3.5 Deviations from Test/QAPlan 8
4.0 Ozone Fumigation Procedures 9
4.1 Test Matrix and Environmental Conditions 9
4.2 Decontamination Technology Description and Procedures 14
5.0 Results 18
5.1 Inactivation of B. anthracis Spores 18
5.2 Inactivation of B. subtilis Spores 33
5.3 Surface Damage to Materials 46
6.0 Summary 47
7.0 References 49
Vlll
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Figures
Figure 2-1. Inoculation of coupon using a micropipette 3
Figure 4-1. Ultrasonic fogging system 10
Figure 4-2. Custom-fabricated hygrometer 11
Figure 4-3. Ozone generator, low concentration ozone analyzer, and low-level
ambient monitor 14
Figure 4-4. Diagram of ozone fumigation setup 15
Figure 5-1. Efficacy (log reduction) against.8. anthracis on glass 27
Figure 5-2. Efficacy (log reduction) against B. anthracis on wood 28
Figure 5-3. Efficacy (log reduction) against B. anthracis on carpet 29
Figure 5-4. Efficacy (log reduction) against.8. anthracis on laminate 30
Figure 5-5. Efficacy (log reduction) against B. anthracis on metal ductwork 31
Figure 5-6. Efficacy (log reduction) against B. anthracis on wallboard paper 32
Figure 5-7. Efficacy (log reduction) against B. subtilis on glass 40
Figure 5-8. Efficacy (log reduction) against.8. subtilis on wood 41
Figure 5-9. Efficacy (log reduction) against B. subtilis on carpet 42
Figure 5-10. Efficacy (log reduction) against.8. subtilis on laminate 43
Figure 5-11. Efficacy (log reduction) against B. subtilis on metal ductwork 44
Figure 5-12. Efficacy (log reduction) against B. subtilis on wallboard paper 45
IX
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Tables
Table ES-1. Inactivation (Log Reduction) of B. anthracis vi
Table ES-2. Inactivation (Log Reduction) of B. subtilis vii
Table 2-1. Test Materials 3
Table 3-1. Performance Evaluation Audits 7
Table 4-1. Target Test Matrix for Ozone Fumigation 9
Table 4-2. Summary of Temperature, Relative Humidity, and Ozone Conditions for
B. anthracis Tests 12
Table 4-3. Summary of Temperature, Relative Humidity, and Ozone Conditions for
B. subtilis Tests 13
Table 4-4. Summary of High- and Low-Level Analyzer Comparisons 17
Table 5-1. Ozone Fumigation Results for B. anthracis Spores at 7,000 ppmv Ozone,
22 °C, 85% RH and 75% RH 20
Table 5-2. Ozone Fumigation Results for B. anthracis Spores at 9,000 ppmv Ozone,
22 °C, 85% RH and 75% RH 21
Table 5-3. Ozone Fumigation Results for B. anthracis Spores at 9,000 ppmv Ozone,
22 °C, 85% RH 22
Table 5-4. Ozone Fumigation Results for B. anthracis Spores at 9,800 ppmv Ozone,
22 °C, 85% RH 23
Table 5-5. Ozone Fumigation Results for B. anthracis Spores at 9,800 ppmv Ozone,
22 °C, 85% RH (with pre-humidification) 24
Table 5-6. Ozone Fumigation Results for B. anthracis Spores at 11,000 ppmv Ozone,
22 °C, 85% RH 25
Table 5-7. Ozone Fumigation Results for B. anthracis Spores at 12,000 ppmv Ozone,
22 °C, 85% RH 26
Table 5-8. Ozone Fumigation Results for B. subtilis Spores at 7,000 ppmv Ozone,
22 °C, 85% RH and 75% RH 34
Table 5-9. Ozone Fumigation Results for B. subtilis Spores at 9,000 ppmv Ozone,
22 °C, 85% RH and 75% RH 35
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Table 5-10. Ozone Fumigation Results for B. subtilis Spores at 9,000 ppmv Ozone,
22 °C, 85% RH 36
Table 5-11. Ozone Fumigation Results for B. subtilis Spores at 9,800 ppmv Ozone,
22 °C, 85% RH 37
Table 5-12. Ozone Fumigation Results for B. subtilis Spores at 9,800 ppmv Ozone,
22 °C, 85% RH (with pre-humidification) 38
Table 5-13. Ozone Fumigation Results for B. subtilis Spores at 12,000 ppmv Ozone,
22 °C, 85% RH 39
XI
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BSC
CPU
CI
EPA
g/Nm3
HEPA
NHSRC
NIST
ORD
ppmv
QA
QC
QMP
RH
rpm
SE
sL/m
ISA
WA
Abbreviations/Acronyms
biological safety cabinet
colony-forming units
confidence interval
U.S. Environmental Protection Agency
gram/Normal meter cubed
high-efficiency particulate air
National Homeland Security Research Center
National Institute of Standards and Technology
Office of Research and Development
parts per million by volume
quality assurance
quality control
quality management plan
relative humidity
revolutions per minute
standard error
standard liters per minute
technical systems audit
work assignment
xn
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1.0 Introduction
The U.S. Environmental Protection
Agency's (EPA's) National Homeland
Security Research Center (NHSRC) is
helping protect human health and the
environment from adverse impacts
resulting from the release of chemical,
biological, or radiological agents. With an
emphasis on decontamination and
consequence management, water
infrastructure protection, and threat and
consequence assessment, NHSRC is
working to develop tools and information
that will help detect the intentional
introduction of chemical or biological
contaminants in buildings or water
systems, contain these contaminants,
decontaminate buildings, outdoor areas
and water systems, and facilitate the
disposal of material resulting from
cleanups.
NHSRC works in partnership with
recognized testing organizations; with
stakeholder groups consisting of buyers,
vendor organizations, and permitters;
and with the participation of individual
technology developers in carrying out
performance tests on homeland security
technologies. NHSRC evaluates the
performance of innovative homeland
security technologies by developing test
plans that are responsive to the needs of
stakeholders, conducting tests, collecting
and analyzing data, and preparing peer-
reviewed reports. All evaluations are
conducted in accordance with rigorous
quality assurance (QA) protocols to
ensure that data of known and high
quality are generated and that the results
are defensible. Through this work,
NHSRC provides high-quality
information that is useful to decision
makers in purchasing or applying the
tested technologies. It provides potential
users with unbiased, third-party
information that can supplement vendor-
provided information. Stakeholder
involvement ensures that user needs and
perspectives are incorporated into the
test design so that useful performance
information is produced for each of the
tested technologies.
In this work, the efficacy of ozone
fumigation against Bacillus anthracis
Ames and Bacillus subtilis spores
applied to glass, wood, carpet, laminate,
galvanized metal, and painted wallboard
paper was evaluated at elevated (75%
and 85%) relative humidity (RH) and
7,000, 9,000, 9,800, 11,000 or 12,000
parts per million by volume (ppmv)
ozone. Decontamination efficacy was
determined based on the log reduction in
viable spores recovered from the
inoculated materials (with and without
exposure to ozone gas).
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2.0 Procedures
This section provides an overview of the
procedures that were used for the bench-
scale evaluation of ozone fumigation to
inactivate B. anthracis and B. subtilis
spores on six different test materials.
Testing was performed in accordance
with a peer reviewed and EPA approved
test and quality assurance plan. The
general test approach and methods are
summarized in this section.
2.1 Biological Agent
Testing was conducted with B. anthracis
Ames spores produced at Battelle
Biomedical Research Center (West
Jefferson, OH); this strain was verified
via genotyping by an independent
laboratory. Details of the method used to
produce these B. anthracis spores are
published in the Journal of Applied
Microbiology.(2) Testing was also
conducted with B. subtilis spores (ATCC
19659) from Battelle stock culture.
2.2 Test Materials
Decontamination testing was conducted
using glass, wood, carpet, laminate,
galvanized metal, and painted wallboard
paper. Information on these materials
and associated sterilization approaches is
presented in Table 2-1. Coupons of the
materials were cut to size (1.9 cm width
by 7.5 cm length) from a larger piece of
material. Coupons were then sterilized
by autoclaving or gamma irradiation; the
selected approach (Table 2-1) was based
on cost-effectiveness and minimization
of the physical alterations of the
material. Autoclaving was performed at
Battelle following an internal standard
operating procedure, and gamma
irradiation at -40 kilogray was
conducted by STERIS Isomedix
Services (Libertyville, IL). Gamma-
irradiated coupons were sealed in 6 mil
Uline® poly tubing (Uline, Chicago, IL)
to preserve sterility until the coupons
were ready for use.
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Table 2-1. Test Materials
Material
Glass
Wood
(untreated pine)
Carpet
Laminate
Metal ductwork
(galvanized metal)
Painted wallboard
Paper
Lot/Batch/
Observation
C1036
Generic modeling
Shaw EcoTek 6
NA
NA
05-16-03; Set-E-493;
Roll-3
Manufacturer/
Supplier Name
Brooks Brothers Glass,
Columbus, OH
West Jefferson
Hardware, West
Jefferson, OH
Grossmans Bargain
Outlet; Columbus, OH
A'Jacklnc.;
Columbus, OH
Adept Products;
West Jefferson, OH
United States Gypsum
Company; Chicago, IL
Coupon Size, Material
Width x Length Preparation
1.9
1.9
1.9
1.9
1.9
1.9
cm x 7.5 cm
cm x 7.5 cm
cm x 7.5 cm
cm x 7.5 cm
cm x 7.5 cm
cm x 7.5 cm
Autoclave
Gamma
irradiation
Gamma
irradiation
Gamma
irradiation
Autoclave
Gamma
irradiation
2.3 Coupon Inoculation
Test and positive control coupons were
placed on a flat surface within a Class II
biological safety cabinet (BSC) and
inoculated with approximately 1 x 108
colony-forming units (CPU) of viable B.
anthracis or B. subtilis spores per
coupon. A 100 jiL aliquot of a stock
suspension of approximately 1x10
CFU/mL was dispensed using a
micropipette applied as 10 jiL droplets
across the coupon surface (see Figure 2-
1). After inoculation, the coupons were
held overnight in a Class III BSC to dry
under ambient conditions, approximately
22 °C and 40% RH.
Figure 2-1. Inoculation of coupon using a micropipette.
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The number and type of replicate
coupons used for each combination of
material, ozone concentration, and %RH
included were:
• 5 replicate test coupons
(inoculated with B. anthracis or
B. subtilis spores and exposed to
ozone) per contact time,
• 5 positive controls (inoculated
with B. anthracis or B. subtilis
spores, but not exposed to
ozone),
• 1 laboratory blank (inoculated
only with sterile water and not
exposed to ozone)
• 1 procedural blank (inoculated
only with sterile water and
exposed to ozone for the longest
contact time).
2.4 Coupon Extraction and Biological
Agent Quantification
For sample extraction, test coupons,
positive controls, and blanks were placed
in 50 mL polypropylene conical vials
containing 10 mL of sterile phosphate-
buffered saline extraction buffer
containing 0.1% Triton X-100 surfactant
(Sigma, St. Louis, MO). The vials were
capped, placed on their side and agitated
on an orbital shaker for 15 minutes at
approximately 200 revolutions per
minute (rpm) at room temperature.
Residual viable spores were determined
using a dilution plating approach.
Following extraction, the extract was
removed and a series of 10-fold dilutions
was prepared in sterile water. An aliquot
(0.1 mL) of either the undiluted extract
and/or each serial dilution were plated
onto tryptic soy agar in triplicate. The
cultures were incubated for 18-24 hrs at
37 °C ± 2 °C for B. anthracis and 35 °C
± 2 °C for B. subtilis. Colonies were
counted manually and CFU/mL were
determined by multiplying the average
number of colonies per plate by the
reciprocal of the dilution. Dilution data
representing the greatest number of
individually definable colonies were
expressed as arithmetic mean ± standard
deviation of the numbers of CFU
observed.
2.5 Calculations
The percent recovery of viable B.
anthracis and B. subtilis spores from
each test and positive control coupon
was determined in order to ascertain the
differential number of spores recovered
from test coupons following initial
inoculation and completing the
fumigation process. Recovery was
defined as the total number of viable
CFU extracted from each test and
positive control coupon relative to the
number of CFU inoculated onto each
coupon.
