EPA 600/R-12/517 | May 2012 | www.epa.gov/ord
United States
Environmental Protection
Agency
Inactivation of Bacillus
anthracis Spores in Soil
Matrices with Chlorine
Dioxide Gas
Off ce of Research and Development
National Homeland Security Research Center
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EPA/600/R-12/517
May 2012
Inactivation of Bacillus
anthracis Spores in Soil
Matrices with Chlorine Dioxide
Gas
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 laboratory study
designed to better understand the effectiveness of chlorine dioxide (C1O2) gas to decontaminate
soil 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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Contents
Foreword iv
Acknowledgments v
Tables viii
Figures viii
Abbreviations/Acronyms ix
Executive Summary xi
1.0 Introduction 1
2.0 C1O2 Gas Generation and Test Matrix 2
2.1 C1O2 Gas Generation 2
2.2 Test Matrix for Soil Fumigation 2
3.0 Summary of Test Procedures 4
3.1 Preparation of Soil Coupons 4
3.2 C1O2 Fumigation of Soil Materials 7
3.3 Decontamination Efficacy 7
3.4 C1O2 Concentration, Temperature, and RH Measurement 9
4.0 Quality Assurance/Quality Control 11
4.1 Equipment Calibration 11
4.2 QC Results 11
4.3 Audits 11
4.3.1 Performance Evaluation Audit 11
4.3.2 Technical Systems Audit 12
4.3.3 Data Quality Audit 12
4.4 Quality Assurance Project Plan Amendments and Deviations 13
4.5 QA/QC Reporting 13
4.6 Data Review 13
5.0 Fumigation of Unsterilized Soils at 1 cm Depth 14
5.1 Fumigation Conditions 14
5.2 Decontamination Efficacy 14
6.0 Unsterilized Soils and Endogenous Flora 22
6.1 Background 22
6.2 Soil Recovery Tests 22
7.0 Fumigant Testing with Sterilized Soils at 1 cm Depth, with B. anthracis 26
7.1 Fumigation Conditions 26
7.2 Decontamination Efficacy 26
7.3 Comparison of Efficacy Results for B. anthracis on Non-Sterile and
Sterile Soils 26
VI
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8.0 Fumigant Testing with Sterilized Soils at 2 cm Depth 31
8.1 Fumigation Conditions 31
8.2 Decontamination Results 31
9.0 Summary of Results 37
10.0 References 40
Appendix A 41
vn
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Tables
Table 2-1. Test Matrix 3
Table 3-1. Summary of Soil Materials used for Decontaminant Testing 6
Table 4-1. Performance Standards for Amperometric Titration 12
Table 4-2. Performance Evaluation Audits 12
Table 5-1. Inactivation of Bacillus anthracis Spores at 75% RH on 1 cm Unsterilized Soils 16
Table 5-2. Inactivation of Bacillus subtilis Spores at 75% RH on 1 cm Unsterilized Soils 17
Table 5-3. Inactivation of Bacillus anthracis Spores at 85% RH on 1 cm Unsterilized Soils 18
Table 5-4. Inactivation of Bacillus subtilis Spores at 85% RH on 1 cm Unsterilized Soils 19
Table 5-5. Summary of Efficacy Values for 3,000 ppmv C1O2 Gas on 1cm Unsterilized Soils 20
Table 6-1. Evaluation of Spore Recovery for Unsterilized and Sterilized Topsoil 24
Table 6-2. Evaluation of Spore Recovery for Unsterilized Arizona Test Dust 25
Table 7-1. Inactivation of Bacillus anthracis Spores at 75% RH on 1 cm Sterilized Soils 27
Table 7-2. Inactivation of Bacillus anthracis Spores at 85% RH on 1 cm Sterilized Soils 28
Table 7-3. Summary of Efficacy Values for 3,000 ppmv C1O2 Gas on 1 cm Sterilized Soils for B.
anthracis 29
Table 8-1. Inactivation of Bacillus anthracis Spores on 2 cm Sterilized Soils at 75% RH 32
Table 8-2. Inactivation of Bacillus subtilis Spores on 2 cm Sterilized Soils at 75% RH 33
Table 8-3. Inactivation of Bacillus anthracis Spores on 2 cm Sterilized Soils at 85% RH 34
Table 8-4. Inactivation of Bacillus subtilis Spores on 2 cm Sterilized Soils at 85% RH 35
Table 8-5. Summary of Decontamination Efficacy Values for 3,000 ppmv C1O2 Gas on 2 cm
Sterilized Soils 36
Table 9-1. Summary of Soil Fumigation Results 39
Figures
Figure 3-1. Topsoil "coupons" made with Parafilm®-lined Petri dishes 6
Figure 5-1. Summary of decontamination efficacies for 3,000 ppmv C1O2 fumigant testing on
1 cm unsterilized AZTD and topsoil 21
Figure 7-1. Comparison of decontamination efficacies for unsterilized vs. sterilized soils with
B. anthracis 30
Vlll
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Abbreviations/Acronyms
7 gamma
ATCC American Type Culture Collection
atm atmosphere
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
CBR chemical, biological, and/or radiological
CPU colony-forming unit(s)
CGB compact glovebox
CI confidence interval
C1O2 chlorine dioxide
cm centimeter(s)
EPA U.S. Environmental Protection Agency
g gram(s)
HDPE high density polyethylene
HC1 hydrochloric acid
hr hour(s)
HS homeland security
L liter(s)
LAL Limulus Amebocyte Lysate
M Molarity
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
PTFE polytetrafluoroethlyene
QA quality assurance
QAPP Quality Assurance Proj ect Plan
QC quality control
RH relative humidity
IX
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rpm revolutions per minute
SD standard deviation
SE standard error
SFW sterile filtered water (cell-culture grade)
STS sodium thiosulfate
TSA technical systems audit(s)
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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 chlorine dioxide
(C1O2) gas in inactivating Bacillus anthracis
(causative agent for anthrax) spores in two
types of soil material. The objective of this
study was to provide an understanding of the
performance of the C1O2 gas
decontamination technology to guide its use
and implementation in HS applications,
particularly for hard-to-decontaminate
materials such as soil. 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 two types of soil
material (topsoil and Arizona Test Dust
[AZTD]). 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. Tests were conducted
with varying relative humidity (RH) levels
and contact times to assess the effect of
these fumigation operational parameters on
decontamination efficacy. In addition, tests
were conducted at two different soil depths
to assess the impact of soil cover on the
spore inactivation efficacy.
Summary of Results
The C1O2 gas decontamination technology
provided complete inactivation of B.
anthracis spores on the AZTD samples for
nearly all tests conducted with this soil type.
In all tests with the AZTD, over a six log
reduction was achieved. In contrast, topsoil
proved to be more difficult to
decontaminate. Although B. anthracis spore
log reductions greater than six were
achieved for all tests conducted using topsoil
at a depth of 1 cm, a maximum log reduction
of only 3.7 was achieved with topsoil at a
depth of 2 cm. Moreover, none of the
topsoil samples were completely
decontaminated in any of the tests conducted
with this material.
With regard to comparing the
decontamination efficacies of B. anthracis
and B. subtilis, all but four test results for
these two microorganisms were statistically
equivalent. Among the four tests whose
results were statistically different, B. subtilis
was inactivated to a lesser degree in two of
the tests.
XI
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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 C1O2 gas to decontaminate
two types of soil materials contaminated
with B. anthracis spores. This report
documents the impact of various factors on
the efficacy of C1O2 fumigation against
spores of B. anthracis and B. subtilis in soil
matrices, the latter organism included to
assess its potential as a surrogate for future
studies related to B. anthracis. Specifically,
tests were conducted to determine the effect
of soil type, soil depth, RH, and contact time
on the inactivation efficacy of B. anthracis
(or B. subtilis) spores.
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2.0 C1O2 Gas Generation and Test Matrix
2.1 C1O2 Gas Generation
Since ClC^is unstable as a compressed gas,
it must be produced on site. Aqueous
solutions of C1O2 were first prepared
(preparation of the aqueous solutions of
C1O2 is described in a previous report1), and
then pumped into a sparging column to
transfer the C1O2 from the liquid to gas
phase. Air from the test chamber was used
in the sparging process and recirculated to
establish the desired gaseous C1O2
concentration of 3,000 ppmv. As the C1O2
concentration in the test chamber dropped,
additional gas from the generator was added
to the test chamber.
2.2 Test Matrix for Soil Fumigation
The soil fumigation tests performed with
C1O2 gas are shown in Table 2-1. All tests
were conducted at ambient temperature (22
°C ± 2 °C) with a target C1O2 concentration
of 3,000 ppmv. The test matrix included
soil type, organism, RH, contact time, and
depth of soil as the experimental variables.
Soil referenced in this report as "1 cm
depth" means that a 1 cm layer soil was
placed in the dish and inoculated. Soil
referenced as "2 cm depth" means that a 1
cm layer of soil was placed in the dish,
inoculated with spores, and allowed to dry
for 1 hour, followed by the addition of
another 1 cm layer of soil on top of the
inoculated layer, forming a "sandwich" of
the inoculum.
