EPA 600/R-12/516 | May 2012 | www.epa.gov/ord
United States
Environmental Protection
Agency
Decontamination of Indoor
and Outdoor Materials with
Aqueous Chlorine Dioxide
Solutions
•••^•iwHiHrHr^H
0 O O O C I
Office of Research and Development
National Homeland Security Research Center
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EPA/600/R/12/516
May 2012
Decontamination of Indoor and
Outdoor Materials with Aqueous
Chlorine Dioxide Solutions
U.S. Environmental Protection Agency
Research Triangle Park, NC 27711
11
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Disclaimer
The U.S. Environmental Protection Agency (EPA), through its Office of Research and
Development's (ORD) National Homeland Security Research Center (NHSRC), funded, directed
and managed this work through Contract Number EP-C-10-001 with Battelle. 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 or its application 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
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 capability 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 comprehensive
laboratory study designed to better understand and maximize the effectiveness of aqueous
solutions of chlorine dioxide (C1O2) to decontaminate materials contaminated with Bacillus
anthracis 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 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 individuals and organization to this report are gratefully
acknowledged:
United States Environmental Protection Agency (EPA)
Office of Research and Development, National Homeland Security Research
Center
Eletha Brady-Roberts (Quality Assurance)
Lukas Oudejans (peer review)
United States Environmental Protection Agency (EPA)
Office of Emergency Management, National Decontamination Team
Michael Ottlinger (peer review)
United States Environmental Protection Agency (EPA)
Office of Research and Development, National Risk Management Research Laboratory
Timothy Dean (peer review)
Battelle
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Executive Summary
The U.S. Environmental Protection Agency
(EPA), Office of Research and Development
is striving to protect human health and the
environment from adverse impacts resulting
from acts of terror by investigating the
effectiveness and applicability of
technologies for homeland security (HS)-
related applications. The purpose of this
investigation was to determine the
decontamination efficacy of aqueous
chlorine dioxide (C1O2) solutions in
inactivating Bacillus anthracis (causative
agent for anthrax) spores, as a function of
material and decontamination parameters
(concentration, contact time, number of
spray applications). The objective of this
study was to provide an understanding of the
performance of the aqueous C1O2
decontamination technology to guide its use
and implementation in HS applications. In
the assessment of options for
decontamination following intentional
release of B. anthracis., it is important to
know whether and to what extent such
factors can impact the decontamination
efficacy.
This investigation focused on
decontamination of both indoor and outdoor
materials, including industrial carpet, treated
wood, unpainted concrete, two types of soil
material (topsoil and Arizona Test Dust
[AZTD]), decorative laminate, galvanized
metal, and glass. Decontamination efficacy
tests were conducted with spores of Bacillus
anthracis or Bacillus subtilis, the latter
organism included to assess its potential as a
surrogate for future studies related to B.
anthracis. Decontamination efficacy was
quantified in terms of log reduction, based
on the difference in the number of bacterial
spores recovered from the positive controls
and test coupons. Efficacy tests were
conducted with increasing levels of C1O2
(1,500 to 4,000 parts per million (ppm)),
contact times (one or two hours) and/or
number of spray applications (two to four
total spray applications), in order to
maximize the decontamination efficacy for
each material (i.e., to achieve a target log
reduction of at least 6.0). In addition to
efficacy, the impact of the decontamination
process on the materials was visually
observed and reported.
Summary of Results
The aqueous C1O2 decontamination
technology provided complete inactivation
of B. anthracis spores on galvanized metal,
decorative laminate, and glass using a 3,000
ppm, three-spray, one-hour contact time
treatment. Complete decontamination was
not achieved for any of the other materials
for any of the C1O2 decontamination
conditions tested. The highest average log
reductions achieved for B. anthracis on
treated wood, industrial carpet, and
unpainted concrete were 2.64, 3.40, and
2.50, respectively. The aqueous C1O2
decontaminant was the least effective on the
soil materials (topsoil and AZTD), with
nearly all log reduction results less than 1.0
for the six C1O2 conditions tested. Overall,
the results show that with robust enough
conditions (i.e., 3,000 - 4,000 ppm C1O2,
one-two hour contact time, and two - four
spray applications), the aqueous C1O2 spray
technology may be an effective
VI
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decontaminant for some nonporous
materials such as glass, galvanized metal,
and laminate. However, aqueous C1O2 was
found to be largely ineffective on porous
materials (e.g., wood, carpet, soils, and
concrete), even under the most robust
conditions tested.
With regard to comparing the
decontamination efficacies of B. anthracis
and B. subtilis, there were no test results in
which B. subtilis was inactivated to a
significantly greater degree than B.
anthracis. Additionally, no visible damage
was observed on any test materials for any
of the decontamination conditions tested.
vn
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Contents
Foreword iv
Acknowledgments v
Executive Summary vi
Tables x
Figures xiii
Abbreviations/Acronyms xiv
1.0 Introduction 1
2.0 Preparation of C1O2 Solutions and Test Matrix 2
2.1 Preparation of Aqueous C1O2 Solutions 2
2.2 Test Matrix 2
3.0 Summary of Test Procedures 6
3.1 Preparation of Test Coupons 6
3.2 Decontaminant Testing 9
3.2.1 Chlorine Dioxide Liquid Spray Neutralization 9
3.2.2 Chlorine Dioxide Liquid Spray Tests 10
3.3 Decontamination Efficacy 11
3.4 Qualitative Assessment of Surface Damage 12
3.5 Chlorine Dioxide Solution Characterization 12
4.0 Quality Assurance/Quality Control 13
4.1 Equipment Calibration 13
4.2 QC Results 13
4.3 Audits 13
4.3.1 Performance Evaluation Audit 13
4.3.2 Technical Systems Audit 14
4.3.3 Data Quality Audit 14
4.4 QAPP Amendments and Deviations 15
4.5 QA/QC Reporting 15
4.6 Data Review 15
5.0 Initial Chlorine Dioxide Spray Scoping Tests 16
5.1 Initial Scoping Decontamination Tests Using Galvanized Metal Coupons 16
5.2 Scoping Decontamination Tests Using Other Materials 19
6.0 Chlorine Dioxide Spray Efficacy Maximization Tests 24
7.0 Liquid Spray Results for Two vs. Four Total Spray Applications 40
7.1 Description 40
7.1.1 Test Material Composition 40
7.1.2 Subjectivity of Spraying Method 40
viii
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7.1.3 Chemistry of C1O2 Solution 41
7.2 Decontamination Efficacy 43
8.0 Summary of Results 47
9.0 References 51
Appendix A. Spray Deposition and Neutralization Tests 52
Appendix B. Effect of Pre-sterilization of Soil Materials on the Decontamination Efficacy for
Aqueous C1O2 Spray 66
IX
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Tables
Table 2-1. Initial Scoping Test Matrix for C1O2 Liquid Spray 3
Table 2-2. Efficacy Maximization Test Matrix for C1O2 Liquid Spray 4
Table 2-3. Matrix of Repeat Liquid Spray Tests to Assess Effect of Increasing Number of Spray
Applications 5
Table 3-1. Summary of Materials used for Decontaminant Testing 9
Table 4-1. Performance Standards for Amperometric Titration 14
Table 4-2. Performance Evaluation Audits 14
Table 5-1. Inactivation of Bacillus anthracis Spores—Initial Scoping Tests on Galvanized Metal 17
Table 5-2. Inactivation of Bacillus subtilis Spores—Initial Scoping Tests on Galvanized Metal 18
Table 5-3. Summary of Efficacy Values with 95% Confidence Intervals for Initial Scoping
Maximization of C1O2 Li quid Sprayed on Galvanized Metal 19
Table 5-4. Inactivation of Bacillus anthracis Spores—3,000 ppm C1O2 Liquid on Building and
Outdoor Materials (One Hour Contact, Three Total Spray Applications) 21
Table 5-5. Inactivation of Bacillus subtilis Spores—3,000 ppm C1O2 Liquid on Building and
Outdoor Materials (One Hour Contact, Three Total Spray Applications) 22
Table 5-6. Summary of Efficacy Values with 95% Confidence Intervals for 3,000 ppm C1O2
Liquid on Building and Outdoor Materials (One Hour Contact, Three Total Spray
Applications) 23
Table 6-1. Inactivation of Bacillus anthracis Spores—3,000 ppm C1O2 Liquid Sprayed on
Building and Outdoor Materials (Four Total Spray Applications, One Hour
Contact Time) 26
Table 6-2. Inactivation of Bacillus subtilis Spores—3,000 ppm C1O2 Liquid Sprayed on
Building and Outdoor Materials (Four Total Spray Applications, One Hour
Contact Time) 27
Table 6-3. Inactivation of Bacillus anthracis Spores—4,000 ppm C1O2 Liquid Sprayed on
Building and Outdoor Materials (Two Total Spray Applications, One Hour
Contact Time) 28
Table 6-4. Inactivation of Bacillus subtilis Spores—4,000 ppm C1O2 Liquid Sprayed on
Building and Outdoor Materials (Two Total Spray Applications, One Hour
Contact Time) 29
Table 6-5. Inactivation of Bacillus anthracis Spores—4,000 ppm C1O2 Liquid Sprayed on
Building and Outdoor Materials (Four Total Spray Applications, One Hour
Contact Time) 30
Table 6-6. Inactivation of Bacillus subtilis Spores—4,000 ppm C1O2 Liquid Sprayed on
Building and Outdoor Materials (Four Total Spray Applications, One Hour
Contact Time) 31
Table 6-7. Inactivation of Bacillus anthracis Spores—3,000 ppm C1O2 Liquid Sprayed on
Building and Outdoor Materials (Four Total Spray Applications, Two Hour
Contact Time) 32
Table 6-8. Inactivation of Bacillus subtilis Spores—3,000 ppm C1O2 Liquid Sprayed on
Building and Outdoor Materials (Four Total Spray Applications, Two Hour
Contact Time) 33
Table 6-9. Inactivation of Bacillus anthracis Spores—4,000 ppm C1O2 Liquid Sprayed on
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Building and Outdoor Materials (Four Total Spray Applications, Two Hour
Contact Time) 34
Table 6-10. Inactivation of Bacillus subtilis Spores—4,000 ppm C1O2 Liquid Sprayed on
Building and Outdoor Materials (Four Total Spray Applications, Two Hour
Contact Time) 35
Table 6-11. Summary of Mean Quantitative Efficacy with 95% Confidence Intervals for C1O2
Liquid Spray Efficacy Maximization Results for B. anthracis 36
Table 6-12. Summary of Mean Quantitative Efficacy with 95% Confidence Intervals for C1O2
Liquid Spray Efficacy Maximization Results for B. subtilis 37
Table 7-1. Chlorine Dioxide Liquid Batch Chemical Comparison 42
Table 7-2. Inactivation of Bacillus anthracis Spores—Two Applications vs. Four Applications
3,000 ppm C1O2 Liquid Sprayed on Materials (One Hour Contact Time) 44
Table 7-3. Inactivation of Bacillus anthracis Spores—Two Applications vs. Four Applications
4,000 ppm C1O2 Liquid Sprayed on Materials (One Hour Contact Time) 45
Table 7-4. Summary of Decontamination Efficacy Values for Two vs. Four Applications 46
Table 7-5. Comparing Decontamination Efficacy Values for Tests that Were Repeated 46
Table 8-1. Summary of Liquid C1O2 Decontamination Efficacy for Initial Scoping Tests 49
Table 8-2. Summary of Liquid C1O2 Liquid Decontamination Efficacy for Maximization Tests 50
Table A-1. Deposition/Runoff Weight of C1O2 Liquid with 60 Minute Contact Time 52
Table A-2. Neutralization Testing with Bacillus anthracis Spores with 3,000 ppm C1O2 Liquid,
One Hour Contact, Two Total Spray Applications 54
Table A-3. Neutralization Testing with Bacillus anthracis Spores with 3,000 ppm C1O2 Liquid,
One Hour Contact, Three Total Spray Applications 54
Table A-4. Neutralization Testing with Bacillus subtilis Spores with 3,000 ppm C1O2 Liquid,
One Hour Contact, Three Total Spray Applications 55
Table A-5. Neutralization Testing with Bacillus anthracis Spores with 1,500 ppm C1O2 Liquid,
One Hour Contact, Four Total Spray Applications 56
Table A-6. Neutralization Testing with Bacillus subtilis Spores with 1,500 ppm C1O2 Liquid,
One Hour Contact, Four Total Spray Applications 56
Table A-7. Neutralization Testing with Bacillus anthracis Spores with 2,000 ppm C1O2 Liquid,
One Hour Contact, Four Total Spray Applications 57
Table A-8. Neutralization Testing with Bacillus subtilis Spores with 2,000 ppm C1O2 Liquid,
One Hour Contact, Four Total Spray Applications 57
Table A-9. Deposition/Runoff Weight of C1O2 Liquid Spray per Maximization Testing Condition
and Material 59
Table A-10. Neutralization Testing with Bacillus anthracis Spores with 3,000 ppm C1O2 Liquid,
One Hour Contact, Four Total Spray Applications 61
Table A-l 1. Neutralization Testing with Bacillus subtilis Spores with 3,000 ppm C1O2 Liquid,
One Hour Contact, Four Total Spray Applications 61
Table A-12. Neutralization Testing with Bacillus anthracis Spores with 4,000 ppm C1O2 Liquid,
One Hour Contact, Two Total Spray Applications 62
Table A-13. Neutralization Testing with Bacillus subtilis Spores with 4,000 ppm C1O2 Liquid,
One Hour Contact, Two Total Spray Applications 62
Table A-14. Neutralization Testing with Bacillus anthracis Spores with 4,000 ppm C1O2 Liquid,
One Hour Contact, Four Total Spray Applications 63
XI
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Table A-15. Neutralization Testing with Bacillus subtilis Spores with 4,000 ppm C1O2 Liquid,
One Hour Contact, Four Total Spray Applications 63
Table A-16. Neutralization Testing with Bacillus anthracis Spores with 3,000 ppm C1O2 Liquid,
Two Hour Contact, Four Total Spray Applications 64
Table A-17. Neutralization Testing with Bacillus subtilis Spores with 3,000 ppm C1O2 Liquid,
Two Hour Contact, Four Total Spray Applications 64
Table A-18. Neutralization Testing with Bacillus anthracis Spores with 4,000 ppm C1O2 Liquid,
Two Hour Contact, Four Total Spray Applications 65
Table A-19. Neutralization Testing with Bacillus subtilis Spores with 4,000 ppm C1O2 Liquid,
Two Hour Contact, Four Total Spray Applications 65
Table B-l. Comparison of Decontamination Efficacy with 95% Confidence Intervals using
Unsterile vs. Sterile Soils for C1O2 Liquid Spray Testing 67
xn
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Figures
Figure 3-1. Inoculation of coupon using a multi-channel micropipette 7
Figure 3-2. Topsoil "coupons" made with Parafilm®-lined Petri dishes 8
Figure 5-1. Summary of efficacies (log reduction) and confidence intervals for initial scoping
tests with C1O2 liquid spray on galvanized metal 19
Figure 5-2. Summary of efficacies (log reduction) and confidence intervals for initial scoping
tests on building and outdoor materials 23
Figure 6-1 a. Summary of efficacies (log reduction) and confidence intervals for liquid spray
testing conditions 38
Figure 6-lb. Summary of efficacies (log reduction) and confidence intervals for liquid spray
testing conditions 39
Xlll
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Abbreviations/Acronyms
ATCC American Type Culture Collection
AZTD Arizona Test Dust
AZ Arizona
B. anthracis Bacillus anthracis (Ames strain)
B. subtilis Bacillus subtilis (ATCC 19659)
BBRC Battelle Biomedical Research Center
BSC III biological safety cabinet, Class III
C Celsius
CGB compact glovebox
CPU colony-forming unit(s)
CI confidence interval
C1O2 chlorine dioxide
cm centimeter(s)
DNA deoxyribonucleic acid
EPA U.S. Environmental Protection Agency
g gram(s)
HDPE high density polyethylene
hr hour(s)
HS homeland security
L liter(s)
LAL Limulus Amebocyte Lysate
min minute(s)
mg milligram(s)
mL milliliter(s)
|iL microliter(s)
N Normal
NA not applicable
NHSRC National Homeland Security Research Center
NIST National Institute of Standards and Technology
ORD EPA Office of Research and Development
PBS phosphate-buffered saline
PCR polymerase chain reaction
ppm parts per million
ppmv parts per million by volume
psi pounds per square inch
QA quality assurance
QAPP Quality Assurance Project Plan
QC quality control
RH relative humidity
rpm revolutions per minute
SD standard deviation
SE standard error
xiv
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SFW sterile filtered water (cell-culture grade)
STS sodium thiosulfate
ISA technical systems audit(s)
xv
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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,
outdoor environments, or water systems;
contain these contaminants; decontaminate
buildings, outdoor environments, or water
systems; and facilitate the disposal of material
resulting from remediation efforts.
As part of the above effort, EPA investigates
the effectiveness and applicability of
technologies for homeland security (HS)-
related applications 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.
EPA provides high-quality information that
is useful to decision makers in purchasing or
applying the tested technologies. EPA
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.
The purpose of this investigation was to
develop an understanding of the
effectiveness of liquid solutions of C1O2 to
decontaminate a range of materials. This
investigation focused on decontamination of
indoor and outdoor surfaces typical of those
found in a public building or outdoors that
could be contaminated by a biological agent
(such as B. anthracis) following an
intentional release. Residual biological agent
on surfaces following decontamination after
an intentional release could present a
potential health risk. This report documents
the impact of various factors on the efficacy
of aqueous C1O2 against spores of B.
anthracis and B. subtilis on surfaces.
Specifically, tests were conducted to
determine the C1O2 liquid spray application
requirements (e.g., in terms of
concentration, number of sprays, contact
time) needed to maximize the inactivation
efficacy of B. anthracis (or B. subtilis)
spores as a function of material.
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2.0 Preparation of C1O2 Solutions and Test Matrix
2.1 Preparation of Aqueous
Solutions
To prepare the aqueous solutions of C1O2, 6
N HC1 was mixed into sterile filtered water
(SFW) in a glass bottle. Commercially-
available Clorox® bleach was added to the
HC1 + SFW mixture and mixed carefully. A
formulation of 25% sodium chlorite solution
(SabreChlor 25, Sabre™) was mixed into
the Clorox® + HC1 + SFW solution which
yielded the C1O2 solution. Target C1O2
concentrations were achieved by adjusting
the amount of Clorox . (Target
concentrations of ClC^were between 3,000
to 4,000 ppm depending on the test). Target
chlorite ranges were achieved by adjusting
the amount of sodium chlorite solution, to
achieve a 50% minimum ratio of chlorite to
C1O2 in the solution. Target pH levels (4 -
7) were achieved by adjusting the amount of
HC1. (See Section 3.5 for further details on
the methods used to measure chlorite and
C1O2 concentrations and pH.) The solution
was then transferred into a commercially-
available, 480 mL (16 ounce) sprayer with a
cylinder style high density polyethylene
(HOPE) bottle (Qorpak® Item No. 733 IX,
Bridgeville, PA). The HOPE bottle was
covered with aluminum foil since C1O2 is
photosensitive.
