Fogging of Chlorine-Based Sporicidal
Liquids for the Inactivation of Bacillus
Anthracis Surrogate Spores
^ i
¦¦
Office of Research and Development
Homeland Security Research Program
United States
Environmental Protection
Agency
I:PA/600/R 16/J34 I July 2017
www.epa.gov/homeland-security-research
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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
investigation through contract EP-C-15-008 with Jacobs Technology Inc. This report has been peer and
administratively reviewed and has been approved for publication as an EPA document. It does not
necessarily reflect the views of the Agency. No official endorsement should be inferred. EPA does not
endorse the purchase or sale of any commercial products or services.
Questions concerning this document or its application should be addressed to:
Joseph Wood
Decontamination and Consequence Management Division
National Homeland Security Research Center
U.S. Environmental Protection Agency (MD-E343-06)
Office of Research and Development
109. T.W. Alexander Drive
Research Triangle Park, NC 27711
Phone:919-541-5029
E-mail: wood.ioe@epa.gov
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Acknowledgments
This effort was directed by the principal investigator from ORD's NHSRC, with support of a project team
consisting of EPA and contract support staff. The contributions of the following individuals have been a
valued asset throughout this effort:
U.S. Environmental Protection Agency (EPA) Project Team
Joseph Wood, Principal Investigator; National Homeland Security Research Center (NHSRC)
Leroy Mickelsen and Shannon Serre, Office of Land and Emergency Management
Worth Calfee and Lukas Oudejans, NHSRC
Richard Rupert, On-Scene Coordinator, Region 3
US EPA Technical Reviewers of Report
Anne Mikelonis, NHSRC
Michael Towle, Region 3
US EPA Quality Assurance
Eletha Brady Roberts, National Homeland Security Research Center
Jacobs Technology Inc.
Stella McDonald
Abderrahmane Touati
Denise Aslett
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Executive Summary
The U.S. Environmental Protection Agency's (EPA) Homeland Security Research Program (HSRP) is
helping protect human health and the environment from adverse impacts resulting from the release of
chemical, biological, or radiological agents. As part of the HSRP, EPA is investigating the effectiveness
and applicability of technologies for homeland security—related applications. The purpose of this
investigation was to determine the sporicidal efficacy of a fogging technology using chlorine-based
sporicidal liquids for inactivating bacterial spores (Bacillus [B.] atrophaeus, a surrogate for B. anthracis) in
an office or indoor environment. The use of fogging technology to disseminate sporicidal solutions via
microscopic droplets has the potential to be a less arduous, more economical volumetric decontamination
alternative to fumigation.
Twenty-seven pilot-scale tests were conducted overall. Test surfaces, or coupons, were typical indoor
and outdoor building materials and included carpet, ceiling tile, concrete, glass, laminate, painted
wallboard (PWB) paper, galvanized metal, and wood. Known amounts of B. atrophaeus spores were
inoculated onto the material coupons, and then the coupons were placed in three locations in a mock
office: under a desk, on top of a desk, and above the ceiling tiles (one ceiling tile was removed to allow for
fog distribution). The chlorine-based decontamination solutions investigated were pH-adjusted bleach
(pAB), diluted bleach (1 in 4 dilution), sodium dichloro-s-triazinetrione (dichlor), and aqueous chlorine
dioxide (CIO2). One or two foggers were used to disseminate the sporicidal solutions throughout the
chamber in the form of an aerosol.
Experimental parameters included the sporicidal solution, active ingredient concentration (AIC) of the
liquid sporicide, disseminated volume of solution, dwell time, and chamber air exchange. The efficacy of
the fogging treatment was characterized in terms of logio reduction (LR), which was calculated as the
difference between the log of the number of bacterial spores (as colony-forming units, or CFU) recovered
from the coupons before (positive controls) and log of the number after decontamination. A
decontaminant is considered to be an effective sporicide if a 6 LR or greater is achieved based upon
appropriate laboratory testing.
Summary of Results
The decontamination efficacy results were variable and depended greatly on the material. The nonporous
materials tested were easier to decontaminate, i.e., had generally higher decontamination efficacies,
while materials that are porous or comprised of organic chemical constituents proved more difficult to
effectively decontaminate. In the majority of the tests, galvanized metal, glass, laminate, and PWB paper
were effectively decontaminated (> 6 LR). Fogging of the chlorinated decontaminants was moderately
effective for concrete, with only one test achieving an average > 6 LR on this material (but several tests in
which > 5 LR was achieved). Ceiling tile, carpet, and wood (porous and organic-based materials) were
the most difficult materials to decontaminate. There were no tests in which ceiling tile or carpet were
effectively decontaminated as defined as > 6 LR.
Statistical analyses of results showed that the disseminated volume of solution proved to have a
significant effect on decontamination efficacy. Further, maximizing the fogged solution quantity (up to
approximately 336 mL per cubic meter volume to be decontaminated) and the AIC generally produced
similar results for all sporicides. More specifically, the average decontamination efficacy for all materials in
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the tests at these optimized operating conditions was generally above 5 LR, and was independent of the
sporicide fogged.
Analysis of the data showed a significant yet minor average improvement (~ 0.5 LR) in the
decontamination efficacy for the coupons placed on the desk location compared to the other locations.
Coupons located under the desk and above the ceiling showed the same average decontamination
efficacy. Overall, these differences in decontamination results as a function of test chamber location were
minor and generally imply the fog was well distributed.
An evaluation of the neutralization requirements for coupon samples containing dichlor residue during the
extraction process determined that there were statistically insignificant differences in spore recovery
between samples extracted with buffer solution plus neutralizer and those extracted with just the buffer.
This was shown to be the case for all materials. Additionally, the recovery of viable bacterial spores
inoculated onto coupons already having a dichlor residue was significantly diminished.
This study has demonstrated the potential of using chlorine-based decontaminants applied with a
commercially available fogging technology for volumetric decontamination of surfaces typical of indoor
environments contaminated by B. anthracis surrogate spores. However, this decontamination approach
may be better suited for areas that do not contain significant quantities of porous or organic materials
such as carpet, ceiling tile, or wood.
