September 2004
Environmental Technology
Verification Report
CDG Research Corporation
Bench-Scale Chlorine Dioxide
Gas:Solid Generator
Prepared by
Battelle
Battelle
The Business of Innovation
Under a contract with
£EPA U.S. Environmental Protection Agency
ETt etV etV
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September 2004
Environmental Technology Verification
Report
ETV Building Decontamination Technology Center
CDG Research Corporation
Bench-Scale Chlorine Dioxide
Gas:Solid Generator
by
James V. Rogers
Carol L. Sabourin
Michael L. Taylor
Karen Riggs
Young W. Choi
William R. Richter
Denise C. Rudnicki
Harry J. Stone
Battelle
Columbus, Ohio 43201
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Notice
The U.S. Environmental Protection Agency (EPA), through its Office of Research and
Development, has financially supported and collaborated in the extramural program
described here. This document has been peer-reviewed by the Agency and recommended for
public release. Mention of trade names or commercial products does not constitute
endorsement or recommendation by the EPA for use.
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Foreword
The U.S. Environmental Protection Agency (EPA) is charged by Congress with protecting
the nation's air, water, and land resources. Under a mandate of national environmental laws,
the Agency strives to formulate and implement actions leading to a compatible balance
between human activities and the ability of natural systems to support and nurture life. To
meet this mandate, the EPA's Office of Research and Development provides data and
science support that can be used to solve environmental problems and to build the scientific
knowledge base needed to manage our ecological resources wisely, to understand how
pollutants affect our health, and to prevent or reduce environmental risks.
The Environmental Technology Verification (ETV) Program has been established by the
EPA to verify the performance characteristics of innovative environmental technologies
across all media and to report this objective information to permitters, buyers, and users of
the technology, thus substantially accelerating the entrance of new environmental
technologies into the marketplace. Verification organizations oversee and report verification
activities based on testing and quality assurance protocols developed with input from major
stakeholders and customer groups associated with the technology area. ETV consists of six
verification centers. Information about each of these centers can be found on the internet at
http://www.epa.gov/etv.
Effective verifications of monitoring technologies are needed to assess environmental
quality and to supply cost and performance data to select the most appropriate technology
for that assessment. In 2002, EPA established the Building Decontamination Technology
Center at Battelle. Battelle plans, coordinates, and conducts verification tests of
decontamination technologies and reports the results to the community at large. Information
concerning this specific environmental technology area can be found on the Internet at
http://www.epa.gov/etv/centers/center9.html.
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Acknowledgments
The authors wish to acknowledge the support of all those who helped plan and conduct the
verification test, analyze the data, and prepare this report. In particular we would like to
thank Dr. John Chang, U.S. Environmental Protection Agency (EPA); Doris Betancourt,
EPA; Shirley Wasson, EPA; Jeff Kempter, EPA; Dr. Dorothy Canter, EPA;
Dr. Greg Knudson, CIA; and John Kyme, Defense Group, Inc., who reviewed
the test/quality assurance plan and/or verification report.
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Contents
Notice ii
Foreword iii
Acknowledgments iv
List of Abbreviations vii
1. Background 1
2. Technology Description 2
3. Test Design and Procedures 4
3.1 Introduction 4
3.2 Test Design 5
3.3 Agents and Surrogates 5
3.4 Test Sequence 6
3.5 Coupon-Scale Testing 7
3.5.1 Preparation of Test Materials 7
3.5.2 Application of Agents to Test Coupons 8
3.5.3 Confirmation of Surface Applications 8
3.5.4 Decontamination 8
3.5.4.1 Verification Testing Apparatus and Parameters 8
3.5.4.2 Chlorine Dioxide Measurement 11
3.5.4.3 Decontamination Efficacy 11
3.5.5 Observation of Surface Damage 13
4. Quality Assurance/Quality Control 14
4.1 Equipment Calibration 14
4.2 Audits 14
4.2.1 Technical Systems Audit 14
4.2.2 Audit of Data Quality 14
4.3 QA/QC Reporting 15
4.4 Data Review 15
5. Statistical Methods 16
5.1 Efficacy Calculations 16
5.2 Statistical Analysis 16
6. Test Results 18
6.1 Efficacy 18
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6.1.1 Bacillus anthracis Ames Spores 18
6.1.2 Bacillus subtilis (ATCC 19659) Spores 21
6.1.3 Geobacillus stearothermophilus (ATCC 12980) Spores 24
6.1.4 Statistical Analysis 27
6.2 Damage to Coupons 28
6.3 Other Factors 28
6.3.1 Operation of the CDG Bench-scale Unit 28
6.3.2 Operator Bias 29
7. Performance Summary 30
8. References 32
Figures
Figure 2-1. CDG Research Corporation Bench-Scale Unit 2
Figure 3-1. Test Materials 4
Figure 3-2. Overview of Plas-Labs Compact Glove Box 9
Figure 3-3. Fans in the Plas-Labs Compact Glove Box 10
Figure 3-4. Nebulizers in the Plas-Labs Compact Glove Box 10
Figure 3-5 SAIC Chlorine Dioxide Monitor 12
Figure 6-1. Representative Chlorine Dioxide Concentration from a Single Experiment.... 28
Tables
Table 3-1. Test Sequence and Parameters 6
Table 3-2. Material Characteristics 7
Table 4-1. Summary of Data Recording Process 15
Table 6-1. CDG Bench-Scale Unit Decontamination of Bacillus anthracis
Ames Spores 19
Table 6-2. Liquid Culture Assessment of Bacillus anthracis Ames Spores 20
Table 6-3. Representative Liquid Culture Assessment of Biological
Indicators/Spore Strips 21
Table 6-4. CDG Bench-Scale Unit Decontamination of Bacillus subtilis Spores 22
Table 6-5. Liquid Culture Assessment of Bacillus subtilis Spores 23
Table 6-6. Representative Liquid Culture Assessment of Biological
Indicators/Spore Strips 24
Table 6-7. CDG Bench-Scale Unit Decontamination of Geobacillus stearothermophilus
Spores 25
Table 6-8. Liquid Culture Assessment of Geobacillus stearothermophilus
Spores 26
Table 6-9. Representative Liquid Culture Assessment of Biological
Indicators/Spore Strips 27
Table 6-10. Statistical Analysis of Mean Efficacy (Log Reduction) for Spores 27
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List of Abbreviations
ANOVA analysis of variance
ATCC American Type Culture Collection
BDT Building Decontamination Technology
BSC biological safety cabinet
BWD bare wood (pine lumber)
CFU colony-forming unit
CIO2 chlorine dioxide
cm centimeter
DL decorative laminate
EPA U.S. Environmental Protection Agency
ETV Environmental Technology Verification
GM galvanized metal ductwork
GS glass
HEPA high-efficiency particulate air
HVAC heating, ventilating, and air conditioning
IC industrial-grade carpet
in inch
mL milliliter
PC painted (latex, semi-gloss) concrete cinder block
ppm part per million
PW painted (latex, flat) wallboard paper
QA quality assurance
QC quality control
QMP Quality Management Plan
SAIC Science Application International Corporation
SD standard deviation
TSA technical systems audit
UV ultraviolet
vii
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Chapter 1
Background
The U.S. Environmental Protection Agency (EPA) supports the Environmental Technology
Verification (ETV) Program to facilitate the deployment of innovative environmental tech-
nologies through performance verification and dissemination of information. The goal of the
ETV Program is to further environmental protection by accelerating the acceptance and use
of improved and cost-effective technologies. The ETV Program seeks to achieve this goal
by providing high-quality, peer-reviewed data on technology performance to those involved
in the design, distribution, financing, permitting, purchase, and use of environmental
technologies.
The ETV Program works in partnership with recognized testing organizations; with
stakeholder groups consisting of buyers, vendor organizations, and permitters; and with the
full participation of individual technology developers. The program evaluates the
performance of innovative technologies by developing test plans that are responsive to the
needs of stakeholders, conducting field or laboratory tests (as appropriate), 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
adequate quality are generated and that the results are defensible.