2.2 Test Matrix
To help target subsequent testing conditions
with additional materials, initial scoping
tests were conducted using only galvanized
metal coupons with aqueous solutions of
C1O2 at concentrations ranging from 1,500
to 3,000 ppm, as shown in Table 2-1.
(Galvanized metal was selected for initial
tests based on results of previous testing.1)
In these initial tests, galvanized metal was
completely decontaminated only with the
3,000 ppm, three-spray treatment. Based on
these results with galvanized metal,
additional scoping tests were performed
using this same 3,000 ppm, three-spray
treatment, with industrial carpet, treated
wood, laminate, topsoil, and glass. Refer to
Chapter 5 for further details.
Since glass and galvanized metal were the
only materials completely decontaminated
for both B. anthracis and B. subtilis spores
in the initial scoping tests, glass and
galvanized metal were eliminated from
further testing, and in their place, AZTD and
unpainted concrete were included in the test
matrix. Thus tests with AZTD and
unpainted concrete, along with the materials
from the initial scoping tests that were not
completely decontaminated, were then
conducted at increasing levels of C1O2,
contact time, and/or number of spray
applications with the goal of improving or
maximizing decontamination efficacy.
Refer to the decontamination
"maximization" test matrix summarized in
Table 2-2 and the details presented in
Chapter 6. An adaptive management
approach was used to incorporate new
knowledge into the testing as
decontamination efficacy results became
available.
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Table 2-1. Initial Scoping Test Matrix for ClOi Liquid Spray
Biological
Agent"
B. anthracis
or
B. subtilis
B. anthracis
or
B. subtilis
B. anthracis
or
B. subtilis
B. anthracis
or
B. subtilis
B. anthracis
or
B. subtilis
A/r , . . C1O2
Material ^ .
Concentration
(ppm)
Galvanized metal
Galvanized metal
Galvanized metal
Galvanized metal
Industrial carpet
Treated wood
Decorative
laminate
Topsoil
Glass
a Tests for B. anthracis and B. subtilis
1,500 ± 150
2,000 ± 200
3,000 ±300
3,000 ±300
3,000 ±300
# Spray Applications
(application times)
4
(Time 0, +15, +30, +45 min)
4
(Time 0, +15, +30, +45 min)
2
(Time 0, +30 min)
3
(Time 0, +20, +40 min)
3
(Time 0, +20, +40 min)
Contact
Time (hr)
1
1
1
1
1
were conducted separately
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Table 2-2. Efficacy Maximization Test Matrix for ClOi Liquid Spray
Biological
Agent3
B. anthracis
or
B. sub tills
B. anthracis
or
B. sub tilts
B. anthracis
or
B. sub tilts
B. anthracis
or
B. sub tilts
B. anthracis
or
B. sub tilts
CIO
Materials „ 2 . # Spray Applications Contact
Concentration / ,• .• .• ^ T. „ ,
, . (application times) lime(hr)
Industrial carpet
Treated wood
Decorative laminate _ „„„ , _„„ 4
TT • + A 4. 3,000 ±300 ™. „ ,_ ,.„ _L/I, . ,
Unpamted concrete (lime 0, +15, +30, +45 mm)
AZ test dust
Topsoil
Industrial carpet
Treated wood
Decorative laminate . „ „ „ , . „ „ 2
TT • + A + 4,000 + 400 ™. „ ,.„ . ,
Unpamted concrete (lime 0, +30 mm)
AZ test dust
Topsoil
Industrial carpet
Treated wood
Decorative laminate . „„„ . „„ 4
Unpainted concrete ' (Time 0, +15, +30, +45 min)
AZ test dust
Topsoil
Industrial carpet
Treated wood
Decorative laminate _ „ „ „ _ „ „ 4
Unpainted concrete ' (Time 0, +15, +30, +45 min)
AZ test dust
Topsoil
Industrial carpet
Treated wood
Decorative laminate 4 QQQ ± 4QQ 4
Unpainted concrete ' (Time 0, +15, +30, +45 min)
AZ test dust
Topsoil
1
1
1
2
2
a Tests for B. anthracis and B. subtilis were conducted separately
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Lastly, due to some variability in results with carpet, wood, and unpainted concrete, a few tests
were repeated with these materials using B. anthracis spores; this test matrix is shown in Table
2-3. These repeat tests were conducted to assess the effect of increasing the number of spray
applications at a given C1O2 concentration while eliminating the possibility that slight differences
in the batch preparation of the aqueous C1O2 solutions were affecting decontamination efficacy
(i.e., causing variability in results). The results of these repeat tests and further details are
discussed in Chapter 7.
Table 2-3. Matrix of Repeat Liquid Spray Tests to Assess Effect of Increasing Number of
Spray Applications
Biological
Agent
B. anthracis
B. anthracis
B. anthracis
B. anthracis
a Tested using the
b Tested using the
CIO
Materials 2 # Spray Applications Contact
Concentration , ,: . . , „. ,, ,
, , (application times) Time (hr)
Industrial carpet ~
Treated wood 3,000 ±300a ,_,. „_,_.„ . ,
TT . , (lime 0, +30 mm)
Unpainted concrete
Industrial carpet .
Treated wood 3,000 ±300a ™. „ _,__ '*_.„ _,_., . ,
TT • * j * (Time 0, +15, +30, +45 mm)
Unpainted concrete
Industrial carpet ,
Treated wood 4,000 ±300b ,„. _ __ . x
TT • . j . (Time 0, +30 mm)
Unpainted concrete
Industrial carpet .
Treated wood 4,000 ±300b ,„. _ 1S __ ., . x
TT • . , . (Time 0, +15, +30, +45 mm)
Unpainted concrete
same batch of 3,000 ppm C1O2.
same batch of 4,000 ppm C1O2.
1
1
1
1
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3.0 Summary of Test Procedures
Test procedures were performed in
accordance with a pre-approved Quality
Assurance Project Plan (QAPP) and are
summarized in this chapter.
3.1 Preparation of Test Coupons
The B. anthracis spores used for this testing
were prepared from a qualified stock of the
Ames strain at the Battelle Biomedical
Research Center (BBRC). All spore lots
were subject to a stringent characterization
and qualification process required by
Battelle's standard operating procedure for
spore production. Specifically, all spore lots
were characterized prior to use by
observation of colony morphology, direct
microscopic observation of spore
morphology and size and determination of
percent refractivity and percent
encapsulation. In addition, the number of
viable spores was determined by colony
count and expressed as colony forming units
per milliliter (CFU/mL). Theoretically, once
plated onto bacterial growth media, each
viable spore germinates and yields one CPU.
Variations in the expected colony
phenotypes were recorded. Endotoxin
concentration of each spore preparation was
determined by the Limulus Amebocyte
Lysate (LAL)2 assay to assess whether
contamination from gram-negative bacteria
occurred during the propagation and
purification process of the spores. Genomic
DNA was extracted from the spores and
DNA fingerprinting by polymerase chain
reaction (PCR) was done to confirm the
genotype. The virulence of the spore lot was
measured by challenging guinea pigs
intradermally with a dilution series of spore
suspensions, and virulence was expressed as
the intradermal median lethal dose. In
addition, testing was conducted for
robustness of the spores via HC1 resistance.
The stock spore suspension was prepared in
SFW at an approximate concentration of 1 x
109 CFU/mL and stored under refrigeration
at 2 to 8 °C. The B. subtilis spores did not
undergo the level of stringency for
characterization (LAL assay, DNA
fingerprinting, and virulence testing
excluded), but qualitative PCR was done
using a custom PCR assay to confirm B.
subtilis. Primers were designed that targeted
a conserved region of B. subtilis
chromosomal DNA, since multiple strains of
this bacterium exist.
B. anthracis or B. subtilis spores were
inoculated onto test coupons in an
appropriate biosafety cabinet Level III (BSC
III) according to established BBRC
procedures. Inoculated coupons and soils
(topsoil and AZTD) were prepared prior to
each day of experimental work. Coupons
were placed flat in the BSC III and
inoculated at approximately 1 x 108 total
spores per coupon. This inoculation on
coupons was accomplished by dispensing a
100 microliter (jiL) aliquot of the spore
stock suspension (approximately 1 x 109
spores/mL) using a multi-channel
micropipette as 10 droplets (each of 10 jiL
volume, Figure 3-1) 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.
-------
Figure 3-1. Inoculation of coupon using a multi-channel micropipette.
The inoculations for the soils were done by
dispensing a 100 jiL aliquot of the spore
stock suspension (approximately 1 x 109
spores/mL) using a single-channel
micropipette as 10 droplets across the
surface of the soils. The reason a single-
channel micropipette was used to inoculate
the soils was that the topsoil and AZTD test
"coupons" consisted of these materials
placed in a Parafilm®-lined, 3.5 cm diameter
x 1.0 cm tall Petri dish to an unpacked depth
of 1.0 cm (Figure 3-2), so the multichannel
micropipette dimensions did not match the
Petri dish. The reason the Petri dishes were
lined with Parafilm was that this inert film
enabled the easy removal of the soils from
the dishes into the extraction tubes in a
single motion without having to scoop the
inoculated decontaminated soils which
presented handling and safety issues.
The commercial topsoil used for this
evaluation was a proprietary mixture of soil,
composted cow manure, sand, and other
ingredients (also proprietary). Analysis of
this topsoil (conducted by a third party
environmental laboratory) showed that it
had an average water content of 16.2%, an
average fraction organic carbon value of
5.20%, an average recalcitrant organic
carbon value of 2.05%, and an average soil
pH of 7.28.
The AZTD was also commercially available,
and its chemical composition was supplied
by the vendor as percent of weight: Silicon
dioxide (68 to 76%), aluminum oxide (10 to
15%), iron (III) oxide (2 to 5%), sodium
oxide (2 to 4%), magnesium oxide (1 to
2%), titanium dioxide (0.5 to 1%), and
potassium oxide (2 to 5%). Analysis of this
AZTD showed that it had an average water
content of 1.19%, an average fraction
organic carbon value of 0.569%, an average
recalcitrant organic carbon value of 1.07%,
and an average soil pH of 8.30.
After inoculation, the test coupons remained
undisturbed overnight in the BSC III to dry
thoroughly. Test coupons were then exposed
to the decontaminant the next day (i.e.,
within 24 hours after inoculation).
-------
Figure 3-2. Topsoil "coupons" made with ParafilmR-lined Petri dishes.
The origin and specifications of the
materials used for test coupons are shown in
Table 3-1. All materials were selected as
representative types of building and outdoor
materials. All test coupons were made from
new materials. The porous and nonporous
test coupons were 1.9 by 7.5 cm in size, and
the soils were 3.5 cm diameter by 1.0 cm tall
in size.
The industrial carpet was comprised of
recycled nylon fiber (90%) that had
undergone the solution dye process, which
made the carpet inherently stain-resistant
(particularly to acid-based substances), so no
additional stain treatment was applied to the
material during its production. The carpet
was treated, however, with an antimicrobial
chemical (zinc omadine). The carpet
backing consisted entirely of thermoplastic
polyolefm compound (also recycled
content).
The porous and nonporous coupons were
sterilized before use by gamma-(^)-
irradiation (treated wood, industrial carpet,
and decorative laminate) or autoclaving
(galvanized metal and glass). In the initial
scoping test with topsoil, the topsoil was not
sterilized prior to use in order to maintain
chemical and physical integrity of the
material. But due to concerns that the high
background levels of the endogenous
organisms could potentially interfere with
quantitative results, all subsequent tests with
the soil materials were sterilized by y-
irradiation. Refer to Appendix B for further
details. The ^-irradiation sterilization
method was chosen for the porous materials
since the pressure (15 pounds per square
inch [psi]) and heat (121 °C) from an
autoclave could physically alter or damage
these coupons. Therefore, the nonporous
coupons were sent to be ^-irradiated at
approximately 40 kilogray by a vendor that
specializes in this type of processing
-------
(STERIS Isomedix Services, Libertyville,
IL). The nonporous materials were
autoclaved at Battelle by following an
internal standard operating procedure.
Table 3-1. Summary of Materials used for Decontaminant Testing
Material
Lot/Batch/
Observation
Manufacturer/
Supplier Name
Coupon Size,
Width x Length
Material
Preparation
NONPOROUS
Glass
Galvanized metal
ductwork
Decorative laminate
C1036
NAa
NA
Brooks Brothers Glass;
Columbus, OH
Adept Products;
West Jefferson, OH
A' Jack Inc.;
Columbus, OH
1.9 cmx7.5 cm
1.9 cmx7.5 cm
1.9 cmx7.5 cm
Autoclave
Autoclave
Y - irradiation
POROUS
Carpet
Painted-wallboard
paper
Treated wood
Unpainted concrete
Shaw EcoTek 6
05-16-03; Set-E-493;
Roll-3
30906, ACQ, 2x6x8
Prime
5 parts sand and
2 parts cement
Grossmans Bargain
Outlet; Columbus, OH
United States Gypsum
Company; Chicago, IL
Lowe's Top Choice;
Springfield, OH
Wysong Concrete;
Fairborn, OH
1.9 cmx7.5 cm
1.9 cmx7.5 cm
1.9 cmx7.5 cm
1.0 cmx 3.0 cm
Y - irradiation
Y - irradiation
Y - irradiation
Y - irradiation
SOILS
Topsoil
Arizona Test Dust
PY1A0597
ISO 121030-1
GardenScape, Inc.;
Eau Claire, PA
Powder Technology, Inc.;
Burnsville, MN
3.5 cm diameter x 1.0
(unpacked)
3.5 cm diameter x 1.0
(unpacked)
nonsterile,
Y - irradiation
Y - irradiation
aNot applicable.
3.2 Decontaminant Testing
3.2.1 Chlorine Dioxide Liquid Spray
Neutralization
Neutralization panels were conducted before
any testing with each aqueous C1O2 liquid
spray batch, using coupons that had not been
inoculated with spores. In these
neutralization panels, the decontaminant was
applied and measurements were made with
multiple coupons of each material type to
determine the amount of the decontaminant
that remained on, or ran off from, each
material (i.e., "spray-and-weigh"). These
C1O2 liquid deposition data were used in
trial runs to determine the amount of
neutralizing agent needed to stop the action
of the decontaminant after the prescribed
contact time. Refer to Appendix A for the
results of these tests.
3.2.2 Chlorine Dioxide Liquid Spray Tests
Five replicate test coupons (inoculated with
B. anthracis or B. subtilis spores and
-------
decontaminated), five replicate positive
control coupons (inoculated and not
decontaminated, i.e., sprayed only with
SFW), one procedural blank (not inoculated,
decontaminated), and one laboratory blank
(not inoculated, not decontaminated) of each
coupon material were used in testing under
each set of testing conditions. All test
coupons were oriented horizontally (i.e.,
lying flat). After the contact time, the C1O2
solution that had pooled on top of each test
coupon was captured, neutralized, and
subjected to spore extraction along with the
associated test coupon.
On the day following inoculation, test
coupons intended for decontamination
(including blanks) were separated from the
positive controls or coupons not exposed to
decontaminant (including blanks) since both
sets were inoculated and dried overnight in
the same BSC III. The C1O2 liquid spray
distance (30.5 cm), humidity (< 70% RH),
and temperature (22 °C ± 2 °C) were the
same for all applications, including the
positive controls. The positive controls
(including blanks) were transferred into a
separate compact glove box, or CGB
(Compact Glove Box, Plas-Labs Model No.
830-ABC, Lansing, MI), where SFW was
sprayed using the same type of sprayer. For
porous materials and soils, more sprays (i.e.,
trigger pulls) were required to wet the
surfaces of the materials since they absorbed
the liquid.
Following the appropriate number of spray
applications and contact time, each coupon
(along with any associated pooled
decontaminant) was aseptically transferred
to a sterile 50 mL conical tube 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) and the appropriate concentration of
sodium thiosulfate (STS) neutralizer needed
to stop the decontamination activity of the
C1O2 liquid. The required concentration of
STS was determined in the neutralization
panels for each C1O2 test solution or number
of applications tested. In each of the
neutralization panels, a range of STS
concentrations was tested to determine the
concentration that most effectively stopped
the action of the C1O2 (as indicated by the
maximum recovery of viable spores in
simulated coupon extracts). The results of
those neutralization panels are shown in
Appendix A.
The coupons were extracted by agitation on
an orbital shaker for 15 minutes at
approximately 200 revolutions per minute
(rpm) at room temperature. (Unsterilized
topsoil was incubated in a water bath for one
hour at 55 to 60 °C to inactivate the
endogenous flora that were susceptible to
heat shock). The unpainted concrete
coupons were sonicated for 1 hour to
increase extraction efficiency. The soils
were transferred from the Petri dishes to the
extraction tubes. Following extraction, a
1 mL aliquot of the coupon extract was
removed, and a series of dilutions up
through 10"7 was prepared in SFW. An
aliquot (0.1 mL) of the undiluted extract and
each serial dilution were 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. The number of
CFU/mL was determined by multiplying the
average number of colonies for the triplicate
plates by the reciprocal of the dilution, and
accounting for the 0.1 mL volume of the
extract or dilution that was plated.
Before further decontamination tests with
the next C1O2 solution, the BSC III and the
compact glove box (CGB) were thoroughly
cleaned (using separate steps involving
bleach, ethanol, water, then drying)
10
-------
following procedures established under the coupons. The blanks were spiked with an
BBRC Facility Safety Plan. equivalent amount of 0.1 mL of "stock
suspension" that did not contain the
Laboratory blanks controlled for sterility biological agent. The target acceptance
and procedural blanks controlled for viable criterion was that extracts of laboratory or
spores inadvertently introduced to test procedural blanks were to contain no CPU.
The mean percent spore recovery from each coupon material was calculated using results from
positive control coupons (spiked, not decontaminated (sprayed with SFW instead of the
decontaminant)), by means of the following equation:
Mean % Recovery = [Mean CFUpc/CFUSpike] x 100 (1)
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 or B. subtilis on each coupon material, and the results
are included in Chapters 5 through 12.
3.3 Decontamination Efficacy
The performance or efficacy of the C1O2 liquid spray was assessed by determining the number of
viable organisms remaining on each test coupon and in any decontaminant run-off from the
coupon (for liquid spray testing only) after decontamination. Those numbers were compared to
the number of viable organisms extracted from the positive control coupons.
The number of viable spores of B. anthracis or B. subtilis in extracts of test and positive control
coupons was determined in order to calculate efficacy of the decontaminant. Efficacy is defined
as the extent (as logic reduction) to which viable spores extracted from test coupons after
decontamination were less numerous than the viable spores extracted from positive control
coupons. The logarithm of the CFU abundance from each coupon extract was determined, and
the mean of those logarithm values was then determined for each set of control and associated
test coupons, respectively. Efficacy of a decontaminant for a test organism/test condition on the
f coupon material was calculated as the difference between those mean log values, i.e.:
Efficacy = (loglo CFUcl}) - (loglo CFUtl}) (2)
where logic CFUcy refers to they individual logarithm values obtained from the positive control
coupons and logic CFUty refers to they individual logarithm values obtained from the
corresponding test coupons, and the overbar designates a mean value. In tests conducted under
this plan, there were five control and five corresponding test coupons (i.e.,7 = 5) for each coupon
material. In the case where no viable spores were found in any of the five test coupon extracts
after decontamination, a CFU abundance of 1 was assigned, resulting in a logic CFU of zero for
that material. This situation occurred when the decontaminant was highly effective, and no
viable spores were found on the decontaminated test coupons. In such cases, the final efficacy on
that material was reported as greater than or equal to (>) the value calculated by Equation 2.