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Table of Contents
Disclaimer i
Acknowledgements ivi
Executive Summary iii
List of Figures vvi
List of Tables vvii
Acronyms and Abbreviations vviii
1 Introduction 1
2 Materials and Methods 2
2.1 Biological Organism and Spore Deposition 2
2.2 Decontamination Chamber and Test Environment 2
2.3 Mock Office 2
2.4 Fogging System and Methods 3
2.5 Sporicidal Solutions 4
2.6 Measurement of CIO2 Gas Levels 6
2.7 Extractive Sampling Method for CI2 Gas Measurement 7
2.8 Coupon Materials and Biological Indicators 8
2.9 Test Sequence 8
2.10 Bacterial Spore Sampling and Analysis 9
2.11 Dichlor Residue Evaluations 11
2.12 Decontamination Efficacy Characterization 13
2.13 Statistical Analyses 14
3 Quality Assurance / Quality Control 15
3.1 Sampling, Monitoring, and Equipment Calibration 15
3.2 Acceptance Criteria for Critical Measurements 15
3.3 Data Quality 16
4 Results and Discussion 19
4.1 Test Matrix Summary and Fumigation Conditions 19
4.2 Results 22
4.2.1 Spore Recovery from Positive Controls 22
4.2.2 Efficacy of Individual Fogging Tests 22
4.2.3 Decontamination Results by Material 253
4.2.4 Decontamination Results for Each Location 25
4.3 Summary of Efficacious Test Conditions 26
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4.3.1 Effect of Disseminated Volume on Fogging Efficacy 29
4.3.2 Effect of Air Exchange on Decontamination Efficacy 30
4.3.3 Effect of Dwell on Decontamination Efficacy 310
4.3.4 Effect of Active Ingredient Concentration on Decontamination Efficacy 31
4.4 Biological Indicator Results 32
4.5 Impact of Dichlor Residue on Spore Recovery 33
4.5.1 Extractive Sample Neutralization Scoping Test 34
4.5.2 Coupon Storage Neutralization Test 35
5 Summary and Conclusions 38
6 References 40
Appendix A: Detailed Decontamination Results 42
Appendix B: Efficacy Charts for Individual Tests 444
List of Figures
Figure 2-1. Mock office setup and fogger placement 3
Figure 2-2. Coupons coated with residue after exposure to dichlor fog 11
Figure 2-3. 14 in. x 14 in. stainless steel coupons 12
Figure 3-1. Cb measurement accuracy assessment 17
Figure 3-2. Field blank test sample results for tested materials 18
Figure 4-1. Average CFU recovery (± SD) from positive controls for each material 22
Figure 4-2. Efficacy results (Avg. LR) grouped by sporicide (±SD) 23
Figure 4-3. Average LR for materials (±SD) 264
Figure 4-4. Average Efficacy for Sporicidal Solutions with Respect to Material (±SD) 25
Figure 4-5. Average LR for mock office locations (± SD) 326
Figure 4-6. Minimum mass of active ingredient needed to achieve effective decontamination 28
Figure 4-7. Percent of Bis inactivated for Each Test 32
Figure 4-8. Bl inactivation percentages in each Mock Office Location 33
Figure 4-9. Average CFU recoveries from stainless steel clean surfaces vs. surfaces loaded with
dichlor residue (±SD) 34
Figure 4-10. Effect of Neutralizer During Extraction on Average CFU Recovery (± SD) 35
Figure 4-11. Effect of Neutralizer During Coupon Storage (± SD) 36
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List of Tables
Table 2-1. Summary of Sporicides 4
Table 2-2. Summary of Samples Used for each Test 10
Table 3-1. Sampling and Monitoring Equipment Calibration Frequency 15
Table 3-2. Analysis Equipment Calibration Frequency 15
Table 3-3. Summary of QA/QC Checks 16
Table 4-1. Summary of Fogging Conditions 20
Table 4-2. Summary of Efficacious (> 6 LR) Decontamination Conditions per Material 28
Table 4-3. Decontamination Efficacy Comparison of Similar Tests with Different Volumes of
Dissemnated Solution 29
Table 4-4. Efficacy Comparison of Similar Tests with Different Rates of Air Exchange 30
Table 4-5. Efficacy Comparison of Similar Tests with Different Dwell Times 310
Table 4-6. Efficacy Comparison for Tests with Varying AlCs 31
Table 4-7. Average CFU Recoveries for Extraction Procedures 35
Table 4-8. Average CFU Recoveries for Alternative Storage Procedures 36
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Acronyms and Abbreviations
ADA
AIC
ANOVA
B.
Bl
BSC
Ch
CIO2
CFU
cm
COMMANDER
Dl
dichlor
EPA
FAC
g
HEPA
Hg
HSRP
i.d.
in.
kg
L
lb
LR
|JL
m
MDI
mg
min
mL
mm
NCASI
NHSRC
NIST
o.d.
pAB
Aerosol Deposition Apparatus
active ingredient concentration
analysis of variance
Bacillus
biological indicator
biological safety cabinet
chlorine
chlorine dioxide
colony-forming unit(s)
centimeter(s)
Consequence Management and Decontamination Evaluation Room
deionized
sodium dichloro-s-triazinetrione
U.S. Environmental Protection Agency
free available chlorine
gram(s)
high-efficiency particulate air
mercury
Homeland Security Research Program
inner diameter
inch(es)
kilogram(s)
liter(s)
pound(s)
log reduction
microliter(s)
meter(s)
metered-dose inhaler
milligram(s)
minute(s)
milliliter(s)
millimeter(s)
National Council of the Paper Industry for Air and Stream Improvement, Inc.
National Homeland Security Research Center (EPA/ORD)
National Institute of Standards and Technology
outer diameter
pH-adjusted bleach
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PBST
phosphate-buffered saline with Tween® 20
PPE
personal protective equipment
PPm
part(s) per million
PVC
polyvinyl chloride
QA
quality assurance
QAPP
quality assurance project plan
QC
quality control
RH
relative humidity
RPM
revolutions per minute
s
second(s)
SCADA
supervisory control and data acquisition
SD
standard deviation
STS
sodium thiosulfate
TNTC
too numerous to count
TSA
tryptic soy agar
VHP®
vaporized hydrogen peroxide
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1 Introduction
The U.S. Environmental Protection Agency's (EPA) Homeland Security Research Program (HSRP) 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, the HSRP is
working to develop tools and information that will help detect, contain, and decontaminate radiological,
chemical, or biological contaminants resulting from an intentional introduction of these agents into
buildings, water systems, or the outdoor environment, as well as facilitate the treatment and disposal of
materials resulting from remediation activities. As part of this effort, and in response to the needs of the
HSRP partners, EPA is investigating the effectiveness and applicability of technologies for homeland
security-related applications by developing test plans, conducting tests, collecting and analyzing data,
and preparing peer-reviewed reports. All evaluations are conducted in accordance with quality assurance
(QA) protocols to ensure that data of known and high quality are generated.
In 2001, the introduction of a few letters containing Bacillus (B.) anthracis (anthrax) spores into the U.S.
Postal Service system resulted in contamination of several facilities. Although most of the facilities in
which these letters were processed or received were heavily contaminated, they were successfully
remediated with approaches such as fumigation with chlorine dioxide (CIO2) or vaporized hydrogen
peroxide (VHP®) (Canter et al., 2005). Large-scale use ofsporicidal chemicals to decontaminate large
buildings was unprecedented (Rastogi et al., 2010), and the overall cost of remediation activities for the
letter attacks was estimated to be approximately $320 million (Schmitt and Zacchia, 2012). It is generally
agreed that additional rapid, effective, and economical decontamination methods that can be employed
over wide areas (outdoor and indoor) are needed to increase preparedness for such a release.
While previous tests have been conducted by EPA to evaluate the inactivation of B. anthracis spores
using peracetic acid solutions (Wood et al., 2013), there are few data available in the literature related to
decontamination efficacy when fogging chlorine-based solutions. Thus to fill this gap, the study reported
here evaluated the effectiveness of different chlorine-based sporicidal liquids, disseminated using a
commercially available fogging device, to inactivate bacterial spores in a pilot-scale decontamination
chamber. (The efficacy of bleach-based decontaminants in inactivating spores has been evaluated on a
number of different materials when applied as a spray (Ryan et al., 2014; U.S. EPA, 2006; U.S. EPA,
2012; U.S. EPA, 2015; Wood et al., 2011), but again, few data are available on efficacy when chorine-
based decontaminants are applied as a fog.) Experimental variables included material, location within test
chamber, sporicidal solution, quantity of solution, AIC, and air exchange. The chlorine-based sporicidal
decontaminants chosen for testing were solutions of diluted bleach (1 in 4 dilution), pH-amended bleach
(pAB), aqueous chlorine dioxide, and a concentrated aqueous solution of sodium dichlor-s-trianzinetrione
dihydrate (a pool sanitizer chemical commonly referred to as dichlor).