The number of CFU/mL (spore density) was calculated from plate counts as:
CFU/mL = CFU counted on plate / volume of spore suspension inoculated onto plate (1)
The number of viable CFU inoculated onto a coupon was calculated as:
CPU/coupon = spore density (CFU/mL) x 0.1 mL inoculation volume/coupon (2)
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The number of detected viable CPU extracted from a coupon was calculated as:
Total CPU = [(arithmetic mean CPU plate count x I/dilution factor)/plated
volume] x (extraction buffer volume)
where:
(3)
CPU plate count = arithmetic mean of triplicate plates
plated volume = portion of total extraction buffer used to prepare dilutions
inoculated onto the plates (0.1 mL)
dilution factor = serial dilution having the greatest number of colonies
extraction buffer volume = 10 mL
%) was calculated for the/11
Recovery
individual positive control (or test)
•Hi
coupon within the /' test material (a
specific test material) as:
where:
Bsoaery
-xlOO
(4)
•th
= CPU values in extract samples for7 of five positive control (or test)
,th
coupons of / test material after drying period
•Hi
= CPU values spiked ontoy of five positive control (or test) coupons of
test material
The number of CPU of B. anthracis
spores in extracts of test and control
coupons was determined to calculate
efficacy of the decontaminant. Efficacy
was defined as the extent (as log
reduction) by which viable B. anthracis
spores extracted from test coupons after
decontamination were less numerous
than the viable B. anthracis spores
extracted from the associated positive
control coupons. The first steps in this
calculation were to determine the
logarithm of the CPU count value from
each coupon, and then the mean of those
logarithm values (geometric mean) for
each set of positive control and
associated test coupons. Efficacy of a
decontaminant for a test organism on the
/ coupon material was calculated as the
difference between those mean log
values, i.e.,:
where:
Efficacy = (log CFUCij) - (log CFUtv)
(I
(5)
log CFUcij = j individual logarithm values obtained from positive control coupons
log CFUtij = j individual logarithm values obtained from corresponding test
coupons
= geometric mean (the overbar designates a mean value)
-------
In each test, five positive control
coupons and five corresponding test
coupons (i.e.,7 = 5) were used. In the
case where no viable CPU were found in
a coupon extract, a CPU count of 1 was
assigned, resulting in a log CPU of zero
for that coupon.
The variances (i.e., the square of the
standard deviation) of the log CFUcy
and log CFUty values were also
calculated for both the control and test
coupons (i.e., S2ctj and -5%), and were
used to calculate the pooled standard
error (SE) for the efficacy value
calculated in Equation 6, as follows:
SE =
(6)
where:
the number 5, again, represents the number y of coupons in both the control and
test data sets.
The significance of differences in
efficacy across different coupon
materials or treatments was assessed
based on the 95% confidence interval
(CI) of each efficacy result. The 95% CI
s:
95% CI = Efficacy ± (1.96 x SE)
(7)
2.6 Surface Damage
The physical effect of the ozone
fumigation on the materials was also
qualitatively monitored during the
evaluation. This approach provided a
gross visual assessment of whether the
ozone fumigation altered the appearance
of the test materials. The procedural
control (coupon that is fumigated, but
has no spores applied) was visually
compared to a laboratory blank coupon
(a coupon not exposed to ozone and that
has no spores applied). Obvious visible
damage might include structural
damage, surface degradation,
discoloration, or other aesthetic impacts.
-------
3.0 Quality Assurance/Quality Control
Quality assurance/quality control (QC)
procedures were performed in
accordance with the test/QA Plan
developed for this study. The QA/QC
procedures and results are summarized
below.
3.1 Performance Evaluation Audit
Performance evaluation audits were
conducted to assess the quality of the
results obtained during these
experiments. Temperatures were
monitored but efforts were not
undertaken to control any of the test
temperatures. A performance evaluation
audit was not conducted for ozone
concentration as appropriate certified
standards were not available. Similarly,
no performance evaluation audits were
performed to confirm the concentration
of the B. anthracis or B. subtilis spores
because quantitative standards for these
biological agents do not exist. However,
the control coupons and blanks support
the spore measurements and the stock
inoculums were confirmed on each day
of testing. Both organisms were
obtained in pure culture as described in
Section 2.1. and were cultured according
to Battelle internal SOPs. Table 3-1
summarizes the performance evaluation
audits that were performed.
Table 3-1. Performance Evaluation Audits
Measurement
Temperature
Relative
Humidity*
Time
Audit
Procedure
Compared to independent
calibrated thermometer
N/A
Compare time to independent
clock or watch value
Allowable
Tolerance
±2°C
N/A
± 2 sec/hr
Actual
Tolerance
All <2 °C for 13
instances
N/A
0 second/hr for 8
instances
*Relative humidity was measured using the wet/dry bulb thermometer and verification of RH was covered
under the temperature Performance Evaluation Audit.
3.2 Technical Systems Audit
Observations and findings from the
technical systems audit (TSA) were
documented and submitted to the test
leader for response. Laboratory QA staff
conducted technical systems audits on
July 19 and 20, and November 4, 9 and
10, 2010 to ensure that the tests were
being conducted in accordance with the
appropriate test/QA plan. As part of the
audit, test procedures were compared to
those specified in the test/QA plan and
data acquisition and handling procedures
were reviewed. None of the findings of
the TSA required corrective action. TSA
records were permanently stored with
the laboratory QA Manager.
3.3 Data Quality Audit
At least 10% of the data acquired during
the evaluation were audited. A 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 QA/QC Reporting
Each assessment and audit was
documented in accordance with the
test/QA plan. For these tests, findings
were noted (none significant) in the data
quality audit, but no follow-up corrective
action was necessary. The findings were
mostly minor data transcriptions errors
requiring some recalculation of efficacy
results, but none were gross errors in
recording. Copies of the assessment
reports were distributed to the EPA QA
Manager and laboratory staff. QA/QC
procedures were performed in
accordance with the test/QA plan.
3.5 Deviations from Test/QA Plan
All mean relative humidity levels were
within the target range, although there
were a few measurements outside the
target range; see Section 4.0, Tables 4-1
and 4-2.
A Performance Evaluation Audit was
not performed for relative humidity
because a hygrometer was not used. The
relative humidity audit was covered by
the temperature audit since the relative
humidity measurements were made
using the wet/dry bulb method.
Lock & Lock (HPL838P, Farmers
Branch, TX) containers were used to
house all positive control and test
materials inside the decontamination
testing chamber. This method was
chosen to ensure that all materials would
be open to the ozone gas at the same
time instead of exposing each coupon
individually.
The positive control recovery (mean
CFU >5% and <120% of spike control)
was not attained for the wood positive
control (1.26% to 4.96%) on all
occasions except for the following:
• 7,000 ppmv ozone, 85% RH, B.
anthracis
• 9,000 ppmv ozone, 85% RH, B.
anthracis
• 9,800 ppmv ozone, 85% RH, B.
anthracis
• 12,000 ppmv ozone, 85% RH, B.
anthracis
Positive control recoveries for wallboard
paper were also below the recovery
criterion on three occasions: 9,000 ppmv
ozone, 85% RH, B. subtilis (3.13%),
9,800 ppmv ozone, 85% RH, B. subtilis
(2.00%), and 12,000 ppmv ozone, 85%
RH, B. subtilis (3.22%).
In previous studies with bare pine wood,
spore recoveries were also low (9% for
B. anthracis, 1% for B. subtilis spores
and 2.2% for Geobacillus
stearothermophilus spores).1 While the
mechanisms which cause spores to be
difficult to recover from porous
materials have not been determined, it is
possible that spores applied in aqueous
suspension are carried via capillary
action acting on the liquid into the pits
and cavities of the porous matrix thereby
making mechanical extraction of the
spores from these materials difficult.
There were not expected to be any
adverse impacts on testing as a sufficient
amount of B. anthracis spores were
recovered (> 106 CFU) from which to
evaluate decontamination efficacy.
Percent recovery of spores from wood
and wallboard paper positive controls
from all other tests was >5%.
-------
4.0 Ozone Fumigation Procedures
4.1 Test Matrix and Environmental
Conditions
As shown in the test matrix (Table 4-1),
ozone fumigation of spores applied to
the six material types (glass, wood,
carpet, laminate, metal ductwork, and
painted wallboard paper) was evaluated
at five different ozone concentrations at
ambient temperature (approximately 25
°C) and under two elevated relative
humidity levels (75% RH and 85% RH)
for 4, 6, 8, 9 and 12 hours. At the 9,000
ppmv concentration, 85% RH was tested
twice in order to investigate longer
contact times (i.e., 9 and 12 hours). At
the 9,800 ppmv concentration, 85% RH
was also tested twice. In one instance the
testing chamber was pre-humidified and
the coupons were held at this condition
immediately following inoculation for
approximately 24 hours prior to
decontamination.
Table 4-1. Target Test Matrix for Ozone Fumigation
Ozone
Concentration,
Temperature
7,
22
9,
22
9,
22
11,
22
12
22
000
°c
000
°c
800
°c
000
°c
ppmv,
±5 °C
ppmv,
±5 °C
ppmv,
±5 °C
ppmv*,
±5 °C
,000 ppmv,
°C±5 °C
%RH ± %,
Contact Time,
full scale
85% ±
75% ±
85% ±
75% ±
85% ±
85% ±
85% ±
85% ±
85% ±
5%
5%
5%J
5%
5%J
5%
5%1
5%
5%
4
4
4
4
6,
6,
6,
6,
6,
hr
,6
,6
,6
,6
9,
9,
9,
9,
9,
,8
,8
,8
,8
12
12
121
12
12
Immediately following inoculation, all materials were kept at 85% ± 5% RH for approximately
24 hours prior to introduction of ozone.
* Conditions tested twice in order to evaluate longer contact times (i.e., 9 and 12 hours).
* This concentration tested with B. anihracis spores only
Testing was performed inside a Class III
BSC (The Baker Company, Sanford,
ME). A 120 mm fan (Cooler Guys,
UF12B12BWL, Kirkland, WA) inside
the Class III BSC was used to mix the
atmosphere inside the cabinet. RH was
increased as needed to achieve the target
RH levels by using an ultrasonic fogger
system. The custom-designed ultrasonic
fogger with a water trap, shown in
Figure 4-1, was developed and used to
humidify the test chamber. The manually
controlled ultrasonic fogger was attached
to a polyvinylchloride pipe. Humidified
air from inside the pipe was then
pumped into the test chamber until the
specified RH was reached.
-------
Figure 4-1. Ultrasonic fogging system.
A wet/dry bulb hygrometer (comprised
of two National Institute of Standards
and Technology [NIST]-traceable
thermometers [Fisher 13-990-270]) was
fabricated (see Figure 4-2) and used to
monitor the temperature (and
subsequently relative humidity) every 2
to 15 minutes. The tops of these two
high quality thermometers were glued to
the backing of an existing hygrometer in
order for the measurements to be made
using NIST-traceable thermometers. The
wick material from the hygrometer was
used to wrap one NIST-traceable
thermometer bulb (on right in
photograph) which was kept constantly
wet by immersion in a siphon reservoir
(-10 mL H2O) while the other NIST-
traceable thermometer (on left in
photograph) gave a dry temperature
reading. The fan described above was
approximately 12 inches behind the
bulbs of the thermometers and provided
constant air flow. The actual bulbs of the
thermometers were open to the airflow
of the test chamber. Differences in the
temperature readings from the wet/dry
bulb thermometers were used to
determine relative humidity with an
online humidity calculator(3).
10
-------
NIST-traceable dry
bulb thermometer
NIST-traceable wet
bulb thermometer
Wet wick
Water reservoir
Figure 4-2. Custom-fabricated hygrometer.
Target test temperatures (22 °C ± 5 °C)
were attained as measured by the dry
bulb thermometer (associated with a
wet/dry bulb hygrometer). Measured by
the wet/dry bulb hygrometer, relative
humidity levels were infrequently above
the upper limits of the targeted relative
humidity levels, although mean relative
humidity levels were all within the target
range of either 75% or 85% ± 5 %. Mean
ozone concentrations met the target
concentration of 7,000, 9,000, 9,800,
11,000 or 12,000 ppmv ozone (±10%).
All temperature, relative humidity and
concentration data for all the testing are
shown in Tables 4-2 and 4-3.
11
-------
Table 4-2. Summary of Temperature, Relative Humidity and Ozone Conditions for
B. anthracis Tests
Target Test Contact
Conditions, Time,
Ozone/%RH+ hr
7,000 ppmv
85%RH±5%
7,000 ppmv
75%RH±5%
9,000 ppmv
85%RH±5%
9,000 ppmv
75%RH±5%
9,000 ppmv
85%RH±5%
9,800 ppmv
85%RH±5%
9,800 ppmv1
85%RH±5%
11, 000 ppmv
85%RH±5%
12,000 ppmv
85%RH±5%
4
6
8
4
6
8
4
6
8
4
6
8
6
9
12
6
9
12
6
9
12
6
9
12
6
9
12
Dry Bulb
Thermometer,
oC*t
25. 3 ±0.5
25.4 ±0.5
25.2 ±0.6
24.4 ±0.4
24.5 ±0.4
24.5 ±0.4
24.7 ±0.5
24.7 ±0.5
24.6 ±0.5
24.4 ±0.7
24.3 ±0.6
24.2 ±0.6
25. 8 ±0.5
25.7 ±0.5
25.4 ±0.8
25. 9 ±0.5
25. 9 ±0.5
25. 8 ±0.6
26.1 ±0.4
26.1 ±0.4
25. 8 ±0.7
25. 3 ±0.3
25. 3 ±0.4
25.0 ±0.7
24.8 ±0.5
24.8 ±0.5
24.6 ±0.6
Wet Bulb
Thermometer,
oC*t
24.0 ±0.5
24.1 ±0.5
23. 8 ±0.8
21.4 ±0.4
21.4 ±0.5
21. 5 ±0.5
22.9 ±0.5
22.8 ±0.6
22.8 ±0.6
21. 3 ±0.6
21.2 ±0.5
21.1±0.5
24.0 ±0.5
23. 8 ±0.5
23.6 ±0.8
24.0 ±0.5
24.0 ±0.5
23. 9 ±0.6
24.2 ±0.5
24.1 ±0.5
23. 8 ±0.7
23. 3 ±0.3
23. 3 ±0.4
23.0 ±0.7
22.9 ±0.6
22.8 ±0.5
22.6 ±0.7
%RH*f
90.0 ±0.5
89.6 ±0.9
88.4 ±2.4
76.9 ±2.8
76.3 ±2.9
76.3 ±2.6
85.6 ±1.7
84.9 ±1.9
85.0 ±1.8
75. 8 ±2.0
75. 9 ±1.8
76.4 ±2.0
85. 9 ±1.6
85. 5 ±1.6
85.4 ±1.6
85.2 ±1.0
85. 3 ±1.0
85.2 ±1.1
85.4 ±1.7
84.7 ±1.8
84.5 ±1.7
84.4 ±2.0
84.2 ±2.1
84.3 ±2.2
84.4 ±1.8
84.7 ±2.3
84.7 ±2.4
Ozone ppmv**
7,000 ±40
7,021 ±71
7,021 ± 102
-------
7,011 ±43
7,011 ±39
-------
9,002 ± 44
9,003 ± 44
-------
9,015 ±75
9,017 ±75
-------
8,995 ± 40
8,982 ± 73
-------
9,798 ± 54
9,798 ± 52
-------
9,796 ± 43
9,798 ± 48
-------^
10,967 ± 83
10,976 ± 80
-------3
11,972 ±73
11,981 ±68
Immediately following inoculation, all materials were kept at 85% ± 5% RH for approximately 24 hours
prior to introduction of ozone.