Initial tests with B. anthracis and B. subtilis
spores, at 75% and 85% RH and 1 cm depth,
were conducted using unsterilized soils;
refer to Chapter 5. But due to concerns that
the presence of endogenous flora in the
unsterilized soils could bias analysis (refer
to Chapter 6, which provides data indicating
that recovery of inoculated spores is affected
by whether the soil is pre-sterilized or not),
this test condition (75% and 85% RH, 1 cm
depth) was repeated for B. anthracis using
sterilized soils; refer to Chapter 7. All
subsequent tests (i.e., all tests with soils at 2
cm depth) were conducted using sterilized
soils; refer to Chapter 8.
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Table 2-1. Test Matrix
Biological
Agent
B. anthracis"
or
Rsubtitt^
B. anthracis"
or
Rsubtiltf
B. anthracis"
or
B. subtilisb
B. anthracis"
or
Rsubtiltf
B. anthracis"
or
Rsubtitt^
B. anthracis"
or
B. subtilisb
B. anthracis0
or
R_subtiM_
B. anthracis0
or
Rsubtiti£
B. anthracis0
or
B. subtilis0
B. anthracis0
or
Rsubtiti£
B. anthracis0
or
R_subtiM_
B. anthracis0
or
B. subtilis0
Materials
(depth)
Topsoil
AZ test dust
(1cm)
Topsoil
AZ test dust
(1cm)
Topsoil
AZ test dust
(1cm)
Topsoil
AZ test dust
(1cm)
Topsoil
AZ test dust
(1cm)
Topsoil
AZ test dust
(1cm)
Topsoil
AZ test dust
(2cm)
Topsoil
AZ test dust
(2cm)
Topsoil
AZ test dust
(2cm)
Topsoil
AZ test dust
(2cm)
Topsoil
AZ test dust
(2cm)
Topsoil
AZ test dust
(2cm)
Target C1O2 ^ , 0/ ~, , , T.
„ , ,. larget % Contact lime
Concentration ™TT ,. x
, , RH (hr)
(ppmv)
3,000 ±300 75 ±7.5
3,000 ±300 75 ±7.5
3,000 ±300 75 ±7.5
3,000 ±300 85 ±8.5
3,000 ±300 85 ±8.5
3,000 ±300 85 ±8.5
3,000 ±300 75 ±7.5
3,000 ±300 75 ±7.5
3,000 ±300 75 ±7.5
3,000 ±300 85 ±8.5
3,000 ±300 85 ±8.5
3,000 ±300 85 ±8.5
2
3
4
2
3
4
2
3
4
2
3
4
a Tested on both unsterilized and sterilized soil materials.
b Tested only on unsterilized soil materials.
c Tested on only sterilized soil materials.
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3.0 Summary of Test Procedures
Test procedures were performed in
accordance with a pre-approved Quality
Assurance Project Plan2 (QAPP) and are
summarized in this chapter.
3.1 Preparation of Soil 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)3 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
sterile filtered water (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.
Spores of B. anthracis or B. subtilis were
inoculated onto the soil samples in an
appropriate biosafety cabinet Level III (BSC
III) according to established BBRC
procedures. The soils were inoculated one
day prior to the day of experimental work,
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. After inoculation, the
soil "coupons" remained undisturbed
overnight in the BSC III to dry thoroughly.
Test coupons were then exposed to the C1O2
fumigant the next day (i.e., within 24 hours
after inoculation).
The topsoil and AZTD materials were
placed unpacked in a Parafilm®-lined, 3.5
cm diameter x 1.0 cm tall Petri dish (for the
1 cm coupons) and in Petri dishes 4.0 cm
diameter by 5.0 cm deep for the 2 cm soil
coupons. Refer to Figure 3-1. The Petri
dishes were lined with Parafilm® to enable
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.
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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. The origin and specifications of
the soil materials used for testing are shown
in Table 3-1.
In the initial tests with topsoil and AZTD
(Chapter 5), the soil materials were 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 7-
irradiation. (Refer to Chapters 5 and 6 for
further details.) The y -irradiation
sterilization method was chosen for the soil
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 soils were sent
to be Y -irradiated at approximately 40
kilogray by a vendor that specializes in this
type of processing (STERIS Isomedix
Services, Libertyville, IL).
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Figure 3-1. Topsoil "coupons" made with Parafilm"-lined Petri dishes.
Table 3-1. Summary of Soil Materials used for Decontaminant Testing
Material
Topsoil
Arizona Test Dust
Lot/Batch/
Observation
PY1A0597
ISO 121030-1
Manufacturer/
Supplier Name
GardenScape, Inc.;
Eau Claire, PA
Powder Technology, Inc.;
Burnsville, MN
Coupon Size,
Width x Length
3.5 cm diameter x 1.0
and 2.0 cm tall
(unpacked)
3.5 cm diameter x 1.0
and 2.0 cm tall
(unpacked)
Material
Preparation
Non-sterile,
y - irradiation
Non-sterile,
Y - irradiation
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3.2 C1O2 Fumigation of Soil Materials
Five replicate test coupons (soils inoculated
with either B. anthracis or B. subtilis spores
and exposed to C1O2 gas), five replicate
positive control coupons (inoculated and not
decontaminated), one procedural blank (not
inoculated, decontaminated), and one
laboratory blank (not inoculated, not
decontaminated) of each soil type were used
in testing with each testing condition.
On the day following inoculation, the soils
intended for decontamination (including
blanks) were separated from the positive
controls or coupons not exposed to
decontaminant (including blanks) because
both sets were inoculated and dried
overnight in the same BSC III. The humidity
(75% ± 10% or 85% ± 10% 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
CGB (Compact Glove Box, Plas-Labs
Model No. 830-ABC, Lansing, MI) that had
RH levels similar to those of the glove box
where fumigation occurred.
After the fumigation, the soils were
extracted by transferring them from the Petri
dishes to the extraction tubes and agitated on
an orbital shaker for 15 minutes at
approximately 200 rpm at room temperature.
Following extraction, a 1 mL aliquot of the
unsterilized soil extract was removed
followed by incubation in a water bath for
one hour at 55 to 60 °C to inactivate the
endogenous flora that may be susceptible to
heat shock. (Despite the heat shock step for
the unsterilized soils, there were instances of
growth of endogenous flora on the plates.)
This heat shock step was not used for
sterilized soils.
•v-7
A series of dilutions up through 10" was
then prepared in SFW. An aliquot (0.1 mL)
of the undiluted extract and/or each serial
dilution was then spread-plated onto tryptic
soy agar (TSA) 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.
After each decontamination test, the BSC III
and the compact glove box (CGB) were
thoroughly cleaned (using separate steps
involving bleach, ethanol, water, then
drying) following procedures established
under the BBRC Facility Safety Plan.
Laboratory blanks controlled for sterility
and procedural blanks controlled for viable
spores inadvertently introduced to test
coupons. The blanks were spiked with an
equivalent amount of 0.1 mL of "stock
suspension" that did not contain the
biological agent. The target acceptance
criterion was that extracts of laboratory or
procedural blanks were to contain no CFU.
3.3 Decontamination Efficacy
The mean percent spore recovery from each
soil coupon was calculated using results
from positive control coupons (spiked, not
decontaminated), 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
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control coupons of a single material, and
CFUspike is the number of CPU 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 soil coupon, and the results are
included in Chapters 5 through 8.
The performance or efficacy of the C1O2
fumigation was assessed by determining the
number of viable organisms remaining on
each soil test coupon 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 logio
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 CPU 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 /'th coupon
material was calculated as the difference
between those mean log values, i.e.:
Efficacy = (loglo CFUc, ) - (loglo CFUt, )
(2)
where logio CFUcy refers to they individual
logarithm values obtained from the positive
control coupons and logio 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
positive controls and five corresponding test
coupons (i.e.,7 = 5) for each soil coupon. A
decontaminant which achieves a 6 log
reduction or greater is considered effective.4
In the case where no viable spores were
found in any of the five test coupon extracts
after decontamination, a CPU abundance of
1 was assigned, resulting in a logio CPU 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.
The variances (i.e., the square of the
standard deviation) of the logio CFUctj and
logio CFUtjj values were also calculated for
both the control and test coupons (i.e., S Cy
and S2tjj), and were used to calculate the
pooled standard error (SE) for the efficacy
value calculated in Equation 2, as follows:
SE =
(3)
where the number 5 again represents the
number y of coupons in both the control and
test data sets. Each efficacy result is reported
as a log reduction value with an associated
95% confidence interval (CI), calculated as
follows:
95% CI = Efficacy ± (1.96 x SE)
(4)
The significance of differences in efficacy
across different test conditions and spore
types was assessed based on the 95%
confidence interval of each efficacy result.
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 C1O2 Concentration, Temperature, and RH measurement
The concentration of C1O2 in the test chamber was measured during each experiment
approximately every 20 minutes. Air from the test chamber was drawn through impingers (at a
rate of 1 L/min for two minutes using an air mass flow controller) that contained 15 mL of 5%
potassium iodide in phosphate buffer (pH 7.0) solution.
The concentrations of C1O2 in the impinger solutions were then measured using a modified
titration method based on the Standard Method 4500-C1O2 E Amperometric Method II5. While
the gas is bubbled through the impinger, C1O2 is captured in the liquid and 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 C1O2 gas
concentration. Using the formula below, the gas phase concentration of C1O2 was calculated as
follows:
C102(ppmv)= 1V 7 x-x 24.45x1000
V2(L) 5
where:
C1O2 = chlorine dioxide (parts per million by volume [ppmv] in air)
Vi = volume of STS titrant (mL)
M = molarity (mol/L) of STS titrant (which for STS is equal to its normality)
¥2 = volume of air (at 25 degrees Celsius [°C], 1 atmosphere [atm]) that passed through
impinger (L)
24.45 = ideal gas constant, L/mol, at 25 °C, 1 atm
1000 = conversion factor = 106 ppmv x 1 L / 1000 mL
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
C1O2 testing to verify accuracy. The pH of the aqueous C1O2 solutions was measured with a
calibrated pH meter (Thermo Scientific, Waltham, MA).