11
-------
The variances (i.e., the square of the standard deviation) of the logic CFUcy and logic 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 2,
as follows:
(3)
where the number 5 again represents the number7 of coupons in both the control and test data
sets. Each efficacy result is thus reported as a log reduction value with an associated SE value.
The significance of differences in efficacy across different coupon materials and spore types was
assessed based on the 95% confidence interval of each efficacy result. The 95% confidence
interval (CI) is:
95% CI = Efficacy ± (1.96 x SE)
(4)
Differences in efficacy were judged to be significant if the 95% CIs of the two efficacy results
did not overlap. Any results based on this formula are hereafter noted as significantly different.
Note this comparison is not applicable when the two efficacy results being compared are both
reported with log reductions as > some value.
3.4 Qualitative Assessment of Surface
Damage
Visual inspection of each coupon surface
took place after the prescribed C1O2 liquid
and fumigant contact time and application
rates, through side-by-side comparison of
the decontaminated test surface and control
coupons of the same test material.
Differences in color, reflectivity, and
roughness were assessed qualitatively, and
observations were documented.
3.5 Chlorine Dioxide Solution
Characterization
The concentrations of C1O2 solutions were
measured using a modified titration method
based on the Standard Method 4500-C1O2 E
Amperometric Method II3. For this titration
method, 5 mL of the C1O2 solution is added
to a 150 mL solution of 5% potassium
iodide in phosphate buffer (pH 7.0). Under
these conditions, C1O2 oxidizes the iodide to
iodine (and C1O2 is converted to chlorite).
The total resulting iodine is reduced back to
iodide when titrated with standard 0.1
Normal (N) [equal to 0.1 molar] sodium
thiosulfate (STS). After this initial reaction
with C1O2, the solution is acidified using 6
N hydrochloric acid (HC1), which forms
additional chlorite, and is titrated further
with STS. The total volume (mL) of STS
solution titrated is proportional to the
amount of iodine generated, which is
proportional to the chlorine dioxide
concentration.
Certified National Institute of Standards and
Technology (NIST)-traceable chlorite
standards, appropriately diluted in solution
comparable to the sampling solution, were
titrated each day of chlorine dioxide testing
to verify accuracy.
The pH of the C1O2 solutions was measured
with a calibrated pH meter (Thermo
Scientific, Waltham, MA).
12
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4.0 Quality Assurance/Quality Control
Quality assurance/quality control (QA/QC)
procedures were performed in accordance
with the QAPP (available upon request).
The QA/QC procedures are summarized
below.
4.1 Equipment Calibration
All equipment (e.g., pipettes, incubators,
biological safety cabinets, pH meter) and
monitoring devices (e.g., thermometer,
hygrometer) used at the time of evaluation
were verified as being certified, calibrated,
or validated.
4.2 QC Results
Quality control efforts conducted during
decontaminant testing included positive
control coupons (inoculated, not
decontaminated), procedural blanks (not
inoculated, decontaminated), laboratory
blanks (not inoculated, not decontaminated),
and spike control samples (analysis of the
stock spore suspension).
All positive control results were within the
target recovery range of 1 to 150% of the
spiked spores, and all procedural and
laboratory blanks met the criterion of no
observed CPU for both organisms.
Spike control samples were taken from the
spore suspension on the day of testing and
serially diluted, nutrient plated and counted
to establish the spore density used to spike
the coupons. The spore density levels met
the QA target criterion of 1 x 109 CFU/mL
(± 25%) for all tests, with the exception of
the following three test conditions:
• B. anthracis and B. subtilis for the
3,000 ppm, 1 hour, 2 total spray
applications. In these tests, the
inoculum spore density was 6.37 x
108 CFU/mL and 6.47 x 108
CFU/mL, respectively.
• B. subtilis for the 3,000 ppm, 1 hour,
3 total spray applications. In this
test, the inoculum spore density was
6.53 x 108 CFU/mL.
Although these few tests did not meet the
QA target criterion, the results are not
expected to be adversely affected.
4.3 Audits
4.3.1 Performance Evaluation Audit
Performance evaluation audits were
conducted to assess the quality of the results
obtained during evaluation.
Performance standards for amperometric
titration for 1000 mg/L, 3000 mg/L, and
4000 mg/L chlorite using sodium chlorite
stock solutions to verify the titration method
were made and tested. The results for these
performance standards are listed in Table 4-
1.
13
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Table 4-1. Performance Standards for Amperometric Titration
Sodium Chlorite
Stock (mg/L)
Measured Chlorite
(mg/L)
1,000 ± 100
1,045
3,000 ±300
3,203
4,000 ± 400
4,113
Temperatures were monitored but no efforts were undertaken to control any of the test
temperatures.
No performance evaluation audits were performed to confirm the concentration and purity of B.
anthracis or B. subtilis spores because quantitative standards do not exist for these organisms.
The control coupons and blanks support the spore measurements.
Table 4-2 summarizes the performance evaluation audits that were performed.
Table 4-2. Performance Evaluation Audits
Measurement
Volume of liquid
from micropipettes
Chlorite
Temperature
Relative Humidity
Time
Audit
Procedure
Gravimetric evaluation
Amperometric titration
Compared to independently
calibrated thermometer
Compared to independently
calibrated hygrometer
Compare time to independent
clock or watch value
Allowable
Tolerance
± 10%
± 10%
±2°C
± 10%
± 2 sec/hr
Actual
Tolerance
±5%
± 10%
±2°C
< 10%
0 sec/hr
4.3.2 Technical Systems Audit
Contractor QA staff conducted technical
systems audits (TSAs) on June 17, July 20,
August 26 and 27, and November 4, 2010,
to ensure that the tests were being conducted
in accordance with the appropriate test
plan/QAPP. As part of the audit, test
procedures were compared to those
specified in the test/QAPP and data
acquisition and handling procedures were
reviewed. Observations and findings from
the TSA were documented and submitted to
the test leader for response. None of the
findings of the TSA required corrective
action. TSA records were permanently
stored with the Contractor QA Manager.
4.3.3 Data Quality Audit
At least 10% of the data acquired during the
evaluation were audited. The contract 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.
14
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4.4 QAPP Amendments and Deviations
Two deviations were prepared, approved,
and retained in the test files for this
evaluation. The first deviation related to the
initiation of testing as described in the two
QAPP amendments without fully-
signed/approved amendments in place. A
second deviation was for the cases during
C1O2 liquid spray testing when the positive
controls exceeded the ±25% criterion from
the target inoculum titer. These deviations
are not expected to adversely impact results;
refer to Section 4.2 for more information.
4.5 QA/QC Reporting
Each assessment and audit was documented
in accordance with the QAPP. For these
tests, findings were noted (none significant)
in the data quality audit, but no followup
corrective action was necessary. The
findings were mostly minor data
transcription errors requiring some
recalculation of efficacy results, but none
were gross errors in recording. Copies of the
assessment reports were distributed to the
EPA and contractor staff. QA/QC
procedures were performed in accordance
with the QAPP.
4.6 Data Review
Records and data generated in the evaluation
received a QC/technical review before they
were utilized in calculating or evaluating
results and prior to incorporation in reports.
All data were recorded by contractor staff.
The staff member 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 staff member who stored the record.
15
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5.0 Initial Chlorine Dioxide Spray Scoping Tests
To help target subsequent testing conditions
with additional materials, initial scoping
tests were performed first using galvanized
metal coupons to assist in focusing the C1O2
concentration, contact time, and number of
spray applications that would potentially be
needed to effectively decontaminate other
materials. Galvanized metal and the initial
decontamination conditions were selected
based on results of previous testing1.
Specifically, C1O2 liquid spray testing with
target concentrations of 1,500; 2,000; and
3,000 ppm; contact time of 1 hour, and
varying number of spray applications was
conducted on galvanized metal inoculated
with either spores of B. anthracis or B.
subtilis.
5.1 Initial Scoping Decontamination Tests
Using Galvanized Metal Coupons
The decontamination efficacies were
evaluated for B. anthracis and B. subtilis on
galvanized metal for the following
conditions:
• 3,000 ppm C1O2, 1 hour contact
time, two total spray applications.
(Actual C1O2 liquid concentration
measured at 3,103 ppm for both B.
anthracis and B. subtilis testing.)
• 3,000 ppm C1O2, 1 hour contact
time, three total spray applications.
(Actual C1O2 liquid concentration
measured at 3,103 ppm for both B.
anthracis and B. subtilis testing.)
• 1,500 ppm C1O2, 1 hour contact
time, four total spray applications.
(Actual C1O2 liquid concentration
measured at 1,484 ppm for both B.
anthracis and B. subtilis testing.)
• 2,000 ppm C1O2, 1 hour contact
time, four total spray applications.
(Actual C1O2 liquid concentration
measured at 2,024 ppm for both B.
anthracis and B. subtilis testing.)
As expected, increasing the concentration
and number of spray applications improved
decontamination efficacy; refer to Tables 5-
1 through 5-3and Figure 5-1. Complete
inactivation within the detection limit was
seen only when testing at the condition of
3,000 ppm C1O2, 1 hour contact time, and
three total spray applications. Both B.
anthracis (> 7.89) and B. subtilis (> 7.70)
were completely inactivated under these
conditions. For three of the four B.
anthracis tests, a 6 log reduction or greater
was achieved, and for the other test with B.
anthracis, a log reduction of nearly 6 (5.99)
was obtained. (A decontaminant which
achieves a 6 log reduction or greater is
considered effective.4)
16
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Table 5-1. Inactivation of Bacillus anthracis Spores—Initial Scoping Tests on Galvanized
Metal
Contact Time,
Applications a
3000 ppm (1 hr contact,
two total sprays)
Positive Controls'3
Test Coupons0
Laboratory Blankd
Procedural Blank6
3000 ppm (1 hr contact ,
three total sprays)
Positive Controls
Test Coupons
Laboratory Blank
Procedural Blank
1500 ppm (1 hr contact,
four total sprays)
Positive Controls
Test Coupons
Laboratory Blank
Procedural Blank
2000 ppm (1 hr contact,
four total sprays)
Positive Controls
Test Coupons
Laboratory Blank
Procedural Blank
Inoculum
(CFU)
6.37 x 107
6.37 x 107
0
0
9.43 x 107
9.43 x 107
0
0
9.90 x 107
9.90 x 107
0
0
9.43 x 107
9.43 x 107
0
0
Mean of Logs of
Observed CFU
7.76 ± 0.070
0.680 ±0.93
0
0
7.89 ±0.030
0
0
0
7.84 ±0.030
1.85 ±1.1
0
0
7.87 ±0.080
0.740 ±1.7
0
0
Mean %
Recovery
91.0 ± 15.3
< 0.0100
-
-
83.3 ±5.90
0
-
-
69.7 ±4.10
<0.01
-
-
79.0 ±15.0
<0.01
-
-
Decontamination
Efficacy ± CIf
_g
7.08 ±0.82
-
-
-
> 7.89 ±0.030
-
-
-
5.99 ±0.99
-
-
-
7.12 ±1.5
-
-
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).
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.
f CI = confidence interval (± 1.96 x SE). Differences in efficacy may be significant if the 95% CIs of the two efficacy results do
not overlap; however, this comparison is not applicable when the two efficacy results being compared are both reported with
log reductions as > some value.
8 "-" Not Applicable.
17
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Table 5-2. Inactivation of Bacillus subtilis Spores—Initial Scoping Tests on Galvanized
Metal
Contact Time,
Applications a
3000 ppm (1 hr contact,
two total sprays)
Positive Controls'3
Test Coupons0
Laboratory Blankd
Procedural Blank6
3000 ppm (1 hr contact,
three total sprays)
Positive Controls
Test Coupons
Laboratory Blank
Procedural Blank
1500 ppm (1 hr contact,
four total sprays)
Positive Controls
Test Coupons
Laboratory Blank
Procedural Blank
2000 ppm (1 hr contact,
four total sprays)
Positive Controls
Test Coupons
Laboratory Blank
Procedural Blank
Inoculum
(CFU)
6.47 x 107
6.47 x 107
0
0
6.53 x 107
6.53 x 107
0
0
1.12xl08
1.12xl08
0
0
1.05 x 108
1.05 x 108
0
0
Mean of Logs of
Observed CFU
7.74 ±0.030
0.610 ±0.84
0
0
7.70 ± 0.080
0
0
0
7.98 ±0.040
7.21 ±0.15
0
0
7.98 ±0.040
6.07 ±0.45
0
0
Mean %
Recovery
85.4 ±5.80
<0.01
-
-
77.4 ±14.0
0
-
-
84.6 ± 8.0
15.4 ±6.0
-
-
92.3 ±9.10
1.75 ±1.80
-
-
Decontamination
Efficacy ± CIf
_g
7.13 ±0.74
-
-
-
> 7.70 ±0.070
-
-
-
0.760 ±0.14
-
-
-
1.91 ±0.40
-
-
1 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).
3 Positive Controls = inoculated, not decontaminated coupons (sprayed with SFW).
: Test Coupons = inoculated, decontaminated coupons.
1 Laboratory Blank = not inoculated, not decontaminated coupon.
: Procedural Blank = not inoculated, decontaminated coupon.
F CI = confidence interval (± 1.96 x SE). Differences in efficacy may be significant if the 95% CIs of the two efficacy results do
not overlap; however, this comparison is not applicable when the two efficacy results being compared are both reported with
log reductions as > some value.
1 "-" Not Applicable.
18
-------
Table 5-3. Summary of Efficacy Values with 95% Confidence Intervals for Initial Scoping
Tests of ClOi Liquid Sprayed on Galvanized Metal
ClOi Liquid, Contact Time,
Applications
3000 ppm, 1 hr, 2 applications
3000 ppm, 1 hr, 3 applications
1500 ppm, 1 hr, 4 applications
2000 ppm, 1 hr, 4 applications
Efficacy for
B. anthracis (Ames)
7.08 ±0.82
>7.89±0.030a
5.99 ±0.99
7.12± 1.50
Efficacy for
B. subtilis
7.13 ±0.74
>7.70±0.070a
0.760 ±0.14
1.91 ±0.40
aResult represents complete inactivation within the detection limit of 33.33 CFU/material. Differences in efficacy may
be significant if the 95% CIs of the two efficacy results do not overlap; however, this comparison is not applicable when the two
efficacy results being compared are both reported with log reductions as > some value.
CIO2 Liquid Spray Optimization on Galvanized Metal
i B. anthracis
B, subiihs
3000 ppm, 1 hr, 2 apps
3000 ppm, 1 hr, 3 apps 1500 ppm, 1 hr, 4 jpps
Test Condition
2000 ppm, 1 hr, 4 apps
Figure 5-1. Summary of efficacies (log reduction) and confidence intervals for initial
scoping tests with ClOi liquid spray on galvanized metal (asterisk indicates complete
inactivation within the detection limit).
5.2 Scoping Decontamination Tests Using
Other Materials
Based on the initial scoping test results with
galvanized metal, a second scoping test was
conducted using additional materials, i.e.,
unsterilized topsoil, glass, treated wood,
industrial carpet, and decorative laminate.
Since galvanized metal was completely
decontaminated for both B. anthracis and B.
subtilis only at the test condition of 3,000
ppm C1O2, three spray applications, and 1
hour contact time, this same
decontamination treatment was used for the
additional materials. The actual C1O2 liquid
concentration was measured at 3,103 ppm
for both B. anthracis and B. subtilis testing.
The results are shown in Tables 5-4 and 5-5
and summarized in Table 5-6 and Figure 5-
2. Overall, log reductions varied by material,
with topsoil decontaminated at the worst
efficacy (less than 0.24 log reduction).
Complete inactivation within the detection
limit was observed only on glass and
19
-------
decorative laminate for B. anthracis (> 7.86 efficacy for wood and carpet ranged from
and > 7.84, respectively) and on glass for B. approximately 1.5-4.0 log reduction.
subtilis (> 7.88). The decontamination
20
-------
Table 5-4. Inactivation of Bacillus anthracis Spores—3,000 ppm ClOi Liquid on Building
and Outdoor Materials (1 Hour Contact, Three Total Spray Applications)
Test Material
Topsoila'f
Positive Controls'3
Test Coupons0
Laboratory Blankd
Procedural Blank6
Glass
Positive Controls
Test Coupons
Laboratory Blank
Procedural Blank
Treated Wood
Positive Controls
Test Coupons
Laboratory Blank
Procedural Blank
Industrial Carpet
Positive Controls
Test Coupons
Laboratory Blank
Procedural Blank
Decorative Laminate
Positive Controls
Test Coupons
Laboratory Blank
Procedural Blank
Inoculum
(CFU)
1.26 xlO8
1.26 x 108
0
0
1.26 x 108
1.26 x 108
0
0
1.26 x 108
1.26 x 108
0
0
1.26 x 108
1.26 x 108
0
0
1.26 x 108
1.26 x 108
0
0
Mean of Logs of
Observed CFU
7.97 ± 0.060
7.72 ± 0.060
0
0
7.86 ±0.080
0
0
0
6.92 ±0.050
4.51±0.18
0
0
8.01 ±0.060
4.61 ±0.84
0
0
7.84 ±0.050
0
0
0
Mean %
Recovery
74.6 ± 11.0
42.4 ±5.80
-
-
58.7 ±11.0
0
-
-
6.61 ±0.76
0.0300 ±0.01
-
-
81.8±11
0.150 ±0.28
-
-
55.4 ±6.10
0
-
-
Decontamination
Efficacy ± CIg
h
0.240 ± 0.080
-
-
-
> 7.86 ± 0.070
-
-
-
2.41 ±0.17
-
-
-
3.40 ±0.73
-
-
.
> 7.84 ±0.040
-
-
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).
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.
f Unsterilized topsoil used for testing.
8 CI = confidence interval (± 1.96 * SE). Differences in efficacy may be significant if the 95% CIs of the two efficacy results
do not overlap; however, this comparison is not applicable when the two efficacy results being compared are both reported
with log reductions as > some value.
h "-"Not Applicable.
21
-------
Table 5-5. Inactivation of Bacillus subtilis Spores—3,000 ppm ClOi Liquid on Building and
Outdoor Materials (1 Hour Contact, Three Total Spray Applications)
Test Material
Topsoila'f
Positive Controls'3
Test Coupons0
Laboratory Blankd
Procedural Blank6
Glass
Positive Controls
Test Coupons
Laboratory Blank
Procedural Blank
Treated Wood
Positive Controls
Test Coupons
Laboratory Blank
Procedural Blank
Industrial Carpet
Positive Controls
Test Coupons
Laboratory Blank
Procedural Blank
Decorative Laminate
Positive Controls
Test Coupons
Laboratory Blank
Procedural Blank
Inoculum
(CFU)
1.16xl08
1.16xl08
0
0
1.16xl08
1.16xl08
0
0
1.16xl08
1.16xl08
0
0
1.16xl08
1.16xl08
0
0
1.16xl08
1.16xl08
0
0
Mean of Logs of
Observed CFU
7.92 ± 0.040
7.83 ±0.030
0
0
7.88±0.11
0
0
0
7.02 ±0.74
5.59 ±0.37
0
0
7.84 ±0.010
3.76 ±0.41
0
0
7.90 ±0.050
2.77 ±1.72
0
0
Mean %
Recovery
72.0 ±6.90
57.9 ±4.20
-
67.4 ±18.0
0
24.9 ±31.0
0.420 ±0.23
59.5 ±2.00
0.01 ±0.01
-
68.4 ±8.0
0.010 ±0.02
-
Decontamination
Efficacy ± CIf
_g
0.0900 ± 0.050
-
-
> 7.88 ±0.10
-
1.43 ±0.72
4.08 ±0.36
-
5. 13 ±1.5
-
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).