Twenty-five tests were conducted to evaluate decontamination efficacy for the fogging of chlorine-based
sporicidal solutions. Two additional tests were conducted to assess the neutralization requirements for
coupon samples containing dichlor residue.
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2 Materials and Methods
2.1 Biological Organism and Spore Deposition
The test organism for this study was B. atrophaeus (American Type Culture Collection 9372) in a dry
powder form of spores mixed with silicon dioxide particles. B. atrophaeus, formerly known as B. subtilis
var. niger or B. globigii, was used as a surrogate for B. anthracis in three decontamination test rounds
(including CIO2 fumigation) of the Bio-response Operational Testing and Evaluation study (U.S. EPA,
2013). The bacterial spores were prepared by the U.S. Army Dugway Proving Ground as reported in
Brown et al. (2007a).
The test surfaces (coupons) were loaded with a target dose of 107 colony-forming units (CFU) of the dry
spore mix using a procedure specifically developed for this purpose. Briefly, each sterilized coupon was
aseptically mounted on the top of a cylindrical coupon holder and topped with a metered-dose inhaler
(MDI) actuator. The pre-weighed MDI was vortexed and hand agitated to evenly distribute the dry spore
mix and then placed inside the actuator, which was activated to disperse the spore mix onto a circular
area in the center of the coupon's surface. Refer to Lee et al. (2011) for further details.
2.2 Decontamination Chamber and Test Environment
All tests were conducted in the Consequence Management and Decontamination Evaluation Room, or
COMMANDER. COMMANDER consists of a stainless steel-lined inner chamber built specifically for
decontamination testing, with internal dimensions of approximately 3.4 m wide, 2.5 m deep, and 2.8 m
high. At the entrance to the chamber is an airlock compartment, and enclosing the chamber and airlock is
an exterior steel shell. When desired, all three components can be kept under cascading slightly negative
pressure (with the greatest negative pressure in the inner chamber) by using separate air streams with
valve controls on the inlet and outlet of each. Air entering the decontamination chamber passes through a
high-efficiency particulate air (HEPA) filter, and exhaust air from the chamber is ducted to an activated
carbon bed and HEPA filter prior to release to the facility exhaust system. Fans were used inside the
chamber to provide internal mixing during fumigation. The inner chamber inlet and outlet duct fans
(blowers) were turned off during fumigation, and the inlet duct valve was closed. Further details and a
diagram of COMMANDER can be found elsewhere (Wood et al., 2013).
Temperature, relative humidity (RH), air pressures, and flow rates within the decontamination chamber
are controlled and/or their data logged continuously using a supervisory control and data acquisition
(SCADA) system. Temperature and RH within the chamber were measured using a temperature and RH
transmitter (model HMD40Y, Vaisala Inc., Helsinki, Finland). This instrument was calibrated prior to each
test by comparing its RH data with known RH values generated in the sealed headspace above individual
saturated solutions of various salt compounds. The RH meters were replaced if calibration criteria could
not be met. During fogging events, the RH and temperature within the chamber were monitored but not
controlled. Typically, RH measurements neared or exceeded the maximum range of the RH meter during
the fogging events.
2.3 Mock Office
The stainless steel surfaces of the decontamination chamber were covered by materials typical of an
indoor office setting. The floor was covered with plywood and then industrial carpet tiles (P/N 54594,
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Multiplicity carpet tile, Shaw Floors, Sherwin-Williams Durham, NC). The rear and side walls were framed
and faced with 1 27-crn-thick drywall (P/N GB4080-0800, GoldBond, Home Depot, Durham, NC). The
drywall was patched with joint compound (P/N 380119048, USG Sheetrock, Lowes, Durham, NC) and
joint tape (P/N 382199010, USG Sheetrock, Lowes, NC) according to typical building practices and then
primed (P/N 20005, Kilz, Lowes, Durham, NC) and painted (P/N 105001, Behr, Lowes, Durham, NC). At
the top of the walls, a drop ceiling was installed and consisted of acoustic ceiling tile panels (P/N
SC1135c, Armstrong, Home Depot, Durham, NC) and two plenum grilles to enable conditioning of the
interior chamber air using the existing RH and temperature controls. The chamber was furnished with
office equipment consisting of a laminated desk, an office chair, a file cabinet, books/catalogs, and an
oscillating fan for chamber mixing. Figure 2-1 shows the mock office with furniture and fogger positions.
2.4 Fogging System arid Methods
Decontamination tests were conducted using an ultra-low volume fogger (SANI-TIZER™, Curtis Dyna-
fog, Ltd., Westfield, IN), which consisted of a motor/blower assembly, nozzle system, nozzle housing,
1-gallon formulation tank, and metering valve. The sporicide was drawn from the formulation tank through
the control valve and into the nozzle system where it was pneumatically sheared into droplets. The
droplets were then disseminated throughout the chamber by ambient air passing through the nozzle
system.
/
Figure 2-1. Mock office setup and fogger placement.
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The foggerwas operated by first transferring a measured volume of sporicidal solution into the
formulation tank and then weighing the fogger. (The formulation tank could accommodate slightly more
than 4 L [1 gallon] of sporicide, so two foggers were used for volumes greater than 4 L.) The foggerwas
transferred to COMMANDER and placed on the floor in front of the chamber door, facing the back wall
with the nozzle positioned at an angle of approximately 70° from horizontal. The metering valve knob
was positioned on the low setting to regulate the mean droplet size at 14-20 microns, according to the
manufacturer. The foggerwas plugged into an unenergized power outlet and the fogger's power switch
moved to the on position. COMMANDER was sealed and the target air exchange set by adjusting the
chamber's air supply valve. The foggerwas activated remotely using the SCADA system by increasing
the voltage output of the power outlet from 0% to 100%. Fogging typically began within 1-2 hours after the
active ingredient concentration was measured. At the completion of testing, the foggerwas removed from
the chamber, weighed, and drained of sporicide. The drained sporicide was collected in a graduated
cylinder where the volume was measured, and then discarded. The empty fogger was purged with
deionized water and reused if the post-test dissemination rate was within 20% of the initial rate at factory
condition. If the criteria were not met, the foggerwas removed from service.
2.5 Sporicidal Solutions
The type, volume, and concentration of sporicidal solution used for fogging were some of the independent
variables for this investigation. The solutions used for this effort are detailed in Table 2-1, and were all
tested at laboratory ambient temperature of approximately 22 °C.
Table 2-1. Summary of Sporicides
Sporicidal Solution
Active Ingredient
Vendor
pH-adjusted bleach (pAB)
Sodium hypochlorite,
hypochlorous acid
Produced on-site with Clorox® concentrated
germicidal bleach (EPA registration 5813-102;
Lowes, Durham, NC) and 5% acetic acid
Diluted bleach
Sodium hypochlorite
Produced on-site with Clorox® concentrated
germicidal bleach
Stabilized chlorinating
granules (dichlor)
Sodium dichloro-s-triazinetrione
Hydrated, hypochlorous acid
Pool Solutions, Pool Supply World, P/N PSW-
CSC158-5; Brilliance for spas, B & G Builders
Pools & Spas, Durham, NC
Aqueous CIO2
Aqueous chlorine dioxide
P/N G0005, G02 International, Buena Park, CA
The pAB was prepared as follows: one part Clorox® concentrated germicidal bleach (Clorox Corp.,
Oakland, CA) was diluted with approximately eight parts of deionized water and one part 5% (v/v) acetic
acid (P/N 13025 or equivalent, Fisher Scientific, Pittsburgh, PA;). This brand of bleach is registered with
EPA as an antimicrobial pesticide and has a hypochlorite concentration of 8.3%. The pH was adjusted to
6.5-7.0 with additional 5% acetic acid, as needed. For the first two tests, the free available chlorine (FAC)
content was adjusted to 6000-6700 ppm with deionized water after preparation. The FAC levels for
subsequent tests with pAB had higher FAC levels that were more consistent with the FAC levels of the
diluted bleach tests.