Temperature and RH were measured with a Fisherbrand Traceable Radio-Signal
Hygrometer/Thermometer, Cat. No. 14-648-52 (Thermo Scientific, Waltham, MA). Relative
humidity levels were controlled during C1O2 fumigant testing to achieve 75% or 85% RH. A
nebulizer filled with sterile water was used to pre-condition the glove box used for
decontamination prior to injection of any C1O2 fumigant. Impinger sampling of the glove box
used for decontamination sometimes caused the RH levels to decrease as air was drawn out. The
-------
nebulizer was then turned on to increase RH. As a result of the nebulization, sometimes the C1O2
levels dropped, resulting in a slight injection of C1O2 fumigant. If the RH got too high (i.e.,
beyond the allowable tolerance), a damper was opened to allow the excess humidity to be
purged. Purging of excess humidity impacted C1O2 fumigant levels, too, so another slight
injection of C1O2 fumigant followed. The positive control glove box had the same type of
nebulization system to increase RH levels. If the RH levels got too high in this glove box, an air
drying system was used (i.e., moist air passed through a column of desiccant).
The average actual C1O2 concentration, temperature, and RH for each test condition are reported
in each results chapter and summarized in Appendix A.
10
-------
4.0 Quality Assurance/Quality Control
Quality assurance/quality control (QA/QC)
procedures were performed in accordance
with the QAPP2 (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).
In testing of the 3,000 ppmv C1O2, two-,
three-, and four-hour contact times, at both
75% and 85% RH, using 1 cm unsterilized
soils, all procedural and laboratory blanks
failed to meet the criterion of no observed
CPU due to the presence of endogenous
flora; refer to Chapters 5 and 6 for additional
details on how this may affect results. In the
remaining tests in which sterilized soils were
used (refer to Chapters 7 and 8), all
procedural and laboratory blanks met the
criterion of no observed CPU.
All positive control results were within the
target recovery range of 1 to 150% of the
spiked spores, except as follows: In the
repeat testing of 1 cm sterilized soils with B.
anthracis only (see chapter 7), all positive
control results were within the target
recovery range of 1 to 150% of the spiked
spores, except for the test with topsoil at the
two-hour contact time and 85% RH, which
had a recovery of 162%. Although this one
test did not meet the QA target criterion on
the basis of average recovery, it was within
the target recovery range based on the 95%
confidence interval.
Spike control samples were taken from the
spore suspension on each day of testing and
serially diluted, nutrient-plated and counted
to establish the spore density used to spike
the coupons. All the spore density levels met
the QA target criterion of 1 x 109 CFU/mL
(± 25%) for all tests.
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.
11
-------
Table 4-1. Performance Standards for Amperometric Titration
Sodium Chlorite
Stock (mg/L)
Measured Chlorite
(mg/L)
1,000 ± 100
3,000 ±300
1,045
3,203
4^113
Temperatures were monitored but no efforts were undertaken to control any of the test
temperatures. All tests were conducted under ambient temperature conditions.
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
n/r ^ Audit
Measurement „ ,
Procedure
Volume of liquid „ , ..
c . . Gravimetric evaluation
from micropipettes
Chlorite Amperometric titration
„ Compared to independent
Temperature ,.£ . , ., r .
calibrated thermometer
„ , „. TT .,., Compared to independent
Relative Humidity ,5, , ,
J calibrated hygrometer
„. Compare time to independent
Tlme 11 4. U V
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 lead 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 contractor 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.
12
-------
4.4 Quality Assurance Project Plan
Amendments and Deviations
One deviation was prepared, approved, and
retained in the test files for this study. The
deviation related to the initiation of testing
as described in the two QAPP amendments
without fully-signed/approved amendments
in place. No adverse impacts were
expected.
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 follow-up
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. 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.
13
-------
5.0 Fumigation of Unsterilized Soils at 1 cm Depth
5.1 Fumigation Conditions
The initial C1O2 fumigation tests were
conducted using 1 cm of unsterilized soil
materials. The decontamination efficacy of
3,000 ppmv C1O2, two-, three-, and four-
hour contact times, at 75% RH was
evaluated for B. anthracis (Ames) and B.
subtilis on two soil types at 1 cm depth.
Measurements of C1O2 inside the testing
chamber were made approximately every 10
to 20 minutes for the duration of testing to
monitor and adjust the C1O2 concentration.
For B. anthracis testing, the average
concentration of C1O2 was measured at
2,805 ppmv ± 399.6 ppmv, the average RH
was measured at 73% ± 2%, and the average
temperature was measured at 23.7 °C ± 0.5
°C. For B. subtilis testing, the average
concentration of C1O2 was measured at
3,028 ppmv ± 240.6 ppmv, the average RH
was measured at 69% ± 2%, and the average
temperature was measured at 23.7 °C ± 0.6
The decontamination efficacy of 3,000
ppmv C1O2, two-, three-, and four-hour
contact times, at 85% RH was evaluated for
B. anthracis (Ames) and B. subtilis on two
soil types. Measurements of C1O2 inside the
testing chamber were made approximately
every 10 to 20 minutes for the duration of
testing to monitor and adjust the C1O2
concentration. For B. anthracis testing, the
average concentration of C1O2 was
measured at 3,019 ppmv ±233.7 ppmv, the
average RH was measured at 87% ± 1%,
and the average temperature was measured
at 24.3 °C ± 0.7 °C. For B. subtilis testing,
the average concentration of C1O2 was
measured at 3,086 ppmv ± 230.8 ppmv, the
average RH was measured at 87% ± 2%,
and the average temperature was measured
at24.7°C±0.8°C.
Fumigation conditions are tabulated in
Appendix A.
5.2 Decontamination Efficacy
The detailed decontamination efficacy
results are shown in Tables 5-1 through 5-4
and are summarized in Table 5-5 and Figure
5-1. All laboratory and procedural blanks
showed the presence of endogenous flora
due to the use of unsterilized soil matrices.
Due to the concern that endogenous flora
may impact the recovery of the target
organisms, tests were conducted to examine
this possibility; please refer to Chapter 6.
The AZTD was effectively decontaminated
(log reduction greater than 6.00) in all 12
tests conducted (both microorganisms, both
RH levels, and the three contact times). In
seven of the 12 tests with AZTD, all spores
were completely inactivated (i.e., no viable
spores were recovered). The topsoil was
effectively decontaminated in five of the 12
tests conducted, although the complete
inactivation of all spores did not occur in
any of topsoil tests.
The effect of increasing RH or contact time
was judged to be insignificant in nearly all
tests; the exception being the B. anthracis
topsoil tests at 85% RH, which showed
significantly improved decontamination
efficacy with increasing contact time.
Of the 12 fumigation tests conducted with 1
cm unsterilized soils, there were three tests
in which the log reduction results for B.
anthracis and B. subtilis were significantly
different. On topsoil at the 75% RH, 2-hour
14
-------
test condition; and on AZTD at the 85% RH, condition, B. anthracis was inactivated to a
2-hour test condition, B. subtilis was significantly higher degree.
inactivated to a significantly higher degree.
On topsoil at the 85% RH, 3-hour test
15
-------
Table 5-1. Inactivation of Bacillus anthracis Spores at 75% RH on 1 cm Unsterilized Soils"
Test Material
(Contact Time)
Topsoil (Two Hours)
Positive Controls'3
Test Coupons0
Laboratory Blankd
Procedural Blank6
Topsoil (Three Hours)
Positive Controls
Test Coupons
Laboratory Blank
Procedural Blank
Topsoil (Four Hours)
Positive Controls
Test Coupons
Laboratory Blank
Procedural Blank
AZ Test Dust
(Two Hours)
Positive Controls
Test Coupons
Laboratory Blank
Procedural Blank
AZ Test Dust
(Three Hours)
Positive Controls
Test Coupons
Laboratory Blank
Procedural Blank
AZ Test Dust
(Four Hours)
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
1.16xl08
1.16xl08
0
0
Mean of Logs of
Observed CFU
±CIg
7.79±0.33f
2.73 ± 1.2f
Of
Of
7.90 ± 0.090f
2.08±1.2f
Of
Of
8.01±0.050f
3.10±1.3f
Of
Of
7.86±0.13f
0.730 ±1.0f
Of
Of
7.67±0.39f
0.300 ±0.68f
Of
Of
7.98 ± 0.070f
Of
Of
Of
Mean %
Recovery
63.7 ±32
0.0100 ±0.02
-
-
69.8 ±14
< 0.0100
-
-
87.8 ±10
0.0240 ± 0.050
-
-
64.0 ±17
<0.01
-
-
50.0 ±25
<0.01
-
-
83.4 ±13
0
-
-
Decontamination
Efficacy ± CI
h
5.06 ±1.0
-
-
.
5.83 ±1.0
-
-
-
4.91 ±1.1
-
-
.
7.13 ±0.88
-
-
.