*Data are presented as mean ± standard deviation.
+ Target temperature was 22 °C ± 5 °C.
* Measurements were recorded manually every 2-15 minutes.
* Measurements were logged automatically every minute.
12
-------
Table 4-3. Summary of Temperature, Relative Humidity and Ozone Conditions for
B. subtitis Tests
Target Test Contact
Conditions, Time,
Ozone/%RH+ hr
7,000 ppmv
85%RH±5%
7,000 ppmv
75%RH±5%
9,000 ppmv
85%RH±5%
9,000 ppmv
75%RH±5%
9,000 ppmv
85%RH±5%
9,800 ppmv
85%RH±5%
9,800 ppmv1
85%RH±5%
12,000 ppmv
85%RH±5%
4
6
8
4
6
8
4
6
8
4
6
8
6
9
12
6
9
12
6
9
12
6
9
12
Dry Bulb
Thermometer,
oC*t
25.6 ±0.7
25.6 ±0.6
25.6 ±0.6
24.4 ±0.4
24.5 ±0.5
24.5 ±0.5
24.3 ±0.5
24.4 ±0.5
24.3 ±0.5
24.7 ±0.4
24.7 ±0.5
24.5 ±0.6
26.0 ±0.4
25.8 ±0.5
25.6 ±0.7
25.6 ±0.5
25.7 ±0.5
25.6 ±0.6
24.9 ±0.4
25.2 ±0.6
25.3 ±0.6
24.8 ±0.4
24.7 ±0.4
24.6 ±0.6
Wet Bulb
Thermometer,
oC*t
23. 8 ±0.7
23. 8 ±0.6
23. 9 ±0.7
21. 3 ±0.5
21.4 ±0.5
21.4 ±0.5
22.3 ±0.6
22.4 ±0.6
22.4 ±0.6
21.6 ±0.3
21.6 ±0.3
21. 5 ±0.5
24.0 ±0.4
23. 8 ±0.5
23. 3 ±1.0
23.7 ±0.6
23. 8 ±0.6
23.6 ±0.7
23.4 ±0.3
23.7 ±0.6
23. 9 ±0.6
22.9 ±0.5
22.8 ±0.5
22.7 ±0.6
%RH*f
86.2 ±1.8
86.4 ±2.3
86.2 ±2.4
75. 5 ±1.9
75. 5 ±2.0
75.7 ±1.9
84.2 ±1.8
84.6 ±1.7
85.2 ±2.1
76.0 ±2.2
76.1 ±2.4
76.0 ±2.3
85.1 ±0.8
85.0 ±0.9
84.8 ±1.1
85.1 ±1.9
85.4 ±2.0
85.1 ±2.0
88.6 ±0.8
88.6 ±0.6
88.5 ±0.6
85.2 ±1.5
84.9 ±1.4
85.1 ±1.7
Ozone ppmv**
6,998 ± 32
7,002 ± 36
7,008 ±51
7,003 ± 18
6,996 ± 33
6,996 ± 29
9,007 ± 33
9,006 ± 35
9,003 ± 35
9,011 ±48
9,011 ±48
9,003 ± 43
9,0 10 ±44
9,009 ± 40
9,009 ± 38
9,791 ± 39
9,786 ± 39
9,785 ± 43
9,790 ± 39
9,799 ± 40
9,799 ± 39
12,009 ± 106
11,999 ±101
12,008 ± 94
Immediately following inoculation, all materials were kept at 85% ± 5% RH for approximately 24 hours
prior to introduction of ozone.
* Data are presented as mean ± standard deviation.
+ Target temperature was 22 °C ± 5 °C.
* Measurements were recorded manually every 2-15 minutes.
* Measurements were logged automatically every minute.
13
-------
4.2 Decontamination Technology
Description and Procedures
An Ozone Generator AC-2045 (IN USA,
Inc., Norwood, MA) and a Low
Concentration Ozone Analyzer IN2000-
L2-LC (IN USA, Inc.), shown in Figure
4-3, were initially used in this
evaluation. Since the low level analyzer
range was applicable for 0-9,999 ppmv,
a Mini Hicon-LR (IN USA, Inc.) was
used for the higher level concentrations
(11,000 and 12,000 ppmv; these ozone
levels were not initially planned for
testing). In addition, a Smart-Irak® 2
flow controller (Sierra Instruments,
Monterey, CA) was used to control the
flow of oxygen into the ozone generator.
The ozone generator was used per the
manufacturer's instructions. Briefly, the
oxygen gas (Praxair, Inc., Danbury, CT;
with 99.5% oxygen and 0.5% nitrogen)
was supplied to the generator through
the flow controller at 10 standard
liters/minute (sL/m) and then subjected
to electrical discharge in the ozone
generating cell which produced the
ozone gas. The ozone generator was run
at maximum power (100%) until the
desired concentration was reached. At
that time, the power on the generator
was reduced to approximately 30% and
the oxygen flow reduced to
approximately 0.4-0.5 sL/m to provide a
small but continuous injection of ozone
to keep the concentration at the desired
level (e.g. 9,000 ppmv± 10%). Because
high concentrations of ozone are
dangerous to humans, ozone levels
around the chamber, especially near
inlets/outlets, were continuously checked
using a low-level ozone monitor (Model
A-22, Ozone Solutions, Inc., Hull, IA)
when the generator was in use (also
shown in Figure 4-3). See Figure 4-4 for
a diagram of the ozone fumigation setup.
Figure 4-3. Ozone generator, low concentration ozone analyzer, and low-level
ambient monitor.
14
-------
i Wet/Dry Bulb Hygrometer
Omega Temperature Sensor
Fisher Temperarure/RH Sensors
Test Coupons on wire rack
Fan
Ultrasonic Fogger
Flow Controller
Supply Air
Return Air
Supply Air Return Air
L _t
O, from tank to O3 generator
O} from generator to chamber
j from chamber to analyzer
O} from analyzer back to chamber
Figure 4-4. Diagram of ozone fumigation setup.
During each fumigation cycle, coupons
inoculated with B. anthracis or B.
subtilis spores were placed in sealed
containers (Lock & Lock, HPL838P,
Farmers Branch, TX) in the Class III
BSC. All containers were opened in the
Class III BSC until the targeted %RH
was achieved. At this point, all
containers were closed during the initial
injection of ozone. The containers
holding the test coupons were opened in
sequence in order to achieve an
appropriate contact time for exposure to
ozone. Contact time was defined as the
time from opening the container to the
time ozone was exhausted from the
Class III BSC. Positive controls and
laboratory blanks were also kept in a
sealed container (same as the test
materials) in the Class III BSC for the
full fumigation cycle. The procedural
blank coupons were opened for exposure
to ozone at the same time as the test
coupons with the longest contact time
(e.g. 8 or 12 hours).
15
-------
At the conclusion of each fumigation
cycle, the ozone generator was shut
down and the ozone was rapidly
exhausted from the Class III BSC.
The Low Concentration Ozone Analyzer
IN-2000-L2-LC was used to measure
ozone concentrations for all testing
except for the 11,000 or 12,000 ppmv
tests, in which the High Concentration
Ozone Analyzer Mini Hicon-LR was
used. The Mini Hicon-LR analyzer reads
in g/Nm3 and is calibrated for use
between 0 and 50 g/Nm3. A conversion
factor (1 gram/Normal meter cubed
[g/Nm3] = 467 ppmv) was used to
determine the concentration in ppmv
during fumigation. After results were
received from the 11,000 ppmv
fumigation test (using B. anthracis), the
analyzers did not appear to be reading
the same concentration of ozone when
drawing from the fumigation chamber at
the same time. IN USA, Inc. was
contacted regarding this issue and
explained the differences in
concentration readings. The Mini Hicon-
LR comes with a pressure and
temperature sensor built in; this
temperature and pressure compensation
is a process that adjusts the measurement
of gas to standardize conditions for
comparison with other measurements.
Therefore, all measurements are
normalized to 273K and 1 atm, making
the conversion of 1 g/Nm3 = 467 ppmv
relevant. The IN-2000-12-LC provides
ozone concentration data directly in
units of ppmv.
In addition, the IN-2000-L2-LC was
initially calibrated on July 9, 2009, and
again on 10/20/2010 by the
manufacturer for a range of 1-9,999
ppmv, and, according to IN-USA,
making the unit the most accurate at the
mid-range level of between 4,000 and
6,000 ppmv. The Mini Hicon-LR was
calibrated by the manufacturer on
1/28/11 for a range of-0-50 g/Nm3, and
11,000-12,000 ppmv (-23.55 - 25.70
g/Nm3) falls in the middle of this range.
According to the manufacturer, the issue
of having one analyzer run at the high
end of its calibration and the other run at
the mid-range of its calibration caused
the differences in concentration output
between the two analyzers.
To address this discrepancy, the two
analyzers were run simultaneously and
data were captured from both at 7,000,
9,000 and 9,800 ppmv (at both 75% and
85% RH) as read on the IN-2000-L2-LC
analyzer. The ozone was held at each
target condition for approximately 15
minutes to capture sufficient data.
Because of this discrepancy, the 11,000
ppmv results obtained by the high-level
analyzer were found to correspond with
the 9,800 ppmv results on the low-level
analyzer. Due to the corresponding
results, B. subtilis was not tested at the
11,000 ppmv concentration. Similarly,
when these data were extrapolated, the
12,000 ppmv results were found to
correspond to -10,800 ppmv if read with
the low-level analyzer. The results of
this comparison are summarized in
Table 4-4.
16
-------
Table 4-4. Summary of High- and Low-Level Analyzer Comparisons
Dry Bulb T1.T „„„„ T „ T ^ ,„. . TT. T „ Average
_ , „ .... + r™ . n/wTT* IN-2000-L2-LC Mini Hicon-LR _..„. 6
Target Condition Thermometer, %RH* „ *± r\ *± Difference,
& orAf Ozone ppmv*+ Ozone ppmv*+
*Data are presented as mean (range).
+ Target temperature was 22 °C ± 5 °C.
t Measurements were taken every 5 minutes.
* Measurements were taken every minute.
ppmv
7,000 ppmv ozone,
85%RH±5%RH
7,000 ppmv ozone,
75% RH ± 5% RH
9,000 ppmv ozone,
85%RH±5%RH
9,000 ppmv ozone,
75% RH ± 5% RH
9,800 ppmv ozone,
85% RH ± 5% RH
9,800 ppmv ozone,
75%RH±5%RH
23.8
(23.8-23.8)
24.6
(24.3-24.8)
24.1
(24.0-24.3)
24.7
(24.5-24.8)
24.4
(24.3-24.5)
24.6
(24.5-24.8)
85.3
(83.9-85.8)
76.3
(75.0-78.4)
82.2
(80.2-84.1)
75.4
(71.4-78.5)
85.6
(84.2-86.1)
73.2
(73.1-73.2)
7,072
(6957-7185)
7,081
(6,985-7,191)
9,030
(8,669-9,121)
9,053
(8,973-9,199)
9825
(9,742-9,966)
9,795
(9,736-9,885)
8,073
(7,892-8,412)
8,013
(7,929-8,145)
10,264
(10,138-10,355)
10,248
(10,168-10,325)
11,123
(11,045-11,276)
11,107
(10,858-11,485)
1,001
932
1,234
1,195
1,298
1,312
17
-------
5.0 Results
5.1 Inactivation of B. anthracis Spores
7,000 ppmv Ozone. Ozone fumigation
results for B. anthracis spores at 7,000
ppmv ozone are presented in Table 5-1
and Figures 5-1 through 5-6. At 85%
RH, no viable B. anthracis spores were
recovered from wood or carpet
following 8 hours of contact time, or
from painted wallboard paper following
the 4 or 8 hour contact times. Viable B.
anthracis spores were recovered from
the materials at all other contact times
and at both 75% and 85% at this
concentration (log reductions for all
materials ranged from 1.44 on metal
ductwork to 6.97 on wallboard paper).