Initially, diluted bleach was prepared by mixing Clorox® concentrated germicidal bleach with deionized
water to reach the target FAC, and the pH was recorded. Most of the tests thereafter with diluted bleach
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used one part bleach and three parts water, to give an FAC level of 20,000 ppm or higher. For tests with
lower FAC concentrations, the ratio of bleach to water was varied to meet the target FAC specification.
The dichlor solutions were prepared by dissolving 0.33 lb of the dichlor granules per 1 gallon of deionized
water, except for Test 24, in which 0.5 lb of the dichlor product was added per gallon of water. Note that
the actual measured FAC level (discussed next) of the dichlor solutions does not correspond directly to
the above reported quantities (mass) of the product added per liter of water. This is because the pool
chemical used does not result in 100% conversion of FAC, as measured by the technique discussed
below.
The aqueous CIO2 solutions were prepared according to the manufacturer's instructions by first dissolving
the active aqueous CIO2 brand component A (52% sodium chlorite) in 5 L of tap water and then adding
component B (97% sodium bisulfate). The solution was gently stirred to promote even mixing, and then
required three hours to complete the reactions to fully produce the CIO2 solution. All CIO2 solutions were
prepared in chemical resistant containers (polyethylene or polypropylene) and used within 1 hour after
completion of the required 3-hour reaction hold time. These aqueous solutions were typically at ambient
temperature (~ 23 °C) prior to fogging. Safety precautions were taken to protect personnel from liberated
chlorine and chlorine dioxide gas.
The FAC concentration of the formulations for pAB, diluted bleach, and dichlor was measured using the
HACH® high-range bleach test kit (Method 10100, [model CN-HRDT], HACH, Loveland, CO) which was
adapted from ASTM Method D2022-89. A 1 or 5-mL aliquot of the decontaminant solution was mixed with
approximately 150 mL of deionized water in a 250 mL glass beaker. The size of aliquot depended on the
expected concentration of the FAC. Usually, for solutions with target FAC concentrations less than 10,000
mg/L, a 5 mL aliquot was used and, for those solutions with target FACs greater than 10,000 mg/L, a 1
mL aliquot was used. A potassium iodide powder pillow (HACH®, P/N 20599-96) was added and mixed
until completely dissolved. The sample was acidified with an acid reagent powder pillow (HACH®, P/N
1042-99) then iodometrically titrated with sodium thiosulfate (STS) to a colorless end point. The bleach
solution aliquot was taken and analyzed immediately after formulation and mixing.
The CIO2 concentration of the aqueous CIO2 solutions was also determined using the HACH® high-range
bleach test kit, but modified as follows to measure only CIO2. A 1 mL aliquot of solution was mixed with
approximately 150 mL of deionized water in a 250 mL glass beaker. A potassium iodide powder pillow
was added and mixed until completely dissolved. A neutral (no acid added) titration was performed
iodometrically with sodium thiosulfate (STS) to a colorless end point.
The pH of each solution was measured with an Oakton Acorn® series pH 5 meter (Oakton Instruments,
Vernon Hills, IL). This meter was calibrated daily.
Sporicide volumes ranged from 1 L to 8 L, but the volume added to the fogger was not necessarily the
volume disseminated. Typically, dissemination limitations caused by equipment efficiency resulted in
approximately 50 mL of sporicide remaining in the fogger after testing. However, equipment malfunctions
in some tests resulted in more than 50 mL of unused sporicide. Two foggers were deployed for tests
requiring more than 4 L of sporicide. The test parameters for each solution are discussed in Section 4.
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2.6 Measurement of CIO2 Gas Levels
Gas samples using a modified version of Method 4500-CI02 E (Standard Methods Online, 2005) were
taken to monitor the CI02gas concentration inside the mock office, when fogging aqueous CIO2 solutions.
This method is an amperometric titration suitable for aqueous CIO2 concentrations between 0.1 and 100
mg/L. This method does not address gas-phase sampling. The full method is quite complex in that a
multi-titration scheme is used to differentiate several chlorine-containing analytes. A modification of this
method to incorporate gas-phase sampling requires the use of a buffered potassium iodide bubbler
sample collection and restricting the official method to a single acidic titration versus a two-step neutral
and acidic titration. The neutral titration analyzes the combined chlorine, chlorine dioxide, and chlorite as
a single value. It can only be applied where chlorite and chlorate are not present. Since the modified
standard method described below is applied to gas-phase samples, the presumption of the absence of
chlorite and chlorate is valid.
The modified method was performed as follows:
• A series of four impingers were assembled: Impingers 1 and 2 contained 20 mL of potassium
iodide (Kl) phosphate buffer solution (KIPB) with a pH of 7.2. The solution was prepared using 25
g of Kl in 500 mL of phosphate buffer). Impinger 3 was empty, and impinger 4 contained silica
desiccant.
• CIO2 gas was impinged from the chamber into the KIPB solution in the impingers in series at a
flow rate 0.5 L/min for a time necessary for the KIPB to turn from clear to a solid yellow color.
• The 20 mL of KIPB solution from each impinger were combined into a 200-mL volumetric flask.
The impingers were rinsed thoroughly with deionized water and the rinse was collected in a 250-
mL beaker.
• 1 mL of 6 N HCI was added to the solution.
• The solution was placed in the dark for 5 min.
• The solution was titrated with 0.01 N STS, and the volume of STS used in the titration was
recorded.
Conversion from titrant volume to CIO2 concentration was based on the modified 4500 E method and
calculated as follows:
Ta x 13490 x N
2 0.025 (fraction of gas titrated)
where
Ta = volume of STS (mL)
N = Normality of STS
This method removed many of the possible interferences listed in Method 4500-CI02 E. The initial
presence of Kl in excess prevents iodate formation, which can occur in the absence of Kl and leads to a
negative bias. The presence of the pH 7 buffer during impinging prevents oxidation of iodide by oxygen,
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which occurs in strongly acidic solutions. Other interferences are unlikely to be a problem in this
application as manganese, copper, and nitrate are unlikely in a gaseous sample.
The second impinger filled with KIPB solution was added in series to reduce the likelihood of
breakthrough. The second impinger was not analyzed independently, but was combined with the first
impinger for analysis.
2.7 Extractive Sampling Method for CI2 Gas Measurement
The CI2 extractive sampling method was used to monitor mock office Cb levels in the gas phase when
fogging pAB, diluted bleach, and dichlor solutions. Extractive samples were collected continuously (one
after another) while the foggers were active. The method was developed by the National Council of the
Paper Industry for Air and Stream Improvement, Inc. (NCASI) for monitoring CI2 and CIO2 in bleach plants
(NCASI, 1997). This method is suitable for CI2 concentrations above 1 mg/L for a 10-L sample. Briefly, an
air sample is extracted from the mock office and passed through impingers containing potassium iodide
phosphate buffer solution. The NCASI method involved an assessment of iodine formed at neutral and
acidic pH for quantitative assessment of CI2. The method was modified, however, to a single, acidic
titration. As mentioned previously, omission of the neutral titration can only be applied where chlorite and
chlorate are not present. Since CIO2 was not present in the air sample, the presumption of the absence of
chlorite and chlorate is, again, quite valid.