7.37 ±0.68
-
-
-
> 7.98 ±0.060
-
-
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 Endogenous flora observed in blank or at least one sample replicate.
g 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 "-" Not Applicable.
16
-------
Table 5-2. Inactivation of Bacillus subtilis Spores at 75% RH on 1 cm Unsterilized Soils"
Test Material
Contact time
Topsoil (Two Hours)
Positive Controls'3
Test Coupons0
Laboratory Blankd
Procedural Blank6
Topsoil (Three Hours)
Positive Controls
Test Coupons
Laboratory Blank
Procedural Blank
Topsoil (Four Hours)
Positive Controls
Test Coupons
Laboratory Blank
Procedural Blank
AZ Test Dust (Two Hours)
Positive Controls
Test Coupons
Laboratory Blank
Procedural Blank
AZ Test Dust
(Three Hours)
Positive Controls
Test Coupons
Laboratory Blank
Procedural Blank
AZ Test Dust
(Four Hours)
Positive Controls
Test Coupons
Laboratory Blank
Procedural Blank
Inoculum
(CFU)
8.83 x 107
8.83 x 107
0
0
8.83 x 107
8.83 x 107
0
0
8.83 x 107
8.83 x 107
0
0
8.83 x 107
8.83 x 107
0
0
8.83 x 107
8.83 x 107
0
0
8.83 x 107
8.83 x 107
0
0
Mean of Logs of
Observed CFU
±CIg
7.86±0.10f
0.76±l.lf
Of
Of
7.85±0.20f
0.61±0.83f
Of
Of
7.87±0.16f
1.87±l.lf
Of
Of
7.88±0.20f
Of
Of
Of
7.95±0.050f
0
Of
Of
7.63 ± 0.26f
Of
Of
Of
Mean %
Recovery
83.2 ±18
<0.01
_h
-
86.2 ± 29
<0.01
-
-
88.0 ± 26
<0.01
-
-
92.6 ±33
0
-
-
100± 12
0
-
-
55.6 ±30
0
-
-
Decontamination
Efficacy ± CI
-
7.09 ±0.95
-
-
-
7.24 ±0.75
-
-
-
6.00 ± 0.94
-
-
.
> 7.88 ±0.18
-
-
.
> 7.95 ±0.050
-
-
.
> 7.63 ±0.23
-
-
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 Endogenous flora observed in blank or at least one sample replicate.
g 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 "-"NotApplicable.
17
-------
Table 5-3. Inactivation of Bacillus anthracis Spores at 85% RH on 1 cm Unsterilized Soils"
Test Material
(Contact Time)
Topsoil 2 Hours
Positive Controls'3
Test Coupons0
Laboratory Blankd
Procedural Blank6
Topsoil 3 Hours
Positive Controls
Test Coupons
Laboratory Blank
Procedural Blank
Topsoil 4 Hours
Positive Controls
Test Coupons
Laboratory Blank
Procedural Blank
AZ Test Dust 2 Hours
Positive Controls
Test Coupons
Laboratory Blank
Procedural Blank
AZ Test Dust 3 Hours
Positive Controls
Test Coupons
Laboratory Blank
Procedural Blank
AZ Test Dust 4 Hours
Positive Controls
Test Coupons
Laboratory Blank
Procedural Blank
Inoculum
(CFU)
1.22 xlO8
1.22 xlO8
0
0
1.22 xlO8
1.22 xlO8
0
0
1.22 xlO8
1.22 xlO8
0
0
1.22 xlO8
1.22 xlO8
0
0
1.22 xlO8
1.22 xlO8
0
0
1.22 xlO8
1.22 xlO8
0
0
Mean of Logs of
Observed CFU
±CIg
7.99 ± 0.040f
3.06±0.39f
Of
Of
7.92 ± 0.070f
2.15±0.11f
Of
Of
8.05 ± 0.040f
l.ll±l.lf
Of
Of
7.94 ± 0.020f
1.55±0.92f
Of
Of
7.94 ± 0.040f
Of
Of
Of
7.98 ± 0.070f
0.500 ±l.lf
Of
Of
Mean %
Recovery
80.1 ±6.4
< 0.010
_h
-
69.0 ±10
< 0.010
-
-
92.6 ±9.1
<0.01
-
-
71.5±4.1
<0.01
-
-
68.1 ±9.8
0
-
-
72.3 ± 16
<0.01
-
-
Decontamination
Efficacy ± CI
-
4.93 ±0.34
-
-
-
5.77 ±0.12
-
-
-
6.94 ±0.96
-
-
.
6.39 ±0.81
-
-
.
> 7.94 ± 0.060
-
-
.
7.43 ±0.99
-
-
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 Endogenous flora observed in blank or at least one sample replicate.
g 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 "-" Not Applicable.
18
-------
Table 5-4. Inactivation of Bacillus subtilis Spores at 85% RH on 1 cm Unsterilized Soils"
Test Material
(Contact Time)
Topsoil (Two Hours)
Positive Controls'3
Test Coupons0
Laboratory Blankd
Procedural Blank6
Topsoil (Three Hours)
Positive Controls
Test Coupons
Laboratory Blank
Procedural Blank
Topsoil (Four Hours)
Positive Controls
Test Coupons
Laboratory Blank
Procedural Blank
AZ Test Dust (Two Hours)
Positive Controls
Test Coupons
Laboratory Blank
Procedural Blank
AZ Test Dust
(Three Hours)
Positive Controls
Test Coupons
Laboratory Blank
Procedural Blank
AZ Test Dust
(Four Hours)
Positive Controls
Test Coupons
Laboratory Blank
Procedural Blank
Inoculum
(CFU)
l.lSxlO8
l.lSxlO8
0
0
l.lSxlO8
l.lSxlO8
0
0
LlSxlO8
LlSxlO8
0
0
LlSxlO8
LlSxlO8
0
0
LlSxlO8
LlSxlO8
0
0
LlSxlO8
LlSxlO8
0
0
Mean of Logs of ,. _ „ ,
y-»i. j /?T,TT Mean %
Observed CFU _
„„ Recovery
7.83±0.080f 58.2 ±10
2.39±1.4f <0.01
Of -h
of
7.86±0.070f 61.3 ±9.0
2.81±0.27f <0.01
Of
Of
7.87±0.020f 62.5 ±3.4
1.60±1.5f <0.01
Of
Of
7.96±0.010f 76.5 ±2.6
Of 0
Of
Of
7.95±0.030f 75. 9 ±5.9
0.580 ±1.3f <0.01
Of
Of
7.86±0.080f 62.2 ±9.9
Of 0
Of
Of
Decontamination
Efficacy ± CI
-
5.44 ±1.2
-
-
-
5.04 ±0.25
-
-
-
6.27 ±1.3
-
-
.
> 7.96 ±0.010
-
-
.
7.37 ±1.1
-
-
.
> 7.86 ± 0.070
-
-
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 Endogenous flora observed in blank or at least one sample replicate.
g 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"-" Not Applicable.
19
-------
Table 5-5. Summary of Efficacy Values for 3,000 ppmv ClOi Gas on 1 cm Unsterilized Soils
RH
Test Material Efficacy for Efficacy for
Contact Time B. anthracis (Ames) B. subtitis
75% RH
Topsoil Two Hours 5.06 ± 1.0 7.09 ± 0.95
Topsoil Three Hours 5.83 ± 1.0 7.24 ± 0.75
Topsoil Four Hours 4.91 ±1.1 6.00 ± 0.94
AZ Test Dust Two Hours 7.13 ±0.88 >7.88±0.18a
AZ Test Dust Three Hours 7.37 ± 0.68 > 7.95 ± 0.050a
AZ Test Dust Four Hours > 7.98 ± 0.060a > 7.63 ± 0.23a
85% RH
Topsoil Two Hours 4.93 ± 0.34 5.44 ± 1.2
Topsoil Three Hours 5.77 ±0.12 5.04 ± 0.25
Topsoil Four Hours 6.94 ± 0.96 6.27 ± 1.3
AZ Test Dust Two Hours 6.39 ±0.81 > 7.96 ± 0.010a
AZ Test Dust Three Hours > 7.94 ± 0.060a 7.37 ± 1.1
AZ Test Dust Four Hours 7.43 ± 0.99 > 7.86 ± 0.070a
a Result represents complete inactivation within the detection limit of 33.33 CPU/soil. Differences in efficacy between
the two microorganisms may be significant if the 95% CIs of the two efficacy results do not overlap (shown in bold above);
however, this comparison is not applicable when the two efficacy results being compared are both reported with log
reductions as > some value.
20
-------
g
4->
U
T3
01
QC
ao
o
o
ro
u
Arizona Test Dust
I B.anthracis
I B.subtilis
2hr, 75% RH 3 hr, 75% RH
4hr, 75%RH 2hr, 85% RH
Test Condition
3hr, 85% RH 4hr, 85%RH
Topsoil
I B. anthracis
i B.subtilis
2hr, 75% RH 3 hr, 75% RH 4hr, 75% RH 2hr, 85% RH 3hr, 85% RH 4hr, 85% RH
Test Condition
Figure 5-1. Summary of decontamination efficacies for 3,000 ppmv ClOi fumigant testing
on 1 cm unsterilized AZTD and topsoil.