9,000 ppmv Ozone. Ozone fumigation
results for B. anthracis spores at 9,000
ppmv ozone are presented in Tables 5-2
and 5-3 and Figures 5-1 through 5-6. At
85% RH, no viable B. anthracis spores
were recovered from carpet or painted
wallboard paper following contact times
of 6, 8, 9 or 12 hours or from wood after
8, 9 or 12 hours. Viable B. anthracis
spores were recovered from the
materials at all other contact times and at
both 75% and 85% at this concentration
(log reductions for all materials ranged
from 1.17 on metal ductwork to 7.17 on
wallboard paper).
9,800 ppmv Ozone (with and without
24 hour pre-humidification). Ozone
fumigation results for B. anthracis
spores at 9,800 ppmv ozone are
presented in Tables 5-4 and 5-5 and
Figures 5-1 through 5-6. At 85% RH, no
viable B. anthracis spores were
recovered from carpet or painted
wallboard paper following any contact
time (6, 9 or 12 hours). After subjecting
the test materials to 85% RH for -24
hours and then introducing 9,800 ppmv
ozone, no viable B. anthracis spores
were recovered from wood, carpet or
painted wallboard paper following 6, 9
or 12 hour contact times. Viable B.
anthracis spores were recovered from
the materials at all other contact times at
this concentration (log reductions for all
materials ranged from 2.58 on metal
ductwork to 7.33 on glass). This
concentration of ozone was not tested at
75% RH. Pre-humidification of the
testing chamber for 24 hours did not
improve efficacy and even reduced
efficacy in most instances for glass,
laminate and metal ductwork.
11,000 ppmv Ozone. Results from
fumigation of B. anthracis spores with
11,000 ppmv ozone are presented in
Table 5-6 and Figures 5-1 through 5-6.
At 85% RH, no viable B. anthracis
spores were recovered from wood or
carpet following 9 and 12 hour contact
times. No spores were recovered from
wallboard paper following any contact
time (6, 9 or 12 hours). Viable B.
anthracis spores were recovered from
the materials at all other contact times at
this concentration (log reductions for all
materials ranged from 2.27 on laminate
to 7.51 on carpet). The concentration for
this test was measured by the high-level
analyzer which corresponds to -9,800
ppmv as measured with the low-level
analyzer (Reference Section 4.2 and
Table 4-4). The results from this test are
similar to the results obtained in the
9,800 ppmv test in that wood, carpet and
wallboard paper had the highest log
18
-------
reductions. This concentration of ozone
was not tested at 75% RH.
12,000 ppmv Ozone. Ozone fumigation
results for B. anthracis spores at 12,000
ppmv ozone are presented in Table 5-7
and Figures 5-1 through 5-6. At 85%
RH, no viable B. anthracis spores were
recovered from wallboard paper and
wood at 6 or 12 hours, carpet at 9 or 12
hours, and glass at 12 hours. Viable B.
anthracis spores were recovered from
the materials at all other contact times at
this concentration (log reductions for all
materials ranged from 5.05 on laminate
to 6.93 on laminate). This concentration
of ozone was not tested at 75% RH.
All associated laboratory and procedural
blanks resulted in 0 CPU recovered for
all B. anthracis tests.
Note that for test conditions in which no
viable spores were recovered from all
five replicate test coupons for a given
material, log reduction results are
reported using a ">" symbol, indicating
the potential for having a higher log
reduction had there been a greater
amount of spores inoculated onto the
coupons. Additionally, we caution the
reader to avoid comparing log reduction
results for two materials when both
materials have been completely
decontaminated, as the difference in log
reduction may be attributed to a
difference in extraction recovery.
19
-------
Table 5-1. Ozone Fumigation Results for B. anthracis
22 °C, 85% RH and 75% RH*
Contact
Time,
hr
Inoculation
Material Amount,
CFU
Spores at 7,000 ppmv Ozone,
Positive Test
Control* Material
Mean Recovered B. anthracis Spores,
CFUf
Inactivation
Efficacy *
85% RH
4
6
8
Glass
Wood
TCarpet 8.20 xlO7
Laminate
Metal Ductwork
Wallboard Paper
Glass
Wood
T Carpet 8.20 x 107
Laminate
Metal Ductwork
Wallboard Paper
Glass
Wood
T Carpet 8.20 x 107
Laminate
Metal Ductwork
Wallboard Paper
3.27 ± 4.25 xlO7
5.16 ± 1.61 xlO6
6.73 ± 1.43 x 107
4.28 ± 0.508 xlO7
4.90 ± 0.818 xlO7
4.13 ± 0.997 xlO7
3.27±"4".25xi07
5.16 ± 1.61 xlO6
6.73 ± 1.43 x 107
4.28 ± 0.508 xlO7
4.90 ± 0.818 xlO7
4.13 ± 0.997 xlO7
3.27±"4".25xi07
5.16 ± 1.61 xlO6
6.73 ± 1.43 x 107
4.28 ± 0.508 xlO7
4.90 ± 0.818 xlO7
4.13 ± 0.997 xlO7
1.03±1.10xl04
2.27 ± 2.40 x 103
1.23 ± 0.917 xlO4
3.55 ± 1.47 xlO4
2.43 ± 0.911 xlO5
0.00 ± 0.00
9.72 ± 1.04 xlO4
4.47 ± 6.19 xlO2
9.27 ± 13.9xl02
7.50 ± 3.07 xlO4
6.71 ± 4.60 xlO5
2.68 ± 3.67 xlO1
6.54 ± 10. 4 x 102"
0.00 ± 0.00
0.00 ± 0.00
2.46 ± 2.65 xlO3
1.86 ± 2.52 xlO5
0.00 ± 0.00
4.39 ±1.29
4.13 ±1.36
4. 10 ±0.86
3.11±0.16
2.33 ±0.17
>7.60±0.10
2.71 ±0.39
5.04 ±1.35
6.13 ±1.46
2.79 ±0.17
1.98 ±0.35
6.87 ±0.88
6.25 ±1.53
>6.70±0.11
>7.82 ± 0.08
4.99 ±1.35
2.95 ±0.72
>7.60±0.10
75% RH
4
6
8
Glass
Wood
TCarpet 1.15 xlO8
Laminate
Metal Ductwork
Wallboard Paper
Glass
Wood
TCarpet 1.15 xlO8
Laminate
Metal Ductwork
Wallboard Paper
Glass
Wood
TCarpet 1.15 xlO8
Laminate
Metal Ductwork
Wallboard Paper
8.03 ± 10.3 x 107
3.74 ± 0.592 xlO6
6.34 ± 1.72 xlO7
3.15±0.402xl07
5.35 ± 0.336 xlO7
2.80 ± 0.826 xlO7
8.03 ± 10.3 x I67
3.74 ± 0.592 xlO6
6.34 ± 1.72 xlO7
3.15±0.402xl07
5.35 ± 0.336 xlO7
2.80 ± 0.826 xlO7
8.03 ± 10.3 x I67
3.74 ± 0.592 xlO6
6.34 ± 1.72 xlO7
3.15±0.402xl07
5.35 ± 0.336 xlO7
2.80 ± 0.826 xlO7
7.13 ± 1.91 xlO5
1.46 ± 1.41 xlO4
5.90 ± 3.00 xlO5
9.75 ± 1.87 xlO5
1. 89 ± 0.820 xlO6
2.73±3.19xl02
2.89 ± 2.06 xlO5
1.08 ± 0.809 xlO4
8.60 ± 10.5 xlO4
7.05 ± 1.06 xlO5
2.19 ± 1.04 xlO6
4.00 ± 8.94 xlO1
3.32 ± 2.66 xlO5
1.67 ± 3.00 xlO4
1.25 ± 1.55 xlO4
9.97 ± 5.21 xlO5
1.09 ± 0.227 xlO6
4.21 ± 8.68 xlO1
1.86 ±0.39
2.53 ±0.31
2.06 ±0.23
1.51 ±0.08
1.48 ±0.16
5.21 ±0.46
2.33 ±0.47
3. 35 ±1.59
3.03 ±0.36
1.65 ±0.08
1.44 ±0.23
6.97 ±0.91
2.33 ±0.52
2.95 ±0.70
4.38 ±1.07
1.58 ±0.30
1.70 ±0.08
6.34 ±1.36
^Data are expressed as mean ± standard deviation of five replicates.
* Positive control coupons were inoculated but not exposed to ozone.
§ Test materials were inoculated and exposed to ozone for the contact time.
*Data are expressed as mean log reduction ± 95% CI of the SE.
* Ozone concentration, temperature and relative humidity listed are target levels. Refer to Table 4-2 for
actual levels.
20
-------
Table 5-2. Ozone Fumigation Results for B. anthracis Spores at 9,000 ppmv Ozone,
22 °C, 85% RH and 75% RH*
Contact
Time,
hr
Inoculation
Material Amount,
CFU
Positive Test
Control* Material
Mean Recovered B. anthracis Spores,
CFUf
Inactivation
Efficacy*
85% RH
4
6
8
Glass
Wood
TCarpet 1.08 xlO8
Laminate
Metal Ductwork
Wallboard Paper
Glass
Wood
TCarpet 1.08 xlO8
Laminate
Metal Ductwork
Wallboard Paper
Glass
Wood
TCarpet 1.08 xlO8
Laminate
Metal Ductwork
Wallboard Paper
1.00±1.10xl08
4.89 ± 1.29 xlO6
8.69 ± 1.38 xlO7
5.38 ± 3.23 xlO7
7.18 ± 1.69 xlO7
4.63 ± 1.03 x 107
1.00±1.10xl08
4.89 ± 1.29 xlO6
8.69 ± 1.38 xlO7
5.38±3.23xl07
7.18 ± 1.69 xlO7
4.63 ± 1.03 x 107
1.00 ± LlOxlO8
4.89 ± 1.29 xlO6
8.69 ± 1.38 xlO7
5.38 ± 3.23 xlO7
7.18 ± 1.69 xlO7
4.63 ± 1.03 x 107
1.67 ± 0.277 xlO5
4.34 ± 9.70 xlO2
4.49 ± 1.83 xlO3
2.88 ± 0.818 xlO5
1. 17 ± 0.341 xlO6
5.34±11.9xl01
9.53 ± 5.72 xlO4
1.34±3.00xl01
0.00 ± 0.00
7.83±1.15xl03
6.39 ± 4.96 xlO6
0.00 ± 0.00
6.79 ± 4.20 xlO4
0.00 ± 0.00
0.00 ± 0.00
3.64 ± 0.747 xlO4
3.77 ± 1.21 xlO5
0.00 ± 0.00
2.64 ±0.31
6.01 ±1.31
4.31 ±0.16
2.21 ±0.30
1.79 ±0.16
7. 17 ±0.96
3.26 ±0.97
6.31 ±0.72
>7.93 ± 0.06
3.76 ±0.28
2.17 ±0.37
>7.66 ± 0.09
3. 92 ±1.95
>6.68±0.10
>7.93 ± 0.06
3. 10 ±0.28
2.28 ±0.15
>7.66 ± 0.09
75% RH
4
6
8
Glass
Wood
Carpet LllxloB
Laminate
Metal Ductwork
Wallboard Paper
Glass
Wood
TCarpet 1.11 xlO8
Laminate
Metal Ductwork
Wallboard Paper
Glass
Wood
TCarpet 1.11 xlO8
Laminate
Metal Ductwork
Wallboard Paper
3.78 ± 0.718 xlO7
5.41 ± 1.69 xlO6
7.05±1.10xl07
3.77 ± 0.481 xlO7
5.98 ± 1.08 xlO7
3.55 ± 0.534 xlO7
3.78 ±0.718x10'
5.41 ±1.69 xlO6
7.05 ± LlOxlO7
3.77 ± 0.481 xlO7
5.98 ± 1.08 xlO7
3.55 ± 0.534 xlO7
3.78 ± 0.718 xlO7
5.41 ±1.69 xlO6
7.05 ± LlOxlO7
3.77 ± 0.481 xlO7
5.98 ± 1.08 xlO7
3.55 ± 0.534 xlO7
8.17±5.00xl05
4.32 ± 3.88 xlO4
3.54±1.43xl05
2.04 ± 0.181 xlO6
1.84 ± 0.909 xlO6
2.35 ± 2.90 xlO4
7.83 ± 2.82 x 10"
3.64 ± 2.12 xlO4
5.23 ± 2.43 xlO4
2.03 ± 0.128 xlO6
2.50 ± 0.930 xlO6
3.42 ± 3.79 xlO3
6.41 ± 4.12 xlO5
2.29 ± 4.59 xlO4
9.18 ± 11.5 xlO3
1.24 ± 0.247 xlO6
3.85±4.91xl06
2.79 ± 4.09 xlO3
1.72 ±0.24
2.31 ±0.52
2.33 ±0.19
1.27 ±0.06
1.55 ±0.20
3.65 ±0.69
1.70 ±0.14
2.21 ±0.24
3.16±0.18
1.27 ±0.06
1.40 ±0.18
5. 32 ±1.79
1.88 ±0.37
2.56 ±0.55
4.29 ±0.76
1.49 ±0.09
1.40 ±0.40
5.42 ±1.72
are expressed as mean ± standard deviation of five replicates.