The EPA's National Risk Management Research Laboratory and its verification
organization partner, Battelle, operate the Building Decontamination Technology (BDT)
Center under the ETV Program. The BDT Center recently evaluated the performance of the
CDG Research Corporation bench-scale chlorine dioxide (CIO2) Gas:Solid generator (unit)
for decontaminating buildings contaminated with a biological agent and surrogates.
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Chapter 2
Technology Description
The objective of the ETV BDT Center is to verify the performance characteristics of
technologies that can be used to decontaminate indoor surfaces in buildings contaminated
with either chemical or biological agents as a result of an intentional attack. This
verification report provides results for testing of the CDG Research Corporation bench-scale
unit. The following description of the CDG bench-scale unit is based on information
provided by the vendor. The information provided below was not verified in this test.
The CDG bench-scale unit generates CIO2
gas for decontaminating a sealed area by
producing a blend of CIO2 gas in nitrogen
or air. A mixture of nitrogen (or air) and
chlorine gas is passed through a reactor
cartridge containing processed pellets of
sodium chlorite. The chlorine reacts with
the sodium chlorite to produce CIO? gas
and sodium chloride.
Figure 2-1. CDG Research Corporation
Bench-Scale Unit
adjusting the flow rate of the nitrogen/chlorine
different chlorine:nitrogen ratio.
For each molecule of chlorine gas, CI2, the
reaction produces two molecules of CIO2.
Therefore the volumetric concentration of
the CIO2 produced by the reaction is
approximately twice the concentration of
the chlorine feed gas. As long as the
chlorine concentration in the feed gas never
exceeds 5%, the concentration of the CIO2
can never enter the range (20% or greater)
in which it can spontaneously undergo a
self-propagating reaction.1 The production
rate of CIO2 is controlled either by
blend or by using a compressed gas with a
The CDG bench-scale unit includes a compressed gas cylinder containing 4% chlorine in
nitrogen (vol/vol), a sodium chlorite cartridge containing Saf-T-Chlor thermally stable
sodium chlorite pellets, a supply of nitrogen for purging the system prior to shutdown, a
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flow meter and valve for controlling the flow rate of nitrogen/chlorine (and thereby
controlling the production rate of CIO2), a pressure regulator and gauge for controlling the
gas pressure in the generator, pressure relief valves to protect against over-pressure, and on-
off valves for nitrogen/chlorine supply and nitrogen purge.
The CDG bench-scale unit consists of a cabinet about 20 inches (in) [51 centimeters (cm)]
high by 16 in (41 cm) wide by 9 in (23 cm) deep, plus the required nitrogen and chlorine
(4% chlorine in nitrogen) gas cylinders. The production rate of CIO2 is controlled and
recorded manually.
The CDG bench-scale unit was attached to a Plas-Labs Compact Glove Box
(Model 830-ABC) modified for this verification test (see Section 3.5.4.1). The connections
between the CDG bench-scale unit and the glove box consisted of flexible supply and
delivery gassing hoses connected to the glove box high-efficiency particulate air (HEPA)
filters. A CIO2 monitor also was placed inside the glove box to measure the concentration of
CIO2 during each run of this verification test. A hygrometer was added inside of the glove
box to measure relative humidity.
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Chapter 3
Test Design and Procedures
3.1 Introduction
This verification test was conducted according to procedures specified in the Test/QA Plan
for Verification of Chlorine Dioxide Gas Technologies for Decontaminating Indoor
Surfaces Contaminated with Biological or Chemical Agents.2 The biological and chemical
agents that pose a threat to buildings include toxic industrial chemicals, chemical warfare
agents, and biological warfare agents
(including biotoxins). The biological agent
selected for this verification test was
Bacillus anthracis (Ames strain). In addi-
tion, two biological surrogates were used:
B. subtilis (ATCC 19659) and GeobaciHus
stearothermophilus (ATCC 12980). The
latter two organisms also were used to
prepare biological indicators that were used
in the tests; spore strips containing
B. atrophaeus also were used. Seven
materials representing indoor surfaces
commonly found in buildings were used for
he verification testing. The indoor surfaces
ested (Figure 3-1) include
Industrial-grade caipet (IC)
Bare wood (pine lumber) (BWD)
Glass (GS)
Decorative laminate (DL)
Galvanized metal ductwork (GM)
Painted (latex, flat) wallboard paper (PW)
Painted (latex, semi-gloss) concrete cinder
block (PC).
BWD
Figure 3-1. Test Materials
The objective of the verification testing was to evaluate the efficacy of the CDG bench-scale
unit to decontaminate a biological agent/surrogate. Efficacy was tested by applying a
biological agent or surrogates to the surfaces of test coupons and, after using the CDG
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bench-scale unit, comparing the number of viable spores on decontaminated and control
(non-decontaminated) samples. Visual inspection of the physical integrity of the test
materials was performed, and observations were recorded before and after using the CDG
bench-scale unit in an effort to detect any degradation or chemical destruction of the
material itself.
3.2 Test Design
Coupons were cut from larger pieces of the representative materials for each of the seven
indoor surfaces (Section 3.1). These coupons measured 3/4 x 3 in (1.9 x 7.5 cm) and varied
in thickness from about 0.02 in (0.05 cm) to 0.28 in (0.71 cm), depending upon the
material. In triplicate, the coupons were placed into a biological safety cabinet (BSC), and
aliquots of an aqueous suspension of the biological agent/surrogate were added to the
surface of each coupon. Based upon the concentration of the spores in the aqueous
suspension, the number of spores added to each coupon was calculated. The coupons were
allowed to dry overnight. After drying, the inoculated coupons intended for decontamination
were transferred into a custom-modified glove box and placed horizontally on a wire rack.
Both blank (uncontaminated; N=2) and control (inoculated with spores, but not
decontaminated; N=3) coupons were prepared, together with the inoculated coupons that
were to be decontaminated (N=3).
Efficacy of the CDG bench-scale unit was determined by comparing the number of viable
spores recovered from the control coupons (not decontaminated) to the number recovered
from the decontaminated coupons, expressed as a log reduction. Following extraction of
spores from the test, control, and blank coupons, efficacy was further evaluated for each
biological agent/surrogate by transferring each coupon into liquid growth medium and
assessing bacterial growth after 1 and 7 days. (Note: The test/QA plan states that bacterial
growth will be assessed at 1 day after extraction; however, growth was also assessed at
7 days. Accordingly, a deviation to the test/QA plan was prepared.)
Physical degradation of the indoor materials used as test surfaces was evaluated informally
in conjunction with the efficacy testing procedure. After decontaminating the test coupons,
the appearance of the decontaminated coupons was observed; and any obvious visible
changes in the color, reflectivity, and apparent roughness of the coupon surfaces were noted.
These observations were preliminary in nature and not meant to be definitive.
3.3 Agents and Surrogates
The following biological agent was used for verification testing:
¦ Bacillus anthracis spores (Ames strain).
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To provide correlations with the biological agent results, two biological surrogates also were
used:
¦ Bacillus subtilis spores [American Type Culture Collection (ATCC) 19659]
¦ Geobacillus stearothermophilus spores (ATCC 12980).
Biological indicators and spore strips that were used to evaluate decontamination efficacy
included:
¦ Biological indicators (Apex Laboratories. Apex, North Carolina), approximately 1 x 106
spores each: Bacillus subtilis (ATCC 19659) and Geobacillus stearothermophilus
(ATCC 12980) spores on steel disks in sealed Tyvek pouches
¦ Spore strips (Raven Biological Laboratories. Omaha, Nebraska): with
Bacillus atrophaeus (ATCC 9372) spores, approximately 1 x 106 spores per strip on a
filter paper matrix in sealed glassine envelopes.