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.
f Unsterilized topsoil used for testing.
8 CI = confidence interval (± 1.96 x SE). Differences in efficacy may be significant if the 95% CIs of the two efficacy results
do not overlap; however, this comparison is not applicable when the two efficacy results being compared are both reported
with log reductions as > some value.
h "-"NotApplicable.
22
-------
Table 5-6. Summary of Efficacy Values with 95% Confidence Intervals for 3,000 ppm ClOi
Liquid on Building and Outdoor Materials (1 Hour Contact, Three Total Spray
Applications)
Test Material
Topsoil
Glass
Treated Wood
Industrial Carpet
Decorative Laminate
Efficacy for
B. anthracis (Ames)
0.24 ±0.08
>7.86±0.07a
2.41 ±0.17
3.40 ±0.73
>7.84±0.04a
Efficacy for
B. subtilis
0.09 ±0.05
>7.88±0.10a
1.43 ±0.72
4.08 ±0.36
5.13 ± 1.50
aResult represents complete inactivation within the detectable limit of 3.33 CFU/mL. Differences in efficacy may be
significant if the 95% CIs of the two efficacy results do not overlap; however, this comparison is not applicable when the two
efficacy results being compared are both reported with log reductions as > some value.
3,000 ppm CIO2 Liquid Spray, 1 Hour Contact, 3 Applications
i B. anthracis
I B. subtilis
TopSoi
Glass
Treated Wood
Material
Industrial Carpet
Decorative Laminate
Figure 5-2. Summary of efficacies (log reduction) and confidence intervals for initial
scoping tests on building and outdoor materials (asterisk indicates complete inactivation
within the detection limit).
23
-------
6.0 Chlorine Dioxide Spray Efficacy Maximization Tests
Additional C1O2 liquid spray testing on
building and outdoor materials was
conducted to determine if increasing the
C1O2 concentration, number of spray
applications, and/or contact time would
improve decontamination efficacy for the
materials not completely decontaminated
under the test conditions described in
Chapter 5. Tests for galvanized metal and
glass were discontinued from further testing
because these materials were completely
decontaminated for the two microorganisms
using 3000 ppm C1O2, three sprays, and one
hour contact time. In place of these
materials, unpainted concrete and AZTD
were added to the efficacy maximization test
matrix.
An adaptive management approach was
used to incorporate new knowledge into the
testing as decontamination efficacy results
became available. Test matrices are
presented in the order in which the tests
were conducted:
• 3,000 ppm C1O2, one hour contact
time, four total spray applications.
(Actual C1O2 liquid concentration
measured at 3,238 ppm for B.
anthracis testing and an average of
3,137 ppm for B. subtilis testing).
• 4,000 ppm C1O2, one hour contact
time, two total spray applications.
(Actual C1O2 liquid concentration
measured at 4,047 ppm for both B.
anthracis and B. subtilis testing.)
• 4,000 ppm C1O2, one hour contact
time, four total spray applications.
(Actual C1O2 liquid concentration
measured at an average of 3,980 ppm
for B. anthracis testing and an
average of 4,047 ppm for B. subtilis
testing).
• 3,000 ppm C1O2, two hour contact
time, four total spray applications.
(Actual C1O2 liquid concentration
measured at 3,170 ppm for B.
anthracis testing and 3,103 ppm for
B. subtilis testing.)
• 4,000 ppm C1O2, two hour contact
time, four total spray applications.
(Actual C1O2 liquid concentration
measured at 3,912 ppm for both B.
anthracis and B. subtilis testing.)
The detailed decontamination efficacy
results are shown in Tables 6-1 through 6-
10, and summarized in Tables 6-11 and 6-
12and in Figure 6-1. Decorative laminate
was the only material to be completely
decontaminated in some of the tests, and a
log reduction > 6.0 was achieved on
laminate for nine of the ten tests. The soil
materials (topsoil and AZTD) were the most
difficult materials to decontaminate, with all
average log reduction results for AZTD less
than 1.5 and all log reduction results for
topsoil less than 0.31. Except for a few tests,
all of the log reduction results for wood,
concrete, and carpet were less than 3.0. The
most robust liquid spray treatment of 4,000
ppm C1O2, four spray applications, and two-
hour contact time did offer improved
decontamination efficacy compared to some
of the other test conditions (all other
24
-------
variables being equal), but did not
significantly improve efficacy compared to
the 4,000 ppm C1O2, four spray applications,
one hour contact time except for a few tests.
Decontamination efficacy results did not
always improve with increasing C1O2
concentration, contact time, or number of
spray applications. This effect was more
pronounced on carpet, wood, and unpainted
concrete, where some test results were much
higher than expected. For example,
industrial carpet was decontaminated at a
log reduction of greater than 7.0 at the 3,000
ppm, one hour, four-spray test condition, but
the log reduction results for carpet were all
less than 3.0 for all the other maximization
tests. A somewhat similar result occurred
with unpainted concrete. A number of
factors could be the cause of these outlier
results, including random experimental
error, but we think these results may also be
associated with using different batches for
the testing of the C1O2 solutions. A few
tests were repeated with these materials to
assess the effect of increasing the number of
spray applications at a given C1O2
concentration while eliminating the
possibility that differences in the batch
preparation of the aqueous C1O2 solutions
were affecting decontamination efficacy
(i.e., causing variability in results). See
Chapter 7 for further discussion of potential
causes of experimental variability and test
results.
25
-------
Table 6-1. Inactivation of Bacillus anthracis Spores—3,000 ppm ClOi Liquid Sprayed on
Building and Outdoor Materials (Four Total Spray Applications, One Hour Contact Time)
Test Material
Industrial Carpet"
Positive Controls'3
Test Coupons0
Laboratory Blankd
Procedural Blank6
Treated Wood
Positive Controls
Test Coupons
Laboratory Blank
Procedural Blank
Decorative Laminate
Positive Controls
Test Coupons
Laboratory Blank
Procedural Blank
Unpainted Concrete
Positive Controls
Test Coupons
Laboratory Blank
Procedural Blank
Arizona Test Dust
Positive Controls
Test Coupons
Laboratory Blank
Procedural Blank
Topsoil
Positive Controls
Test Coupons
Laboratory Blank
Procedural Blank
Inoculum
(CFU)
1.12xl08
1.12xl08
0
0
1.12xl08
1.12xl08
0
0
1.12xl08
1.12xl08
0
0
1.02 xlO8
1.02 xlO8
0
0
1.14xl08
1.14xl08
0
0
1.14xl08
1.14xl08
0
0
Mean of Logs of Mean % Decontamination
Observed CFU Recovery Efficacy ± CIf
7.94 ±0.05 77.4 ±8.80
0.82 ±1.8 <0.01
0
0
7.66 ±0.43 57.0 ±39
4.82 ±0.04 0.06 ±0.01
0
0
7.84 ±0.09 62.8 ±13
0.68 ±1.5 <0.01
0
0
7.32±0.19 21.9±8.1
1.19 ±2.70 0.180 ±0.39
0
0
7.97 ±0.05 82.2 ±8.8
7.85 ±0.07 62.5 ±10
0
0
7.98 ±0.06 84.1 ±10
7.93 ±0.06 75.0 ±11
0
0
_g
7.12 ±1.6
-
-
2.85 ±0.38
-
7.16 ±1.30
6.13 ±2.30
-
0.12 ±0.08
-
0.05 ±0.07
1 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).15 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.
f CI = confidence interval (± 1.96 x SE). Differences in efficacy may be significant if the 95% CIs of the two efficacy results do
not overlap; however, this comparison is not applicable when the two efficacy results being compared are both reported with
log reductions as > some value.
g "-" Not Applicable.
26
-------
Table 6-2. Inactivation of Bacillus subtilis Spores—3,000 ppm ClOi Liquid Sprayed on
Building and Outdoor Materials (Four Total Spray Applications, One Hour Contact Time)
Test Material
Industrial Carpet"
Positive Controls'3
Test Coupons0
Laboratory Blankd
Procedural Blank6
Treated Wood
Positive Controls
Test Coupons
Laboratory Blank
Procedural Blank
Decorative Laminate
Positive Controls
Test Coupons
Laboratory Blank
Procedural Blank
Unpainted Concrete
Positive Controls
Test Coupons
Laboratory Blank
Procedural Blank
Arizona Test Dust
Positive Controls
Test Coupons
Laboratory Blank
Procedural Blank
Topsoil
Positive Controls
Test Coupons
Laboratory Blank
Procedural Blank
Inoculum
(CFU)
1.20 x 108
1.20 x 108
0
0
1.20 x 108
1.20 x 108
0
0
1.20 x 108
1.20 x 108
0
0
9.33 x 107
9.33 x 107
0
0
1.02 x 108
1.02 x 108
0
0
1.02 x 108
1.02 x 108
0
0
Mean of Logs of
Observed CFU
7.86 ±0.07
6.59 ±0.16
0
0
6.88 ±0.08
4.84 ±2.70
0
0
7.82 ±0.04
0
0
0
7.49 ±0.12
2. 10 ±2.0
0
0
7.94 ±0.03
7.86 ±0.06
0
0
7.89 ±0.05
7.90 ±0.04
0
0
Mean %
Recovery
61.2 ±9.0
3.44 ±1.1
-
6.43 ±1.10
0.80 ±0.60
55.3 ±5.70
0
34.3 ±8.50
<0.01
-
85.4 ±5.60
71.4 ± 10.0
-
75.8 ±9.60
78.8 ±7.80
Decontamination
Efficacy ± CIf
_g
1.27 ±0.16
-
-
2.04 ± 2.40
-
> 7.82 ±0.04
5.39 ± 1.80
-
0.08 ±0.06
-
0.005 ± 0.06
1 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).15 Positive Controls = inoculated,
not decontaminated coupons (sprayed with SFW).
: Test Coupons = inoculated, decontaminated coupons.
1 Laboratory Blank = not inoculated, not decontaminated coupon.
: Procedural Blank = not inoculated, decontaminated coupon.
F CI = confidence interval (± 1.96 x SE). Differences in efficacy may be significant if the 95% CIs of the two efficacy results do
not overlap; however, this comparison is not applicable when the two efficacy results being compared are both reported with
log reductions as > some value.
1 "-" Not Applicable.
27
-------
Table 6-3. Inactivation of Bacillus anthracis Spores—4,000 ppm ClOi Liquid Sprayed on
Building and Outdoor Materials (Two Total Spray Applications, One Hour Contact Time)
Test Material
Industrial Carpet"
Positive Controls'3
Test Coupons0
Laboratory Blankd
Procedural Blank6
Treated Wood
Positive Controls
Test Coupons
Laboratory Blank
Procedural Blank
Decorative Laminate
Positive Controls
Test Coupons
Laboratory Blank
Procedural Blank
Unpainted Concrete
Positive Controls
Test Coupons
Laboratory Blank
Procedural Blank
Arizona Test Dust
Positive Controls
Test Coupons
Laboratory Blank
Procedural Blank
Topsoil
Positive Controls
Test Coupons
Laboratory Blank
Procedural Blank
Inoculum
(CFU)
1.05 xlO8
1.05 x 108
0
0
1.05 x 108
1.05 x 108
0
0
1.05 x 108
1.05 x 108
0
0
1.22 xlO8
1.22 xlO8
0
0
1.21 xlO8
1.21 xlO8
0
0
1.21 xlO8
1.21 xlO8
0
0
Mean of Logs of
Observed CFU
8.04 ±0.05
5.22 ±0.60
0
0
7.16 ±0.26
5.21 ±0.50
0
0
7.76 ±0.07
0
0
0
7.27 ±0.24
0.68 ±0.93
0
0
7.99 ±0.01
7.05 ±0.12
0
0
7.98 ±0.01
7.93 ±0.01
0
0
Mean %
Recovery
106 ±11.0
0.31 ±0.34
-
16.3 ±12.0
0.24 ±0.21
55.8 ±9.60
0
17.3 ±9.30
<0.01
-
81.6 ±2.40
9.59 ±2.50
-
78.9 ±1.10
70.1 ±1.60
Decontamination
Efficacy ± CIf
_g
2.82 ±0.52
-
-
1.95 ±0.49
-
> 7.76 ±0.06
6.60 ±0.84
-
0.94 ±0.11
-
0.05 ±0.01
1 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).15 Positive Controls = inoculated,
not decontaminated coupons (sprayed with SFW).
: Test Coupons = inoculated, decontaminated coupons.
1 Laboratory Blank = not inoculated, not decontaminated coupon.
: Procedural Blank = not inoculated, decontaminated coupon.
F CI = confidence interval (± 1.96 x SE). Differences in efficacy may be significant if the 95% CIs of the two efficacy results do
not overlap; however, this comparison is not applicable when the two efficacy results being compared are both reported with
log reductions as > some value.
1 "-" Not Applicable.
28
-------
Table 6-4. Inactivation of Bacillus subtilis Spores—4,000 ppm ClOi Liquid Sprayed on
Building and Outdoor Materials (Two Total Spray Applications, One Hour Contact Time)
Test Material
Industrial Carpet"
Positive Controls'3
Test Coupons0
Laboratory Blankd
Procedural Blank6
Treated Wood
Positive Controls
Test Coupons
Laboratory Blank
Procedural Blank
Decorative Laminate
Positive Controls
Test Coupons
Laboratory Blank
Procedural Blank
Unpainted Concrete
Positive Controls
Test Coupons
Laboratory Blank
Procedural Blank
Arizona Test Dust
Positive Controls
Test Coupons
Laboratory Blank
Procedural Blank
Topsoil
Positive Controls
Test Coupons
Laboratory Blank
Procedural Blank
Inoculum
(CFU)
8.93 x 107
8.93 x 107
0
0
8.93 x 107
8.93 x 107
0
0
8.93 x 107
8.93 x 107
0
0
8.93 x 107
8.93 x 107
0
0
1.33 xlO8
1.33 xlO8
0
0
1.33 xlO8
1.33 xlO8
0
0
Mean of Logs of
Observed CFU
7.68 ±0.06
5.17±0.19
0
0
6.59 ±0.12
5.00 ±0.22
0
0
7.63 ±0.08
1.16±1.1
0
0
7.06 ±0.16
3.42 ±0.16
0
0
7.96 ±0.04
7.83 ±0.06
0
0
7.96 ±0.03
7.92 ±0.05
0
0
Mean %
Recovery
53.7 ±7.30
0.18±0.10
-
4.50 ±1.40
0.12 ±0.06
48.8 ±9.10
<0.01
13.7 ±5.60
<0.01
-
69.5 ± 6.20
51.5 ±6.90
-
69.5 ±5.20
63.4 ±6.80
Decontamination
Efficacy ± CIf
_g
2.51 ±0.17
-
-
1.59 ±0.22
-
6.47 ±1.40
3.64 ±0.20
-
0.13 ±0.06
-
0.04 ±0.05
1 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).15 Positive Controls = inoculated,
not decontaminated coupons (sprayed with SFW).
: Test Coupons = inoculated, decontaminated coupons.
1 Laboratory Blank = not inoculated, not decontaminated coupon.
: Procedural Blank = not inoculated, decontaminated coupon.
F CI = confidence interval (± 1.96 x SE). Differences in efficacy may be significant if the 95% CIs of the two efficacy results do
not overlap; however, this comparison is not applicable when the two efficacy results being compared are both reported with
log reductions as > some value.
1 "-" Not Applicable.
29
-------
Table 6-5. Inactivation of Bacillus anthracis Spores—4,000 ppm ClOi Liquid Sprayed on
Building and Outdoor Materials (Four Total Spray Applications, One Hour Contact Time)
Test Material
Industrial Carpet"
Positive Controls'3
Test Coupons0
Laboratory Blankd
Procedural Blank6
Treated Wood
Positive Controls
Test Coupons
Laboratory Blank
Procedural Blank
Decorative Laminate
Positive Controls
Test Coupons
Laboratory Blank
Procedural Blank
Unpainted Concrete
Positive Controls
Test Coupons
Laboratory Blank
Procedural Blank
Arizona Test Dust
Positive Controls
Test Coupons
Laboratory Blank
Procedural Blank
Topsoil
Positive Controls
Test Coupons
Laboratory Blank
Procedural Blank
Inoculum
(CFU)
1.21 xlO8
1.21 xlO8
0
0
1.21 xlO8
1.21 xlO8
0
0
1.21 xlO8
1.21 xlO8
0
0
1.16xl08
1.16xl08
0
0
1.16xl08
1.16xl08
0
0
1.16xl08
1.16xl08
0
0
Mean of Logs of
Observed CFU
7.97 ±0.02
6.69 ±0.10
0
0
7.01 ±0.10
5.80 ±0.09
0
0
7.90 ±0.02
0
0
0
7.74 ±0.21
5. 14 ±0.69
0
0
7.96 ±0.02
6.50 ±0.09
0
0
7.98 ±0.04
7.81 ±0.04
0
0
Mean %
Recovery
76.5 ±3.60
4.18 ±1.00
-
8.62 ±1.90
0.530±0.11
66.1 ±3.30
0
52.4 ±26.0
0.310 ±0.48
-
78.8 ±3.40
2.79 ±0.66
-
82.2 ±7.30
55.7 ±5. 30
Decontamination
Efficacy ± CIf
_g
1.27 ±0.09
-
-
1.21 ±0.12
-
> 7.90 ±0.02
2.61 ±0.63
-
1.46 ±0.08
-
0.17 ±0.05
1 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).15 Positive Controls = inoculated,
not decontaminated coupons (sprayed with SFW).
: Test Coupons = inoculated, decontaminated coupons.
1 Laboratory Blank = not inoculated, not decontaminated coupon.
: Procedural Blank = not inoculated, decontaminated coupon.
F CI = confidence interval (± 1.96 x SE). Differences in efficacy may be significant if the 95% CIs of the two efficacy results do
not overlap; however, this comparison is not applicable when the two efficacy results being compared are both reported with
log reductions as > some value.
1 "-" Not Applicable.