* Positive control coupons were inoculated but not exposed to ozone.
§ Test materials were inoculated and exposed to ozone for the contact time.
*Data are expressed as mean log reduction ± 95% CI of the SE.
* Ozone concentration, temperature and relative humidity listed are target levels. Refer to Table 4-2 for
actual levels.
21
-------
Table 5-3. Ozone Fumigation Results for B. anthracis Spores at 9,000 ppmv Ozone,
22 °C, 85% RH*
Contact
Time,
hr
Inoculation
Material Amount,
CFU
Positive Test
Control* Material
Mean Recovered B. anthracis Spores,
CFUf
Inactivation
Efficacy *
85% RH
6
9
12
Glass
Wood
T CarPett 7.57 x 107
Laminate
Metal Ductwork
Wallboard Paper
Glass
Wood
TCarpet 7.57 xlO7
Laminate
Metal Ductwork
Wallboard Paper
Glass
Wood
T Carpett 7.57 x 107
Laminate
Metal Ductwork
Wallboard Paper
4.13 ±0.702x10'
5.20 ± 1.09 xlO6
8.02 ± 1.43 xlO7
4.86 ± 0.791 xlO7
4.36 ± 1.71 xlO7
5.55 ± 0.824 xlO7
4.13 ±0.702x10'
5.20 ± 1.09 xlO6
8.02 ± 1.43 xlO7
4.86 ± 0.791 xlO7
4.36 ± 1.71 xlO7
5.55 ± 0.824 xlO7
4.13 ±0.702x10'
5.20 ± 1.09 xlO6
8.02 ± 1.43 xlO7
4.86 ± 0.791 xlO7
4.36 ± 1.71 xlO7
5.55 ± 0.824 xlO7
1.71 ± 3.01 xlO4
1.34±3.00xl01
0.00 ± 0.00
8.72 ± 2.25 xlO4
8.90 ± 7.00 xlO4
0.00 ± 0.00
8.17 ± 18.2 xlO3
0.00 ± 0.00
0.00 ± 0.00
1.74 ± 1.66 xlO4
5.38±1.55xl03
0.00 ± 0.00
6.66 ±14.9x10'
0.00 ± 0.00
0.00 ± 0.00
8.80±1.16xl04
1.47 ± 0.758 xlO5
0.00 ± 0.00
4.81 ±1.67
6.34 ±0.72
>7.90 ± 0.07
2.75 ±0.13
2.81 ±0.41
>7.74 ± 0.05
5.68 ±1.47
>6.71±0.08
>7.90 ± 0.07
3.78 ±0.72
3. 90 ±0.19
>7.74 ± 0.05
7.11 ±0.99
>6.71±0.08
>7.90 ± 0.07
2.74 ±0.08
2.55 ±0.40
>7.74 ± 0.05
are expressed as mean ± standard deviation of five replicates.
* Positive control coupons were inoculated but not exposed to ozone.
§ Test materials were inoculated and exposed to ozone for the contact time.
*Data are expressed as mean log reduction ± 95% CI of the SE.
* Ozone concentration, temperature and relative humidity listed are target levels. Refer to Table 4-2 for
actual levels.
22
-------
Table 5-4. Ozone Fumigation Results for B. anthracis Spores at 9,800 ppmv Ozone,
22 °C, 85% RH*
Contact
Time,
hr
6
9
12
Inoculation
Material Amount,
CFU
Glass
Wood
Carpet l Q4 x 1()8
Laminate
Metal Ductwork
Wallboard Paper
Glass
Wood
Carpet 8
1 04 x 10
Laminate
Metal Ductwork
Wallboard Paper
Glass
Wood
Carpet , „ . Irt8
1 04 x 10
Laminate
Metal Ductwork
Wallboard Paper
Positive
Test
Control* Material
Mean Recovered B. anthracis Spores,
CFU"
5.41 ± 2.53 xlO7
1.02 ± 0.958 xlO7
8.37 ± 1.63 xlO7
4.25 ± 1.06 xlO7
6.19 ± 1.41 xlO7
1. 10 ± 0.917 xlO8
5.41 ±2.53x10'
1.02 ± 0.958 xlO7
8.37 ± 1.63 xlO7
4.25 ± 1.06 xlO7
6.19 ± 1.41 xlO7
1. 10 ± 0.917 xlO8
5.41 ±2.53x10'
1.02 ± 0.958 xlO7
8.37 ± 1.63 xlO7
4.25 ± 1.06 xlO7
6.19 ± 1.41 xlO7
1. 10 ± 0.917 xlO8
I
7.93 ± 17.4 x 102
0.00 ± 0.00
0.00 ± 0.00
1.3 1± 0.447 xlO4
4.70 ± 4.77 xlO4
0.00 ± 0.00
1.34 ±3.00x10'
0.00 ± 0.00
0.00 ± 0.00
2.28 ± 1.69 xlO4
3.17±6.23xl04
0.00 ± 0.00
1.14 ±2.55x10"
6.60 ± 14.8
0.00 ± 0.00
5.27 ± 3.91 xlO2
1.31±1.77xl03
0.00 ± 0.00
Inactivation
Efficacy *
6.61 ±1.42
>6.90 ± 0.27
>7.92 ± 0.08
3.52 ±0.17
3.34 ±0.48
>7.84 ± 0.48
7.33 ±0.74
>6.90 ± 0.27
>7.92 ± 0.08
3.35 ±0.30
4.00 ±0.75
>7.84 ± 0.48
6.95 ±1.48
6.60 ±0.65
>7.92 ± 0.08
5.39±1.11
4.97 ±0.51
>7.84 ± 0.48
Data are expressed as mean ± standard deviation of five replicates.
* Positive control coupons were inoculated but not exposed to ozone.
§ Test materials were inoculated and exposed to ozone for the contact time.
*Data are expressed as mean log reduction ± 95% CI of the SE.
* Ozone concentration, temperature and relative humidity listed are target levels. Refer to Table 4-2 for
actual levels.
23
-------
Table 5-5. Ozone Fumigation Results for B. anthracis Spores at 9,800 ppmv Ozone,
22 °C, 85% RH (with pre-humidification)*1
Contact Inoculation
Time, Material Amount,
hr CFU
Glass
Wood
6 TCarpett 9.03 xlO7
Laminate
Metal Ductwork
Wallboard Paper
Glass
Wood
9 TCarpet 9.03 xlO7
Laminate
Metal Ductwork
Wallboard Paper
Glass
Wood
12 TCarpet 9.03 xlO7
Laminate
Metal Ductwork
Wallboard Paper
Positive Test
Control* Material
Mean Recovered B. anthracis Spores,
CFUf
2.74±1.10xl07
3.77 ± 1.48 xlO6
5.87 ± 1.30 xlO7
2.20±1.17xl07
2.47 ± 1.46 xlO7
3.20 ± 1.09 xlO7
2.74 ±1.10x10'
3.77 ± 1.48 xlO6
5.87 ± 1.30 xlO7
2.20±1.17xl07
2.47 ± 1.46 xlO7
3.20 ± 1.09 xlO7
2.74±1.10xl07
3.77 ± 1.48 xlO6
5.87 ± 1.30 xlO7
2.20±1.17xl07
2.47 ± 1.46 xlO7
3.20 ± 1.09 xlO7
2.47 ± 1.43 xlO2
0.00 ± 0.00
0.00 ± 0.00
1.81±1.24xl04
5.61 ± 2.02 xlO4
0.00 ± 0.00
9.54 ± 12.4 xlO2
0.00 ± 0.00
0.00 ± 0.00
4.29 ± 1.77 xlO3
1.00 ± 2.07 xlO5
0.00 ± 0.00
9.34 ± 11.2 xlO1
0.00 ± 0.00
0.00 ± 0.00
1.88 ± 0.720 xlO3
1.12±1.57xl04
0.00 ± 0.00
Inactivation
Efficacy *
5. 13 ±0.42
>6.54±0.19
>7.76 ± 0.09
3. 10 ±0.30
2.58 ±0.32
>7.48±0.17
5. 11 ±1.20
>6.54±0.19
>7.76 ± 0.09
3.70 ±0.28
3.07 ±0.77
>7.48±0.17
6.13 ±1.05
>6.54±0.19
>7.76 ± 0.09
4.05 ±0.26
3.85 ±0.97
>7.48±0.17
Immediately following inoculation, all materials were kept at 85% ± 5% RH for approximately 24 hours
prior to introduction of ozone.
t Data are expressed as mean ± standard deviation of five replicates.
* Positive control coupons were inoculated but not exposed to ozone.
§ Test materials were inoculated and exposed to ozone for the contact time.
*Data are expressed as mean log reduction ± 95% CI of the SE.
* Ozone concentration, temperature and relative humidity listed are target levels. Refer to Table 4-2 for
actual levels.
24
-------
Table 5-6. Ozone Fumigation Results for B. anthracis Spores at 11,000+ ppmv
Ozone, 22 °C, 85% RH*
Contact
Time,
hr
6
9
12
Inoculation
Material Amount,
CFU
Glass
Wood
T Carpet 8.60 x 107
Laminate
Metal Ductwork
Wallboard Paper
Glass
Wood
TCarpett 8.60 xlO7
Laminate
Metal Ductwork
Wallboard Paper
Glass
Wood
TCarpet 8.60 xlO7
Laminate
Metal Ductwork
Wallboard Paper
Positive Test
Control* Material
Mean Recovered B. anthracis Spores,
CFUf
3.21 ± 0.476 xlO7
2.87 ± 1.07 xlO6
6.57 ± 0.745 xlO7
3.16±0.516xl07
4.02 ± 0.583 xlO7
3.43 ± 0.720 xlO7
3.21 ±0.476x10'
2.87 ± 1.07 xlO6
6.57 ± 0.745 xlO7
3.16±0.516xl07
4.02 ± 0.583 xlO7
3.43 ± 0.720 xlO7
3.21 ±0.476x10'
2.87 ± 1.07 xlO6
6.57 ± 0.745 xlO7
3.16±0.516xl07
4.02 ± 0.583 xlO7
3.43 ± 0.720 xlO7
8.15 ± 7.61 xlO3
1.07 ± 1.88 xlO2
6.60 ±14.8
1.81 ± 0.853 xlO5
1.74 ± 1.44 xlO5
0.00 ± 0.00
3.59±3.29xl03
0.00 ± 0.00
0.00 ± 0.00
1.43 ± 0.901 xlO3
1.12±1.13xl04
0.00 ± 0.00
2.00 ± 2.74 xlO2
0.00 ± 0.00
0.00 ± 0.00
5.93±5.28xl02
1.59±1.81xl03
0.00 ± 0.00
Inactivation
Efficacy *
4.04 ±0.82
5.50 ±1.14
7.51 ±0.60
2.27 ±0.16
2.50 ±0.35
>7.53±0.08
4.16 ±0.50
>6.43±0.16
>7.82 ± 0.04
4.43 ±0.31
3.70 ±0.35
>7.53±0.08
6.05 ±1.18
>6.43±0.16
>7.82 ± 0.04
4.92 ±0.46
4.72 ±0.57
>7.53±0.08
are expressed as mean ± standard deviation of five replicates.
* Positive control coupons were inoculated but not exposed to ozone.
§ Test materials were inoculated and exposed to ozone for the contact time.
*Data are expressed as mean log reduction ± 95% CI of the SE.
+ This concentration was measured by the Mini Hicon-LR analyzer and corresponds to -9,800 ppmv on the
IN2000-L2-LC analyzer.
* Ozone concentration, temperature and relative humidity listed are target levels. Refer to Table 4-2 for
actual levels.
25
-------
Table 5-7. Ozone Fumigation Results for B. anthracis Spores at 12,000+ ppmv
Ozone, 22 °C, 85% RH*
Contact
Time,
hr
6
9
12
Inoculation
Material Amount,
CFU
Glass
Wood
TCarpet 1.29 xlO8
Laminate
Metal Ductwork
Wallboard Paper
Glass
Wood
TCarpett 1.29 xlO8
Laminate
Metal Ductwork
Wallboard Paper
Glass
Wood
TCarpet 1.29 xlO8
Laminate
Metal Ductwork
Wallboard Paper
Positive Test
Control* Material
Mean Recovered B. anthracis Spores,
CFUf
6.19 ± 3.29 xlO7
2.21 ± 3.32 xlO7
9.38 ± 0.750 xlO7
5.93 ± 0.727 x 107
5.29 ± 2.00 xlO7
3.13±0.268xl07
6.19 ±3.29x10'
2.21 ± 3.32 xlO7
9.38 ± 0.750 xlO7
5.93 ± 0.727 x 107
5.29 ± 2.00 xlO7
3.13±0.268xl07
6.19 ±3.29x10'
2.21 ± 3.32 xlO7
9.38 ± 0.750 xlO7
5.93 ± 0.727 x 107
5.29 ± 2.00 xlO7
3.13±0.268xl07
6.01 ± 7.24 xlO1
0.00 ± 0.00
I.ll±1.60xl03
9.12±9.45xl02
2.06 ± 1.88 xlO2
0.00 ± 0.00
2.67 ±2.79x10'
6.60 ±14.8
0.00 ± 0.00
1.00 ± 2.06 xlO2
3.12±3.66xl02
6.60 ±14.8
0.00 ± 0.00
0.00 ± 0.00
0.00 ± 0.00
9.61 ± 19.1 xlO2
7.33 ± 8.94 xlO1
0.00 ± 0.00
Inactivation
Efficacy *
6.52 ±1.03
>7.04 ± 0.46
6.21 ±1.50
5.05 ±0.48
5.88 ±0.98
>7.49 ± 0.03
6.69 ±0.89
6.74 ±0.75
>7.97 ± 0.03
6.93 ± 1.07
5.79 ±1.03
7.19 ±0.60
>7.66±0.41
>7.04 ± 0.46
>7.97 ± 0.03
5.82 ±1.16
6.50 ±1.00
>7.49 ± 0.03
are expressed as mean ± standard deviation of five replicates.