3.4 Test Sequence
In Table 3-1, a summary of the verification testing of the CDG bench-scale unit is
presented. Verification testing was performed during a 7-week period that commenced in
March 2004 and concluded in April 2004.
Table 3-1. Test Sequence and Parameters
lost
I'roccdu re
1'aramclcrs l\\alualcd
Data Produced
Biological
Enumerations
Log reduction (efficacy)
Efficacy Test
B. anthracis
B. subtilis
G. stearothermophilus
Liquid culture assessment of coupons
Positive/negative bacterial growth (1 and 7 days)
B. anthracis
B. subtilis
G. stearothermophilus
Biological indicators/spore strips
Positive/negative bacterial growth (1 and 7 days)
B. subtilis
G. stearothermophilus
B. atrophaeus
Coupon
Damage to test coupons
Visual observation of every test coupon in all
Damage
biological efficacy tests before and after
decontamination
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3.5 Coupon-Scale Testing
Coupon-scale testing was used to evaluate the decontamination efficacy of the CDG bench-
scale unit by extracting and measuring the viable biological spores on test coupons.
3.5.1 Preparation of Test Materials
Coupons used for biological agent decontamination were cut to about 3/4 x 3 in (1.9 x
7.5 cm) and prepared as shown in Table 3-2 by Battelle staff. Test coupons were visually
inspected, and the condition of each coupon was recorded. The length, width, and thickness
of the test coupons were measured and recorded. Chain-of-custody forms were used to
ensure that the test coupons were traceable throughout all phases of testing.
Table 3-2. Material Characteristics
Miiloriiil
l.ol. Itiitcli. or , ApproxiniiiU'
Miinuhicluri'iV ' ... .. ( . .
AS 1 M No., or .. .. .. ( oupon Size. M;iUti;iI rm);ir;ilion
Supplier Nsiiik' . ... ....
OI)mt\;iIioii 1. x \\ x II (inch)
Industrial-
grade Carpet
ShawTek,
EcoTek 6
http://www.shawcont
Shaw Industries,
Inc.
ract.com/html/html/
3 x 3/4 x 0.244
technical/technical.asp
Wiped with 70% isopropanol
Wood
Screen Molding
(Pine Wood)
Kingswood
Lumber
3 x 3/4 x 0.220
Wiped with 70% isopropanol
Glass
C1036
Brooks Brothers
3 x 3/4x0.114
Cleaned with acetone; wiped
with 70% isopropanol
Decorative
Laminate
Laminate/ Formica/
White Matte Finish
Solid Surface
Design
3 x 3/4 x 0.047
Wiped with 70% isopropanol
Galvanized
Metal
Ductwork
Industry HVAC
standard 24 Gauge
Galvanized Steel
Accurate
Fabrication
3 x 3/4 x 0.028
Cleaned with acetone; wiped
with 70% isopropanol
Wallboard
Paper
05-16-03; Set-E-
493; Roll-3
United States
Gypsum
Company
3 x 3/4 x 0.020
Roller painted on one side using
Martin Senour Paints. One
primer (#71 -1185) and two
finish (flat, #70-1001) coats;
wiped with 70% isopropanol
Concrete,
Cinder Block
ASTM C90
Wellnitz
3 x 3/4 x 0.280
Brush and roller painted all
sides. One coat Martin Senour
latex primer (#71-1185) and one
coat Porter Paints latex semi-
gloss finish (#919); wiped with
70% isopropanol
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The test materials were not autoclaved due to the risk of the materials being damaged during
the autoclaving process. Therefore, to maintain equivalent treatment/handling, each test
material was wiped with 70% isopropanol prior to inoculation (non-inoculated blanks were
also wiped with 70% isopropanol) with spores; however, this isopropanol wipe does not
guarantee sterility, especially with the porous materials.
3.5.2 Application of Agents to Test Coupons
Test coupons were laid flat in a BSC Class III and contaminated at challenge levels of
approximately 1 x 108 spores per coupon. Working stock suspensions of the spores at the
required concentration were transferred to the coupon using a micropipet by placing the
suspension (a 100-microliter aliquot of the suspension was applied) over the surface as
small droplets. (Note that spore suspension tended to form discrete droplets on the surface
of the glass, decorative laminate, painted wallboard paper, and painted concrete, but rapidly
penetrated into carpet and wood.) After contamination with biological agent or surrogate
suspension, the test coupons were allowed to dry overnight, undisturbed. The next day, the
inoculated test materials intended for decontamination (and one blank) were transferred to
the glove box that was attached to the CDG bench-scale unit (see Section 3.5.4.1). The
control inoculated test materials (not intended for decontamination) and one blank were left
undisturbed in a BSC Class n.
3.5.3 Confirmation of Surface Applications
To confirm the application density of the biological agents and surrogates, the B. anthracis
and surrogate spore suspensions used to contaminate the coupons were re-enumerated on
each day of use. This enumeration was carried out as described in Section 3.5.4.3.
3.5.4 Decontamination
3.5.4.1 Verification Testing Apparatus and Parameters
A Plas-Labs Compact Glove Box (Model 830-ABC) was utilized as the test chamber
(Figure 3-2). The inner dimensions of the glove box are 28 inches wide by 23 inches deep
by 29 inches high (71 cm by 59 cm by 74 cm). The glove box has a total volume of
11.2 cubic feet (317 liters). This glove box was modified and equipped to enable proper
generation of relative humidity and venting during operation of the liquid scrubber. Two
computer fans were mounted inside the glove box (Figure 3-3) to promote circulation. CIO2
is light-sensitive; therefore, the modified test chamber also was wrapped in brown paper.
During testing, the lights in the laboratory were turned off, and the only light source used
was a flashlight. The parameters used for this test, as specified by CDG Research
Corporation, were as follows:
• CIO2 Concentration: 2,000 parts per million (ppm)
• Exposure Time: 6 hours
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^ Liquid
Scrubber
Figure 3-2. Overview of Plas-Labs Compact Glove Box
• Relative Humidity: 70% minimum
• Temperature: room temperature (Actual temperature inside the glove box ranged
from 23°C to 27°C during testing.)
For this verification test, the CDG bench-scale unit was not designed to generate the
minimum 70% relative humidity required in the test chamber. To solve this problem,
Battelle staff configured a series of six nebulizers (Figure 3-4) inside the glove box
(nebulizers located inside the box near the top center) to generate water vapor. These
nebulizers were joined to a HEP A filter that was connected to an air pump. Air was pumped
through the nebulizers at 5 to 7 pounds per square inch gauge (gauge pressure), and a
relative humidity of 70 to 80% was achieved within 5 minutes and remained at this level
(with no maintenance or changes to the nebulizers) for the duration of the test. A traceable
hygrometer was placed inside the glove box to monitor relative humidity.
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Figure 3-3. Fans in the Plas-Labs Compact Glove Box
Figure 3-4. Nebulizers in the Plas-Labs Compact Glove Box
To neutralize the CIO2 removed from the glove box, the test chamber was attached to a
liquid scrubber containing 10% sodium hydroxide/10% sodium thiosulfate in water. The
liquid scrubber was connected to the facility heating, ventilating, and air conditioning
(HVAC) system for subsequent evacuation of the neutralized CIO2. At the end of each run,
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the liquid scrubber was turned on and run for approximately 1 hour. The liquid scrubber was
then turned off, but the facility HVAC system continued to draw air through the test
chamber. Since running the liquid scrubber as well as the facility HVAC system pulled a
vacuum on the testing chamber, the glove box was modified with HEPA-filtered valves
(Figure 3-2) that were opened during liquid scrubber operation for appropriate venting. The
next morning, CIO2 was not detectable in the test chamber.