30
-------
Table 6-6. Inactivation of Bacillus subtilis Spores—4,000 ppm ClOi Liquid Sprayed on
Building and Outdoor Materials (Four Total Spray Applications, One Hour Contact Time)
Test Material
Industrial Carpet"
Positive Controls'3
Test Coupons0
Laboratory Blankd
Procedural Blank6
Treated Wood
Positive Controls
Test Coupons
Laboratory Blank
Procedural Blank
Decorative Laminate
Positive Controls
Test Coupons
Laboratory Blank
Procedural Blank
Unpainted Concrete
Positive Controls
Test Coupons
Laboratory Blank
Procedural Blank
Arizona Test Dust
Positive Controls
Test Coupons
Laboratory Blank
Procedural Blank
Topsoil
Positive Controls
Test Coupons
Laboratory Blank
Procedural Blank
Inoculum
(CFU)
1.14xl08
1.14xl08
0
0
1.14xl08
1.14xl08
0
0
1.14xl08
1.14xl08
0
0
7.67 x 107
7.67 x 107
0
0
7.67 x 107
7.67 x 107
0
0
7.67 x 107
7.67 x 107
0
0
Mean of Logs of
Observed CFU
7.92 ±0.03
7.19 ±0.04
0
0
6.94 ±0.05
6.01 ±0.32
0
0
7.86 ±0.06
0
0
0
7.29 ±0.14
5.76 ±0.39
0
0
7.79 ±0.04
7.57 ±0.16
0
0
7.86 ±0.09
7.74 ±0.08
0
0
Mean %
Recovery
73.5 ±5.50
13.8 ±1.40
-
7.70 ±0.93
1.13 ±0.94
64.4 ±9.10
0
26.4 ±9.30
0.940 ±0.48
-
80.6 ±7.10
51.0 ± 18.0
-
96.2 ± 20.0
73.3 ±13.0
Decontamination
Efficacy ± CIf
_g
0.73 ±0.05
-
-
0.930 ±0.29
-
> 7.86 ±0.06
1.52 ±0.36
-
0.22 ±0.14
-
0.12±0.10
1 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).15 Positive Controls = inoculated,
not decontaminated coupons (sprayed with SFW).
: Test Coupons = inoculated, decontaminated coupons.
1 Laboratory Blank = not inoculated, not decontaminated coupon.
: Procedural Blank = not inoculated, decontaminated coupon.
F CI = confidence interval (± 1.96 x SE). Differences in efficacy may be significant if the 95% CIs of the two efficacy results do
not overlap; however, this comparison is not applicable when the two efficacy results being compared are both reported with
log reductions as > some value.
1 "-" Not Applicable.
31
-------
Table 6-7. Inactivation of Bacillus anthracis Spores—3,000 ppm ClOi Liquid Sprayed on
Building and Outdoor Materials (Four Total Spray Applications, Two Hour Contact Time)
Test Material
Industrial Carpet"
Positive Controls'3
Test Coupons0
Laboratory Blankd
Procedural Blank6
Treated Wood
Positive Controls
Test Coupons
Laboratory Blank
Procedural Blank
Decorative Laminate
Positive Controls
Test Coupons
Laboratory Blank
Procedural Blank
Unpainted Concrete
Positive Controls
Test Coupons
Laboratory Blank
Procedural Blank
Arizona Test Dust
Positive Controls
Test Coupons
Laboratory Blank
Procedural Blank
Topsoil
Positive Controls
Test Coupons
Laboratory Blank
Procedural Blank
Inoculum
(CFU)
1.15xl08
1.15xl08
0
0
1.15xl08
1.15xl08
0
0
1.15xl08
1.15xl08
0
0
1.15xl08
1.15xl08
0
0
1.15xl08
1.15xl08
0
0
1.15xl08
1.15xl08
0
0
Mean of Logs of
Observed CFU
7.96 ±0.07
5.45 ±0.22
0
0
7.11 ±0.09
5.05 ±0.44
0
0
7.88 ±0.07
1.24 ±1.2
0
0
7.94 ±0.03
5.72 ±0.31
0
0
7.87 ±0.03
7.21 ±0.27
0
0
7.98 ±0.02
7.88 ±0.05
0
0
Mean %
Recovery
80.4 ± 13.0
0.270 ±0.12
-
11.4 ±2.00
0.150±0.19
67.0 ±11.0
<0.01
75.6 ±5.60
0.55 ±0.33
-
65.1 ±4.50
16.3 ±8.50
-
82.9 ±4.30
67.0 ±8.60
Decontamination
Efficacy ± CIf
_g
2.51 ±0.20
-
-
2.06 ±0.39
-
6.64 ±1.00
2.22 ±0.28
-
0.66 ± 0.24
-
0.09 ±0.05
1 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).15 Positive Controls = inoculated,
not decontaminated coupons (sprayed with SFW).
: Test Coupons = inoculated, decontaminated coupons.
1 Laboratory Blank = not inoculated, not decontaminated coupon.
: Procedural Blank = not inoculated, decontaminated coupon.
F CI = confidence interval (± 1.96 x SE). Differences in efficacy may be significant if the 95% CIs of the two efficacy results do
not overlap; however, this comparison is not applicable when the two efficacy results being compared are both reported with
log reductions as > some value.
1 "-" Not Applicable.
32
-------
Table 6-8. Inactivation of Bacillus subtilis Spores—3,000 ppm ClOi Liquid Sprayed on
Building and Outdoor Materials (Four Total Spray Applications, 2 Hour Contact Time)
Test Material
Industrial Carpet"
Positive Controls'3
Test Coupons0
Laboratory Blankd
Procedural Blank6
Treated Wood
Positive Controls
Test Coupons
Laboratory Blank
Procedural Blank
Decorative Laminate
Positive Controls
Test Coupons
Laboratory Blank
Procedural Blank
Unpainted Concrete
Positive Controls
Test Coupons
Laboratory Blank
Procedural Blank
Arizona Test Dust
Positive Controls
Test Coupons
Laboratory Blank
Procedural Blank
Topsoil
Positive Controls
Test Coupons
Laboratory Blank
Procedural Blank
Inoculum
(CFU)
9.50 x 107
9.50 x 107
0
0
9.50 x 107
9.50 x 107
0
0
9.50 x 107
9.50 x 107
0
0
9.50 x 107
9.50 x 107
0
0
9.50 x 107
9.50 x 107
0
0
9.50 x 107
9.50 x 107
0
0
Mean of Logs of
Observed CFU
7.85 ±0.09
7.05 ±0.07
0
0
6.98 ±0.09
6.01 ±0.44
0
0
7.94 ±0.04
5.06 ±0.86
0
0
7.47 ± 0.20
6.47 ±0.39
0
0
7.98 ±0.04
7.80 ±0.09
0
0
7.98 ±0.04
7.93 ±0.02
0
0
Mean %
Recovery
76.5 ±
12.0 ±
-
10.3 ±
1.56 ±
91.9±
15.0
1.90
2.50
1.40
8.50
0.390 ±0.48
33.7 ±
4.37 ±
-
99.9 ±
67.5 ±
-
100±
89.5 ±
15.0
4.20
9.10
13.0
9.80
3.90
Decontamination
Efficacy ± CIf
_g
0.80 ±0.10
-
-
0.97 ±0.39
-
2.88 ±0.75
1.00 ±0.38
-
0.18 ±0.08
-
0.05 ±0.04
1 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).15 Positive Controls = inoculated,
not decontaminated coupons (sprayed with SFW).
: Test Coupons = inoculated, decontaminated coupons.
1 Laboratory Blank = not inoculated, not decontaminated coupon.
: Procedural Blank = not inoculated, decontaminated coupon.
F CI = confidence interval (± 1.96 x SE). Differences in efficacy may be significant if the 95% CIs of the two efficacy results do
not overlap; however, this comparison is not applicable when the two efficacy results being compared are both reported with
log reductions as > some value.
1 "-" Not Applicable.
33
-------
Table 6-9. Inactivation of Bacillus anthracis Spores—4,000 ppm ClOi Liquid Sprayed on
Building and Outdoor Materials (Four Total Spray Applications, Two Hour Contact Time)
Test Material
Industrial Carpet"
Positive Controls'3
Test Coupons0
Laboratory Blankd
Procedural Blank6
Treated Wood
Positive Controls
Test Coupons
Laboratory Blank
Procedural Blank
Decorative Laminate
Positive Controls
Test Coupons
Laboratory Blank
Procedural Blank
Unpainted Concrete
Positive Controls
Test Coupons
Laboratory Blank
Procedural Blank
Arizona Test Dust
Positive Controls
Test Coupons
Laboratory Blank
Procedural Blank
Topsoil
Positive Controls
Test Coupons
Laboratory Blank
Procedural Blank
Inoculum
(CFU)
1.33 xlO8
1.33 xlO8
0
0
1.33 xlO8
1.33 xlO8
0
0
1.33 xlO8
1.33 xlO8
0
0
1.15xl08
1.15xl08
0
0
7.87 x 107
7.87 x 107
0
0
1.33 xlO8
1.33 xlO8
0
0
Mean of Logs of
Observed CFU
7.90 ±0.09
5.06 ±0.17
0
0
7.22 ± 0.20
4.59 ±0.71
0
0
7.89 ±0.05
0.310 ±0.69
0
0
7.65 ±0.16
5.15±0.11
0
0
7.90 ±0.03
6.62 ± 0.24
0
0
7.97 ±0.04
7.66 ±0.03
0
0
Mean %
Recovery
60.79 ± 13
0.09 ±0.04
-
13.8 ±7.60
0.05 ±0.03
58.7 ±7.20
<0.01
40.6 ± 16.0
0.12 ±0.03
-
100 ± 5.90
5.97 ±3.70
-
70.6 ±6.80
34.2 ±2.70
Decontamination
Efficacy ± CIf
_g
2.84 ±0.17
-
-
2.64 ±0.65
-
7.58 ±0.61
2.50 ±0.16
-
1.28 ±0.21
-
0.31 ±0.05
1 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).15 Positive Controls = inoculated,
not decontaminated coupons (sprayed with SFW).
: Test Coupons = inoculated, decontaminated coupons.
1 Laboratory Blank = not inoculated, not decontaminated coupon.
: Procedural Blank = not inoculated, decontaminated coupon.
F CI = confidence interval (± 1.96 x SE). Differences in efficacy may be significant if the 95% CIs of the two efficacy results do
not overlap; however, this comparison is not applicable when the two efficacy results being compared are both reported with
log reductions as > some value.
1 "-" Not Applicable
34
-------
Table 6-10. Inactivation of Bacillus subtilis Spores—4,000 ppm ClOi Liquid Sprayed on
Building and Outdoor Materials (Four Total Spray Applications, Two Hour Contact Time)
Test Material
Industrial Carpet"
Positive Controls'3
Test Coupons0
Laboratory Blankd
Procedural Blank6
Treated Wood
Positive Controls
Test Coupons
Laboratory Blank
Procedural Blank
Decorative Laminate
Positive Controls
Test Coupons
Laboratory Blank
Procedural Blank
Unpainted Concrete
Positive Controls
Test Coupons
Laboratory Blank
Procedural Blank
Arizona Test Dust
Positive Controls
Test Coupons
Laboratory Blank
Procedural Blank
Topsoil
Positive Controls
Test Coupons
Laboratory Blank
Procedural Blank
Inoculum
(CFU)
1.30 xlO8
1.30 xlO8
0
0
1.30 xlO8
1.30 xlO8
0
0
1.30 xlO8
1.30 xlO8
0
0
1.30 xlO8
1.30 xlO8
0
0
1.30 xlO8
1.30 xlO8
0
0
1.30 xlO8
1.30 xlO8
0
0
Mean of Logs of
Observed CFU
7.89 ±0.06
5.58 ±0.24
0
0
6.95 ±0.12
4.95 ±0.77
0
0
7.98 ±0.02
0
0
0
6.65 ±0.18
5.60 ±0.37
0
0
7.96 ±0.02
7.27 ±0.25
0
0
8.03 ±0.02
7.96 ±0.03
0
0
Mean %
Recovery
60.5 ± 8.00
0.33 ±0.13
-
7.11 ±2.20
0.18 ±0.26
73.4 ±3. 10
0
3.72 ± 1.80
0.38 ±0.22
-
70.4 ±3.60
16.3 ± 9.70
-
83.3 ±3.80
69.5 ± 4.70
Decontamination
Efficacy ± CIf
_g
2.31 ±0.22
-
-
2.00 ±0.69
-
> 7.98 ±0.02
1.05 ±0.36
-
0.69 ±0.22
-
0.08 ±0.03
1 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).15 Positive Controls = inoculated,
not decontaminated coupons (sprayed with SFW).
: Test Coupons = inoculated, decontaminated coupons.
1 Laboratory Blank = not inoculated, not decontaminated coupon.
: Procedural Blank = not inoculated, decontaminated coupon.
F CI = confidence interval (± 1.96 x SE). Differences in efficacy may be significant if the 95% CIs of the two efficacy results do
not overlap; however, this comparison is not applicable when the two efficacy results being compared are both reported with
log reductions as > some value.
1 "-" Not Applicable
35
-------
Table 6-11. Summary of Mean Quantitative Efficacy with 95% Confidence Intervals for ClOi Liquid Spray Efficacy
Maximization Results for B. anthracis
Quantitative Efficacy (mean log reduction)
Test Material
Industrial Carpet
Treated Wood
Decorative
Laminate
Unpainted
Concrete
Arizona Test Dust
Topsoil
3000 ppm,
One hr contact,
four total spray
applications
7.12± 1.60
2.85 ±0.38
7.16 ±1.34
6.13 ±2.34
0.12 ±0.08
0.05 ±0.07
4000 ppm,
One hr contact,
two total spray
applications
2.82 ±0.52
1.95 ±0.49
>7.76a±0.06
6.60 ±0.84
0.940 ±0.11
0.05 ±0.01
4000 ppm,
One hr contact,
four total spray
applications
1.27 ±0.09
1.21 ±0.12
>7.90a±0.02
2.61 ±0.63
1.46 ±0.08
0.17 ±0.05
3000 ppm,
Two hr contact,
four total spray
applications
2.51 ±0.20
2.06 ±0.39
6.64 ± 1.0
2.22 ±0.28
0.66 ±0.24
0.09 ±0.05
4000 ppm,
Two hr contact,
four total spray
applications
2.84 ±0.17
2.64 ±2.6
7.58 ±0.61
2.50 ±0.16
1.28 ±0.21
0.31 ±0.05
aResult represents complete inactivation within the detectable limit of 33.33 CPU/material. Differences in efficacy may be significant if the 95% CIs of the
two efficacy results do not overlap; however, this comparison is not applicable when the two efficacy results being compared are both reported with log
reductions as > some value.
36
-------
Table 6-12. Summary of Mean Quantitative Efficacy with 95% Confidence Intervals for ClOi Liquid Spray Efficacy
Maximization Results for B. subtitis
Quantitative Efficacy (mean log reduction)
Test Material
Industrial Carpet
Treated Wood
Decorative Laminate
Unpainted Concrete
Arizona Test Dust
Topsoil
3000 ppm,
One hr contact,
four total spray
applications
1.27±0.16
2.04 ±2.40
>7.82a±0.04
5.39 ±1.80
0.08 ±0.06
0.005 ± 0.06
4000 ppm,
One hr contact,
two total spray
applications
2.51 ±0.17
1.59 ±0.22
6.47 ± 1.40
3.64 ±0.20
0.13 ±0.06
0.04 ±0.05
4000 ppm,
One hr contact,
four total spray
applications
0.73 ±0.05
0.93 ± 0.29
>7.86a±0.06
1.52 ±0.36
0.22 ±0.14
0.12±0.10
3000 ppm,
Two hr contact,
four total spray
applications
0.80 ±0.10
0.97 ±0.39
2.88 ±0.75
1.00 ±0.38
0.18 ±0.08
0.05 ± 0.04
4000 ppm,
Two hr contact,
four total spray
applications
2.31 ±0.22
2.00 ±0.69
>7.98a±0.02
1.05 ±0.36
0.69 ±0.22
0.08 ±0.03
aResult represents complete inactivation within the detectable limit of 33.33 CPU/material. Differences in efficacy may be significant if the 95% CIs of the
two efficacy results do not overlap; however, this comparison is not applicable when the two efficacy results being compared are both reported with log
reductions as > some value.
37
-------
Industrial Carpet
ICarpet-B.a.
ICarpet-B.sub
3000ppm,60',4apps 4000ppm,60',2app
4000ppm,60',4apps 3000ppm,120',4apps 4000ppm, 120',4apps
Test Condition
Treated Wood
ITrtdWood-B.j.
ITrtdWood-B.sub
3000ppm, 60', 4jpps 4000ppm, 60', 2apps
4000ppm,60',4apps
Test Condition
SOOOppm, 120', 4apps 4000ppm, 120', 4apps
Decorative Laminate
• Laminate-B.a.
• Laminate-B.sub
3000ppm,60',4apps 4000ppm, 60', 2apps 4000ppm,60',4apps 3000ppm, 120',4apps 4000ppm, 120',4apps
Test Condition
Figure 6-la. Summary of efficacies (log reduction) and confidence intervals for liquid spray
testing conditions (asterisk indicates complete inactivation within the detection limit).
38
-------
Unpainted Concrete
lUnptdConc-B.a.
lUnptdConc-B.sub
3000ppm,60',4apps 4000ppm,60',2apps 4000ppm,60',4apps 3000ppm, 120',4apps 4000ppm, 120',4apps
Test Condition
AZ Test Dust
lAZTD-B.a.
lAZTD-B.sub
o 4-
3000ppm,60',4apps 4000ppm,60',2apps 4000ppm,60',4apps SOOOppm, 120',4apps 4000ppm, 120',4apps
Test Condition
Topsoil
_ 8
I 7
ITopsoil-B.a.
ITopsoil-B.sub
3000ppm,60',4apps 4000ppm,60',2apps 4000ppm,60',4apps BOOOppm, 120',4apps 4000ppm, 120',4apps
Test Condition
Figure 6-lb. Summary of efficacies (log reduction) and confidence intervals for liquid
spray testing conditions.
39
-------
7.0 Liquid Spray Results for Two vs. Four Total Spray Applications
7.1 Description
During the course of the C1O2 liquid spray
efficacy maximization testing (refer to
Chapter 6), the decontamination efficacy
results obtained for some of the porous
materials (industrial carpet, treated wood,
and unpainted concrete) were variable, i.e.,
did not follow expected trends and in some
cases, were much higher than expected. For
example, as more spray applications were
involved (e.g., from two total spray
applications to four total spray applications,
but at the same contact time and
concentration), a significantly lower efficacy
(log reduction) would not be expected.
Similarly, as testing progressed from a lower
C1O2 concentration to a higher concentration
(but with same contact time and number of
spray applications), a significantly lower
efficacy (log reduction) would not be
expected. But for some of these materials
and tests, lower decontamination efficacies
were indeed observed. Several factors may
have contributed to this variability and are
discussed below. We do note that some of
these factors may have had minimal impact
on efficacy for the materials already easily
decontaminated at most or all of the test
conditions (e.g., decorative laminate), or for
materials that were minimally
decontaminated at all of the test conditions
(soil materials).
7.1.1 Test Material Composition
The porous materials that exhibited highly
variable results (industrial carpet, treated
wood, and unpainted concrete) have,
historically, been variable in terms of
efficacy (log reduction) once decontaminant
has been applied. The reason may be
attributed to the porous nature of the
materials themselves. Not only are the spore
suspensions being absorbed, but so is the
sprayed C1O2 liquid. No evaluations have
been made to determine what happens to
decontaminants or how the materials
themselves affect the fate (e.g., activity) of
the decontaminants once applied and
absorbed into the coupons. Nonporous
materials (e.g., laminate) allow the spore
suspensions to dry as droplets in the place
where they were inoculated. These materials
also allow the liquid decontaminants to pool
and stay visibly wetted for the duration of
the contact times.