* Positive control coupons were inoculated but not exposed to ozone.
§ Test materials were inoculated and exposed to ozone for the contact time.
*Data are expressed as mean log reduction ± 95% CI of the SE.
+ This concentration was measured by the Mini Hicon-LR analyzer and corresponds to -10,800 ppmv on
the IN2000-L2-LC analyzer.
* Ozone concentration, temperature and relative humidity listed are target levels. Refer to Table 4-2 for
actual levels.
26
-------
= 6
I
i i I I
75«o.4 -5V6 75°o,S S5»o.4 85«6.6 85«o.J
RH. Time (lir)
7,000 ppm O3, 75% and 85% RH
i
i
m
4 6 8 6 9 12
Time (lir)
9,000 ppm O3, 85% RH
Time (In)
9,000 ppm O3, 75% RH
i: e*
Time flu)
^Coupons were pre-humidified for - 24 hours
9,800 ppm O3, 85% RH
12
11,000 ppm O3, 85% RH
12,000 ppm O3, 85% RH
Figure 5-1. Efficacy (log reduction) against B. anthracis on glass.
(Data are expressed as mean log reduction ± 95% CI of the SE)
27
-------
!
RH, Time (In)
7,000 ppm O3, 75% and 85% RH
I
1 4
8 6
Tim* (In)
9,000 ppm O3, 85% RH
i:
Time (In)
9,000 ppm O3, 75% RH
12 6*
I Hill- (111 )
*Conpons were pi e-luunidifiecl for 24 hours
9,800 ppm O3, 85% RH
12*
Time (In)
11,000 ppm O3, 85% RH
12,000 ppm O3, 85% RH
Figure 5-2. Efficacy (log reduction) against B. anthracis on wood.
(Data are expressed as mean log reduction ± 95% CI of the SE)
28
-------
8
7
I 6
'•5 5
t
S5°o,4 S5°o,6
RH, Tim* (hi )
7,000 ppm O3, 75% and 85% RH
I
M
5
0
8 6
Time (In)
12
9,000 ppm O3, 85% RH
Time (hi)
9,000 ppm O3, 75% RH
I
1
i: e*
Time (In)
*Coupons were pre-hiimidified for -24 hours
9,800 ppm O5, 85% RH
12*
j
9
Time (In)
11,000 ppm O3, 85% RH
Time (hi)
12,000 ppm O3, 85% RH
Figure 5-3. Efficacy (log reduction) against B. anthracis on carpet.
(Data are expressed as mean log reduction ± 95% CI of the SE)
29
-------
8
7 -
I'1
i-l i
1 -
0 -
Ii §
I I
RH
, Time (hi )
85V 6
S?°o.S
7,000 ppm O3, 75% and 85% RH
i
i;
Time dm
9,000 ppm O3, 75% RH
11,000 ppm O3, 85% RH
t
6869
Time (hi)
9,000 ppm O3, 85% RH
_ 6
S
V
I -t
1,
-I 1
FFFFFt
12 6* 9*
Time (hi)
*Coupons were pre-huniidified for 24 hours
9,800 ppm O3, 85% RH
9
Time (hi >
12,000 ppm O3, 85% RH
Figure 5-4. Efficacy (log reduction) against B. anthracis on laminate.
(Data are expressed as mean log reduction ± 95% CI of the SE)
30
-------
'5%.8 S5V4
RH. Time (hi)
S?°o.6 85%.S
7,000 ppm O3, 75% mid 85% RH
8 6
Time (lir)
9,000 ppm O3, 85% RH
I
'€ ?
I4
9,000 ppm O3, 75% RH
8
-
. 6
f 5
i: 6'
Time (In)
^Coupons were pre-hiiniidified for --24 hours
9,800 ppm O3, 85% RH
12'
Time (hi)
11,000 ppm O3, 85% RH
Time (In)
12,000 ppm O3, 85% RH
Figure 5-5. Efficacy (log reduction) against B. anthracis on metal ductwork.
(Data are expressed as mean log reduction ± 95% CI of the SE)
31
-------
"5° o.4 ?5° 0,6 75°o.S 85° o. 4 8?° 0.6 S5°o.S
RH, Time (In)
7,000 ppm O3, 75% and 85% RH
8 6
Tim* (hi)
9,000 ppm O3, 85% RH
12
Time (In)
9,000 ppm O3, 75% RH
in e*
Time (lu)
""Couponswere pre-huniidifiedfor 24 hours
9,800 ppm O3, 85% RH
9
Time (In)
11,000 ppm O3, 85% RH
12
12,000 ppm O3, 85% RH
Figure 5-6. Efficacy (log reduction) against B. anthmcis on wallboard paper.
(Data are expressed as mean log reduction ± 95% CI of the SE)
32
-------
5.2 Inactivation of B. subtilis Spores
7,000 ppmv Ozone. Ozone fumigation
results for B. subtilis spores at 7,000
ppmv ozone are presented in Table 5-8
and Figures 5-7 through 5-12. At 85%
RH, no viable B. subtilis spores were
recovered from wallboard paper at 6 or 8
hours or on laminate at 8 hours. Viable
B. subtilis spores were recovered from
the materials at all other contact times at
this concentration and at both 85% and
75% RH (log reductions for all materials
ranged from 0.38 on wood to 7.60 on
glass).
9,000 ppmv Ozone. Ozone fumigation
results for B. subtilis spores at 9,000
ppmv ozone are presented in Tables 5-9
and 5-10 and Figures 5-7 through 5-12.
At 85% RH, no viable B. subtilis spores
were recovered from metal ductwork at
12 hours, from laminate at 6 or 9 hours,
or from wallboard paper at 6, 9 or 12
hours. Viable B. subtilis spores were
recovered from the materials at all other
contact times at this concentration and at
both 85% and 75% RH (log reductions
for all materials ranged from 0.69 on
wood to 7.17 on metal ductwork).
9,800 ppmv Ozone (with and without
24 hour pre-humidification). Ozone
fumigation results for B. subtilis spores
at 9,800 ppmv ozone are presented in
Tables 5-11 and 5-12 and Figures 5-7
through 5-12. At 85% RH, no viable B.
subtilis spores were recovered from
painted wallboard paper following the 6,
9 and 12 hour contact times, laminate
following the 9 and 12 hour contact
times, and galvanized metal following
the 9 hour contact time. After subjecting
the test materials to 85% RH for -24
hours and then introducing 9,800 ppmv,
no viable B. subtilis spores were
recovered from wood or pained
wallboard paper after 6, 9 or 12 hours,
glass after 6 hours or laminate after 12
hours. Spores were recovered from the
materials at all other contact times at this
concentration (log reductions for all
materials ranged from 3.23 to 7.82 on
laminate). Pre-humidification of the
chamber for 24 hours increased the
efficacy of the ozone gas on both wood
and carpet, but did not increase the
efficacy on the other materials in most
instances.
12,000 ppmv Ozone. Ozone fumigation
results for B. subtilis spores at 12,000
ppmv ozone are presented in Table 5-13
and Figures 5-7 through 5-12. At 85%
RH, no viable B. subtilis spores were
recovered from glass at 12 hours or from
laminate and wallboard paper at 6, 9 or
12 hours. Viable B. subtilis spores were
recovered from the materials at all other
contact times at this concentration (log
reductions for all materials ranged from
2.80 on wood to 7.80 on laminate).
All associated laboratory and procedural
blanks resulted in 0 CFU recovered for
all B. subtilis tests.
33
-------
Table 5-8. Ozone Fumigation Results for B. subtilis Spores at 7,000 ppmv Ozone,
22 °C, 85% RH and 75% RH
Contact
Time,
hr
Inoculation
Material Amount,
CFU
Positive Test
Control* Material
Mean Recovered B. anthrads Spores,
CFUf
Inactivation
Efficacy *
85% RH
4
6
8
Glass
Wood
TCarpet 3.17 xlO8
Laminate
Metal Ductwork
Wallboard Paper
Glass
Wood
TCarpet 3.17xl08
Laminate
Metal Ductwork
Wallboard Paper
Glass
Wood
TCarpet 3.17xl08
Laminate
Metal Ductwork
Wallboard Paper
1.30 ± 1.46 xlO8
1.42 ± 2.04 xlO7
1.61 ± 1.87 xlO8
1.07 ± 0.252 xlO8
2.36 ± 0.811 xlO8
3.08 ± 1.53 xlO7
1.30 ± 1.46 xlO8
1.42 ± 2.04 xlO7
1.61 ± 1.87 xlO8
1.07 ± 0.252 xlO8
2.36 ± 0.811 xlO8
3.08 ± 1.53 xlO7
1.30 ±1.46x10"
1.42 ± 2.04 xlO7
1.61 ± 1.87 xlO8
1.07 ± 0.252 xlO8
2.36 ± 0.811 xlO8
3.08 ± 1.53 xlO7
1.54 ± 2.74 xlO4
2.84 ± 1.00 xlO5
3.12±2.40xl06
6.67±12.1xl02
9.68 ± 5.36 xlO5
1.32 ± 1.81 xlO1
1.05 ± 2.34 xlO5
9.65 ± 12.3 xlO3
4.22±3.13xl05
5.67±11.8xl02
7.53±3.13xl05
0.00 ± 0.00
1.34 ±3.00x10'
1.30 ± 1.58 xlO4
1. 17 ± 0.498 xlO6
0.00 ± 0.00
1.05 ± 1.06 xlO5
0.00 ± 0.00
4.51 ±0.93
1.48 ±0.44
1.72 ±0.56
6.49 ±1.37
2.42 ± 0.27
6.84 ±0.76
6.51 ±2.19
4.00 ±1.66
2.82 ±0.99
6.57 ±1.28
2.51 ±0.23
>7.44 ± 0.20
7.60 ±0.78
3.30 ±0.87
2.01 ±0.38
>8.02 ± 0.09
3.45 ±0.32
>7.44 ± 0.20
75% RH
4
6
8
Glass
Wood
T Carpet 7.67 x 107
Laminate
Metal Ductwork
Wallboard Paper
Glass
Wood
T Carpet 7.67 x 107
Laminate
Metal Ductwork
Wallboard Paper
Glass
Wood
TCarpet 7.67 xlO7
Laminate
Metal Ductwork
Wallboard Paper
4.31 ± 1.07 xlO7
1.49 ± 0.794 xlO6
3.02±1.18xl07
4.77 ± 0.425 x 107
3.23±3.17xl07
4.17 ± 1.25 xlO6
4.31 ± 1.07 xlO7
1.49 ± 0.794 xlO6
3.02±1.18xl07
4.77 ± 0.425 x 107
3.23±3.17xl07
4.17 ± 1.25 xlO6
4.31 ± 1.07 xlO7
1.49 ± 0.794 xlO6
3.02±1.18xl07
4.77 ± 0.425 x 107
3.23±3.17xl07
4.17 ± 1.25 xlO6
3.08 ± 1.36 xlO6
5.83 ± 1.56 xlO5
4.22 ± 1.22 xlO6
4.63 ± 0.534 xlO6
8.31 ± 1.22 xlO6
5.37 ± 4.27 xlO3
7.30 ± 5.52 xlO5
3.82 ± 1.05 xlO5
3.70 ± 0.0915 xlO6
3.57 ± 2.44 xlO6
7.56 ± 1.58 xlO6
2.08 ± 4.11 xlO3
1.63 ± 0.267 xlO5
5.54 ± 2.17 xlO5
7.11 ± 1.78 xlO5
1.43 ± 0.210 xlO6
5.80 ± 1.21 xlO6
2.97 ± 6.61 xlO3
1.18 ±0.22
0.38 ±0.21
0.83 ±0.24
1.01 ±0.06
0.39 ±0.42
3.07 ±0.49
1.88 ±0.35
1.71 ±2.17
0.87 ±0.21
1.25 ±0.36
0.44 ± 0.42
5.81 ±1.56
2.42 ±0.11
0.42 ±0.25
1.60 ±0.23
1.53 ±0.06
0.55 ±0.42
5. 17 ±1.50
are expressed as mean ± standard deviation of five replicates.