3.5.4.2 Chlorine Dioxide Measurement
Spectroscopic monitoring of the concentration of CIO2 gas in the test chamber was
conducted during the decontamination to enable a real-time measurement of the CIO2
concentration. This monitoring was accomplished using customized CIO2 monitoring
devices manufactured by Science Application International Corporation (SAIC) under a
Defense Advanced Research Projects Agency contract (Figure 3-5). The monitors obtained
from SAIC measure the 360-nanometer ultraviolet (UV) absorption of CIO2 gas. The UV
optical beam is produced by a low-power light-emitting diode and detected by a photodiode.
The monitor provided an analog (4 to 20 milliamp) signal that was displayed using a Fluke
IV multimeter and recorded manually every 15 minutes during the 6-hour exposure time.
The analog signal was converted manually to a concentration value (ppm) using a
standardized table. Only one monitor was placed inside the box, sitting on the bottom in the
back right corner. Note that the monitor is a prototype; although Battelle did ascertain that
the monitor yielded data that compared well with data obtained using a published method,
the SAIC method has not been fully validated.
3.5.4.3 Decontamination Efficacy
Biological agent/surrogate decontamination efficacy was quantified by measuring the viable
spores on both exposed (test) and unexposed (control) coupons. Each coupon was placed in
a 50 milliliter (mL) test tube containing 10 mL of sterile phosphate-buffered saline to which
0.1% Triton X-100 had been added. The purpose of the Triton X-100 was to minimize
clumping of spores. For spore extraction, the tubes were agitated on an orbital shaker for
15 minutes at room temperature. Each tube was then heat-shocked at 60 to 65 °C for 1 hour
to kill vegetative bacteria. Following the heat-shock, 1.0 mL of each extract was removed,
and a series of dilutions through 10"7 were prepared in sterile water.
Spore viability was determined by dilution plating, using both the undiluted extracts and the
successive dilutions of each extract. One hundred microliters of the undiluted extract and of
each serial dilution were plated onto tryptic soy agar plates in triplicate, allowed to dry, and
incubated overnight at 35 to 37 °C for B. anthracis and B. subtilis and at 55 to 60 °C for
G. stearothermophilus. [Note: The incubation of B. anthracis Ames for 24 hours is based
upon in-house standard operating procedures and practical laboratory experience. Within
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Figure 3-5. SAIC Chlorine Dioxide Monitor
24 hours, the B. anthracis Ames colonies are large (about 0.5 cm in diameter); therefore,
incubating the tryptic soy agar plates for an additional 12 to 24 hours would potentially
decrease the sensitivity of counting colonies due to the potential for overgrowth on the
plates.] Plates were enumerated the next day, and the colony-forming units (CFU)/mL were
determined by multiplying the average number of colonies per plate by the reciprocal of the
dilution. Data were expressed as a mean ± standard deviation (SD) of the number of CFUs
observed. To calculate the efficacy of the decontamination treatment, the number of spores
remaining on the decontaminated test coupons was compared to the number of spores on the
control coupons. Efficacy for biological agents was expressed in terms of a log reduction.
The percent recovery of spores on all seven test materials ranged from 83 to 4%, with an
average of 37% recovery; therefore, it was assumed that viable spores could remain on the
test materials. After the extraction process described above, each coupon was transferred to
a sterile 50-mL tube containing 20 mL of tryptic soy broth culture medium to promote spore
germination, thereby enabling the vegetative bacteria to proliferate. The vials were sealed
and incubated on an orbital shaker at the appropriate temperatures (see above) for
B. anthracis or the surrogate organism. At 1 and 7 days post-decontamination, the tubes
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were visually assessed qualitatively for viability. Viability, "growth," was determined if the
liquid culture medium turned cloudy, while "no growth" was determined when the liquid
medium remained clear. However, since the test materials were not sterilized by auto-
claving, this type of assessment may not discriminate between the growth of B. anthracis
and other microorganisms. (Therefore, growth in these liquid culture samples should not be
interpreted as decreased efficacy.)
The biological indicators and spore strips were also evaluated in a similar manner at 1 and
7 days post-decontamination for "growth" or "no growth."
3.5.5 Observation of Surface Damage
Following decontamination, each test surface was examined visually to establish whether
decontamination using the CDG bench-scale unit caused any obvious damage to the surface.
The coupons were observed immediately after completing the decontamination process, but
before post-decontamination sampling. The surface was visually inspected by comparing the
decontaminated test surface with control coupons of the same test material. Differences in
color, reflectivity, contrast, and roughness were assessed and recorded. These assessments,
as stated previously, are qualitative in nature and not intended to be rigorous.
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Chapter 4
Quality Assurance/Quality Control
QA/quality control (QC) procedures were performed in accordance with the Quality
Management Plan (QMP) for the BDT Center3 and the test/QA plan for this verification
test.2 QA/QC procedures and results are described below.
4.1 Equipment Calibration
All equipment (e.g., pipets, incubators, BSCs) used at the time of testing was verified as
being certified, calibrated, or validated.
4.2 Audits
Two types of audit were performed during the verification test: a technical systems audit
(TSA) of the verification test performance and an audit of data quality. Audit procedures are
described below.
4.2.1 Technical Systems Audit
The Battelle Quality Assurance Unit conducted a TSA on March 24, 2004, to ensure that the
verification test was being conducted in accordance with the test/QA plan2 and the BDT
Center QMP.3 As part of the TSA, test procedures were compared to those specified in the
test/QA plan, and data acquisition and handling procedures were reviewed. Observations
and findings from the TSA were documented and submitted to the Battelle verification test
coordinator for response. None of the findings of the TSA required corrective action. TSA
records are permanently stored with the ETV quality assurance manager.
4.2.2 Audit of Data Quality
At least 10% of the data acquired during the verification test were audited. A Battelle
quality assurance 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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4.3 QA/QC Reporting
Each audit was documented in accordance with Section 3.3.4 of the QMP for the ETV BDT
Center.3 Once the audit reports were prepared, the Battelle verification test coordinator
ensured that a response was provided for each adverse finding or potential problem and
implemented any necessary follow-up corrective action. A Battelle quality assurance auditor
ensured that follow-up corrective action was taken. The results of the TS A were submitted
to the EPA.
4.4 Data Review
Records generated in the verification test received a QC/technical review and a QA review
before they were used to calculate, evaluate, or report verification results. Table 4-1
summarizes the types of data recorded and reviewed. All data were recorded by Battelle
staff. The person performing the review was involved in the experiments and added his/her
initials and the date to a hard copy of the record being reviewed.
Table 4-1. Summary of Data Recording Process
Diilii 1<> IU'
Kccortktl
1 >akv link's..!'kM
events
WIUTl'
Ki-cortk-tl
1 >;il;i li'i'ins
Mow on en
Ki'conktl
Slai l.viul nf k'M, aiul al
each change of a test
parameter
Disposition ol'
Diilii
1 sed In ni'gaiii/e.vlkvk k'M
results; manually incorporated
into spreadsheets as necessary
Test parameters
(agent/surrogate identi-
ties, concentrations, test
surfaces, test conditions,
etc.)
Data forms
When set or changed, or
as needed to document the
sequence of test
Used to organize/check test
results; manually incorporated
in data spreadsheets as
necessary
Sampling data
Data forms
At least at start/end of
reference sample, and at
each change of a test
parameter
Used to organize/check test
results; manually incorporated
into spreadsheets as necessary
Biological enumeration
and liquid culture assess-
ment, chain of custody,
and results
Data forms
Throughout sample
handling and analysis
process
Transferred to spreadsheets
Records and observa-
tions of CDG bench-
scale unit use
Reading from the
SAIC monitor;
data forms
Throughout use of the
CDG bench-scale unit
Reviewed and summarized to
support data interpretation
Surface damage
Data forms
Start/end of test
Used to assess damage of test
materials following use of the
CDG bench-scale unit
15
-------
Chapter 5
Statistical Methods
The statistical methods for evaluating the efficacy of the CDG bench-scale unit are
presented in this chapter. Qualitative observations also were used to evaluate verification
test data.