Cb extractive sampling was performed as follows:
• A series of four impingers were assembled: Impingers 1 and 2 contained 20 mL of potassium
iodide (Kl) phosphate buffer solution (KIPB) with a pH of 7.2. The solution was prepared using 25
g of Kl in 500 mL of phosphate buffer). Impinger 3 was empty, and impinger 4 contained silica
desiccant.
• Cb gas was impinged from the chamber into the KIPB solution in the impingers in series at a flow
rate 0.5 L/min for a time necessary for the KIPB to turn from clear to a solid yellow color.
• The 20 mL of KIPB solution from each impinger were combined into a 200-mL volumetric flask.
The impingers were rinsed thoroughly with deionized water and the rinse was collected in the
250-mL beaker.
• 10 mL of 10% sulfuric acid was added to the solution.
• The solution was titrated with 0.01 N STS, and the volume of STS used in the titration was
recorded.
Conversion from titrant volume to CI2 concentration was calculated as follows:
Cl2 Moles x 24.04—-i~x 106
Cl2(ppm) = — ^le
J Scxts
where
Sc =corrected sampling flow rate
7
-------
Ts = time sampled, min
Possible interferences for this method include sulfur dioxide and hydrogen peroxide, neither of which
were present during testing. Results of an accuracy assessment performed for this sampling method are
discussed in Section 3.3.
2.8 Coupon Materials and Biological Indicators
All eight materials used for this study were fabricated so that they could be mounted onto circular
aluminum scanning electron microscopy stubs (18 mm in diameter). Oak wood coupons were 8-mm-thick
plugs (part SPO750, Woodworks Ltd, Haltom City, TX). The borosilicate glass coupons were 3.3-mm-
thick plugs (Prism Research Glass, Inc., Research Triangle Park, NC). The 18-mm discs were cut from
26-gauge galvanized metal (East Coast Metal, Durham, NC), carpet (Multiplicity 54594, Shaw Industries
Group, Dalton, GA), laminate flooring (Pergo Estate Oak Laminate Flooring, Home Depot SKU 257063 -
no longer available), and ceiling tile (Armstrong, P/N 949, Lowes SKU 40684). Concrete coupons were
cast from sand and cement mix (P/N 110360, Quikrete, Lowes, Durham, NC). The front facing of drywall
(P/N GB00090800, GoldBond, Lowes SKU 34137) was primed (Kilz, 2-gallon, P/N 20005, Home Depot
SKU 317390) and painted (Behr Premium Plus flat white latex, P/N 105001, Home Depot SKU 923827).
The paper was then removed from the gypsum before the 18-mm discs were cut from the paper. All
materials were then mounted to 18-mm aluminum stubs (P/N 16119, Ted Pella, Inc., Redding, CA) using
double-sided adhesive tape (P/N 16073-2, Ted Pella, Inc., Redding, CA) and placed in holder trays. To
prevent contamination and bias of results due to non-target organisms, all coupons and stubs were
sterilized with ethylene oxide using an EOGas AN333 sterilization system (Andersen Products, Haw
River, NC).
Biological indicators (Bl) were also used to assess the effectiveness of fogging in inactivating bacterial
spores. The Bis comprised nominally 106 B. atrophaeus spores inoculated onto stainless steel discs and
wrapped in Tyvek envelopes. The Bis, obtained from Mesa Labs (model 1-6100ST, Lakewood, CO), were
placed in triplicate in each of the three locations inside the mock office and in the enclosure (the
unexposed location just outside the exposure chamber). The Bis were collected upon completion of each
test and analyzed according to manufacturer instructions to determine whether any of the Bis exhibited
growth of bacteria (survival of any spores).
2.9 Test Sequence
Each fogging event consisted of three phases: 1) Active fogging, characterized as the segment of the test
during which the fogger(s) were powered on. During active fogging, chamber conditions such as
temperature, RH, and sporicide concentration were monitored. 2) The dwell phase began when the
fogger(s) were turned off. Monitoring of control chamber conditions continued during the dwell phase;
however, the wet chemistry samples were collected for a relatively short time during the beginning of the
phase. The dwell phase continued overnight. 3) The aeration phase began the next morning when the
inner chamber valves were opened and the exhaust duct blower was turned on. Chamber aeration
continued until the concentration of sporicide was safe for reentry.
The following general test sequence was used for all 27 mock office tests:
8
-------
• Material coupons were prepared for testing as follows: Coupons designated as positive controls
(not exposed to the fog) and test coupons were inoculated within 24 hours of testing, with the
exception of Tests 14 and 15. Tests 14 and 15 coupons were inoculated 72 hours prior to
exposure and stored in a refrigerator at 4 °C. Procedural blanks, field blanks, and negative
control coupons (not inoculated with spores) were stored in the same manner as the inoculated
coupons.
• Test coupons and Bis were placed in COMMANDER in three predetermined locations: 1) on the
floor underneath the office desk, 2) on top of the office desk, and 3) in the ceiling on the ceiling
tile (one ceiling tile was removed from the tile framing to allow for fog to reach above the
remaining tiles). Procedural blank coupons were placed on the floor underneath the office desk.
Positive and negative control coupons remained outside the chamber and were not exposed to
fogging conditions. Bis were also placed in the unexposed enclosure area just outside the
exposure chamber.
• The chamber was sealed and fogging initiated (including operation of fan). Chamber conditions
during each test are detailed in Section 4.1. The fogger flow (dissemination) rate varied
somewhat, but averaged about 175 mL per minute when using two foggers. Thus for an eight-
liter decontaminant solution, this would require approximately 45 minutes to disseminate.
• Dwell was typically overnight, although there were a few tests with a dwell of only 2 hours, to
assess the effect of this operating parameter.
• After the dwell, the chamber was aerated for the time required to achieve a safe level of
decontaminant concentration prior to reentry. The aeration duration was typically only about five
minutes following fogging of bleach solutions, and was approximately an hour for the fogging of
dichlor solutions.
• Upon completion of the aeration phase, the chamber was entered and the test and procedural
blank coupons collected. In some instances, it was necessary to enter using supplied air
respirators to collect samples in a timely manner.
• Positive control, field blank, and material blank coupons were collected in empty sterile sample
tubes.
• The coupons were transferred to the National Homeland Security Research Center (NHSRC)
biocontaminant laboratory (biolab) for storage in a refrigerator over the weekend until subsequent
analysis.
2.10 Bacterial Spore Sampling and Analysis
Numerous microbiological samples and assays were used to characterize bacterial spore presence or
absence in the mock office for each experiment (116 total for each test). Coupons were collected and
spores were extracted, serial plated, filter plated (if needed) and enumerated as CFU as described in
Wood et al. (2016). Samples or assays were either quantitative (providing a numerical result) or
qualitative (indicating either presence or absence of bacterial growth). Laboratory blanks of items such as
growth media and sampling materials were also employed in each experiment to check for aseptic
conditions. A summary of the number and type of samples/assays for each experiment is shown in Table
2-2. Each sample or assay is further described in the narrative below.