* Positive control coupons were inoculated but not exposed to ozone.
§ Test materials were inoculated and exposed to ozone for the contact time.
*Data are expressed as mean log reduction ± 95% CI of the SE.
* Ozone concentration, temperature and relative humidity listed are target levels. Refer to Table 4-3 for
actual levels.
34
-------
Table 5-9. Ozone Fumigation Results for B. subtilis Spores at 9,000 ppmv Ozone,
22 °C, 85% RH and 75% RH
Contact
Time,
hr
Inoculation
Material Amount,
CFU
Positive
Test
Control* Material
Mean Recovered B. anthrads Spores,
CFU"
I
Inactivation
Efficacy *
85% RH
4
6
8
Glass
Wood
Carpet : 04 x 108
Laminate
Metal Ductwork
Wallboard Paper
Glass
Wood
Carpet 104 xlO8
Laminate
Metal Ductwork
Wallboard Paper
Glass
Wood
Carpet 104 xlO8
Laminate
Metal Ductwork
Wallboard Paper
3.96 ± 0.646 xlO7
1.31 ± 0.106 xlO6
3.67 ± 0.655 xlO7
4.39 ± 0.270 xlO7
5.63 ± 1.51 xlO7
3.25 ± 0.666 xlO6
3.96 ± 0.646 xlO7
1.31 ± 0.106 xlO6
3.67 ± 0.655 xlO7
4.39 ± 0.270 xlO7
5.63 ± 1.51 xlO7
3.25 ± 0.666 xlO6
3.96 ± 0.646 xlO7
1.31 ± 0.106 xlO6
3.67 ± 0.655 xlO7
4.39 ± 0.270 xlO7
5.63 ± 1.51 xlO7
3.25 ± 0.666 xlO6
8.19 ± 6.28 xlO4
2.72 ± 1.46 xlO5
3.86 ± 2.53 xlO6
7.49 ± 1.64 xlO4
4.28 ± 3.71 xlO6
1.16±1.79xl04
6.55 ± 4.08 xlO3
1.25 ± 0.456 xlO5
2.94 ± 1.58 xlO5
1. 55 ± 0.631 xlO5
2.82 ± 1.01 xlO6
4.72 ± 6.23 xlO2
1.09 ± 0.614 xlO3
6.33 ± 4.78 xlO4
5.02 ± 2.17 xlO4
5.59 ± 1.59 xlO4
5.79 ± 5.45 xlO5
1.87 ± 3.64 xlO2
2.84 ±0.42
0.74 ±0.23
2.06 ±0.29
2.78 ±0.09
1.23 ±0.34
3. 17 ±0.99
3.87 ±0.30
1.04 ±0.14
2.15 ±0.25
2.48 ±0.14
1.32 ±0.21
5.30 ±1.45
4.60 ±0.21
1.53 ±0.51
2.89 ±0.18
2.91 ±0.13
2.15 ±0.43
5.52 ±1.22
75% RH
4
6
8
Glass
Wood
Carpet 8 83 x 107
Laminate
Metal Ductwork
Wallboard Paper
Glass
Wood
Carpet „ „„ , _7
8 83 x 10
Laminate
Metal Ductwork
Wallboard Paper
Glass
Wood
Carpet 8 83 x 107
Laminate
Metal Ductwork
Wallboard Paper
3.05 ±0.890x10'
1.52 ± 0.236 xlO6
3.63 ± 1.02 x 107
4.43 ± 0.328 x 107
6.50 ± 1.69 xlO7
1.59 ± 1.31 xlO7
3.05 ±0.890x10'
1.52 ± 0.236 xlO6
3.63 ± 1.02 x 107
4.43 ± 0.328 x 107
6.50 ± 1.69 xlO7
1.59 ± 1.31 xlO7
3.05 ±0.890x10'
1.52 ± 0.236 xlO6
3.63 ± 1.02 x 107
4.43 ± 0.328 xlO7
6.50 ± 1.69 xlO7
1.59 ± 1.31 xlO7
1.23 ±1.60x10"
3.50 ± 1.55 xlO5
6.16 ± 4.45 xlO5
9.61 ± 4.05 xlO4
1.46 ± 0.938 xlO6
1.18±2.54xl03
4.49 ± 2.57 xlO3
2.74±1.14xl05
4.40 ± 2.90 xlO5
1.86 ± 1.47 xlO5
1. 33 ± 0.938 xlO6
3.47 ± 6.30 xlO3
1.00 ± 1.70 xlO2
1.25 ± 0.795 xlO5
2.03 ± 1.35 xlO5
1.19±1.65xl04
1. 19 ± 0.909 xlO5
2.00 ± 4.47 xlO1
2.77 ± 0.67
0.69 ±0.24
1.87 ±0.35
2.70 ±0.17
1.71 ±0.26
5.87 ±1.55
3.91 ±0.35
0.78 ±0.23
2.02 ±0.37
2.50 ±0.34
1.75 ±0.26
4.08 ±0.74
6.28 ±1.02
1.14 ±022
2.32 ±0.29
3.84 ±0.44
2.80 ±0.26
6.67 ±0.86
rData are expressed as mean ± standard deviation of five replicates.
* Positive control coupons were inoculated but not exposed to ozone.
§ Test materials were inoculated and exposed to ozone for the contact time.
*Data are expressed as mean log reduction ± 95% CI of the SE.
* Ozone concentration, temperature and relative humidity listed are target levels. Refer to Table 4-3 for
actual levels.
35
-------
Table 5-10. Ozone Fumigation Results for B. subtilis Spores at 9,000 ppmv Ozone,
22 °C, 85% RH
Contact
Time,
hr
Inoculation
Material Amount,
CFU
Positive
Control*
Mean Recovered B.
Test
Material
anthracis Spores,
_*
Inactivation
Efficacy *
CFU1
85% RH
6
9
12
Glass
Wood
Carpet 7
9 03 x 10
Laminate
Metal Ductwork
Wallboard Paper
Glass
Wood
Carpet 9 03 xlO7
Laminate
Metal Ductwork
Wallboard Paper
Glass
Wood
Carpet 9 03 xlO7
Laminate
Metal Ductwork
Wallboard Paper
2.71 ± 0.741 xlO7
1.54 ± 0.366 xlO6
4.98 ± 1.06 xlO7
3.95 ± 0.498 xlO7
3.09 ± 0.945 xlO7
5.90 ± 2.32 xlO6
2.71 ± 0.741 xlO7
1.54 ± 0.366 xlO6
4.98 ± 1.06 xlO7
3.95 ± 0.498 xlO7
3.09 ± 0.945 xlO7
5.90 ± 2.32 xlO6
2.71 ± 0.741 xlO7
1.54 ± 0.366 xlO6
4.98 ± 1.06 xlO7
3.95 ± 0.498 xlO7
3.09 ± 0.495 xlO7
5.90 ± 2.32 xlO6
1.00 ± 1.88 xlO2
6.63 ± 8.04 x 103
1.30 ± 1.51 xlO4
0.00 ± 0.00
6.60 ±14.8
0.00 ± 0.00
8.66 ± 19.4 xlO1
6.51 ± 8.38 xlO3
9.32 ± 12.9 xlO2
0.00 ± 0.00
1.32 ± 1.81 xlO1
0.00 ± 0.00
6.60 ±14.8
6.60 ±14.8
1.93 ± 3.72 xlO3
6.60 ±14.8
0.00 ± 0.00
0.00 ± 0.00
6.53 ±1.11
2.62 ±0.49
3.84 ±0.52
>7.59±0.05
7.17 ±0.61
>6.75±0.14
6.89 ±1.04
3.41 ±1.53
5. 14 ±0.65
>7.59±0.05
6.86 ±0.74
>6.75±0.14
7.11±0.61
5.87 ±0.60
5.99 ±1.56
7.29 ±0.60
>7.47±0.13
>6.75±0.14
are expressed as mean ± standard deviation of five replicates.
* Positive control coupons were inoculated but not exposed to ozone.
§ Test materials were inoculated and exposed to ozone for the contact time.
*Data are expressed as mean log reduction ± 95% CI of the SE.
* Ozone concentration, temperature and relative humidity listed are target levels. Refer to Table 4-3 for
actual levels.
36
-------
Table 5-11. Ozone Fumigation Results for B. subtilis Spores at 9,800 ppmv Ozone,
22 °C, 85% RH
Contact
Time,
hr
6
9
12
Inoculation
Material Amount,
CFU
Glass
Wood
Carpet _ ._ ,.7
9 47 x 10
Laminate
Metal Ductwork
Wallboard Paper
Glass
Wood
Carpet _ ._ ,.7
947x10
Laminate
Metal Ductwork
Wallboard Paper
Glass
Wood
Carpet 9 47 xlO7
Laminate
Metal Ductwork
Wallboard Paper
Positive
Test
Control* Material
Mean Recovered B. anthracis Spores,
CFU"
2.88 ±0.800x10'
2.53 ± 0.958 xlO6
4.63±1.18xl07
6.65 ± 1.30 xlO7
4.69 ± 1.77 xlO7
7.79 ± 2.69 xlO6
2.88 ±0.800x10'
2.53 ± 0.958 xlO6
4.63±1.18xl07
6.65 ± 1.30 xlO7
4.69 ± 1.77 xlO7
7.79 ± 2.69 xlO6
2.88 ± 0.800 xlO7
2.53 ± 0.958 xlO6
4.63±1.18xl07
6.65 ± 1.30 xlO7
4.69 ± 1.77 xlO7
7.79 ± 2.69 xlO6
I
1.87 ± 2.18 xlO2
3.21 ± 6.71 xlO3
3.07 ± 2.59 xlO3
6.60 ±14.8
6.68 ± 6.25 xlO1
0.00 ± 0.00
1.20 ± 2.50 xlO2
9.34 ± 16.7 xlO3
4.19 ± 5.31 xlO2
0.00 ± 0.00
0.00 ± 0.00
0.00 ± 0.00
1.34±3.00xl01
4.86 ± 10.9 xlO2
4.07 ± 8.91 xlO2
0.00 ± 0.00
4.00 ± 2.81 xlO1
0.00 ± 0.00
Inactivation
Efficacy *
5.68 ±0.94
4.54 ±1.61
4.35 ±0.47
7.51 ±0.60
6. 17 ±0.77
>6.87±0.14
6.59 ±1.10
3.64 ±1.58
5. 34 ±0.54
>7.82 ± 0.08
>7.65±0.15
>6.87±0.14
7.08 ±0.73
5.70 ±1.34
6.69 ±1.29
>7.82 ± 0.08
6.31 ±0.69
>6.87±0.14
rData are expressed as mean ± standard deviation of five replicates.
* Positive control coupons were inoculated but not exposed to ozone.
§ Test materials were inoculated and exposed to ozone for the contact time.
*Data are expressed as mean log reduction ± 95% CI of the SE.
* Ozone concentration, temperature and relative humidity listed are target levels. Refer to Table 4-3 for
actual levels.
37
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Table 5-12. Ozone Fumigation Results for B. subtilis Spores at 9,800 ppmv Ozone,
22 °C, 85% RH (with pre-humidification)!
Contact
Time,
hr
6
9
12
Inoculation
Material Amount,
CFU
Glass
Wood
Carpet . „_ ,.7
4 87 x 10
Laminate
Metal Ductwork
Wallboard Paper
Glass
Wood
Carpet 7
4 87 x 10
Laminate
Metal Ductwork
Wallboard Paper
Glass
Wood
Carpet 487xl07
Laminate
Metal Ductwork
Wallboard Paper
Positive
Test
Control* Material
Mean Recovered B. anthracis Spores,
CFU"
2.01 ± 0.794 xlO7
2.41 ± 1.27 xlO6
3.02±1.16xl07
1.93 ± 0.781 xlO7
1.40 ± 0.198 xlO7
9.74 ± 3.26 xlO5
2.01 ± 0.794 xlO7
2.41 ± 1.27 xlO6
3.02±1.16xl07
1.93 ± 0.781 xlO7
1.40 ± 0.198 xlO7
9.74 ± 3.26 xlO5
2.01 ± 0.794 xlO7
2.41 ± 1.27 xlO6
3.02±1.16xl07
1.93 ± 0.781 xlO7
1.40 ± 0.198 xlO7
9.74 ± 3.26 xlO5
I
0.00 ± 0.00
0.00 ± 0.00
5.34±5.76xl02
1. 19 ± 0.613 xlO4
1.07 ± 1.52 xlO2
0.00 ± 0.00
6.66 ± 8.50 xlO1
0.00 ± 0.00
1.32 ± 1.81 xlO1
3.32±2.37xl01
2.00 ± 2.99 xlO1
0.00 ± 0.00
4.68 ± 7.32 xlO1
0.00 ± 0.00
2.00 ± 4.47 xlO1
0.00 ± 0.00
5.34±11.9xl01
0.00 ± 0.00
Inactivation
Efficacy *
>7.28±0.15
>6.32±0.25
4.97 ±0.52
3.23 ±0.26
5. 86 ±1.05
>5.97±0.13
6.11 ±0.97
>6.32±0.25
6.84 ±0.75
5. 98 ±0.65
6.47 ±0.81
>5.97±0.13
6.47 ±0.99
>6.32±0.25
7.05 ±0.80
>7.26±0.15
6.66 ±0.95
>5.97±0.13
Immediately following inoculation, all materials were kept at 85% ± 5% RH for approximately 24 hours
prior to introduction of ozone.
t Data are expressed as mean ± standard deviation of five replicates.