7.1.2 Subjectivity of Spraying Method
The C1O2 solution is sprayed using a
commercially-available trigger sprayer as
described in Chapter 2. Prior to spraying, the
sprayer was tipped so that it was horizontal
(i.e., facing the inoculated material coupons
in the horizontal configuration) at a distance
of 30.5 cm. When sprayed, the trigger was
pulled while simultaneously sweeping the
sprayer from side-to-side to cover the five
inoculated replicates of one material. The
number of trigger pulls was counted to
render the materials "fully wetted" for that
spray application at that time point. The
same procedure was followed for each
subsequent material until all materials were
sprayed with the C1O2 solution for that time
40
-------
point (e.g., time 0). This same procedure
was repeated for additional time points (e.g.,
+30 minutes). The subjectivity involved
with this method is apparent, but this
method was the most reasonable method
when previous tasks that involved
specialized sprayers/applicators (depending
on the vendor) were taken into
consideration. Any development or
implementation of an automated system was
precluded due to the number of variables
(e.g., nozzle type, pressure requirements,
number of decontaminant component
reservoirs) associated with each
decontaminant formulation.
Since a commercially-available trigger
sprayer was used to dispense and apply the
C1O2 liquid, there was no form of precise
control of where the sprayed liquid
deposited. Due to space limitations
associated with working in a Class III BSC
glove box, two material types were placed
per elevated grill. Despite efforts taken to
control the conical spray pattern from the
trigger sprayer, a "shadowing effect" may
have resulted in over-spraying one material
type that landed on the other material on the
same elevated grill, possibly yielding one
material actually getting more sprayed C1O2
liquid than intended.
7.1.3 Chemistry of CIO2 Solution
Batch-to-batch differences in the pH and
other chemical characteristics of the aqueous
C1O2 solutions may also have been a factor
in the variable decontamination efficacy
results. When a C1O2 solution batch was
made, the pH, chlorite, C1O2, and ratio of
chlorite to C1O2 were measured or
calculated. According to the manufacturer,
the pH of the C1O2 solution must be between
4 and 7. Also, the chlorite concentration
(ppm) must be within a minimum of 50% of
the target C1O2 concentration (e.g., if a
3,000 ppm C1O2 liquid is needed, the
chlorite can be no less than 1,500 ppm).
Every effort, however, was taken during
C1O2 liquid spray testing to bring the pH of
the solution as close to 7 as possible because
the measured pH was near 7, so, for
consistency's sake, every batch of C1O2
solution was adjusted to be pH 7. Of the 22
times that the C1O2 solution was made for
liquid spray testing, the average pH was
6.73 ± 0.56, and the average chlorite to C1O2
ratio was 84.42 ± 13.42%. See Table 12-1
for a summary of the chemical
characteristics and their variability for each
batch of aqueous C1O2 used in testing.
To eliminate the possibility that some of the
variability in efficacy results for the wood,
concrete and carpet materials was due to
batch-to-batch variability of the aqueous
solutions, tests were conducted to see
whether two total spray applications versus
four total spray applications using the same
C1O2 liquid batch (either 3000 ppm or 4000
ppm C1O2) and contact time would yield the
expected results (i.e., higher efficacy with
increased number of sprays) on industrial
carpet, treated wood, and unpainted concrete
inoculated with B. anthracis spores. With
these additional tests, we would be better
able to determine whether increasing the
number of spray applications improved
decontamination efficacy.
41
-------
Table 7-1. Chlorine Dioxide Liquid Batch Chemical Comparison
Test Condition
(C1O2 Liquid Prep #)
3000 ppm, 60', three apps
(1)
(2)
(3)
(4)
Test Condition Average
Test Condition Standard
Deviation
3000 ppm, 60', four apps
(1)
(2)
(3)
(4)
(5)
(6)
(7)
Test Condition Average
Test Condition Standard
Deviation
4000 ppm, 60', two apps (1)
(2)
(3)
(4)
(5)
Test Condition Average
Test Condition Standard
Deviation
4000 ppm, 60', four apps
(1)
(2)
(3)
(4)
(5)
(6)
Test Condition Average
Test Condition Standard
Deviation
Total AVG
Total STDEV
Measured pH
(4-7)
6.86
6.97
6.57
6.82
6.81
0.170
6.92
6.93
6.91
6.60
6.98
6.88
6.95
6.89
0.130
6.90
4.35
6.84
6.77
6.96
6.36
1.13
6.30
6.98
6.93
6.89
6.81
6.85
6.79
0.250
6.73
0.560
Titrated C1O2 (ppm)
3102.7
3102.7
3102.7
3237.6
3136.4
67.450
3237.6
3237.6
3170.2
3102.7
3237.6
3102.7
3102.7
3170.2
67.450
4181.9
4181.9
4047.0
4047.0
4047.0
4101.0
73.890
3777.2
4115.5
4181.9
3912.1
4047.0
3912.1
3991.0
150.48
N/A
N/A
Titrated Chlorite
(ppm)
3169.7
3203.4
3540.6
3270.8
3296.1
168.32
1719.7
2529.0
2562.7
2663.9
2158.1
2630.2
2427.8
2384.5
338.16
3473.2
3102.2
3237.1
3169.7
3102.2
3216.9
153.79
3608.0
3304.6
3203.4
3709.2
3270.8
3473.2
3428.2
201.94
N/A
N/A
Ratio of
[Chlorite] vs. [C1O2]
(50% minimum)
102%
103%
114%
101%
105%
6.05%
53.1%
78.1%
80.8%
85.9%
66.7%
84.8%
78.2%
75.4%
11.7%
83.0%
74.2%
80.0%
78.3%
76.7%
78.4%
3.35%
95.5%
80.3%
76.6%
94.8%
80.8%
88.8%
86.1%
8.05%
84.4%
13.4%
N/A = Not applicable
42
-------
7.2 Decontamination Efficacy
For the 3,000 ppm C1O2 repeat test (actual
C1O2 liquid concentration was 3,238 ppm),
the decontamination efficacies for all
materials improved when increasing the
number of sprays from two to four, as
shown in Table 7-2 and summarized in
Table 7-4. Although increasing the number
of sprays did improve efficacy at this test
condition, the log reduction results were still
very poor and were all below 2.27.
An increase in decontamination efficacy
between the two spray applications versus
the four spray applications for 4,000 ppm
C1O2 batch was seen only for industrial
carpet, as shown in Table 7-3 and
summarized in Table 7-4. (Actual C1O2
liquid concentration measured at 4,182
ppm.) Regardless, the highest log reduction
achieved in this repeat test condition was
2.31.
A comparison of the "maximization" test
results (refer to Chapter 6) and those that
were repeated is summarized in Table 7-5.
At the 3,000 ppm (four sprays) test
condition and the 4,000 ppm (two sprays)
test condition, the repeat test log reduction
results are lower than the initial test results
reported in Chapter 6. At the 4,000 ppm
(four sprays) test condition, the repeat test
results were slightly higher or equivalent. In
general, the repeat test results are consistent
with the other test results for each material
(see Chapter 6). Thus the outlying results
for industrial carpet and unpainted concrete
may have been due to experimental error or
some uncontrolled parameter, possibly
related to the C1O2 solution chemistry.
43
-------
Table 7-2. Inactivation of Bacillus anthracis Spores—Two Applications vs. Four
Applications 3,000 ppm ClOi Liquid Sprayed on Materials (One Hour Contact Time)
Test Material
Inoculum
(CFU)
Mean of Logs of
Observed CFU
Mean %
Recovery
Decontamination
Efficacy ± CIf
Two Applications
Industrial Carpet"
Positive Controls'3
Test Coupons0
Laboratory Blankd
Procedural Blank6
Treated Wood
Positive Controls
Test Coupons
Laboratory Blank
Procedural Blank
Unpainted Concrete
Positive Controls
Test Coupons
Laboratory Blank
Procedural Blank
1.16xl08
1.16xl08
0
0
1.16xl08
1.16xl08
0
0
1.16xl08
1.16xl08
0
0
7.78 ±0.04
7.15 ±0.20
0
0
7.08 ±0.03
5.58 ±0.39
0
0
7.62 ±0.05
6.45 ±0.15
0
0
52.5 ± 4.70
13.2 ±6.90
-
-
10.4 ±0.66
0.440 ±0.35
-
-
36.2 ±4.10
2.55 ±0.71
-
-
_g
0.64 ±0.18
-
-
-
1.50 ±0.35
-
-
-
1.17±0.14
-
-
Four Applications
Industrial Carpet
Positive Controls
Test Coupons
Laboratory Blank
Procedural Blank
Treated Wood
Positive Controls
Test Coupons
Laboratory Blank
Procedural Blank
Unpainted Concrete
Positive Controls
Test Coupons
Laboratory Blank
Procedural Blank
1.16xl08
1.16xl08
0
0
1.16xl08
1.16xl08
0
0
1.16xl08
1.16xl08
0
0
7.75 ±0.06
5.78 ±0.32
0
0
7.37 ±0.24
5. 10 ±0.09
0
0
7.83 ±0.06
5.72 ±0.32
0
0
49.3 ±6.50
0.650 ±0.47
-
-
22.0 ± 8.40
0.110 ±0.02
-
-
58.7 ±8.3
0.55 ±0.30
-
-
-
1.97 ±0.29
-
-
-
2.27 ±0.22
-
-
-
2.11 ±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).15 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.
f CI = confidence interval (± 1.96 x SE). Differences in efficacy may be significant if the 95% CIs of the two efficacy results do
not overlap; however, this comparison is not applicable when the two efficacy results being compared are both reported with
log reductions as > some value.
g "-" Not Applicable.
44
-------
Table 7-3. Inactivation of Bacillus anthracis Spores—Two Applications vs. Four
Applications 4,000 ppm ClOi Liquid Sprayed on Materials (One Hour Contact Time)
Test Material
Inoculum
(CFU)
Mean of Logs of
Observed CFU
Mean %
Recovery
Decontamination
Efficacy ± CIf
Two Applications
Industrial Carpet"
Positive Controls'3
Test Coupons0
Laboratory Blankd
Procedural Blank6
Treated Wood
Positive Controls
Test Coupons
Laboratory Blank
Procedural Blank
Unpainted Concrete
Positive Controls
Test Coupons
Laboratory Blank
Procedural Blank
1.19xl08
1.19xl08
0
0
1.19xl08
1.19xl08
0
0
1.19xl08
1.19xl08
0
0
8.07 ±0.03
6.95 ±0.15
0
0
7.47 ±0.31
5.80 ±0.48
0
0
8.03 ±0.15
6.41 ±0.16
0
0
100 ±7.30
7.87±2.50
-
-
29.6 ±16.0
0.780 ±0.57
-
-
93.8 ±26.0
2.29 ±0.74
-
-
_g
1.12±0.13
-
-
-
1.67 ±0.50
-
-
-
1.62 ±0.19
-
-
Four Applications
Industrial Carpet
Positive Controls
Test Coupons
Laboratory Blank
Procedural Blank
Treated Wood
Positive Controls
Test Coupons
Laboratory Blank
Procedural Blank
Unpainted Concrete
Positive Controls
Test Coupons
Laboratory Blank
Procedural Blank
1.19xl08
1.19xl08
0
0
1.19xl08
1.19xl08
0
0
1.19xl08
1.19xl08
0
0
8.02 ±0.04
6.25 ± 0.43
0
0
7.33 ±0.40
5.02 ±0.51
0
0
7.91 ±0.12
6.14 ±0.43
0
0
88.6 ±7.70
2.04 ±1.30
-
-
27.0 ±31.0
70.2 ±0.15
-
-
70.4 ± 22.0
1.59 ± 1.20
-
-
-
1.77 ±0.38
-
-
-
2.31 ±0.57
-
-
-
1.77 ±0.39
-
-
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).15 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.
f CI = confidence interval (± 1.96 x SE). Differences in efficacy may be significant if the 95% CIs of the two efficacy results do
not overlap; however, this comparison is not applicable when the two efficacy results being compared are both reported with
log reductions as > some value.
8 "-" Not Applicable.
45
-------
Table 7-4. Summary of Decontamination Efficacy Values for Two vs. Four Applications
Test Material
3,000 ppm C1O2
Industrial Carpet
Treated Wood
Unpainted Concrete
Efficacy for
B. anthracis
Two sprays
0.64 ±0.18
1.50 ±0.35
1.17±0.14
Efficacy for
B. anthracis
Four sprays
1.97 ±0.29
2.27 ±0.22
2.11 ±0.29
4,000 ppm C1O2
Industrial Carpet
Treated Wood
Unpainted Concrete
1.12±0.13
1.67 ±0.50
1.62 ±0.19
1.77 ±0.38
2.31 ±0.57
1.77 ±0.39
All efficacy results reported in terms of mean log reduction ± 95% confidence interval
Table 7-5. Comparing Decontamination Efficacy Values for Tests that Were Repeated
Efficacy for Efficacy for
Test Condition B. anthracis B. anthracis
Initial Test (Ch. 6) Repeat Test
3,000 ppm C1O2 - Four sprays,
One hr
Industrial Carpet
Treated Wood
Unpainted Concrete
7.12 ± 1.60
2.85 ±0.38
5.42 ±2.40
1.97 ±0.29
2.27 ±0.22
2.11 ±0.29
4,000 ppm C1O2 - Two sprays,
One hr
Industrial Carpet
Treated Wood
Unpainted Concrete
2.82 ±0.52
1.95 ±0.49
6.60 ±0.84
1.12±0.13
1.67 ±0.50
1.62±0.19
4,000 ppm C1O2 - Four sprays,
One hr
Industrial Carpet
Treated Wood
Unpainted Concrete
1.27 ±0.09
1.21 ±0.12
2.61 ±0.63
1.77 ±0.38
2.31 ±0.57
1.77 ±0.39
All efficacy results reported in terms of mean log reduction ± 95% confidence interval
46
-------
8.0 Summary of Results
A summary of the decontamination efficacy
results for the initial C1O2 scoping tests is
shown in Table 8-1. Galvanized metal was
effectively decontaminated (having a 6 log
reduction or greater) in three of the four B.
anthracis tests. For the other test with B.
anthracis, a log reduction of nearly 6 (5.99)
was obtained. Complete inactivation of
spores for both organisms on galvanized
metal was observed only under the test
condition utilizing 3,000 ppm C1O2, one
hour contact time and three spray
applications. With this same
decontamination treatment on the additional
test materials, glass was the only other
material completely decontaminated for both
B. anthracis and B. subtilis spores. The
decorative laminate material was completely
decontaminated under this same test
condition, but only with regard to B.
anthracis. Topsoil, treated wood, and
industrial carpet were all ineffectively
decontaminated (i.e., having a log reduction
< 6.0), with the highest log reduction
achieved on industrial carpet (4.08) with B.
subtilis.
Based on these initial scoping results, glass
as well as galvanized metal was eliminated
from further testing, and in their place,
AZTD and unpainted concrete were
included in the test matrix. Further
"maximization" testing continued with
increasing C1O2 levels, number of spray
applications, and contact times, to determine
the conditions necessary to maximize
decontamination efficacy on the more
difficult to decontaminate materials. The
results for the tests to increase
decontamination efficacy with aqueous C1O2
are summarized in Table 8-2. In these tests,
the highest decontamination efficacies were
observed on the nonporous decorative
laminate; laminate was the only other
material to be completely decontaminated
under some of the test conditions. The soil
materials (topsoil and AZTD) were the most
difficult materials to decontaminate, with all
log reduction results for AZTD less than 1.5
and all log reduction results for topsoil less
than 0.31. Except for a few tests (including
some outlying results; discussed below), all
of the log reduction results for treated wood,
concrete, and industrial carpet were less than
3.0.
Unexpectedly, the decontamination efficacy
results did not always improve with
increasing concentration, contact time, or
number of spray applications. This effect
was more pronounced on carpet, wood, and
unpainted concrete. A few tests were
repeated (noted in Table 8-2 as "Repeat
tests") with these materials to assess the
effect of increasing the number of spray
applications at a given C1O2 concentration
while eliminating the possibility that
differences in the batch
preparation/chemistry of the aqueous C1O2
solutions were affecting decontamination
efficacy (i.e., causing variability in results).
At the 3,000 ppm level, the decontamination
efficacies for all materials improved
significantly when increasing the number of
47
-------
sprays from two to four. At the 4,000 ppm
level, efficacy improved significantly only
on industrial carpet when increasing the
number of sprays. In general, the results for
the tests that were repeated were more
consistent with the other test results for each
material. The outlying results for industrial
carpet and unpainted concrete may therefore
have been due to unknown experimental
error or some uncontrolled parameter, with
some possible association with the C1O2
solution chemistry.
The most robust liquid spray treatment of
4,000 ppm C1O2, four spray applications,
and two hour contact time did offer
improved decontamination efficacy
compared to some of the other test
conditions (all other variables being equal),
but did not significantly improve efficacy
compared to the 4,000 ppm C1O2, four spray
applications, one hour contact time except
for a few tests.
Comparing Log Reductions for B.
anthracis and B. subtitis
Of the 39 liquid spray tests in which
decontamination efficacies could be
compared between B. anthracis and B.
subtilis (see Tables 8-1 and 8-2), 21 of the
test results were significantly different. For
all of these 21 tests, B. subtilis was
inactivated to a lesser degree.
Material Compatibility
No visible damage was observed on any test
materials for any of the tests conducted.
48
-------
Table 8-1. Summary of Liquid ClOi Decontamination Efficacy for Initial Scoping Tests
,T, ,-..-,., T ,,-,,.,. Efficacy for Efficacy for
Test Material Test Condition „ ., • „ /•,•
B. anthracis B. subtihs
Galvanized Metal
Galvanized Metal
Galvanized Metal
Galvanized Metal
Topsoil0
Glass
Treated Wood
Industrial Carpet
Decorative Laminate
1500 ppm,
2000 ppm,
3000 ppm,
3000 ppm,
3000 ppm,
3000 ppm,
3000 ppm,
3000 ppm,
3000 ppm,
1 hr, 4 apps
1 hr, 4 apps
1 hr, 2 apps
1 hr, 3 apps
1 hr, 3 apps
1 hr, 3 apps
1 hr, 3 apps
1 hr, 3 apps
1 hr, 3 apps
5.99 ±0.99
7.12± 1.50
7.08 ±0.82
>7.89±0.03a
0.24 ±0.08
>7.86±0.07a
2.41 ±0.17
3.40 ±0.73
> 7.84 ± 0.04a
0.76±0.14b
1.91 ± 0.40b
7.13 ±0.74
>7.70±0.07a
0.09 ± 0.05b
>7.88±0.10a
1.43 ± 0.72b
4.08 ±0.36
5.13±1.50b
All efficacy results reported in terms of mean log reduction ± 95% confidence interval Differences in efficacy may be
significant if the 95% CIs of the two efficacy results do not overlap; however, this comparison is not applicable when the two
efficacy results being compared are both reported with log reductions as > some value.
aResult represents complete inactivation within the detection limit of 33.33 CPU/material.
b Values in bold for B. subtilis by testing condition are significantly different from corresponding values for B.
anthracis.
c Tested with unsterilized soil.