9
-------
Table 2-2. Summary of Samples Used for each Test
Sample or Assay Type
No. Procedural Blanks
No. Positive Controls
No. Exposed Samples
Biological indicators
0
3
9
Material coupons
8
24
72
Total
8
27
81
CFU counts per coupon were calculated by multiplying the number of counted colonies by the dilution
factor and by the volume of the sample extract. Efficacy is defined as the extent (by log reduction, or LR)
to which the agent extracted from the material surface after the treatment with the decontamination
procedure is reduced below that extracted from a similar material coupon before decontamination.
Efficacy was calculated for each material (j) (eight materials were used for each fog test (i)) as follows:
SGogioQJ
LR. = ^ lo^
/ \
vijk ~ nijc ^X^ijk;
(2-1)
where
Cijc is the number of viable organisms recovered from control coupons for the /th test and yth
material,
riijc is the number of control coupons for the /th test and yth test material (n = 3 positive controls
were used for each material, each test), and
Nyk is number of viable organisms recovered on the /cth replicate test coupon for the /th test and
yth test material (9 replicates were used for each material, each test; 3 replicates were placed in
each of the 3 locations within the test chamber).
If no viable spores were detected, then the detection limit of the sample was used for Nyk and the efficacy
reported as greater than or equal to the value calculated by Eqn. 2-1. The detection limit of a sample
depends on the analysis method and so might vary. The detection limit of a spread-plate is 1 CFU, but
half of this value was used in calculating the detection limit of the sample. For instance, the detection
limit of a 0.1-mL plating of a 20-mL sample suspension is 100 CFU (0.5 CFU / 0.1 mL * 20 mL), but if all
20 mL of the sample is filter plated, the detection limit would be 0.5 CFU.
The standard deviation (SD) of the LR values for a particular material and test was calculated with MS
Excel as the standard deviation of the sample (STDEV function).
10
-------
2.11 Dichlor Residue Evaluations
2.11.1 Spore Recovery Evaluations
During the process of collecting samples exposed to dichlor fog, residue was observed on the coupon
surfaces and in COMMANDER itself. Figure 2-2 shows the residue formed on the surfaces of the material
coupons and the desk after exposure to dichlor fog.
Figure 2-2. Coupons coated with residue after exposure to dichlor fog.
In response, tests were conducted to determine the impact, if any, of dichlor residue on the recovery of
spores subsequently inoculated onto coupons. During one test with dichlor solution, six sterile stainless
steel coupons (14 in. x 14 in.) were placed in various locations throughout the chamber (the majority were
located on the floor in sterile trays for handling) in addition to the 18-mm material coupons. Figure 2-3
shows the placement of these coupons during exposure. The fog test was performed as described in
Section 2.9, but the stainless steel coupons remained in the chamber for 72 hours after treatment to allow
them to dry. (The chamber was entered as usual the day following treatment to retrieve the 18-mm
coupons and Bis, but the stainless steel coupons were left behind). After the 72-hour drying time, the
stainless steel coupons were removed from the chamber and inoculated with spores via an MDI as
follows: three positive control coupons (inoculated onto coupons not unexposed to dichlor treatment), five
test coupons (inoculated onto coupons that were exposed to dichlor treatment), one procedural blank
coupon (uninoculated and exposed to dichlor treatment), and one laboratory blank coupon (uninoculated
and unexposed to dichlor treatment). After inoculation, the spores were allowed 48 hours of contact with
the coupon surfaces. After the contact time, coupons were sampled using a sponge wipe method and
relinquished for extraction, serial plating, and enumeration.
11
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Figure 2-3.14 in. x 14 in. stainless steel coupons.
2.11.2 Neutralization Evaluations
Two additional, separate experiments were conducted to assess whether neutralization was needed to
stop any potential lingering sporicidal activity of dichlor residue when processing samples.
The purpose of the first neutralization test was to identify the need, if any, to neutralize the exposed
sample coupons during the extraction process. During the coupon extraction process, dichlor residue may
dissolve into the extraction solution with the extracted spores. There was some concern that potentially
elevated FAC levels in the extraction solution could result in residual inactivation of viable spores, or
assay conditions that prevent the germination and outgrowth of viable spores.
Four material types were selected for this experiment to represent differing levels of material porosity:
carpet and concrete (porous) and galvanized metal and laminate (nonporous). Positive control and test
coupons were inoculated as before. Two sets of each material were placed in triplicate in each of the
three sample locations inside the mock office and positive controls were placed in a location immediately
outside COMMANDER. The sample collection procedure remained consistent with that of previous
fogging tests. But now samples were extracted using two procedures. The extraction procedure used for
the first set of coupons remained unchanged from that of other tests with the use of 10 mL of phosphate-
buffered saline with Tween® 20 (PBST) as the extraction solution. The extraction procedure used for the
second set of coupons differed with the addition of an STS neutralizing solution to PBST in stoichiometric
equivalent quantities.
12
-------
The purpose of the second neutralization test was to identify the need to neutralize the exposed sample
coupons during the 72-hour period over the weekend when coupons were stored until they could be
extracted. As outlined in Section 2.9, at the completion of a fog treatment, exposed coupons were
collected in empty sterile sample tubes and then refrigerated for approximately 72 hours prior to the
extraction process. Thus for this second neutralization test, three material types were selected for this
test to represent varying levels of material porosity: carpet, laminate, and concrete. Positive control and
test coupons were inoculated as in the initial neutralization test. Three sets of each inoculated material
was placed in triplicate at each of the three sample locations inside the mock office, and positive controls
(in triplicate) were placed in a location immediately outside the chamber. The sample collection/storage
procedure was modified from fogging tests for two of the three sets of samples. The procedure used to
collect and store the first set of samples was consistent with that used for previous fogging tests. The
samples were aseptically transferred into empty sterile sampling tubes. To determine the effect of simply
diluting any dichlor residue, the coupons from the second set were transferred into sampling tubes
preloaded with 10 mL of PBST. Finally, to assess neutralization requirements during the 72-hour hold
time, coupons from the third set were transferred into sampling tubes preloaded with PBST and STS
neutralizer.
2.12 Decontamination Efficacy Characterization
Spore loading (i.e., positive control spore levels) was quantified by taking the logarithmioof the CFU count
for each material coupon and then calculating the mean and standard deviation of the log values (the
mean of a series of log values is equivalent to the log of the geometric mean for the same series) for each
set of triplicates (positive controls were inoculated in sets of three per material). Post-decontamination
results are presented in terms of spore recovery as well and were calculated in the same manner as the
positive control results. Results are also presented in terms of decontamination efficacy, which was
quantified as LR. The LR was calculated as the mean of the log values for each positive control average
CFU count minus the mean of the log values for each test sample average CFU count. Occasionally
results were reported by noting whether the average LR for a particular coupon or surface test was > 6.0,
since a decontaminant that achieves > 6 LR is considered effective as a sporicidal decontaminant based
upon appropriate laboratory testing (U.S. EPA, 2010). We note, however, that while a decontamination
efficacy > 6 LR may be considered "effective" when reporting test results, in an actual B. anthracis release
event, the goal for decontamination would be to minimize the number of recoverable viable spores,
regardless of LR. Hence, we also report results in terms of the number of samples in which spores were
not detected.
When no spores were detected for a sample, this result implied the highest decontamination efficacy
quantifiable and achievable, and the LR was reported as > the positive control recovery minus the
recovery from the test sample (calculated based on imputing a 0.5 CFU value on the filter plate and
adjusting for the filter plate volume)
13
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2.13 Statistical Analyses
Decontamination efficacies associated with study parameters were compared using one-way analysis of
variance (ANOVA) in MS Excel. The p-value from two-sided (non-directional) tests were used to test the
hypotheses (a = 0.05). Note, all of the tests contained the same distribution of materials and locations
within the room therefore it was assumed that a material or location by volume fogged, air exchange,
dwell, or AIC interaction did not occur. This could be evaluated further and may influence the significance
testing reported here.