* Positive control coupons were inoculated but not exposed to ozone.
§ Test materials were inoculated and exposed to ozone for the contact time.
*Data are expressed as mean log reduction ± 95% CI of the SE.
* Ozone concentration, temperature and relative humidity listed are target levels. Refer to Table 4-3 for
actual levels.
38
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Table 5-13. Ozone Fumigation Results for B. subtitis Spores at 12,000+ ppmv Ozone,
22 °C, 85% RH
Contact
Time,
hr
Inoculation
Material Amount,
CFU
Positive
Control*
Mean Recovered B.
Test
Material
anthracis Spores,
._*
Inactivation
Efficacy *
CFU'
6
9
12
Glass
Wood
Carpet in i /-.s
1 17 x 10
Laminate
Metal Ductwork
Wallboard Paper
Glass
Wood
Carpet 117xl08
Laminate
Metal Ductwork
Wallboard Paper
Glass
Wood
Carpet 117xl08
Laminate
Metal Ductwork
Wallboard Paper
4.08 ±0.829x10'
5.09 ± 2.75 xlO6
5.56 ± 1.49 xlO7
6.41±1.12xl07
5.50 ± 1.32 xlO7
3.76 ± 0.634 xlO6
4.08 ± 0.829 xlO7
5.09 ± 2.75 xlO6
5.56 ± 1.49 xlO7
6.41±1.12xl07
5.50 ± 1.32 xlO7
3.76 ± 0.634 xlO6
4.08 ± 0.829 xlO7
5.09 ± 2.75 xlO6
5.56 ± 1.49 xlO7
6.41±1.12xl07
5.50 ± 1.32 xlO7
3.76 ± 0.634 xlO6
3.06 ± 4.37 xlO2
7.45 ± 1.42 xlO3
1. 11 ± 0.254 xlO4
0.00 ± 0.00
1.29 ± 0.707 xlO3
0.00 ±0.00
6.66 ± 14.9
5.01 ± 8.01 xlO3
7.58 ± 6.84 xlO3
0.00 ± 0.00
1.47 ± 2.04 xlO2
0.00 ± 0.00
0.00 ± 0.00
4.20 ± 2.42 xlO3
2.11 ± 2.21 xlO3
0.00 ± 0.00
1.29 ± 2.82 xlO3
0.00 ± 0.00
6.08 ±1.26
2.80 ±0.19
3.69 ±0.14
>7.80 ± 0.07
4.69 ±0.29
>6.57 ± 0.06
7.30 ±0.60
3.77 ±0.95
4.05 ±0.47
>7.80 ± 0.07
6.42 ±1.12
>6.57 ± 0.06
>7.60 ± 0.08
3. 13 ±0.35
4.72 ±0.65
>7.80 ± 0.07
6.26 ±1.39
>6.57 ± 0.06
are expressed as mean ± standard deviation of five replicates.
* Positive control coupons were inoculated but not exposed to ozone.
§ Test materials were inoculated and exposed to ozone for the contact time.
*Data are expressed as mean log reduction ± 95% CI of the SE.
+ This concentration was measured by the Mini Hicon-LR analyzer and corresponds to -10,800 ppmv on
the IN2000-L2-LC analyzer.
* Ozone concentration, temperature and relative humidity listed are target levels. Refer to Table 4-3 for
actual levels.
39
-------
.2 6
5 *
I
5VS S5°o,4
RH, Time (In )
S5VS
7,000 ppm O3, 75% and 85% RH
- 6
9
4 6 S 6 9
Time (Iir)
9,000 ppm O3, 85% RH
12
6
Time (In)
9,000 ppm O3, 75% RH
12 6*
Time (In)
^Coupons were pi e-huniidified for -24 hours
9,800 ppm O3, 85% RH
12*
12,000 ppm O3, 85% RH
Figure 5-7. Efficacy (log reduction) against B. subtitis on glass.
(Data are expressed as mean log reduction ± 95% CI of the SE)
40
-------
8
7 •
§ 6
I !
i
0 -
:
Jl
I
•
~5°o,4 ""5° 0.6 75»o.S S5°o.4 8?°o,6 S-^o.S
RH, Time (In)
7,000 ppm O3, 75% and 85% RH
- 6
w ^
•3 2
i
ii
i It
i
Tim* (In)
9,000 ppm O?, 85% RH
i:
8
7
i 6
'€ ?
11
6
Time (hi)
9,000 ppm O3, 75% RH
I '
M 3
i:
9. 12t
Time (In )
*Couponswere pre-himiidified for 24 hours
9,800 ppm O?, 85% RH
12
12,000 ppm O3, 85% RH
Figure 5-8. Efficacy (log reduction) against B. subtilis on wood.
(Data are expressed as mean log reduction ± 95% CI of the SE)
41
-------
s
7 •
i 6
1
0
~5°o.4 ~?°o.6 ~5"o,S 85»o,4
RH. Time (hi )
S5«6,6
85° o,S
7,000 ppm O3, 75% and 85% RH
_ 6
S
• -
it
4686
Time (hi)
9 i:
9,000 ppm O3, 85% RH
Tim* (hi)
9,000 ppm O3, 75% RH
^Coupons were pre-humidified for -24 hours
9,800 ppm O3, 85% RH
12
12,000 ppm O3, 85% RH
Figure 5-9. Efficacy (log reduction) against B. subtitis on carpet.
(Data are expressed as mean log reduction ± 95% CI of the SE)
42
-------
75° o,3 85°o,4 85° c.,6 S5«o,8
RH, Time (lu)
7,000 ppm O3, 75% and 85% RH
I
ppt
S 6
Time (hi)
12
9,000 ppm O3, 85% RH
9,000 ppm O3, 75% RH
I l
'€ '
I;
i: 6*
Time (In >
12*
*Couponswere pre-humidifiedfor 24 hours
9,800 ppm O3, 85% RH
12,000 ppm O3, 85% RH
Figure 5-10. Efficacy (log reduction) against B. subtitis on laminate.
(Data are expressed as mean log reduction ± 95% CI of the SE)
43
-------
75«6.4
S50o.6 S5VS
RH, Time (hi )
7,000 ppm O3, 75% and 85% RH
- 6
1 *
t
4 6 S 6 9
Tiin? (In)
9,000 ppm O3, 85% RH
i:
9,000 ppm O3, 75% RH
-> ,
i: 6*
Time (lir)
""Couponswere pre-humidified for 24 hours
9,800 ppm O3, 85% RH
12*
Time
-------
,3 ^ .
o o.4
5%.8 S5°o.4
RH, Time (In)
S5°o,S
7,000 ppm O3, 75% and 85% RH
_ 6
1 <
s
i
Time i In I
9,000 ppm O3, 85% RH
12
9,000 ppm O3, 75% RH
\
12 6*
Time (lit)
""Couponswere pre-hiunidified for 24 hours
9,800 ppm O3, 85% RH
12,000 ppm O3, 85% RH
Figure 5-12. Efficacy (log reduction) against B. subtitis on wallboard paper.
(Data are expressed as mean log reduction ± 95% CI of the SE)
45
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5.3 Surface Damage to Materials material type. Based on the visual
appearance of the decontaminated
At the end of each fumigation test, the coupons, there were no apparent changes
procedural blanks were visually in the color, reflectivity, or roughness of
compared to the laboratory blanks, and any of the six material surfaces after
test coupons were visually compared to being exposed to ozone gas for up to
positive controls, to assess any impact 12,000 ppmv and 12 hours.
the ozone gas may have had on each
46
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6.0 Summary
Viable B. anthracis and B. subtilis
spores were recovered from all coupon
types at 7,000 and 9,000 ppmv ozone
when tested at 75% RH. At these same
concentrations and contact times, but at
85% RH, there were several materials
that were completely decontaminated,
and generally higher log reductions were
achieved when compared to testing at
75% RH. Following inoculation and
prior to decontamination using 9,800
ppmv, a set of samples was held at
-85% RH for -24 hours. Log reductions
for these pre-humidified samples were
slightly lower in most instances
compared to samples tested at the same
concentration (9,800 ppmv ozone) and
contact times, but not pre-humidified for
the 24 hours at 85% RH.
For B. anthracis, wallboard paper, carpet
and wood were easiest to decontaminate
with ozone gas (85%, 70% and 67% of
tests yielded >6 log reduction,
respectively) versus glass, metal
ductwork, and laminate (37%, 4% and
4% of tests yielded >6 log reduction,
respectively). B. anthracis spores were
recovered from laminate and metal
ductwork following fumigation at all
ozone concentrations and contact times
(log reductions ranged from 1.27 to
6.93) and from glass at all
concentrations and contact times except
for 12,000 ppmv and 12 hours (log
reductions ranged from 1.70 to 7.66). No
B. anthracis spores were recovered from
carpet at contact times >8 hours. There
were also no B. anthracis spores
recovered from wood or carpet at 9,800
ppmv or 12,000 ppmv after 6 hours. Few
to no B. anthracis spores were recovered
from wallboard paper at all
concentrations and contact times tested
at 85% RH (0 to 5.34 x 101 CFU
recovered).
For B. subtilis, glass, wallboard paper
and laminate were easiest to
decontaminate with ozone gas (58%,
54% and 54% of tests yielded >6 log
reduction, respectively) versus metal
ductwork, wood and carpet (42%, 13%
and 13% of tests yielded >6 log
reduction, respectively). B. subtilis
spores were recovered from glass
following fumigation at all
concentrations except for 12,000 ppmv
after a 12 hour contact time (log
reductions ranged from 1.18 to 7.60). B.
subtilis spores were recovered from
wood at every contact time and every
concentration (log reductions ranged
from 0.38 to 5.87) except for the
samples subjected to the 24 hour
elevated %RH in which no viable spores
were recovered. Spores were recovered
from carpet at every concentration and
contact time (log reductions ranged from
0.83 to 7.05). At 85% RH, no viable B.
subtilis spores were recovered from the
laminate coupons at 7,000 ppmv after 8
hours, 9,000 and 12,000 ppmv after 6, 9
or 12 hours or at 9,800 ppmv after 9 (not
subjected to the 24 hour elevated %RH)
or 12 hours. B. subtilis spores were also
recovered at all contact times and
concentrations from metal ductwork
except for 9,000 ppmv after 12 hours
(85% RH) and 9,800 ppmv after 9 hours
(log reductions ranged from 0.39 to
7.17). Very few to no B. subtilis spores
were recovered from wallboard paper at
all concentrations and contact times
tested at 85% RH (overall, 0 to 1.16 x
104 CFU recovered).
47
-------
Two different analyzers were used to
measure the target ozone concentrations
at low (7,000 to 9,800 ppmv) and high
(11,000 and 12,000 ppmv) levels. A
comparison of these analyzers indicated
that the high concentration analyzer
yielded measurements approximately
1,000 to 1,300 ppmv higher than those
measured by the low level analyzer.
This may explain the observed lack of
improvement in efficacy for some
materials when increasing the ozone
concentration from 9,800 to 11,000
ppmv (or from 9,800 to 12,000 ppmv for
B. subtilis).
Ozone fumigation did not cause visible
damage to any of the materials tested
(glass, wood, carpet, laminate, metal
ductwork and painted wallboard paper)
for any test, including exposure for up to
12 hours at 12,000 ppmv ozone.
In comparing the decontamination
efficacies (log reduction) for B. subtilis
and B. anthracis for each test condition,
contact time and material, in
approximately 23% out of a total of 144
tests, the log reductions were
significantly higher for B. subtilis.
Differences were determined to be
significant if the 95% confidence
intervals for the log reduction results for
the two microorganisms did not overlap.
In conclusion, ozone gas is a promising
fumigant decontamination technology
for the inactivation of anthrax spores on
building materials, provided that
sufficient concentration, contact time,
temperature and relative humidity are
achieved for the various materials being
decontaminated. In general,
decontamination efficacy improved with
increasing ozone concentration and RH,
and was affected by the material.
48
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7.0 References
1. US Environmental Protection
Agency. Product Performance
Test Guidelines,
OPPTS 810.2000: General
Considerations for Public Health
Uses of Antimicrobial Agents,
Public Review Draft. EPA 712-
C-07-005. November 5, 2009.
2. Rogers, J.V., C.L. Sabourin,
Y.W. Choi, W.R. Richter, D.C.
Rudnicki, K.B. Riggs, M.L.
Taylor, and J. Chang,
Decontamination assessment of
Bacillus anthracis, Bacillus
subtilis, and Geobacillus
stearothermophilus spores on
indoor surfaces using a hydrogen
peroxide gas generator. Journal
of Applied Microbiology,
2005(99): p. 739-748.
3. http://www.ringbell.co.Uk/info/h
umid.htm
49
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United States
Environmental Protection
Agency
PRESORTED STANDARD
POSTAGE & FEES PAID
EPA
PERMIT NO. G-35
Office of Research and Development (8101R)
Washington, DC 20460
Official Business
Penalty for Private Use
$300
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