49
-------
Table 8-2. Summary of Liquid ClOi Decontamination Efficacy for Maximization Tests
3000 ppm,
one hr contact,
Test two total spray
Material applications
B.a.
Industrial
Carpet
Industrial 0.64
Carpet (±0.18)
(Repeat)
Treated Wood
Treated Wood 1.50
(Repeat) (±0.35)
Decorative
Laminate
Unpainted
Concrete
Unpainted 1.17
Concrete (±°-l4)
(Repeat)
AZ Test Dust
Top Soil
3000 ppm,
one hr contact,
four total spray
applications
B.a. B.s.
7.12 1.27
(±1.60) (±0.16)"
1.97
(±0.29)
2.85 0.67
(±0.38) (±0.35)"
2.27
(±0.22)
7.16 >7.82
(±1.30) (±0.04)a
5.42 5.39
(±2.40) (±1.80)
2.11
(±0.29)
0.12 0.08
(±0.08) (±0.06)
0.05 <0.01
(±0.070) (±0.06)
4000 ppm,
one hr contact,
two total spray
applications
B.a. B.s.
2.82 2.51
(±0.52) (±0.17)
1.12
(±0.13)
1.95 2.55
(±0.49) (±2.0)
1.67
(±0.50)
> 7.76 6.47
(±0.06)a (±0.94)"
6.60 3.64
(±0.84) (±0.20)"
1.62
(±0.19)
0.94 0.13
(±0.11) (±0.06)"
0.05 0.04
(±0.01) (±0.05)
4000 ppm,
one hr contact,
four total spray
applications
B.a. B.s.
1.27 0.730
(±0.09) (±0.05)"
1.77
(±0.38)
1.21 0.93
(±0.12) (±0.29)
2.31
(±0.57)
>7.90 >7.86
(±0.02)a (±0.06)a
2.61 1.52
(±0.63) (±0.36)"
1.77
(±0.39)
1.46 0.22
(±0.08) (±0.14)"
0.170 0.12
(±0.05) (±0.10)
3000 ppm,
two hr contact,
four total spray
applications
B.a. B.s.
2.51 0.80
(±0.20) (±0.10)"
-
2.06 0.97
(±0.39) (±0.39)"
-
6.64 2.88
(±1.0) (±0.75)"
2.22 1.00
(±0.28) (±0.38)"
-
0.66 0.18
(±0.24) (±0.08)"
0.09 0.05
(±0.05) (±0.04)
4000 ppm,
two hr contact,
four total spray
applications
B.a. B.s.
2.84 2.31
(±0.17) (±0.22)"
-
2.64 2.00
(±0.65) (±0.69)
-
7.58 >7.98
(±0.61) (±0.02)a
2.50 1.05
(±1.1) (±0.36)
-
1.28 0.69
(±0.03) (±0.22)"
0.310 0.08
(±0.05) (±0.03)"
All efficacy results reported in terms of mean log reduction ± 95% confidence interval. Differences in efficacy may be significant if the 95% CIs of the two efficacy
results do not overlap; however, this comparison is not applicable when the two efficacy results being compared are both reported with log reductions as > some value.
a Result represents complete inactivation within the detection limit of 33.33 CPU/material.
b Values in bold for B. subtilis (B.s.) by testing condition are significantly different from corresponding values for B. anihracis (B. a.).
"-" Not tested.
50
-------
9.0 References
Evaluation of Liquid and Foam
Technologies for the
Decontamination of B. anthracis and
B. subtilis Spores on Building and
Outdoor Materials. US EPA Report
600/R-09/150, November 2009.
Associates of Cape Cod, Inc.,
Limulus Amebocyte Lysate
CHROMO-LAL Method, Part No.
PN001087, RevOOO, East Falmouth,
Massachusetts, November 2007
http ://www. acciusa. com/pdfs/accPro
duct/pi sheets/Chromo-
LAL%20Insert%20English.pdf
Accessed February 22, 2012.
4500-C1O2 Chlorine Dioxide, E.
Amperometric Method II. In
Standard Methods for the
Examination of Water and Waste
Water, 20th Edition; Clesceri, L.S;
Greenberg, A.E.; Eaton, A.D., Eds.;
American Public Health Association,
American Water Works Association,
Water Environment Federation:
Baltimore, MD, 1995; 4-77 to 4-79.
Determining the Efficacy of Liquids
and Fumigants in Systematic
Decontamination Studies for
Bacillus anthracis Using Multiple
Test Methods. US EPA Report
600/R-10/088, December 2010.
51
-------
Appendix A: Spray Deposition and Neutralization Tests
Spray Deposition Results for Initial Scoping Tests (Refer to Chapter 5)
The C1O2 solution was applied from a distance of 30.5 cm (12 inches) to the horizontally-
oriented galvanized metal until the materials were fully wetted. Re-application of the C1O2 was
made on all coupon surfaces at 30 minutes after the initial application, when the coupon surfaces
were tested with two spray applications; at 20 minutes after the initial application, when the
coupon surfaces were tested with three spray applications; or at 15 minutes after the initial
application, when the coupon surfaces were tested with four spray applications. At 60 minutes
after the initial application, each coupon was placed in the 50 mL conical tube that also served to
collect excess C1O2 runoff. The test coupons stayed in their horizontal orientation throughout the
60-minute contact time.
To assess C1O2 spray deposition, triplicate coupons of the galvanized metal were weighed prior
to application of the C1O2 in the trial runs, and these values were recorded. The triplicate
coupons were then sprayed with C1O2 until fully wetted in their horizontal orientations, re-
application^) were made at the appropriate time(s), and, after the 60 minute contact time, each
coupon was weighed again. The pre-application weights were then subtracted from the post-
application weights, and that difference was added to the weight of decontaminant runoff
captured separately from each coupon. The average deposition/runoff weight for each testing
condition is shown in Table Al-1.
Table A-l. Deposition/runoff Weight of ClOi Liquid with 60 Minute Contact Time
T ,„ .... Average Deposition/Runoff
Test Condition & „, " , x , ,
Weight (g)
3,000 ppm C1O2, 0.38 ±0.10
two applications
3,000 ppm C1O2,
three applications
1,500 ppm C1O2,
four applications
2,000 ppm C1O2,
four applications
0.52 ±0.066
0.61 ±0.13
0.61 ±0.13
Neutralization Methodology for Initial Scoping Tests (Refer to Chapter 5)
Neutralizations for the 3,000 ppm (actual concentrations measured at 2,833 ppm for two
applications and 3,238 ppm for three applications), 1,500 ppm (actual concentration measured at
1,484 ppm), and 2,000 ppm (actual concentration measured at 2,023 ppm) C1O2 solutions for the
52
-------
initial scoping tests were achieved with STS. The concentration range of STS used during the
neutralization panels was 0.5, 1.0, 1.5, and 2.0% (for 3,000 ppm and two total spray applications
only) in the extraction solution. These STS concentrations were based on historical data. The
results of the neutralization panels are shown in Tables A-2 to A-8. From these trials, the
following STS concentrations were determined to be sufficient for neutralization in the following
scoping tests:
• 1.0% STS for Bacillus anthracis spores with 3,000 ppm C1O2 liquid, one hour contact,
two total spray applications. The same STS concentration was used for B. subtilis for these
conditions. Subsequent neutralization panels included B. subtilis.
• 1.0% STS for Bacillus anthracis spores with 3,000 ppm C1O2 liquid, one hour contact,
three total spray applications.
• 0.5% STS for Bacillus subtilis spores with 3,000 ppm C1O2 liquid, one hour contact, three
total spray applications.
• 1.0% STS for Bacillus anthracis spores with 3,000 ppm C1O2 liquid, one hour contact,
three total spray applications.
• 0.5% STS for Bacillus anthracis spores with 1,500 ppm C1O2 liquid, one hour contact,
four total spray applications.
• 1.0% STS for Bacillus subtilis spores with 1,500 ppm C1O2 liquid, one hour contact, four
total spray applications.
• 1.0% STS for Bacillus anthracis and B. subtilis spores with 2,000 ppm C1O2 liquid, one
hour contact, four total spray applications.
53
-------
Table A-2. Neutralization Testing with Bacillus anthracis Spores with 3,000 ppm
Liquid, One Hour Contact, Two Total Spray Applications
Treatment
C1O2 + Spores"
C1O2+
PBS +
C1O2+
C1O2+
C1O2+
C1O2+
PBS +
Triton
PBS +
PBS +
PBS +
PBS +
Triton
X-100
Triton
Triton
Triton
Triton
X-100 +
Sporesa'b
+ Spores (Control)b
X-100 +
X-100 +
X-100 +
X-100 +
0
1
1
2.
.5%
.0%
.5%
.0%
STS +
STS +
STS +
STS +
Sporesa'b
Sporesa'b
Sporesa'b
Sporesa'b
T , Total
Inoculum _, ,
rrFin Observed
(CFU) (CFU)
6
.43 x
6.43 x
6.43 x
6
6
6
6
.43 x
.43 x
.43 x
.43 x
107 0
107 0
107 6.50 xlO7
107 6.98 xlO7
107 7.31 xlO7
107 7.29 xlO7
107 6.49 xlO7
%of
Control
0
0
100
107
113
112
99.9
a C1O2 volume of 0.38 mL corresponds to mean gravimetric deposition on test materials and density of
approximately 1.0 g/mL.
b Volume of PBS (10 mL) includes 0.1% of Triton X-100 surfactant and indicated % of STS; total volume for all
samples with C1O2= 10.38 mL (10 mL PBS/Triton X-100/STS + 0.38 mL C1O2).
Table A-3. Neutralization Testing with Bacillus anthracis Spores with 3,000 ppm
Liquid, One Hour Contact, Three Total Spray Applications
Treatment
C1O2 + Spores3
C1O2+ PBS + Triton X-100 + Sporesa'b
PBS + Triton X-100 + Spores (Control)b
C1O2+ PBS + Triton X-100 + 0.5% STS + Sporesa'b
C1O2+ PBS + Triton X-100 + 1.0% STS + Sporesa'b
C1O2+ PBS + Triton X-100 + 1.5% STS + Sporesa'b
Inoculum
(CFU)
9.63 x 107
9.63 x 107
9.63 x 107
9.63 x 107
9.63 x 107
9.63 x 107
Total
Observed
(CFU)
0
0
9.05 xlO7
8.66 xlO7
8.89xl07
8.77 xlO7
%of
Control
0
0
100
95.8
98.3
96.9
C1O2 volume of 0.52 mL corresponds to mean gravimetric deposition on test materials and density of
approximately 1.0 g/mL.
Volume of PBS (10 mL) includes 0.1% of Triton X-100 surfactant and indicated % of STS; total volume for all
samples with C1O2= 10.52 mL (10 mL PBS/Triton X-100/STS + 0.52 mL C1O2).
54
-------
Table A-4. Neutralization Testing with Bacillus subtilis Spores with 3,000 ppm ClOi
Liquid, One Hour Contact, Three Total Spray Applications
Treatment
C1O2 + Spores"
C1O2+ PBS + Triton X-100 + Sporesa'b
PBS + Triton X-100 + Spores (Control)b
C1O2+ PBS + Triton X-100 + 0.5% STS + Sporesa'b
C1O2+ PBS + Triton X-100 + 1.0% STS + Sporesa'b
C1O2+ PBS + Triton X-100 + 1.5% STS + Sporesa'b
Inoculum
(CFU)
4.07 xlO7
4.07 xlO7
4.07 xlO7
4.07 xlO7
4.07 xlO7
4.07 xlO7
Total
Observed
(CFU)
0
0
8.56 xlO7
8.54 xlO7
8.20 xlO7
7.91 xlO7
%of
Control
0
0
100
99.8
95.8
92.5
a C1O2 volume of 0.52 mL corresponds to mean gravimetric deposition on test materials and density of
approximately 1.0 g/mL.
b Volume of PBS (10 mL) includes 0.1% of Triton X-100 surfactant and indicated % of STS; total volume for all
samples with C1O2= 10.52 mL (10 mL PBS/Triton X-100/STS + 0.52 mL C1O2).
55
-------
Table A-5. Neutralization Testing with Bacillus anthracis Spores with 1,500 ppm ClOi
Liquid, One Hour Contact, Four Total Spray Applications
Treatment
C1O2 + Spores"
C1O2+ PBS + Triton X-100 + Sporesa'b
PBS + Triton X-100 + Spores (Control)b
C1O2+ PBS + Triton X-100 + 0.5% STS + Sporesa'b
C1O2+ PBS + Triton X-100 + 1.0% STS + Sporesa'b
C1O2+ PBS + Triton X-100 + 1.5% STS + Sporesa'b
Inoculum
(CFU)
1.08 xlO8
1.08 xlO8
1.08 xlO8
1.08 xlO8
1.08 xlO8
1.08 xlO8
Total
Observed
(CFU)
0
0
9.88 xlO7
9.87 xlO7
9.37 xlO7
9.60 xlO7
%of
Control
0
0
100
99.9
94.9
97.2
a C1O2 volume of 0.61 mL corresponds to mean gravimetric deposition on test materials and density of
approximately 1.0 g/mL.
b Volume of PBS (10 mL) includes 0.1% of Triton X-100 surfactant and indicated % of STS; total volume for all
samples with C1O2= 10.61 mL (10 mL PBS/TritonX-100/STS + 0.61 mL C1O2).
Table A-6. Neutralization Testing with Bacillus subtilis Spores with 1,500 ppm ClOi
Liquid, One Hour Contact, Four Total Spray Applications
Treatment
C1O2 + Spores"
C1O2+ PBS + Triton X-100 + Sporesa'b
PBS + Triton X-100 + Spores (Control)b
C1O2+ PBS + Triton X-100 + 0.5% STS + Sporesa'b
C1O2+ PBS + Triton X-100 + 1.0% STS + Sporesa'b
C1O2+ PBS + Triton X-100 + 1.5% STS + Sporesa'b
Inoculum
(CFU)
8.60 xlO7
8.60 xlO7
8.60 xlO7
8.60 xlO7
8.60 xlO7
8.60 xlO7
Total
Observed
(CFU)
0
0
7.15 xlO7
7.73 xlO7
8.48 xlO7
8.17xl07
%of
Control
0
0
100
108
119
114
a C1O2 volume of 0.61 mL corresponds to mean gravimetric deposition on test materials and density of
approximately 1.0 g/mL.
b Volume of PBS (10 mL) includes 0.1% of Triton X-100 surfactant and indicated % of STS; total volume for all
samples with C1O2= 10.61 mL (10 mL PBS/TritonX-100/STS + 0.61 mL C1O2).
56
-------
Table A-7. Neutralization Testing with Bacillus anthracis Spores with 2,000 ppm ClOi
Liquid, One Hour Contact, Four Total Spray Applications
Treatment
C1O2 + Spores"
C1O2+ PBS + Triton X-100 + Sporesa'b
PBS + Triton X-100 + Spores (Control)b
C1O2+ PBS + Triton X-100 + 0.5% STS + Sporesa'b
C1O2+ PBS + Triton X-100 + 1.0% STS + Sporesa'b
C1O2+ PBS + Triton X-100 + 1.5% STS + Sporesa'b
Inoculum
(CFU)
1.16xl08
1.16xl08
1.16xl08
1.16xl08
1.16xl08
1.16xl08
Total
Observed
(CFU)
0
0
1.05 xlO8
1.07 xlO8
1.14xl08
1.02 xlO8
%of
Control
0
0
100
102
108
97.5
a C1O2 volume of 0.61 mL corresponds to mean gravimetric deposition on test materials and density of
approximately 1.0 g/mL.
b Volume of PBS (10 mL) includes 0.1% of Triton X-100 surfactant and indicated % of STS; total volume for all
samples with C1O2= 10.61 mL (10 mL PBS/TritonX-100/STS + 0.61 mL C1O2).
Table A-8. Neutralization Testing with Bacillus subtilis Spores with 2,000 ppm ClOi
Liquid, One Hour Contact, Four Total Spray Applications
Treatment
C1O2 + Spores"
C1O2+ PBS + Triton X-100 + Sporesa'b
PBS + Triton X-100 + Spores (Control)b
C1O2+ PBS + Triton X-100 + 0.5% STS + Sporesa'b
C1O2+ PBS + Triton X-100 + 1.0% STS + Sporesa'b
C1O2+ PBS + Triton X-100 + 1.5% STS + Sporesa'b
Inoculum
(CFU)
LlOxlO8
LlOxlO8
LlOxlO8
LlOxlO8
LlOxlO8
LlOxlO8
Total
Observed
(CFU)
0
0
1.16xl08
1.12xl08
1.12xl08
1.12xl08
%of
Control
0
0
100
96.2
96.7
96.4
a C1O2 volume of 0.61 mL corresponds to mean gravimetric deposition on test materials and density of
approximately 1.0 g/mL.
b Volume of PBS (10 mL) includes 0.1% of Triton X-100 surfactant and indicated % of STS; total volume for all
samples with C1O2= 10.61 mL (10 mL PBS/TritonX-100/STS + 0.61 mL C1O2).
57
-------
Liquid Spray Deposition Results for Efficacy Maximization Tests (Refer to Chapter 6)
The C1O2 solution was applied from a distance of 30.5 cm (12 inches) to the horizontally
oriented test material coupons until they appeared fully wetted. The C1O2 solution was re-applied
on all coupon surfaces at the following times for each corresponding testing condition:
• 3,000 ppm C1O2 liquid, one hour contact time, four total spray applications at time 0,
+15, +30, and +45 minutes (contact time ends 15 minutes after the +45 minute
application).
• 4,000 ppm C1O2 liquid, one hour contact time, two total spray applications at time 0 and
+30 minutes (contact time ends 30 minutes after the +30 minute application).
• 4,000 ppm C1O2 liquid, one hour contact time, four total spray applications at time 0,
+15, +30, and +45 minutes (contact time ends 15 minutes after the +45 minute
application).
• 3,000 ppm C1O2 liquid, two hour contact time, four total spray applications at time 0,
+30, +60, and +90 minutes (contact time ends 30 minutes after the +90 minute
application).
• 4,000 ppm C1O2 liquid, two hour contact time, four total spray applications at time 0,
+30, +60, and +90 minutes(contact time ends 30 minutes after the +90 minute
application).
After the required total contact time, each coupon was placed in the 50 mL conical tube that also
served to collect the pooled C1O2 solution. The test coupons stayed in their horizontal orientation
throughout the respective contact times.
To assess C1O2 spray deposition, triplicate coupons of each test material were weighed prior to
application of the C1O2 in the trial runs, and these values were recorded. Then the triplicate
coupons were sprayed with C1O2 solution until fully wetted in their horizontal orientations, re-
application^) was made at the appropriate time(s), and, after the respective contact times (60 or
120 minutes), each coupon was weighed again. The pre-application weights were then subtracted
from the post-application weights, and that difference was added to the weight of decontaminant
runoff captured separately from each coupon. The average deposition/runoff weight of the C1O2
solution from each of the test materials is shown in Table A-9. The total averaged value (0.57 g)
over all six materials was then used to estimate the amount of STS needed to neutralize the C1O2
effectively under this testing condition.