14
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3 Quality Assurance I Quality Control
Quality assurance/quality control (QC) procedures were performed in accordance with the NHSRC
Quality Management Plan and the Quality Assurance Project Plan (QAPP-J15-011.0 and J15-011.A1).
The QA/QC procedures and results are summarized below.
3.1 Sampling, Monitoring, and Equipment Calibration
Approved operating procedures were used for the maintenance and calibration of all laboratory
equipment. All equipment was verified as being certified calibrated or having the calibration validated by
EPA's metrology laboratory at the time of use. Standard laboratory equipment such as balances, pH
meters, biological safety cabinets (BSC), and incubators were routinely monitored for proper
performance. Calibration of instruments was done at the frequency shown in Tables 3-1 and 3-2. Any
deficiencies were noted. Any deficient instrument was adjusted to meet calibration tolerances and
recalibrated within 24 hours. If tolerances were not met after recalibration, additional corrective action was
taken, including recalibration or/and replacement of the equipment.
Table 3-1. Sampling and Monitoring Equipment Calibration Frequency
Equipment
Calibration/Certification
Expected
Tolerance
Meter box
Volume of gas is compared to National Institute of Standards and
Technology (NIST)-traceable dry gas meter annually
± 2%
Flow meter
Calibration using a flow hood and a Shortridge manometer
± 5%
RH and temperature
sensor
Compare RH to the head space of three calibration salt solutions in
an enclosed space once a week; thermistor (for temperature) part
of RH sensor and calibrated by manufacturer
± 5%
Stopwatch
Compare against NIST Official U.S. time at
http://nist.time.qov/timezone.cqi?Eastern/d/-5/iava once everv 30
days
± 1 min/30 days
Table 3-2. Analysis Equipment Calibration Frequency
Equipment
Calibration
Frequency
Calibration Method
Responsible Party
Acceptance Criteria
Pipettes
Annually
Gravimetric
Carter Calibrations,
Manassas, VA
± 1% target value
Incubator
Thermometers
Annually
Compared to NIST-
traceable thermometer
Metrology Laboratory
± 0.2 °C
Scale
Before each
use
Compared to Class S
weights
Laboratory staff
± 0.01% target
3.2 Acceptance Criteria for Critical Measurements
QA/QC checks associated with this project were established in the QAPP. A summary of these checks is
provided in Table 3-3.
15
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Table 3-3. Summary of QA/QC Checks
Matrix
Measurement
QA/QC Check
Frequency
Acceptance
Criteria
Corrective Action
Negative test
coupon samples
(field blank)
CFU/sample
Field blank
One per
material type
per test
0 CFU
(no detection)
Revise handling
procedures;
investigate sources
of contamination;
reject results of the
same order of
magnitude
Biolab materials
CFU/sample
Biocontaminant material
blanks of PBST, dilution
tubes, and plating beads
(check that plating
materials are not
contaminated)
3 per each
material used
per test
0 CFU
(no detection)
Investigate
sterilization
procedure;
investigate sources
of contamination
Positive test
samples
CFU
Positive controls
(inoculated w/ spores,
but not subject to any
treatment)
3 per material
per test
5 x 106 to
5 x 107 CFU
Revise deposition or
sampling protocol if
mandated by
WACOR
Test coupon
samples
CFU
Agreement of triplicate
plates of single coupon
at each dilution
Each sample
Each CFU count
must be within
50% of the other
two replicates
Replate or filter
samples
Chamber air
RH
2-point calibration
Once per test
±5%
Replace Vaisala
sensor
Chamber air
Temperature
5-point calibration
Annually
±1 °C accuracy
Replace
thermocouple
Chamber air
Cl2 concentration
2-point calibration
Annually
Factory
calibration with
ACD Cal 2000
chlorine
generator, ±5%
Replace sensor
Chamber air
CI02
concentration
2-point calibration
Prior to each
use
NIST-traceable
transmission
band-pass
optical filters
Replace sensor
Sporicidal
solutions
pH,
effective
concentration of
hydrogen ions in
solution
Oakton Acorn meter
1 per use
> 6.5 and < 7.0
for fresh pAB
Reject solution;
replace reagents
and prepare a new
solution
Sporicidal
solutions
containing
bleach or dichlor
Concentration of
FAC in fresh
pAB, diluted
bleach, and,
dichlor solutions
HACH test kit, model
CN-HRDT
Once upon
production
±10% of target
concentration
Reject solution;
replace reagents
and prepare a new
solution
Sporicidal
solutions
containing CI02
Concentration of
CI02
HACH test kit, model
CN-HRDT
Once upon
production
±10% of target
concentration
Reject solution;
replace reagents
and prepare a new
solution
Exposure/dwell
Time
NIST-traceable timer,
comparison with official
NIST U.S. time
Once per 1
second
± 0.5 second
Synchronize timer
with official NIST
U.S. time
3.3 Data Quality
Temperature and RH measurement devices were maintained within the calibration tolerances listed in
Table 3-1. The extractive sampling method was validated against a known concentration of certified Cb
gas (Airgas Specialty Gases, Durham, NC). During extractive sampling, Cb concentrations were sampled
16
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using impingers, and the impinger liquid was analyzed using wet chemistry techniques. An accuracy
assessment was performed by comparing the wet chemistry measurement to the certified CI2 gas
concentration (target of 53.21 ppm ± 2%). Average wet chemistry values were shown to be within 5%
(0.51 SD) of the certified gas. Figure 3-1 shows the results of the assessment.
100
90
80
£
S" 70
CL
I 60
1 50
c
| 40
o
a 30
o
20
10
0
-Cert. C!2 (53.21 ppm)
-Wet Chemistry (ppm)
0 2 1
Sample
Figure 3-1. CI2 measurement accuracy assessment
Sporicide solution pH levels were measured using a high-accuracy (± 0.01 pH) waterproof pH meter.
Three-point calibrations using certified buffer solutions were performed on the pH measurement device
prior to each use. The device was maintained within ± 0.02 pH of each point prior to use.
Figure 3-2 shows the number of field blank samples that returned CFU counts (11 out of approximately
200 field blanks overall for the study) for the target organism for the overall study. Field blank samples
were handled the same as test samples except that they were not inoculated with spores. For each test,
the field blank coupons (one coupon per material) were placed in separate stages from the test coupons,
inside COMMANDER underneath the desk.
Although the intention was to minimize the presence of contamination, the levels detected on field blanks
were considered minor (in the rare occurrence the target organism was found on a field blank, it was
typically less than five CFU on a filter plate) and were not expected to impact study results. Spores
present on the field blanks could indicate cross-contamination during sampling or sample collection,
confounding post-fumigation results of the same order of magnitude.
17
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4 Results and Discussion
4.1 Test Matrix Summary and Fumigation Conditions
A summary of the conditions for each fogging test is shown in Table 4-1. (Note there were a total of 28
tests conducted in the study, although Test 5 is not included in the table since it was used for method
development. In addition, the last two tests were conducted to investigate issues related to dichlor
residue, which are further discussed below.) These values include the actual level of the active ingredient
(either FAC or CIO2) in the sporicidal solution, the actual volume of liquid sporicide disseminated, the
mass of AIC disseminated (concentration of AIC times volume disseminated), dwell time, RH, and
temperature. All fogging tests were conducted at ambient temperature and RH. Additionally, chamber
chlorine gas and CIO2 gas concentrations were monitored in the air but not controlled. Except as noted in
the table, tests were performed in a closed system; four of the last five decontamination tests were
conducted with controlled room air exchange to test its effect on decontamination efficacy.