5.1 Efficacy Calculations
For biological agents and surrogates, decontamination efficacy was calculated as the log
reduction in viable organisms achieved by the CDG bench-scale unit. The efficacy (E), or
log reduction, for the biological agent, or surrogates was calculated as
E = log (N°/N)
where N° is the mean number of viable organisms recovered from the control coupons (i.e.,
those not subjected to decontamination), and N is the number of viable organisms recovered
from each test coupon after decontamination. For decontaminated samples where viable
organisms were not detected, the efficacy was calculated as the log of the mean number of
viable organisms on the control coupons. Using the calculated log reduction for each test
coupon, the mean log reduction (efficacy) ± SD was calculated.
Percent recovery was calculated for each type of test material inoculated with each
biological agent/surrogate. Percent recovery (mean ± SD) was calculated by dividing the
number of biological organisms in the treated sample by the number of biological organisms
in the controls (non-decontaminated).
5.2 Statistical Analysis
For each material and species combination, log reduction was calculated as described above,
resulting in a total of 63 log reduction values (3 coupons for each of seven materials
analyzed in triplicate). In cases where no viable colonies remained after decontamination,
one colony was assumed to be present for the purpose of this calculation. A two-way
analysis of variance (ANOVA) model with main effects for Bacillus species and test
material and interactions was fitted to the log reduction data. This model was used to
compare each mean to zero, compare each surrogate to B. anthracis (for each material), and
16
-------
compare each surrogate to B. anthracis for porous and non-porous materials. T-tests or
statistical contrasts were used for the comparisons, with no adjustment for multiple
comparisons. The ANOVA model was fitted using the SAS (Version 8.2) GLM procedure.
17
-------
Chapter 6
Test Results
The results of the verification test of the CDG bench-scale unit are presented in this section.
6.1 Efficacy
6.1.1 Bacillus anthracis Ames Spores
Exposure of material test coupons contaminated with B. anthracis Ames spores to the CDG
bench-scale unit resulted in decontamination that varied according to the type of the test
material (Table 6-1). The mean log reduction of detectable viable B. anthracis Ames spores
ranged from 4.33 to >7.79 across all seven test materials. Three of these test materials (IC,
BWD, PC) can be considered porous (on the inoculated surface), while the other four test
materials (GS, DL, GM, PW) can be considered non-porous (on the inoculated surface). The
log reduction in viable spores detected on the porous materials was 4.62, 4.33, and 7.25 for
IC, BWD, and PC, respectively. The log reduction in viable spores detected on the non-
porous materials was 5.70, 4.57, > 7.79, and > 7.68 for GS, DL, GM, and PW, respectively.
Results from the liquid culture growth assessment at 1 and 7 days post-decontamination, to
evaluate whether viable B. anthracis Ames spores may remain on the test materials
following the extraction step, are provided in Table 6-2. Clear liquid medium indicates that
no growth of B. anthracis Ames or other microorganisms in or on the test material occurred
during the incubation period. (Note: This type of assessment may not discriminate between
the growth of B. anthracis or other microorganisms. The presence of growth in media
containing blanks indicates that viable microorganisms, other than the spiked B. anthracis,
may have been present on or in the test material and not killed by either 70% isopropanol
wipe or the decontamination treatment.)
None of the liquid culture samples for IC (both control and decontaminated) exhibited
bacterial growth. Although it was not known prior to the start of testing, the brand of IC
used for this test contains a product known as FlorSept, which is considered a broad
spectrum antimicrobial that is effective against Gram-positive and Gram-negative bacteria,
as well as mold and fungi. It appears that, under the conditions employed for this
verification test, the FlorSept may not be sporicidal since viable B. anthracis Ames spores
were extracted from the IC and cultured on tryptic soy agar plates. Therefore, it is possible
18
-------
that, in the liquid cultures, FlorSept may inhibit growth of vegetative cells derived from
germination of the B. anthracis Ames spores. This growth inhibition was also observed for
B. subtilis (Table 6-5) and G. stearothermophilus (Table 6-8).
After decontamination, GM and PC, in addition to IC, exhibited no growth after seven days
incubation in nutrient broth. After decontamination, GS and PW each exhibited growth in
only one of the three replicate culture media. After decontamination, BWD and DL each
exhibited growth in each of the three replicate culture media.
Table 6-1. CDG Bench-Scale Unit Decontamination of Bacillus anthracis Ames Spores3
Test Mali-rial
Inoculum
Total No. Spores
'( Ucco\cr\
l-llTicac\
Industrial-Grade Carpet (IC)
Control
9.40 x 107
5.29 ± 0.36 x 107
56.2 ±3.85
b
Decontaminated
9.40 x 107
2.41 ± 2.03 x 103
<0.01
4.62 ±0.76 (4.11-5.50)
Blank (control)
0
0
0
-
Blank (decontaminated)
0
0
0
-
Bare Wood (BWD)
Control
9.27 x 107
9.14 ± 0.72 x 106
9.86 ±0.78
Decontaminated
9.27 x 107
4.67 ± 2.33 x 102
<0.001
4.33 ± 0.20 (4.10-4.48)
Blank (control)
0
0
0
-
Blank (decontaminated)
0
0
0
-
Glass (GS)
Control
9.40 x 107
7.77 ± 0.75 x 107
82.6 ± 8.03
-
Decontaminated
9.40 x 107
1.89 ± 1.34 x 102
<0.001
5.70 ± 0.35 (5.35-6.06)
Blank (control)
0
0
0
-
Blank (decontaminated)
0
0
0
-
Decorative Laminate (DL)
Control
9.27 x 107
5.42 ± 0.75 x 107
58.5 ± 8.03
-
Decontaminated
9.27 x 107
1.81 ± 1.48 x 103
<0.01
4.57 ± 0.34 (4.19-4.85)
Blank (control)
0
0
0
-
Blank (decontaminated)
0
0
0
-
Galvanized Metal Ductwork (GM)
Control
9.27 x 107
6.16 ± 0.02 x 107
66.4 ± 0.25
-
Decontaminated
9.27 x 107
0
0
> 7.79 ± 0 (7.79)
Blank (control)
0
0
0
-
Blank (decontaminated)
0
0
0
-
Painted Wallboard Paper (PW)
Control
9.40 x 107
4.78 ± 0.49 x 107
50.8 ±5.21
Decontaminated
9.40 x 107
0
0
> 7.68 ± 0 (7.68)
Blank (control)
0
0
0
-
Blank (decontaminated)
0
0
0
-
Painted Concrete (PC)
Control
9.27 x 107
5.73 ± 1.73 x 107
61.8 ±18.7
-
Decontaminated
9.27 x 107
1.10 ±1.91x10
<0.0001
7.25 ± 0.88 (6.24-7.76)
Blank (control)
0
0
0
-
Blank (decontaminated)
0
0
0
-
a Data are expressed as mean (± SD) total number of spores, percent recovery, and efficacy (log reduction).
The efficacy range is shown in parentheses.
b Not Applicable
19
-------
Table 6-2. Liquid Culture Assessment of Bacillus anthracis Ames Spores
1 >:i\ 1 1 >:i\ 7
MiiliTiiil s| s: s, |}| s| s: s, |}|
Industrial-Grade Carpet (IC) Control
Decontaminated
Bare Wood (BWD) Control
Decontaminated
+ + + -
+ + + +
+ + + -
Glass (GS) Control
Decontaminated
+ + + -
1 1
+
+ +
+
Decorative Laminate (DL) Control
Decontaminated
+ + + -
+ + + -
+ + + +
Galvanized Metal Ductwork (GM) Control
Decontaminated
+ + + -
+ + + -
Painted Wallboard Paper (PW) Control
Decontaminated
+ + + -
1 1
+
+ +
+
Painted Concrete (PC) Control
Decontaminated
+ + + -
+ + + -
51 = Sample 1
52 = Sample 2
53 = Sample 3
B1 = Blank (not inoculated with B. anthracis Ames spores)
"+" = growth; = no growth
For all tests using B. anthracis, the control (not exposed to CIO2) biological indicators and
spore strips exhibited growth in the liquid cultures at both 1 and 7 days. No growth in the
liquid cultures was observed at 1 and 7 days for the biological indicators and spore strips
subject to CIO2 exposure using the CDG bench-scale unit. A representation of the data from
a single test day is shown in Table 6-3.