58
-------
Table A-9. Deposition/runoff Weight of ClOi Liquid Spray per Maximization Testing Condition and Material
Average Deposition/Runoff Weight (g)
Test Material
Industrial Carpet
Treated Wood
Decorative Laminate
Unpainted Concrete
Topsoil
Arizona Test Dust
Total Average
3000 ppm,
one hr contact,
four total spray
applications
0.92 ±0.32
0.46 ±0.082
0.77 ±0.18
0.41 ±0.089
0.19 ±0.090
0.65 ± 0.067
0.57 ±0.26
4000 ppm,
one hr contact,
two total spray
applications
0.43 ±0.83
0.29 ±0.062
0.12 ±0.035
0.28 ±0.051
0.73 ±0.18
0.43 ± 0.040
0.38 ±0.21
4000 ppm,
one hr contact,
four total spray
applications
0.56±0.18
0.56 ±0.070
0.21 ±0.035
0.52 ±0.14
0.85 ±0.28
0.84 ±0.28
0.59 ±0.24
3000 ppm,
two hr contact,
four total spray
applications
1.0±0.17
0.46 ± 0.076
0.39 ±0.042
0.68 ±0.15
0.42 ±0.13
0.53 ±0.10
0.58 ±0.23
4000 ppm,
two hr contact,
four total spray
applications"
1.0±0.17
0.46 ±0.076
0.39 ±0.042
0.68 ±0.15
0.42 ±0.13
0.53 ±0.10
0.58 ±0.2 3
aThe same deposition/runoff determined from 3,000 ppm, two hr contact time, four total spray applications used.
59
-------
Neutralization Methodology for Scoping Tests (Refer to Chapter 6)
Neutralization for the 3,000 ppm and 4,000 ppm C1O2 solutions for the liquid spray
maximization tests was achieved with STS. The concentration range of STS used during the
neutralization panels was 0.5, 1.0, and 1.5% in the extraction solution. These STS concentration
ranges were based on historical data. The results of the neutralization panels are shown in Tables
A10 to A19. The results of these trials demonstrated that the following STS concentrations were
sufficient for neutralization for the following maximization tests:
• 1.5% and 0.5% STS for B. anthracis and B. subtilis spores, respectively, with 3,000 ppm
C1O2 liquid, one hour contact, four total spray applications. (Actual C1O2 liquid
concentration measured at 3,238 ppm for both B. anthracis and B. subtilis testing.)
• 0.5% STS for both B. anthracis and B. subtilis spores with 4,000 ppm C1O2 liquid, one
hour contact, two total spray applications. (Actual C1O2 liquid concentration measured at
4,182 ppm for both B. anthracis and B. subtilis testing.)
• 1.5% STS for both B. subtilis and B. subtilis spores with 4,000 ppm C1O2 liquid, one hour
contact, four total spray applications. (Actual C1O2 liquid concentration measured at
4,182 ppm for both B. anthracis and B. subtilis testing.)
• 1.5% and 1.0% STS for B. anthracis and B. subtilis spores, respectively, with 3,000 ppm
C1O2 liquid, two hour contact, four total spray applications. (Actual C1O2 liquid
concentration measured at 3,238 ppm for both B. anthracis and B. subtilis testing.)
• 1.5% and 0.5% STS for B. anthracis and B. subtilis spores, respectively, with 4,000 ppm
C1O2 liquid, two hour contact, four total spray applications. (Actual C1O2 liquid
concentration measured at 4,114 ppm for both B. anthracis and B. subtilis testing.)
60
-------
Table A-10. Neutralization Testing with Bacillus anthracis Spores with 3,000 ppm ClOi
Liquid, One Hour Contact Time, Four Total Spray Applications
Treatment
C1O2 + Spores"
C1O2+ PBS + Triton X-100 + Sporesa'b
PBS + Triton X-100 + Spores (Control)b
C1O2+ PBS + Triton X-100 + 0.5% STS + Sporesa'b
C1O2+ PBS + Triton X-100 + 1.0% STS + Sporesa'b
C1O2+ PBS + Triton X-100 + 1.5% STS + Sporesa'b
Inoculum
(CFU)
1.03 xlO8
1.03 xlO8
1.03 xlO8
1.03 xlO8
1.03 xlO8
1.03 xlO8
Total
Observed
(CFU)
0
0
8.88xl07
9.36 xlO7
9.13 xlO7
8.93 xlO7
%of
Control
0
0
100
105
103
101
a C1O2 volume of 0.57 mL corresponds to mean gravimetric deposition on test materials and density of
approximately 1.0 g/mL.
b Volume of PBS (10 mL) includes 0. 1% of Triton X-100 surfactant and indicated % of STS; total volume for all
samples with C1O2= 10.57 mL (10 mL PBS/Triton X-100/STS + 0.57 mL C1O2).
Table A-ll. Neutralization Testing with Bacillus subtilis Spores with 3,000 ppm
Liquid, One Hour Contact Time, Four Total Spray Applications
Treatment
C1O2 + Spores3
C1O2+
PBS +
C1O2+
C1O2+
C1O2+
PBS +
Triton
PBS +
PBS +
PBS +
Triton
X-100
Triton
Triton
Triton
X-100 +
Sporesa'b
+ Spores (Control)b
X-100 +
X-100 +
X-100 +
0
1
1
.5%
.0%
.5%
STS + Sporesa'b
STS + Sporesa'b
STS + Sporesa'b
T , Total
Inoculum _, ,
rrprn Observed
(CFU) (CFU)
1.11
1.11
1.11
1.11
1.11
1.11
xlO8 0
xlO8 0
xlO8 7.78 xlO7
xlO8 7.68 xlO7
xlO8 7.40 xlO7
xlO8 8.07 xlO7
%of
Control
0
0
100
98.7
95.2
104
C1O2 volume of 0.57 mL corresponds to mean gravimetric deposition on test materials and density of
approximately 1.0 g/mL.
Volume of PBS (10 mL) includes 0.1% of Triton X-100 surfactant and indicated % of STS; total volume for all
samples with C1O2= 10.57 mL (10 mL PBS/Triton X-100/STS + 0.57 mL C1O2).
61
-------
Table A-12. Neutralization Testing with Bacillus anthracis Spores with 4,000 ppm ClOi
Liquid, One Hour Contact Time, Two Total Spray Applications
Treatment
C1O2 + Spores"
C1O2+ PBS + Triton X-100 + Sporesa'b
PBS + Triton X-100 + Spores (Control)b
C1O2+ PBS + Triton X-100 + 0.5% STS + Sporesa'b
C1O2+ PBS + Triton X-100 + 1.0% STS + Sporesa'b
C1O2+ PBS + Triton X-100 + 1.5% STS + Sporesa'b
Inoculum
(CFU)
1.03 xlO8
1.03 xlO8
1.03 xlO8
1.03 xlO8
1.03 xlO8
1.03 xlO8
Total
Observed
(CFU)
0
0
1.04 xlO8
1.15xl08
1.09 xlO8
9.87 xlO7
%of
Control
0
0
100
110
105
95.1
a C1O2 volume of 0.38 mL corresponds to mean gravimetric deposition on test materials and density of
approximately 1.0 g/mL.
b Volume of PBS (10 mL) includes 0. 1% of Triton X-100 surfactant and indicated % of STS; total volume for all
samples with C1O2= 10.38 mL (10 mL PBS/Triton X-100/STS + 0.38 mL C1O2).
Table A-13. Neutralization Testing with Bacillus subtilis Spores with 4,000 ppm
Liquid, One Hour Contact Time, Two Total Spray Applications
Treatment
C1O2 + Spores3
C1O2+ PBS + Triton X-100 + Sporesa'b
PBS + Triton X-100 + Spores (Control)b
C1O2+ PBS + Triton X-100 + 0.5% STS + Sporesa'b
C1O2+ PBS + Triton X-100 + 1.0% STS + Sporesa'b
C1O2+ PBS + Triton X-100 + 1.5% STS + Sporesa'b
Inoculum
(CFU)
9.43 x 107
9.43 x 107
9.43 x 107
9.43 x 107
9.43 x 107
9.43 x 107
Total
Observed
(CFU)
0
0
1.05 xlO8
9.41 xlO7
9.13 xlO7
9.39 xlO7
%of
Control
0
0
100
90.0
87.4
89.8
C1O2 volume of 0.38 mL corresponds to mean gravimetric deposition on test materials and density of
approximately 1.0 g/mL.
Volume of PBS (10 mL) includes 0.1% of Triton X-100 surfactant and indicated % of STS; total volume for all
samples with C1O2= 10.38 mL (10 mL PBS/Triton X-100/STS + 0.38 mL C1O2).
62
-------
Table A-14. Neutralization Testing with Bacillus anthracis Spores with 4,000 ppm ClOi
Liquid, One Hour Contact Time, Four Total Spray Applications
Treatment
C1O2 + Spores"
C1O2+ PBS + Triton X-100 + Sporesa'b
PBS + Triton X-100 + Spores (Control)b
C1O2+ PBS + Triton X-100 + 0.5% STS + Sporesa'b
C1O2+ PBS + Triton X-100 + 1.0% STS + Sporesa'b
C1O2+ PBS + Triton X-100 + 1.5% STS + Sporesa'b
Inoculum
(CFU)
1.32 xlO8
1.32 xlO8
1.32 xlO8
1.32 xlO8
1.32 xlO8
1.32 xlO8
Total
Observed
(CFU)
0
0
1.21 xlO8
1.13xl08
1.23 xlO8
1.25 xlO8
%of
Control
0
0
100
93.1
102
103
a C1O2 volume of 0.59 mL corresponds to mean gravimetric deposition on test materials and density of
approximately 1.0 g/mL.
b Volume of PBS (10 mL) includes 0. 1% of Triton X-100 surfactant and indicated % of STS; total volume for all
samples with C1O2= 10.59 mL (10 mL PBS/Triton X-100/STS + 0.59 mL C1O2).
Table A- 15. Neutralization Testing with Bacillus subtilis Spores with 4,000 ppm
Liquid, One Hour Contact Time, Four Total Spray Applications
Treatment
C1O2 + Spores3
C1O2+ PBS + Triton X-100 + Sporesa'b
PBS + Triton X-100 + Spores (Control)b
C1O2+ PBS + Triton X-100 + 0.5% STS + Sporesa'b
C1O2+ PBS + Triton X-100 + 1.0% STS + Sporesa'b
C1O2+ PBS + Triton X-100 + 1.5% STS + Sporesa'b
Inoculum
(CFU)
1.25 xlO8
1.25 xlO8
1.25 xlO8
1.25 xlO8
1.25 xlO8
1.25 xlO8
Total
Observed
(CFU)
0
0
1.19xl08
1.15xl08
1.05 xlO8
1.18xl08
%of
Control
0
0
100
96.7
88.3
98.5
C1O2 volume of 0.59 mL corresponds to mean gravimetric deposition on test materials and density of
approximately 1.0 g/mL.
Volume of PBS (10 mL) includes 0.1% of Triton X-100 surfactant and indicated % of STS; total volume for all
samples with C1O2= 10.59 mL (10 mL PBS/Triton X-100/STS + 0.59 mL C1O2).
63
-------
Table A-16. Neutralization Testing with Bacillus anthracis Spores with 3,000 ppm ClOi
Liquid, Two Hour Contact Time, Four Total Spray Applications
Treatment
C1O2 + Spores"
C1O2+ PBS + Triton X-100 + Sporesa'b
PBS + Triton X-100 + Spores (Control)b
C1O2+ PBS + Triton X-100 + 0.5% STS + Sporesa'b
C1O2+ PBS + Triton X-100 + 1.0% STS + Sporesa'b
C1O2+ PBS + Triton X-100 + 1.5% STS + Sporesa'b
Inoculum
(CFU)
1.54 xlO8
1.54 xlO8
1.54 xlO8
1.54 xlO8
1.54 xlO8
1.54 xlO8
Total
Observed
(CFU)
0
0
1.16xl08
LlOxlO8
1.14xl08
1.21 xlO8
%of
Control
0
0
100
95.1
98.6
105
a C1O2 volume of 0.59 mL corresponds to mean gravimetric deposition on test materials and density of
approximately 1.0 g/mL.
b Volume of PBS (10 mL) includes 0. 1% of Triton X-100 surfactant and indicated % of STS; total volume for all
samples with C1O2= 10.59 mL (10 mL PBS/Triton X-100/STS + 0.59 mL C1O2).
Table A- 17. Neutralization Testing with Bacillus subtilis Spores with 3,000 ppm
Liquid, Two Hour Contact Time, Four Total Spray Applications
Treatment
C1O2 + Spores3
C1O2+ PBS + Triton X-100 + Sporesa'b
PBS + Triton X-100 + Spores (Control)b
C1O2+ PBS + Triton X-100 + 0.5% STS + Sporesa'b
C1O2+ PBS + Triton X-100 + 1.0% STS + Sporesa'b
C1O2+ PBS + Triton X-100 + 1.5% STS + Sporesa'b
Inoculum
(CFU)
1.55 xlO8
1.55 xlO8
1.55 xlO8
1.55 xlO8
1.55 xlO8
1.55 xlO8
Total
Observed
(CFU)
0
0
1.16xl08
1.12xl08
1.19xl08
1.15xl08
%of
Control
0
0
100
96.8
103
99.5
C1O2 volume of 0.59 mL corresponds to mean gravimetric deposition on test materials and density of
approximately 1.0 g/mL.
Volume of PBS (10 mL) includes 0.1% of Triton X-100 surfactant and indicated % of STS; total volume for all
samples with C1O2= 10.59 mL (10 mL PBS/Triton X-100/STS + 0.59 mL C1O2).
64
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Table A-18. Neutralization Testing with Bacillus anthracis Spores with 4,000 ppm ClOi
Liquid, Two Hour Contact Time, Four Total Spray Applications
Treatment
C1O2 + Spores"
C1O2+ PBS + Triton X-100 + Spores*
PBS + Triton X-100 + Spores (Control)b
C1O2+ PBS + Triton X-100 + 0.5% STS + Spores*
C1O2+ PBS + Triton X-100 + 1.0% STS + Spores*
C1O2+ PBS + Triton X-100 + 1.5% STS + Spores*
Inoculum
(CFU)
1.50 xlO8
1.50 xlO8
1.50 xlO8
1.50 xlO8
1.50 xlO8
1.50 xlO8
Total
Observed
(CFU)
0
0
1.12xl08
1.09 xlO8
1.09 xlO8
1.16xl08
%of
Control
0
0
100
97.1
97.1
103
a C1O2 volume of 0.59 mL corresponds to mean gravimetric deposition on test materials and density of
approximately 1.0 g/mL.
b Volume of PBS (10 mL) includes 0. 1% of Triton X-100 surfactant and indicated % of STS; total volume for all
samples with C1O2= 10.59 mL (10 mL PBS/Triton X-100/STS + 0.59 mL C1O2).
Table A- 19. Neutralization Testing with Bacillus subtilis Spores with 4,000 ppm
Liquid, Two Hour Contact Time, Four Total Spray Applications
Treatment
C1O2 + Spores3
C1O2+ PBS + Triton X-100 + Sporesa'b
PBS + Triton X-100 + Spores (Control)b
C1O2+ PBS + Triton X-100 + 0.5% STS + Sporesa'b
C1O2+ PBS + Triton X-100 + 1.0% STS + Sporesa'b
C1O2+ PBS + Triton X-100 + 1.5% STS + Sporesa'b
Inoculum
(CFU)
1.56 xlO8
1.56 xlO8
1.56 xlO8
1.56 xlO8
1.56 xlO8
1.56 xlO8
Total
Observed
(CFU)
0
0
1.25 xlO8
1.30 xlO8
1.26 xlO8
1.23 xlO8
%of
Control
0
0
100
104
101
98.6
C1O2 volume of 0.59 mL corresponds to mean gravimetric deposition on test materials and density of
approximately 1.0 g/mL.
Volume of PBS (10 mL) includes 0.1% of Triton X-100 surfactant and indicated % of STS; total volume for all
samples with C1O2= 10.59 mL (10 mL PBS/Triton X-100/STS + 0.59 mL C1O2).
65
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Appendix B: Effect of Pre-Sterilization of Soil Materials on the Decontamination Efficacy
for Aqueous ClOi Spray
We planned initially to test the topsoil and AZTD in an unsterilized state, to maintain the
physical and chemical integrity of the soil materials. However, due to concerns that endogenous
flora in the unsterilized topsoil and AZTD could affect our test results, we decided to sterilize the
soil materials using gamma irradiation. We did find that sterilizing these soils via gamma
irradiation did not change their physical appearance or integrity from an observational standpoint
(e.g., no change in color).
To examine the effect that sterilizing the soil materials would have on decontamination efficacy,
the following tests were conducted using sterilized and unsterilized topsoil and AZTD materials:
• 3,000 ppm C1O2 liquid spray, 60 minute contact time, four total spray applications, B.
anthracis and B. subtilis. (Actual C1O2 liquid concentration measured at 3,103 ppm for
both B. anthracis and B. subtilis testing using unsterilized soils. Actual C1O2 liquid
concentration measured at 3,238 ppm for both B. anthracis and B. subtilis testing using
sterilized soils.)
• 4,000 ppm C1O2 liquid spray, 60 minute contact time, two total spray applications, B.
anthracis and B. subtilis. (Actual C1O2 liquid concentration measured at 4,047 ppm for
both B. anthracis and B. subtilis testing using unsterilized soils. Actual C1O2 liquid
concentration measured at 4,181 ppm for both B. anthracis and B. subtilis testing using
sterilized soils.)
The results between the sterilized and unsterilized soil are compared in Table B-l. Although
sterilization of soil materials does make a significant difference in the majority of results (based
on whether the 95% confidence intervals for the log reduction results overlapped), the
decontamination efficacy for these materials remains dismal. Only one out of the 16 tests showed
an average log reduction greater than 1.0.
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Table B-l. Comparison of Decontamination Efficacy with 95% Confidence Intervals using
Unsterile vs. Sterile Soils for ClOi Liquid Spray Testing
Testing Condition,
Soil Type
B. anthracis Efficacy
(Log Reduction ± CI)
B. subtilis Efficacy
(Log Reduction ± CI)
3,000 ppm C1O2, one hr,
four total spray
applications
Sterilized AZTD
Non-Sterile AZTD
Sterilized Topsoil
Non-Sterile Topsoil
0.120±0.080a
1.22±0.040a
0.0500 ± 0.070
0.160 ±0.17
0.0800 ± 0.060b
0.680 ±0.11b
<0.0100C
0.150 ±0.13C
4,000 ppm C1O2, one hr,
two total spray
applications
Sterilized AZTD
Non-Sterile AZTD
Sterilized Topsoil
Non-Sterile Topsoil
0.940 ±0.11a
0.300 ±0.11a
0.0500 ±0.010
0.0800 ±0.15
0.130 ±0.060b
0.960 ±0.21b
0.0400 ± 0.050
0.130 ±0.10
aResults significantly different for B. anthracis between sterilized and unsterilized AZTD.
Results significantly different for B. subtilis between sterilized and unsterilized AZTD.
"Results significantly different for B. subtilis between sterilized and unsterilized Topsoil
67
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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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