* indicates complete inactivation within the detection limit; all results shown with 95% confidence
intervals
21
-------
6.0 Unsterilized Soils and Endogenous Flora
6.1 Background
The topsoil and AZTD matrices were not
sterilized during the initial testing in order to
maintain the physical and chemical integrity
of the soil materials. As a result, none of the
laboratory or procedural blanks detailed in
Chapter 5 (Tables 5-1 to 5-4) passed the
criterion of no observed CPU, due to
contamination caused by the naturally-
occurring organisms in the soils such as
bacteria, fungi and molds (i.e., endogenous
flora). However, we subsequently found 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).
Based on previous test experience with
unsterilized soils, when these B. anthracis-
or B. subtilis-rnoculated unsterilized soils
were subjected to a heat shock (55 to 65 °C)
for one hour in a water bath prior to sample
extraction, this heat treatment would suffice
to inactivate or suppress colony formation of
the non-spore-forming, non-specific
organisms. In the present study, the viable
target organism (B. anthracis or B. subtilis)
may have inhibited endogenous flora growth
once the sample extracts were plated onto
nutrient media. We believe this possible
inhibition of endogenous flora growth
depends on the number of viable target
organisms (B. anthracis or B. subtilis
spores) in the soil. For example, the positive
controls for the soil samples, which initially
have ~ 108 CPU, showed only minimal
growth of endogenous flora and allowed the
manual count to occur that resulted in
quantitative data reproducible between the
test sample replicates. Additionally,
previous decontamination testing with
unsterilized soils has largely been
ineffective, resulting in remaining spore
levels typically above 5 to 6 log CPU per
sample. Previous decontamination testing
with unsterilized soils may also have led to
endogenous flora growth suppression.
We also hypothesize that just as it is
possible for the target organism to inhibit
the growth of endogenous flora, the opposite
can occur, i.e., the endogenous flora can
prevent target organism growth. For
example, if decontamination efficacy of B.
anthracis or B. subtilis exceeds 1 log
reduction, sensitivity may be impacted since
it would not be known whether the target
inoculum was still viable post-
decontamination or whether any remaining
viable flora inhibited the growth of the
target inoculum.
6.2 Soil Recovery Tests
Because of the aforementioned
considerations in Section 6.1, tests were
conducted to determine the percent recovery
of inoculated spores as a function of the
inoculum level and whether the soil material
was sterilized or not. Gamma-irradiated
sterilized topsoil was available for these
recovery tests, whereas sterilized AZTD was
not yet available, so evaluation of AZTD
was performed using only unsterilized
samples. For the tests, a 1 cm depth of
unsterilized topsoil and AZTD was
inoculated with approximately 1 x 108 to 1 x
103 CFU of either B. anthracis or B.
subtilis spore suspensions. This same
approach was applied with sterilized topsoil.
Both sets were allowed to dry overnight.
The unsterilized topsoil samples (and
AZTD) were heat-shocked as usual in a
water bath set at 55 to 65 °C for 1 hour,
whereas the sterilized topsoil samples did
not undergo heat shock.
22
-------
The results in Tables 6-1 and 6-2 show how
endogenous flora affect target organism
recovery. For the unsterilized soil materials
inoculated with B. anthracis or B. subtilis,
the percent recovery of the inoculated
amount decreased by at least 1 log for each
log decrease in the inoculation level. In
comparison, the sterilized topsoil exhibited
no greater than 2.44-fold difference in
percent recovery (for example, with B.
subtilis, the percent recovery only
diminished from 83% to 34%) when the
inoculation level decreased from 8 log to 3
log CPU.
As a result of inactivating the endogenous
flora (and possibly removing their inhibition
effect on the target organisms) via the
sterilization of the topsoil, the recoveries of
B. anthracis and B. subtilis increased
significantly, thereby increasing the
sensitivity of detection of viable spores
following decontamination of this material.
The previous tests described in Chapter 5
using B. anthracis in 1 cm unsterilized soil
materials were therefore repeated with
sterilized topsoil and AZTD; see Chapter 7.
The presence of the endogenous flora may
have made it difficult to observe or count the
target organisms. An overgrowth of
endogenous flora or "lawn" on a nutrient
agar plate would not only interfere with the
counts but also would make identification of
the target organism impossible due to the
complete coverage.
23
-------
Table 6-1. Evaluation of Spore Recovery for Unsterilized and Sterilized Topsoil
„ , Qualitative Amount
Recovered
Target Inoculum (CFU) „„„ % Recovery of Endogenous
„, a
Flora
Bacillus anthracis in Unsterilized
Topsoil
Ixl08d
IxlO7
IxlO6
IxlO5
IxlO4
IxlO3
9.26 x 107
5.17xl05
4.02 x 103
b
-
-
83
4.6
0.36
.
.
-
+
++
++++
++++++0
++++++
++++++
Bacillus subtilis in Unsterilized
Topsoil
Ixl08e
IxlO7
IxlO6
IxlO5
IxlO4
IxlO3
4.36 x 107
3.47 x 105
2.85 x 103
-
-
-
40
3.2
0.26
.
.
-
+
++
++++
++++++
++++++
++++++
Bacillus anthracis in Sterilized
Topsoil
Ixl08f
IxlO7
IxlO6
IxlO5
IxlO4
IxlO3
9.23 x 107
1.32 xlO7
8.79 x 105
8.19 xlO4
7.3 IxlO3
7.00 x 102
66
95
63
59
53
50
0
0
0
0
0
0
Bacillus subtilis in Sterilized
Topsoil
Ixl08g
IxlO7
IxlO6
IxlO5
IxlO4
IxlO3
8.48 x 107
6.93 x 106
5.03 x 105
5.63 x 104
3.91 xlO3
3.43 x 102
83
68
49
55
38
34
0
0
0
0
0
0
a Density of endogenous flora observed on plates presented in qualitative terms, as actual amount not quantifiable. Number of "+"
denotes qualitative density level of endogenous flora, "0" denotes no endogenous flora observed.
b Recovery not determined due to high levels of endogenous flora.
0 Complete coverage or "lawn" of endogenous flora on nutrient agar plates.
d Actual B. anthracis titer verified asl.l2x!08 CFU. Ten-fold dilutions from 107 to 103 not verified, so % Recovery calculations
based on theoretical titer from 1.12 x 108 CFU starting value.
e Actual B. subtilis titer verified as 1.08 x 108 CFU. Ten-fold dilutions from 107 to 103 not verified, so % Recovery calculations
based on theoretical titer from l.OSx 108 CFU starting value.
f Actual B. anthracis titer verified asl.39x!08 CFU. Ten-fold dilutions from 107 to 103 not verified, so % Recovery calculations
based on theoretical titer from 1.3 9 x 108 CFU starting value.
g Actual B. anthracis titer verified as 1.02 x 108 CFU. Ten-fold dilutions from 107 to 103 not verified, so % Recovery calculations
based on theoretical titer from 1.02 x 108 CFU starting value.
24
-------
Table 6-2. Evaluation of Spore Recovery for Unsterilized Arizona Test Dust
rr ^ T i ,n™n Recovered „. „ Qualitative
Target Inoculum (CFU) „„„ % Recovery _ , _, a
^ ^ ' CFU J Endogenous Flora
Bacillus anthracis in Unsterilized
Arizona Test Dust
Ixl08d 9.07 xlO7 81
1 x 107 6.09 x 105 5.4
IxlO6 3.32 xlO3 0.30
1 x 105 -b
1 x 104
1 x 103
Bacillus subtilis in Unsterilized
Arizona Test Dust
Ixl08e 5.54 xlO7 51
1 x 107 3.97 x 105 3.7
IxlO6 2.19 xlO3 0.20
1 x 105
1 x 104
1 x 103
a Density of endogenous flora observed on plates. Not quantifiable. "+" denotes qualitative density level of endogenous flora, "0"
denotes no endogenous flora observed.
b Recovery not determined due to high levels of endogenous flora.
0 Complete coverage or "lawn" of endogenous flora on nutrient agar plates.
d Actual B. anthracis titer verified asl.l2x!08 CFU. Ten-fold dilutions from 107 to 103 not verified, so % Recovery calculations
based on theoretical titer from 1.12 x 108 CFU starting value.
e Actual B. subtilis titer verified as 1.08 x 108 CFU. Ten-fold dilutions from 107 to 103 not verified, so % Recovery calculations
based on theoretical titer from 1.08 x 108 CFU starting value.
25
-------
7.0 Fumigant Testing with Sterilized
Soils at 1 cm Depth, with B. anthmcis
C1O2 fumigation with a target of 3,000 ppmv
and contact times of two, three, and four
hours was repeated using sterilized, 1-cm
depth topsoil and AZTD inoculated with B.
anthracis inside a testing chamber at 75%
RH and 85% RH. (These same tests were
not repeated using B. subtilis.) These tests
were repeated because of the potential for
endogenous flora to inhibit growth of the
target organism.
7.1 Fumigation Conditions
Measurements of C1O2 inside the testing
chamber were made approximately every
five to ten minutes for the duration of testing
to monitor and adjust the C1O2
concentration. For B. anthracis testing with
the target RH of 75%, the average
concentration of C1O2 was measured at
3,004 ppmv ± 378.4 ppmv, the average RH
inside the testing chamber was measured at
77% ± 2%, and the average temperature
inside the testing chamber was measured at
23.2°C±0.6°C.