20
-------
Table 6-3. Representative Liquid Culture Assessment of Biological Indicators/Spore
Strips
Indicator (Opiumism)
l)a\ 1
si s: S3
l):i\ 7
sis: s3
Biological Indicator (B. subtilis ATCC 19659) Control
Biological Indicator (G. stearothermophilus ATCC 12980) Control
Spore Strip (B. atrophaeus ATCC 9372) Control
+ + +
+ + +
+ + +
+ + +
+ + +
+ + +
Biological Indicator (B. subtilis ATCC 19659) Decontaminated
Biological Indicator (G. stearothermophilus ATCC 12980) Decontaminated
Spore Strip (B. atrophaeus ATCC 9372) Decontaminated
51 = Sample 1
52 = Sample 2
53 = Sample 3
"+" = growth; = no growth
6.1.2 Bacillus subtilis (ATCC19659) Spores
Exposure of test coupons contaminated with B. subtilis spores to the CDG bench-scale unit
resulted in decontamination that varied according to the type of test material. The log
reduction of detectable viable B. subtilis spores ranged from approximately 4.44 to 5.57 for
all seven test materials (Table 6-4). The log reduction in viable spores detected on the
porous materials was 4.44, 4.48, and 4.74 for IC, BWD, and PC, respectively. The log
reduction in viable spores detected on the non-porous materials was 5.23, 5.14, 5.57, and
4.62 for GS, DL, GM, and PW, respectively.
21
-------
Table 6-4. CDG Bench-Scale Unit Decontamination of Bacillus subtilis Spores3
Test Material
Inoculum
Total No. Spores
'( UcCO\ IT\
Ll'l'icacx
Industrial-Grade Carpet (IC)
Control
8.43 x 107
3.60 ± 1.18 x 107
42.7 ± 14.1
b
Decontaminated
8.43 x 107
1.37 ± 0.52 xlO3
<0.01
4.44 ±0.17 (4.28-4.62)
Blank (control)
0
0
0
-
Blank (decontaminated)
0
0
0
-
Bare Wood (BWD)
Control
8.80 x 107
4.15 ± 0.20 x 106
4.72 ± 0.22
-
Decontaminated
8.80 x 107
1.67 ± 1.20 x 102
<0.001
4.48 ± 0.33 (4.14-4.79)
Blank (control)
0
0
0
-
Blank (decontaminated)
0
0
0
-
Glass (GS)
Control
8.43 x 107
4.10 ± 1.37 x 107
48.6 ± 16.3
Decontaminated
8.43 x 107
2.89 ± 2.14 x 102
<0.001
5.23 ±0.31 (4.89-5.49)
Blank (control)
0
0
0
-
Blank (decontaminated)
0
0
0
-
Decorative Laminate (DL)
Control
8.80 x 107
6.51 ± 0.60 x 107
74.0 ± 6.83
-
Decontaminated
8.80 x 107
5.45 ± 3.67 x 102
<0.001
5.14 ±0.27 (4.83-5.34)
Blank (control)
0
0
0
-
Blank (decontaminated)
0
0
0
-
Galvanized Metal Ductwork (GM)
Control
8.80 x 107
7.04 ± 0.43 x 107
80.0 ±4.85
-
Decontaminated
8.80 x 107
1.89 ± 0.19 x 102
<0.001
5.57 ± 0.05 (5.55-5.63)
Blank (control)
0
0
0
-
Blank (decontaminated)
0
0
0
-
Painted Wallboard (PW)
Control
8.43 x 107
9.63 ± 0.35 x 106
11.4 ±0.42
-
Decontaminated
8.43 x 107
1.87 ± 3.15 xlO3
<0.01
4.62 ± 1.20 (3.24-5.47)
Blank (control)
0
0
0
-
Blank (decontaminated)
0
0
0
-
Painted Concrete (PC)
Control
8.80 x 107
2.85 ±0.11 x 107
32.4 ± 1.23
-
Decontaminated
8.80 x 107
5.88 ± 3.85 xlO2
<0.001
4.74 ± 0.26 (4.44-4.93)
Blank (control)
0
0
0
-
Blank (decontaminated)
0
0
0
-
a Data are expressed as mean (± SD) total number of spores, percent recovery, and efficacy (log reduction).
The efficacy range is shown in parentheses.
b Not Applicable
A liquid culture growth assessment at 1 and 7 days post-decontamination was performed to
determine whether viable B. subtilis spores remained on the test materials following the
extraction step (Table 6-5). Only GS and IC exhibited no growth after decontamination.
(Note that IC also showed no growth in control samples. This is attributed to the presence of
FlorSept® broad spectrum antibacterial treatment in the carpet samples.) All samples of PC,
DL, BWD, and GM (except one decontaminated case), including both control and treatment
blanks, exhibited growth in the liquid culture medium. The presence of growth in media
containing blanks indicates that viable microorganisms, other than the spiked surrogate, may
22
-------
have been present on or in the test material (microbes could be indigenous or introduced by
handling coupons) and not killed by either the 70% isopropanol wipe or the
decontamination treatment.
Table 6-5. Liquid Culture Assessment of Bacillus subtilis Spores
1 est Mali-rial
1 >:i\ 1 1 >:i\ 7
SI S2 S3 B1
SI S2 S3 III
Industrial-Grade Carpet (IC) Control
Decontaminated
Bare Wood (BWD) Control
Decontaminated
+ + + +
+ +
+ + + +
+ + + +
Glass (GS) Control
Decontaminated
+ + + -
+ + + -
Decorative Laminate (DL) Control
Decontaminated
+ + + -
+ +
+ + + +
+ + + +
Galvanized Metal Ductwork (GM) Control
Decontaminated
+ + + -
+ + + +
+ + - +
Painted Wallboard Paper (PW) Control
Decontaminated
+ + + -
+ + + +
+
Painted Concrete (PC) Control
Decontaminated
+ + + -
+ + + +
+ + + +
51 = Sample 1
52 = Sample 2
53 = Sample 3
B1 = Blank (not inoculated with B. subtilis spores)
"+" = growth; = no growth
For all tests using B. subtilis ATCC (19659) and B. atrophaeus (ATCC 9372) the control (not
exposed to CIO2) biological indicators and spore strips exhibited growth in the liquid
cultures at both 1 and 7 days. No growth in the liquid cultures was observed at 1 and 7 days
for the biological indicators and spore strips subject to CIO2 exposure using the CDG bench-
scale unit. A representation of the data from a single test day is shown in Table 6-6.
23
-------
Table 6-6. Representative Liquid Culture Assessment of Biological Indicators/Spore
Strips
Indicator (Opiumism)
l)a\ 1
si s: S3
l);i\ 7
sis: s3
Biological Indicator (B. sublilis ATCC 19659) Control
Spore Strip (B. atrophaeus ATCC 9372) Control
+ + +
+ + +
+ + +
+ + +
Biological Indicator (B. subtilis ATCC 19659) Decontaminated
Spore Strip (B. atrophaeus ATCC 9372) Decontaminated
51 = Sample 1
52 = Sample 2
53 = Sample 3
"+" = growth; = no growth
6.1.3 Geobacillus stearothermophilus (ATCC12980) Spores
Exposure of test coupons contaminated with G. stearothermophilus (ATCC 12980) spores
to the CDG bench-scale unit resulted in variable decontamination. The log reduction of
detectable viable G. stearothermophilus spores (ATCC 12980) ranged from approximately
3.22 to 5.79 for all seven test materials (Table 6-7). The log reduction in viable spores
detected on the porous materials was 3.22, 3.78, and 5.79 for IC, BWD, and PC,
respectively. The log reduction in viable spores detected on the non-porous materials was
3.87, 4.44, 3.43, and 5.62 for GS, DL, GM, and PW, respectively.