Measurements of C1O2 inside the testing
chamber were made approximately every
five to ten minutes for the duration of testing
to monitor and adjust the C1O2
concentration. For B. anthracis testing with
the target RH of 85%, the average
concentration of C1O2 was measured at
3,066 ppmv ± 484.7 ppmv, the average RH
inside the testing chamber was measured at
88% ± 2%, and the average temperature
inside the testing chamber was measured at
22.7°C±0.6°C.
Fumigation conditions are tabulated in
Appendix A.
7.2 Decontamination Efficacy
The detailed decontamination efficacy
results are shown in Tables 7-1 and 7-2, and
are summarized in Table 7-3. None of the
laboratory and procedural blanks showed the
presence of endogenous flora.
Both the AZTD and topsoil samples were
effectively decontaminated (log reduction
greater than 6.00) in all tests. In five of the
six tests with AZTD, the spores were
completely inactivated, whereas the
complete inactivation of spores did not
occur in any of topsoil tests.
7.3 Comparison of Efficacy Results for B.
anthracis on Non-Sterile and Sterile Soils
The mean log reduction results were
generally higher for the sterile soil materials
compared to the unsterilized soils, although
there were only four test conditions (out of
12) in which the results were judged to be
significantly different (based on whether the
95% confidence intervals overlap); refer to
Figure 7-1. The higher log reductions for
the sterilized soils may be related to higher
spore recovery.
26
-------
Table 7-1. Inactivation of Bacillus anthracis Spores at 75% RH on 1 cm Sterilized Soils
Test Material
(Contact Time)
Topsoil (Two Hours)3
Positive Controls'3
Test Coupons0
Laboratory Blankd
Procedural Blank6
Topsoil (Three Hours)
Positive Controls
Test Coupons
Laboratory Blank
Procedural Blank
Topsoil (Four Hours)
Positive Controls
Test Coupons
Laboratory Blank
Procedural Blank
AZ Test Dust (Two Hours)
Positive Controls
Test Coupons
Laboratory Blank
Procedural Blank
AZ Test Dust
(Three Hours)
Positive Controls
Test Coupons
Laboratory Blank
Procedural Blank
AZ Test Dust
(Four Hours)
Positive Controls
Test Coupons
Laboratory Blank
Procedural Blank
Inoculum
(CFU)
1.24 xlO8
1.24 xlO8
0
0
1.24 xlO8
1.24 xlO8
0
0
1.24 xlO8
1.24 xlO8
0
0
1.24 xlO8
1.24 xlO8
0
0
1.24 xlO8
1.24 xlO8
0
0
1.24 xlO8
1.24 xlO8
0
0
Mean of Logs of ,. _ „ ,
y-»i. j /?T,TT Mean %
Observed CFU _
rjf Recovery
8.02 ±0.02 85.2 ±3.7
1.46 ±1.4 <0.01
0
0
7.99 ±0.040 78.6 ±8.2
1.65 ±0.98 <0.01
0
0
7.96 ±0.040 74.6 ±7.6
0.610 ±0.83 <0.01
0
0
7.95 ±0.040 71.5 ±6.4
0 0
0
0
7.94 ±0.030 71.0 ±4.1
0 0
0
0
7.97 ±0.030 75.7 ±5.8
0 0
0
0
Decontamination
Efficacy ± CI
_g
6.56 ±1.3
-
-
-
6.34 ±0.86
-
-
-
7.36 ±0.73
-
-
-
> 7.95 ±0.040
-
-
.
> 7.94 ± 0.020
-
-
.
> 7.97 ±0.030
-
-
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.
g "-" Not Applicable.
27
-------
Table 7-2. Inactivation of Bacillus anthracis Spores at 85% RH on 1cm Sterilized Soils
Test Material
(Contact Time)
Topsoil (Two Hours)
Positive Controls'3
Test Coupons0
Laboratory Blankd
Procedural Blank6
Topsoil (Three Hours)
Positive Controls
Test Coupons
Laboratory Blank
Procedural Blank
Topsoil (Four Hours)
Positive Controls
Test Coupons
Laboratory Blank
Procedural Blank
AZ Test Dust (Two Hours)
Positive Controls
Test Coupons
Laboratory Blank
Procedural Blank
AZ Test Dust
(Three Hours)
Positive Controls
Test Coupons
Laboratory Blank
Procedural Blank
AZ Test Dust
(Four Hours)
Positive Controls
Test Coupons
Laboratory Blank
Procedural Blank
Inoculum
(CFU)
9.87 x 107
9.87 x 107
0
0
9.87 x 107
9.87 x 107
0
0
9.87 x 107
9.87 x 107
0
0
9.87 x 107
9.87 x 107
0
0
9.87 x 107
9.87 x 107
0
0
9.87 x 107
9.87 x 107
0
0
Mean of Logs of
Observed CFU
±CIf
8.16 ±0.20
0.56 ±1.3
0
0
8.01 ±0.040
0.67 ±0.92
0
0
7.99 ±0.010
0.30 ±0.68
0
0
7.96 ± 0.040
0
0
0
7.97 ± 0.040
0.70 ±0.98
0
0
7.96 ± 0.060
0
0
0
Mean %
Recovery
162± 45
< 0.0100
-
-
104± 10
< 0.0100
-
-
100± 1.8
<0.01
-
-
92.7 ±9.0
0
-
-
95.0 ±9.0
< 0.0100
-
-
93.6 ±12
0
-
-
Decontamination
Efficacy ± CI
h
7.60 ±1.1
-
-
-
7.34 ±0.81
-
-
-
7.69 ±0.60
-
-
-
> 7.96 ± 0.040
-
-
.
> 7.27 ±0.86
-
-
.
> 7.96 ±0.050
-
-
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 * 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.
28
-------
Table 7-3. Summary of Efficacy Values for 3,000 ppmv C1O2 Gas on 1 cm Sterilized Soils
for B. anthracis
RH Efficacy for
Test Material B. anthracis (Ames)
Contact time ±CIb
75% RH
Topsoil Two Hours 6.56 ±1.3
Topsoil Three Hours 6.34 ± 0.86
Topsoil Four Hours 7.36 ± 0.73
AZ Test Dust Two Hours > 7.95 ± 0.040a
AZ Test Dust Three Hours > 7.94 ± 0.020a
AZ Test Dust Four Hours > 7.97 ± 0.030a
85% RH
Topsoil Two Hours 7.60 ± 1.1
Topsoil Three Hours 7.34 ± 0.81
Topsoil Four Hours 7.69 ± 0.60
AZ Test Dust Two Hours > 7.96 ± 0.040a
AZ Test Dust Three Hours 7.27 ± 0.86
AZ Test Dust Four Hours > 7.96 ± 0.050a
a Result represents complete inactivation within the detection limit of 33.33 CPU/soil. Differences in efficacy may
besignificant 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.
b 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.
29
-------
Non-Sterile vs. Sterile AZ Test Dust
I Non-Sterile AZTD
Sterile AZTD
3000ppm,2hr,
75%RH
3000ppm,2hr,
85%RH
3000ppm,3hr, 3000ppm,4hr,
85%RH 85%RH
Test Condition
Non-Sterile vs. Sterile Topsoil
3000ppm,2hr,
75%RH
3000ppm,3hr,
75%RH
Test Condition
I Non-Sterile Topsoil
Sterile Topsoil
Figure 7-1. Comparison of decontamination efficacies for unsterilized vs. sterilized soils
with B. anthracis.
* indicates complete inactivation within the detection limit; all results shown with 95% confidence intervals
30
-------
8.0 Fumigant Testing with Sterilized Soils at 2 cm Depth
C1O2 fumigation tests with a target of 3,000
ppmv and contact times of two, three, and
four hours were conducted using sterilized
2-cm depth topsoil and AZTD inoculated
with either B. anthracis or B. subtilis inside
a testing chamber at 75% RH or 85% RH.
8.1 Fumigation Conditions
For the tests conducted at the target 75%
RH, measurements of C1O2 inside the testing
chamber were made approximately every 10
to 20 minutes for the duration of testing to
monitor and adjust the C1O2 concentration.
For B. anthracis testing, the average
concentration of C1O2 was measured at
3,240 ppmv ± 327.8 ppmv, the average RH
was measured at 78% ± 2%, and the average
temperature was measured at 20.5 °C ± 0.3
°C. For B. subtilis testing, the average
concentration of C1O2 was measured at
3,089 ppmv ±259.1 ppmv, the average RH
was measured at 76% ±1%, and the average
temperature was measured at 20.9 °C ± 0.3
When testing at the 85% RH target,
measurements of C1O2 inside the testing
chamber were made approximately every 10
to 20 minutes for the duration of testing to
monitor and adjust the C1O2 concentration.
For B. anthracis testing, the average
concentration of C1O2 was measured at
3,108 ppmv ±357.8 ppmv, the average RH
was measured at 86% ± 2%, and the average
temperature was measured at 20.9 °C ± 0.2
°C. For B. subtilis testing, the average
concentration of C1O2 was measured at
3,170 ppmv ±233.9 ppmv, the average RH
was measured at 86% ±1%, and the average
temperature was measured at 20.1 °C ± 0.3
Fumigation conditions are tabulated in
Appendix A.
8.2 Decontamination Results
For the tests conducted at a depth of 2 cm,
the detailed decontamination results are
shown in Tables 8-1 through 8-4 and
summarized in Table 8-5. The AZTD was
completely decontaminated in all 12 tests
conducted with this material. The mean log
reductions for the topsoil material ranged
from a low of 0.59 (75% RH, B. subtilis) to
a high of 3.72 (85% RH, B. subtilis).
Increasing contact time did significantly
improve efficacy for topsoil for both
microorganisms and RH conditions.
Changing the RH level did not significantly
affect the log reduction results.
Of the 12 fumigation tests in which
decontamination efficacies could be
compared between B. anthracis and B.
subtilis, only one of the tests had results for
the two microorganisms which were
significantly different. On topsoil at the
85% RH, 3-hour test condition, B. anthracis
was inactivated to a significantly higher
degree.
-------
Table 8-1. Inactivation of Bacillus anthracis Spores on 2 cm Sterilized Soils at 75% RH
Test Material
(Contact Time)
Topsoil (Two Hours)3
Positive Controls'3
Test Coupons0
Laboratory Blankd
Procedural Blank6
Topsoil (Three Hours)
Positive Controls
Test Coupons
Laboratory Blank
Procedural Blank
Topsoil (Four Hours)
Positive Controls
Test Coupons
Laboratory Blank
Procedural Blank
AZ Test Dust (Two Hours)
Positive Controls
Test Coupons
Laboratory Blank
Procedural Blank
AZ Test Dust
(Three Hours)
Positive Controls
Test Coupons
Laboratory Blank
Procedural Blank
AZ Test Dust
(Four Hours)
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
±CIf
7.77 ±0.030
7.10 ±0.12
0
0
7.71 ±0.050
6.27 ± 0.42
0
0
7.71 ±0.020
4.84 ± 1.1
0
0
7.64 ±0.050
0
0
0
7.68 ±0.050
0
0
0
7.59 ±0.050
0
0
0
Mean %
Recovery
51.2 ±3.8
11.7±2.8
-
-
45. 3 ±5.8
2.10 ±1.3
-
-
44.4 ±1.9
0.210 ±0.25
-
-
38.0 ±4.9
0
-
-
41.9 ±4.8
0
-
-
34.1 ±4.2
0
-
-
Decontamination
Efficacy ± CI
_g
0.670 ±0.10
-
-
-
1.45 ±0.37
-
-
-
2.86 ±0.94
-
-
-
> 7.64 ±0.050
-
-
-
> 7.68 ± 0.040
-
-
.
> 7.59 ±0.050
-
-
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.
g "-"NotApplicable.
32
-------
Table 8-2. Inactivation of Bacillus subtilis Spores on 2 cm Sterilized Soils at 75% RH
Test Material
(Contact Time)
Topsoil (Two Hours)3
Positive Controls'3
Test Coupons0
Laboratory Blankd
Procedural Blank6
Topsoil (Three Hours)
Positive Controls
Test Coupons
Laboratory Blank
Procedural Blank
Topsoil (Four Hours)
Positive Controls
Test Coupons
Laboratory Blank
Procedural Blank
AZ Test Dust (Two Hours)
Positive Controls
Test Coupons
Laboratory Blank
Procedural Blank
AZ Test Dust
(Three Hours)
Positive Controls
Test Coupons
Laboratory Blank
Procedural Blank
AZ Test Dust
(Four Hours)
Positive Controls
Test Coupons
Laboratory Blank
Procedural Blank
Inoculum
(CFU)
9.67 x 107
9.67 x 107
0
0
9.67 x 107
9.67 x 107
0
0
9.67 x 107
9.67 x 107
0
0
9.67 x 107
9.67 x 107
0
0
9.67 x 107
9.67 x 107
0
0
9.67 x 107
9.67 x 107
0
0
Mean of Logs of
Observed CFU
±CIf
7.72 ± 0.090
7.13±0.31
0
0
7.73 ± 0.020
5.85 ±0.99
0
0
7.78 ± 0.040
4.85 ±0.75
0
0
7.73 ± 0.040
0
0
0
7.57 ±0.13
0
0
0
7.77 ±0.030
0
0
0
Mean %
Recovery
55.1 ±10
17.7 ±16
-
-
56.0 ±2.4
2.42 ±2.6
-
-
62.3 ±6.3
0.200 ± 0.27
-
-
56.2 ±5.3
0
-
-
39.6 ±12
0
-
-
61. 3 ±3.6
0
-
-
Decontamination
Efficacy ± CI
_g
0.590 ±0.28
-
-
-
1.88 ±0.86
-
-
-
2.93 ±0.66
-
-
-
> 7.73 ± 0.040
-
-
.
> 7.57 ±0.11
-
-
.
> 7.77 ± 0.020
-
-
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 * 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.
33
-------
Table 8-3. Inactivation of Bacillus anthracis Spores on 2 cm Sterilized Soils at 85% RH
Test Material
(Contact Time)
Topsoil (Two Hours)3
Positive Controls'3
Test Coupons0
Laboratory Blankd
Procedural Blank6
Topsoil (Three Hours)
Positive Controls
Test Coupons
Laboratory Blank
Procedural Blank
Topsoil (Four Hours)
Positive Controls
Test Coupons
Laboratory Blank
Procedural Blank
AZ Test Dust (Two Hours)
Positive Controls
Test Coupons
Laboratory Blank
Procedural Blank
AZ Test Dust
(Three Hours)
Positive Controls
Test Coupons
Laboratory Blank
Procedural Blank
AZ Test Dust
(Four Hours)
Positive Controls
Test Coupons
Laboratory Blank
Procedural Blank
Inoculum
(CFU)
7.50 xlO7
7.50 xlO7
0
0
7.50 xlO7
7.50 xlO7
0
0
7.50 xlO7
7.50 xlO7
0
0
7.50 xlO7
7.50 xlO7
0
0
7.50 xlO7
7.50 xlO7
0
0
7.50 xlO7
7.50 xlO7
0
0
Mean of Logs of
Observed CFU
±CIf
7.66 ± 0.080
6.82 ±0.25
0
0
7.66 ± 0.060
5.36 ±0.62
0
0
7.68 ± 0.040
4.00 ±0.94
0
0
7.51 ±0.050
0
0
0
7.58 ±0.060
0
0
0
7.57 ±0.060
0
0
0
Mean %
Recovery
61.4 ±10.7
9.99 ±5.2
-
-
61.0 ±8.4
0.720 ±1.1
-
-
64.0 ±6.4
0.0600 ±0.11
-
-
43.1 ±4.5
0
-
-
51.4 ±7.0
0
-
-
50.0 ±7.4
0
-
-
Decontamination
Efficacy ± CI
_g
0.840 ±0.23
-
-
-
2.30 ±0.55
-
-
-
3.68 ±0.83
-
-
-
> 7.51 ±0.040
-
-
.
> 7.58 ±0.050
-
-
.
> 7.57 ±0.050
-
-
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.
g "-" Not Applicable.
34
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Table 8-4. Inactivation of Bacillus subtilis Spores on 2 cm Sterilized Soils at 85% RH
Test Material
(Contact Time)
Topsoil (Two Hours)3
Positive Controls'3
Test Coupons0
Laboratory Blankd
Procedural Blank6
Topsoil (Three Hours)
Positive Controls
Test Coupons
Laboratory Blank
Procedural Blank
Topsoil (Four Hours)
Positive Controls
Test Coupons
Laboratory Blank
Procedural Blank
AZ Test Dust (Two Hours)
Positive Controls
Test Coupons
Laboratory Blank
Procedural Blank
AZ Test Dust
(Three Hours)
Positive Controls
Test Coupons
Laboratory Blank
Procedural Blank
AZ Test Dust
(Four Hours)
Positive Controls
Test Coupons
Laboratory Blank
Procedural Blank
Inoculum
(CFU)
1.05 x 108
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
1.05 x 108
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
Mean of Logs of
Observed CFU
±CIf
7.73 ± 0.020
7.04 ±0.16
0
0
7.72 ± 0.060
6.33 ±0.40
0
0
7.73 ±0.050
4.01 ±0.90
0
0
7.68 ± 0.060
0
0
0
7.73 ±0.030
0
0
0
7.72 ±0.030
0
0
0
Mean %
Recovery
51.1 ±2.0
10.9 ±3.0
-
-
49.8 ±6.7
2.77 ±2.2
-
-
51.7 ±5.8
0.0450 ± 0.078
-
-
45. 8 ±6.0
0
-
-
51.6 ±3.2
0
-
-
50.4 ±3.8
0
-
-
Decontamination
Efficacy ± CI
_g
0.690 ±0.14
-
-
-
1.38 ±0.36
-
-
-
3.72 ±0.79
-
-
-
> 7.68 ±0.050
-
-
.
> 7.73 ± 0.020
-
-
.