Results from the liquid culture growth assessment at 1 and 7 days post-decontamination,
performed to determine whether viable G. stearothermophilus spores remained on the test
materials following the extraction step, are provided in Table 6-8. Similar to findings with
other spores, IC exhibited no growth in liquid culture media at 1 or 7 days for controls or
decontaminated test coupons. This is likely due to the IC being manufactured with a broad
spectrum antimicrobial treatment. All other controls for all materials exhibited growth at 1
and 7 days. Decontaminated test coupons of PC and PW (except one 7-day case) exhibited
no growth. After decontamination, BWD, GL (except in one case), DL, and GM exhibited
growth after seven day incubation in liquid culture media. In most cases
G. stearothermophilus or other microbial growth may occur after treatment of test samples.
24
-------
Table 6-7. CDG Bench-Scale Unit Decontamination of Geobacillus stearothermophilus
Spores3
Test Mali-rial
Inoculum
Total No. Spores
'( Ucco\cr\
l-lN'icac\
Industrial-Grade Carpet (IC)
Control
1.13 x 10s
8.70 ± 0.67 x 106
7.70 ± 0.60
b
Decontaminated
1.13 x 10s
5.30 ± 0.65 x 103
<0.01
3.22 ±0.05 (3.17-3.28)
Blank (control)
0
0
0
-
Blank (decontaminated)
0
0
0
-
Bare wood (BWD)
Control
1.17 xlO8
4.71 ± 0.64 x 106
4.03 ± 0.55
-
Decontaminated
1.17 xlO8
7.89 ± 1.50 xlO2
<0.001
3.78 ± 0.08 (3.70-3.87)
Blank (control)
0
0
0
-
Blank (decontaminated)
0
0
0
-
Glass (GS)
Control
1.13 x 108
3.19 ± 0.39 xlO7
28.2 ±3.41
Decontaminated
1.13 x 108
4.85 ± 2.63 x 103
<0.01
3.87 ± 0.29 (3.64-4.20)
Blank (control)
0
0
0
-
Blank (decontaminated)
0
0
0
-
Decorative Laminate (DL)
Control
1.17 xlO8
2.87 ± 0.14 x 107
24.5 ± 1.22
-
Decontaminated
1.17 xlO8
1.08 ± 0.37 xlO3
<0.001
4.44 ±0.15 (4.29-4.59)
Blank (control)
0
0
0
-
Blank (decontaminated)
0
0
0
-
Galvanized Metal Ductwork (GM)
Control
1.17 xlO8
3.67 ± 0.82 x 107
31.3 ±7.00
-
Decontaminated
1.17 xlO8
1.39 ± 0.35 xlO4
0.01 ± 0.003
3.43 ±0.11 (3.33-3.56)
Blank (control)
0
0
0
-
Blank (decontaminated)
0
0
0
-
Painted Wallboard Paper (PW)
Control
1.13 x 108
7.42 ± 1.04 x 106
6.57 ± 0.92
Decontaminated
1.13 x 108
6.67 ± 8.84
<0.0001
5.62 ±1.14 (4.65-6.87)
Blank (control)
0
0
0
-
Blank (decontaminated)
0
0
0
-
Painted Concrete (PC)
Control
1.17 xlO8
8.01 ± 0.30 xlO6
6.85 ±0.33
-
Decontaminated
1.17 xlO8
3.33 ± 3.35 x 10
<0.0001
5.79 ± 0.98 (5.08-6.90)
Blank (control)
0
0
0
-
Blank (decontaminated)
0
0
0
-
a Data are expressed as mean (± SD) total number of spores, percent recovery, and efficacy (log reduction).
The efficacy range is shown in parentheses.
b Not Applicable
25
-------
Table 6-8. Liquid Culture Assessment of Geobacillus stearothermophilus Spores
1 >:i\ 1 1 >:i\ 7
MiiliTiiil s| s: s, |}| s| s: s, |}|
Industrial-Grade Carpet (IC) Control
Decontaminated
Bare Wood (BWD) Control
Decontaminated
+ + + +
+
+ + + +
+ + + +
Glass (GS) Control
Decontaminated
1 1
+ +
+
+
1 1
+ +
+ +
+
Decorative Laminate (DL) Control
Decontaminated
+ + + -
1 1
+ +
+ +
+ +
Galvanized Metal Ductwork (GM) Control
Decontaminated
1 1
+ +
+ +
+ +
1 1
+ +
+ +
+ +
Painted Wallboard Paper (PW) Control
Decontaminated
+ + + -
1 1
+ +
+
+
Painted Concrete (PC) Control
Decontaminated
+ + + -
+ + + -
51 = Sample 1
52 = Sample 2
53 = Sample 3
B1 = Blank (not inoculated with G. stearothermophilus spores)
"+" = growth; = no growth
Analysis of indicators containing G. stearothermophilus, with and without decontamination,
provided consistent results. For all tests using G. stearothermophilus, the control (not
exposed to CIO2) biological indicators exhibited growth in the liquid cultures at both 1 and
7 days. No growth in the liquid cultures was observed at 1 and 7 days for the biological
indicators subjected to CIO2 exposure using the CDG bench-scale unit. A representation of
the data from a single test day is shown in Table 6-9.
26
-------
Table 6-9. Representative Liquid Culture Assessment of Biological Indicators/Spore
Strips
1 >:i\ 1 1 >:i\ 7
liidkiitur (Orjiiiiiism)
Itiolo^icul Imliculor {(i. slciirolhcriiiopliiliis \T( ( 129X01 Cunli'ul
Spore Strip (B. atrophaeus ATCC 9372) Control
+ + +
+ + +
+ + +
+ + +
Biological Indicator (G. stearothermophilus ATCC 12980) Decontaminated
Spore Strip (B. atrophaeus ATCC 9372) Decontaminated
51 = Sample 1
52 = Sample 2
53 = Sample 3
"+" = growth; = no growth
6.1.4 Statistical Analysis
Table 6-10 presents the mean log reduction in spores sorted by material type. Significant
differences are denoted in the table as well. All means were significantly different from
zero, indicating that the CDG bench-scale unit decontaminated statistically significant
numbers of spores on these materials.
Table 6-10. Statistical Analysis of Mean Efficacy (Log Reduction) for Spores
Material
B. tmlhrticis
B. \ublili\
(i. stcarothcrinophilus
Industrial-Grade Carpet (IC)
4.62a
4.44a
3.22a'b
Porous
Painted Concrete (PC)
7.25a
4.74a'b
5.79a'b
Bare Wood (BWD)
4.33a
4.48a
3.78a
Glass (GS)
5.70a
5.23a
3.87a'b
Non-Porous
Decorative Laminate (DL)
4.57a
5.14a
4.44a
Painted Wallboard Paper (PW)
> 7.68a
4.62a'b
5.62a'b
Galvanized Metal Ductwork (GM)
> 7.79a
5.57a'b
3.43a,b
"Mean significantly different from 0 (P < 0.05).
bSurrogate significantly different from B. anthracis for specified material (P < 0.05).
Comparisons within each material indicated that the CDG bench-scale unit decontaminated
significantly fewer B. subtilis and G. stearothermophilus spores than B. anthracis spores for
PC, PW, and GM. Significantly fewer G. stearothermophilus spores were decontaminated
by the CDG bench-scale unit compared to B. anthracis spores on IC and GS.
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6.2 Damage to Coupons
Subsequent to decontamination, the test coupons were evaluated qualitatively for visible
surface damage. No damage (e.g., change in surface texture, color) and no visible changes
to any of the test materials were observed during this verification test with the exception of
IC. Exposure to the CIO2 appeared to produce a bleaching effect (all colors in the multicolor
design were affected) of the IC.