> 7.72 ±0.030
-
-
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 * 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
35
-------
Table 8-5. Summary of Decontamination Efficacy Values for 3,000 ppmv ClOi Gas on 2 cm
Sterilized Soils
RH Efficacy for Efficacy for
Test Material B. anthrads (Ames) B. subtilis
(Contact Time) ±CIb ±CI
75% RH
Topsoil (Two Hours) 0.670 ±0.10 0.590 ± 0.28
Topsoil (Three Hours) 1.45 ±0.37 1.88 ±0.86
Topsoil (Four Hours) 2.86 ± 0.94 2.93 ± 0.66
AZ Test Dust (Two Hours) >7.64±0.050a > 7.73 ± 0.040a
AZ Test Dust (Three Hours) > 7.68 ± 0.040a > 7.57 ± 0.1 la
AZ Test Dust (Four Hours) > 7.59 ± 0.050a >7.77±0.020a
85% RH
Topsoil (Two Hours) 0.840 ± 0.23 0.690 ± 0.14
Topsoil (Three Hours) 2.30 ± 0.55 1.38 ± 0.36
Topsoil (Four Hours) 3.68 ± 0.83 3.72 ± 0.79
AZ Test Dust (Two Hours) >7.51±0.040a > 7.68 ± 0.050a
AZ Test Dust (Three Hours) >7.58±0.050a > 7.73 ± 0.020a
AZ Test Dust (Four Hours) >7.57±0.050a >7.72±0.030a
a Result represents complete inactivation within the detection limit of 33.33 CPU/soil. Differences in efficacy between
the two microorganisms may be significant if the 95% CIs of the two efficacy results do not overlap (shown in bold above);
however, this comparison is not applicable when the two efficacy results being compared are both reported with log
reductions as > some value.
b CI = confidence interval (± 1.96 x SE).
36
-------
9.0 Summary of Results
Decontamination Efficacy
In this study, all C1O2 fumigation tests were
conducted at a target concentration of 3,000
ppmv, with an RH of either 75% or 85%,
and at indoor ambient temperature (~ 22
°C). The decontamination efficacy results
for the soil fumigation test are
summarized in Table 13-1. The AZTD
material was easier to decontaminate
compared to the topsoil material. Out of the
30 tests conducted with AZTD, there were
only six test conditions in which the AZTD
was not completely decontaminated, and all
AZTD was effectively decontaminated, i.e.,
with a log reduction > 6.0.
For the topsoil, there were no tests in which
this material was completely
decontaminated. In addition, the
decontamination efficacy diminished
significantly when tests were conducted
with topsoil at a 2 cm depth, when compared
to the tests with topsoil at 1 cm depth. At
the 1 cm depth, topsoil was effectively
decontaminated in 11 of 18 tests, but at the 2
cm depth of topsoil, the highest average log
reduction achieved out of 12 tests was 3.72.
The soil depth did not affect
decontamination efficacy for the AZTD
material.
Increasing RH from 75% to 85% (all other
test conditions being equal) did not
significantly improve efficacy. Increasing
contact time for the topsoil tests at 2 cm
depth did significantly improve efficacy for
a number of tests under this condition.
Effect of Soil Sterilization on Spore
Recovery
The soil materials were initially not
sterilized prior to testing due to concerns
that sterilization processes could alter
physical and chemical characteristics of the
soils. However, interferences resulting from
the presence of endogenous flora in the soils
became apparent in the first set of tests
conducted using 1 cm of soil materials.
Tests were therefore conducted to assess the
recovery of spores as a function of inoculum
levels and whether the soils were pre-
sterilized or not. These tests did confirm that
the recovery of target spores was affected by
whether the soil materials were pre-
sterilized or not, i.e., that endogenous flora
affected target organism recovery. In each
case, for the unsterilized soil materials
inoculated with B. anthracis or B. subtilis,
the percent recoveries decreased by at least
1 log for each log decrease in the inoculation
levels, while the sterilized topsoils exhibited
no greater than a 2.44-fold difference in
recoveries as the inoculation levels
decreased five orders of magnitude.
Effect of Soil Sterilization on
Decontamination Efficacy for B. anthracis
Because of the effect of sterilization on
spore recovery, the 1 cm soil fumigation
tests were repeated with B. anthracis spores.
(The efficacy data shown in Table 13-1
include these repeat tests for B. anthracis, as
well as the initial test results using
unsterilized soil.) The mean log reduction
results for B. anthracis spores were
generally higher for the sterile soil materials
compared to the unsterilized soils, although
there were only four test conditions (out of
12) in which the sterilized vs. unsterilized
soil results were judged to be significantly
different.
37
-------
Comparing Log Reductions for B.
anthracis and B. subtilis
Of the 24 fumigation tests in which
decontamination efficacies could be
compared between B. anthracis and B.
subtilis (for tests conducted with soils at 1
cm depth, comparisons were made between
the unsterilized soils), four of the tests had
results which were significantly different.
Among those four tests, B. subtilis was
inactivated to a lesser degree in two of the
tests.
38
-------
Table 9-1. Summary of ClOi Soil Fumigation Results
Quantitative Efficacy
(± 95% CI)
Test Material
Topsoil
Topsoil
Topsoil
AZ Test Dust
AZ Test Dust
AZ Test Dust
Contact Time
(hours)
2
3
4
2
3
4
B.a.c
5.06
(±1.0)
5.83
(±1.0)
4.91
(±1.1)
7.13
(±0.88)
7.37
(±0.68)
>7.98
(±0.06)
1 cm Depth,
75% RH
B.a.
6.56
(±1.3)
6.34
(±0.86)
7.36
(±0.73)d
>7.95
(±0.040)a
>7.94
(±0.020)a
>7.97
(±0.030)a
B.s.c
7.09
(±0.95)"
7.24
(±0.75)
6.00
(±0.94)
>7.88
(±0.1 8)a
>7.95
(±0.050)a
>7.63
(±0.23)a
1 cm Depth,
85% RH
B.a.c
4.93
(±0.34)
5.77
(±0.12)
6.94
(±0.96)
6.39
(±0.81)
>7.94
(±0.06)
7.43
(±0.99)
B.a.
7.60
(±1.1 )d
7.34
(±0.81)d
7.69
(±0.60)
>7.96
(±0.040)a'd
7.27
(±0.86)
>7.96
(±0.050)a
B.s.c
5.44
(±1.2)
5.04
(±0.25)"
6.27
(±1.3)
>7.96
(±0.010)"'
b
7.37
(±1.1)
>7.86
(±0.070)a
2 cm Depth,
75% RH
B.a.
0.670
(±0.10)
1.45
(±0.37)
2.86
(±0.94)
>7.64
(±0.050)a
>7.68
(±0.050)a
>7.59
(±0.050)a
B.s.
0.590
(±0.28)
1.88
(±0.86)
2.93
(±0.66)
>7.73
(±0.040)a
>7.57
(±0.1 l)a
>7.77
(±0.020)a'b
2 cm Depth,
85% RH
B.a.
0.840
(±0.23)
2.30
(±0.55)
3.68
(±0.83)
>7.51
(±0.040)a
>7.58
(±0.050)a
>7.57
(±0.050)a
B.s.
0.690
(±0.14)
1.38
(±0.36)
3.72
(±0.79)
>7.68
(±0.050)a
>"7?73'
(±0.020)a
>7.72
(±0.030)a
a Result represents complete inactivation within the detection limit of 33.33 CPU/material.
b Values in bold for B. subtilis are significantly different from corresponding values for B. anthracis (for 1 cm depth, comparison is made with results using
unsterilized soils); i.e., 95% CIs of the two efficacy results do not overlap. This comparison based on overlap of 95% CI is not applicable when the two efficacy results
being compared are both reported with log reductions as > some value.
0 Unsterilized soil materials.
d Log reduction for B. anthracis in sterilized soil significantly different from log reduction in unsterilized soil. 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.
39
-------
10.0 References
Decontamination of Indoor and
Outdoor Materials with Aqueous
Chlorine Dioxide Solutions. US
EPA Report 600/R/12/516, May
2012.
Test/Quality Assurance Plan for
Systematic Investigation of
Fumigant Technologies for
Decontamination of Biological
Agents from Contaminated Building
Materials. Version 1 (5/15/2007);
Amendment 8 (May 26, 2010); and
Amendment 9 (November 19, 2010).
(Available upon request.)
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.
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.
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.
40
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Appendix A: C1O2 Fumigation Conditions
Table A-l is a summary of the actual C1O2 fumigation conditions for all soil decontamination
tests conducted in the study. Presented below are the actual C1O2 concentrations, RH, and
temperature levels (mean ± SD) for each test condition. All tests were conducted at ambient
laboratory temperature, with a target C1O2 gas concentration of 3,000 ppmv, and either at 75% or
85% RH. Refer to the individual chapters for additional information.
Table A-l. Soil fumigation conditions
Soil Test
Condition
1 cm unsterilized
1 cm unsterilized
1 cm unsterilized
1 cm unsterilized
1 cm sterilized
1 cm sterilized
2 cm sterilized
2 cm sterilized
2 cm sterilized
2 cm sterilized
Target
%RH
75
75
85
85
75
85
75
75
85
85
Microorganism
B. anthracis
B. subtilis
B. anthracis
B. subtilis
B. anthracis
B. anthracis
B. anthracis
B. subtilis
B. anthracis
B. subtilis
ClOi concentration
ppmv
2,805 ± 400
3,028 ±241
3,019 ±234
3,086 ±231
3,004 ±378
3,066 ±485
3,240 ± 328
3,089 ±259
3, 108 ±358
3, 170 ±234
RH%
73 ±
69 ±
87 ±
87 ±
77 ±
88 ±
78 ±
76 ±
86 ±
86 ±
2
2
1
2
2
2
2
1
2
1
T(°C)
23.7 ±0.5
23.7 ±0.6
24.3 ±0.7
24.7 ±0.8
23.2 ±0.6
22.7 ±0.6
20.5 ±0.3
20.9 ±0.3
20.9 ±0.2
20.1 ±0.3
Data for C1O2 concentration, RH, and T are expressed as the mean ± SD
41
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