6.3 Other Factors
6.3.1 Operation of the CDG Bench-scale Unit
The CDG bench-scale unit was operated for approximately 50 hours during this verification
test. The CDG bench-scale unit can be set up for operation within minutes. For this
verification test, the liquid scrubber took hours to setup due to connections to the glove box
and facility exhaust system. As described in Section 3.5.4.1, a nebulizer system had to be
utilized to achieve the appropriate relative humidity (75%) within the glove box for each run
of the CDG bench-scale unit. At the end of each run, the CIO2 was drawn out of the glove
box using the liquid scrubber as described in Section 3.5.4.1. No maintenance was required
for the CDG bench-scale unit or liquid scrubber.
The CIO2 concentration was monitored in real time, and the data were recorded manually
(see Section 3.5.4.2). Figure 6-1 is a graphical representation of CIO2 concentrations in the
glove box (measured in real time using the SAIC spectrometer) during a typical 6-hour run.
Chlorine Dioxide Concentration
Time (hours)
Figure 6-1. Representative Chlorine Dioxide Concentration from a Single Experiment
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6.3.2 Operator Bias
The CDG bench-scale unit is operated by manually regulating the introduction of CIO2 into
the exposure chamber. The real-time measurement of CIO2 enabled the operator to maintain
the desired concentration of CIO2 within the glove box. During the 6-hour contact time, the
C102 concentration would decrease slightly over time (concentration of CIO2 in the glove
box during the 6-hour contact time is shown in Figure 6-1). This decrease in the CIO2
concentration was counteracted by the operator manually introducing additional CIO2 gas
into the glove box by temporarily increasing the flow rate. The decontamination and
neutralization steps were run the same day of testing; therefore, a total run time from start to
finish was approximately 8 hours. Note that the duration of the humidification phase was
5 minutes, the neutralization phase was 30 to 60 minutes, and further aeration of the box
occurred overnight.
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Chapter 7
Performance Summary
For this verification test, the CDG bench-scale unit demonstrated a range of decontamina-
tion efficacy for B. anthracis Ames, B. subtilis (ATCC 19659), and G. stearothermophilus
(ATCC 12980) on all seven test materials. Based on these results, it did not appear that the
porosity of the different material types influenced the efficacy of decontamination for the
three organisms. However, IC and BWD consistently exhibited the lowest level of spore
decontamination for B. anthracis Ames, B. subtilis (ATCC 19659), and G. stearothermo-
philus (ATCC 12980). The CDG bench-scale unit decontaminated significantly fewer
B. subtilis and G. stearothermophilus spores than B. anthracis spores for PC, PW, and GM.
Significantly fewer G. stearothermophilus spores were decontaminated by the CDG bench-
scale unit compared to B. anthracis spores on IC and GS.
A quantitative evaluation of the results indicates that the log reduction values for detectable
viable B. anthracis Ames spores ranged from 4.33 to > 7.79 across all seven test materials.
The log reduction values for detectable viable B. subtilis spores ranged from 4.44 to 5.57 for
all seven test materials. The log reduction values for detectable viable G. stearothermo-
philus spores (ATCC 12980) ranged from 3.22 to 5.79 for all seven test materials. Signifi-
cant differences in efficacy were observed between B. anthracis and B. subtilis on PC, PW,
and GM. The only damage observed for any of the test materials subjected to the CDG
bench-scale unit was a bleaching effect on the IC. The differences in decontamination
efficacy across the seven test materials could be a result of the interactions of the different
spore types with each substrate. The observed differences in log reductions and recovery
rates of B. anthracis, B. subtilis, and G. stearothermophilus spores suggest that the test
material composition and/or porosity affect decontamination efficacy and spore recovery.
Although clumping or non-homogenous distribution of spores can occur during the
inoculation and subsequent drying on the non-porous materials, it is assumed that the spores
remain predominantly at the material surface. However, in addition to clumping or non-
homogenous distribution of spores at the surface, the porous characteristics of the industrial
carpet, bare pine wood, and painted concrete materials can also lead to spores penetrating
and embedding into the test material. Such penetration and embedding of spores into the test
materials could preclude the interaction of the decontaminant with the spores, thereby
decreasing the potential for inactivation and affecting spore recovery. Therefore, the
observed differences in log reductions of all three bacterial spore species across all materials
may reflect chlorine dioxide-induced killing of spores at the material surface with little to no
effect on spores that may have penetrated into the test material. For differences in recovery,
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the penetration of bacterial spores into the porous test materials is plausible and differences
in spore coat composition may affect the interactions of the three types of spores with the
different materials. Further work in evaluating spore deposition and material matrix
interactions would be useful to support these conclusions.
A qualitative evaluation of the performance of the CDG bench-scale unit showed that the
control (not exposed to the CDG bench-scale unit) biological indicators and spore strips
used in this test displayed growth in the liquid cultures at both 1 and 7 days. When the
biological indicators and spore strips were exposed to the CDG bench-scale unit, no growth
was observed at 1 and 7 days. Based on these results, the CDG bench-scale unit inactivated
both the biological indicators (containing B. subtilis and G. stearothermophilus) and
sporesstrips (containing B. atrophaeus), all of which contained spore loads of approximately
1 x 106 spores per indicator or spore strip. On the basis of the biological indicator and spore
strip results, the technology effectively inactivated the surrogate and spore strip organisms.
However, the results obtained using porous materials indicates that the performance of the
technology may be influenced by the matrix in which or on which the microorganisms and
dispersed.
In an effort to assess whether viable spores remained in or on the coupons following
decontamination and subsequent extraction, both control and decontaminated coupons were
placed in tubes containing nutrient broth (as called for in the test/QA plan) and incubated for
7 days. The tubes were examined at 1 and 7 days for cloudiness as an indicator of growth.
Besides the controls and decontaminated samples, growth was observed in many of the
tubes containing blank coupons (excluding industrial carpet coupons); therefore these
results were inconclusive. The unexpected growth may have been due to ineffective
sterilization (the 70% isopropanol wipe did not sterilize the internal portions of the coupons)
prior to inoculating the coupons. Identification of organisms causing cloudiness in the
nutrient broth was beyond the scope of the verification testing and not specified in the test
QA plan; therefore, analyses were not performed to identify the organisms that grew in the
broth. Thus, the question of whether or not viable spores remained on the coupons after the
initial extraction remains unanswered. In the case of industrial carpet coupons, however, no
growth was observed when the carpet coupons were incubated following initial extraction of
control and decontaminated coupons. The lack of growth was most likely due to the
presence of an antimicrobial treatment that was incorporated into the carpet during
manufacture.
The CDG bench-scale unit can be set up and ready for operation within minutes. The CDG
bench-scale unit cannot measure parameters such as relative humidity and CIO2 concen-
tration. Within the glove box, the relative humidity was determined using a traceable
hygrometer, and the CIO2 was measured using a CIO2 monitor. The effect of operator skill
level on using the CDG bench-scale unit, while not verified in this test, should be minimal.
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Chapter 8
References
1. Knapp, J. E., Battisti, D. L. "Chlorine Dioxide," in S. S. Block, ed., Disinfection,
Sterilization, and Preservation, Fifth Edition. Philadelphia: Lippencott Williams and
Wilkins; 2001; pp. 215-227.
2. Test/QA Plan for Verification of Chlorine Dioxide Gas Technologies for
Decontaminating Indoor Surfaces Contaminated with Biological or Chemical Agents,
Battelle, Columbus, Ohio, September, 2003.
3. Quality Management Plan (QMP)for the Building Decontamination Technology
Center, Version 2, prepared by Battelle, Columbus, Ohio, March 2004. (This reference
is posted on the ETY web site at: http://www.epa.gov/etv/centers/center9.html.)
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