Initially in the study, tests were conducted with solution volumes less than the fogger capacity of 1 gallon.
However, as testing progressed and with the intent to improve decontamination efficacy, two foggers
were used in some tests, which allowed fogging up to 2 gallons of liquid sporicide. After conducting tests
with 2-gallon fogging, it was decided that 2 gallons would be the maximum amount to fog due to
substantial wetting and dripping of sporicide from the ceiling.
The actual volumes of solution disseminated via the fogger(s) were, in general, within 12% of the target
volume and, in most cases, within 5%. Tests 6, 24, and 25 were exceptions in that the volumes
disseminated were 21%, 87%, and 39% less than the target volumes, respectively. In these cases,
equipment malfunctions occurred during fogging, reducing the flow of solution through the fogger.
Seven tests were performed with pAB solution. Initially, the target FAC was 6000 mg/L to 6800 mg/L
(Tests 1 and 2). During subsequent tests, a new procedure for preparing pAB was adopted that allowed
for higher FAC concentration targets. The FAC for these tests ranged from 7,840 mg/L to 18,701 mg/L.
Eight tests were performed with diluted bleach solution. FAC levels ranged from 15,920 mg/L to 24,201
mg/L.
Seven decontamination tests were performed with dichlor solution. FAC levels of the dichlor solutions
ranged from 20,601 mg/L to 21,901 mg/L with the exception of Test 24. The dichlor solution formulation
was modified for Test 24, from 0.33 lb of stabilized chlorine granules per 1 gallon of Dl water to 0.5
lb/gallon, resulting in an increased FAC level of 32,502 mg/L. Two additional tests (Tests 27 and 28) were
conducted with dichlor to investigate issues related to the residue affecting CFU counts, as discussed in
section 2.11.
Three tests were performed with the CIO2 solution. CIO2 levels in the aqueous phase ranged from 4,763
mg/L to 5,907 mg/L.
The pH levels for dichlor and pAB were in the range of 6-7; diluted bleach pH levels ranged from around
11-12; and aqueous CIO2 pH levels were rather acidic (~ pH of 2).
19
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Table 4-1. Summary of Fogging Conditions
Test
Number
Sporicidal
Solution
Actual
Sporicidal
Solution
Volume
Disseminat
ed (mL)
AlCin
Aqueous
Solution
(mg/L)
Mass of AIC
disseminat
ed (grams)
PH
Mean AIC in
Chamber Air
(ppm)
Max AIC in
Chamber
Air (ppm)
Dwell Time
(h)
Mean RH
(%)
Max RH (%)
Mean T (°C)
Air
Exchange
(fraction of
chamber
volume
replaced
each hour)
1
pAB
887
6440
5.71
6.8
3
5
20
68
71
28
0
2
pAB
2921
6480
18.93
7.0
7
12
24
67
78
26
0
3
Diluted
bleach
2891
17401
50.31
11.4
8
17
23
77
82
28
0
4
pAB
3941
7840
30.90
6.8
7
16
20
64
90
27
0
6
Diluted
bleach
4840
16721
80.93
NA
10
18
20
42
35
28
0
7
Dichlor
5873
20601
120.99
NA
4
8
37
76
90
22
0
8
Diluted
bleach
5300
15920
84.38
12.01
27
40
2
64
90
26
0
9*
pAB
5891
15701
92.49
7.20
35
40
0
69
87
26
0
10
CI02
1910
5906
11.28
2.04
59
72
19
72
73
25
0
11
CI02
3960
4763
18.86
2.24
73
97
19
96
98
24
0
12
pAB
5817
18301
106.46
6.67
89
125
19
97
98
28
0
13
Dichlor
7165
20701
148.32
7.82
12
22
17
91
100
28
0
14*
Diluted
bleach
7642
19001
145.21
11.14
34
93
19
100
100
26
0
15
CI02
7738
5907
45.71
1.66
36
155
19
90
100
26
0
16
pAB
7229
17401
125.79
6.24
131
219
19
80
88
27
0
17*
Diluted
bleach
7776
24201
188.19
11.15
48
103
16
87
102
29
0
18
Dichlor
7915
21301
168.60
6.61
20
32
16
81
86
30
0
19"
Diluted
Bleach
7766
23701
184.06
11.12
46
106
19
90
32
27
0
20
pAB
7860
18701
146.99
6.28
52
224
20
68
95
26
0
21***
Diluted
bleach
7780
23001
178.95
11.31
9
78
18
73
27
24
0
22
Dichlor
7778
21901
170.35
6.52
11
26
21
69
79
25
0.75
23
Dichlor
7396
20701
153.10
NA
14
22
18
84
85
25
0
24*
Dichlor
3141
32502
102.09
6.74
8
15
17
56
73
27
0.75
25
Dichlor
5406
20801
112.45
6.57
9
16
19
65
92
27
0.75
20
-------
Test
Number
Sporicidal
Solution
Actual
Sporicidal
Solution
Volume
Disseminat
ed (mL)
AIC in
Aqueous
Solution
(mg/L)
Mass of AIC
disseminat
ed (grams)
PH
Mean AIC in
Chamber Air
(ppm)
Max AIC in
Chamber
Air (ppm)
Dwell Time
(h)
Mean RH
(%)
Max RH (%)
Mean T (°C)
Air
Exchange
(fraction of
chamber
volume
replaced
each hour)
26
Diluted
bleach
7674
22201
170.37
11.13
16
27
20
66
76
29
0.75
27
Dichlor
7948
22501
178.84
6.74
17.2
21.6
18
97.8
101.1
26.2
0
28
Dichlor
7897
23104
182.45
6.77
11.4
18
19
82.1
84.0
27.6
0
'Insufficient air circulation due to mixing fan malfunction. "Replaced mock office ceiling tiles prior to experiment. ***Fogger nozzles inadvertently positioned toward back wall instead of ceiling.
The active ingredient for liquids were measured as FAC for tests performed with diluted bleach, dichlor, and pAB. CI02 was the active ingredient measured for tests performed with aqueous CI02.
The AIC as measured in air was Cl2 gas when fogging diluted bleach, dichlor, and pAB; and CI02 gas when fogging aqueous CI02
Test 5 not included in table since this test was used for method development, in which peracetic acid (a non-chlorine based decontaminant) was fogged.
21
-------
4.2 Results
This section presents results for the overall effectiveness of each chlorine-based sporicide (in terms of
LR) in inactivating B. atrophaeus spores on contaminated material surfaces. Effectiveness was
determined by comparing the viable spore recoveries of fogged material coupons to their unexposed
positive control counterparts. A 7 log spore challenge (inoculation of test and positive control materials
with ~5 x 107 spores) was used across all tests and materials. This study utilized the generally accepted
criterion of 6 LR to consider an approach effective. Recovery of no viable spores following treatment was
considered highly effective.
4.2.1 Spore Recovery from Positive Controls
Spore recoveries of positive control samples for each material for the entire study using the bacterial
spore sampling and analysis method detailed in Section 2.10 are shown in Figure 4-1. On average,
2.83E+07 (± 9.38E+06) CFU were recovered from coupon materials. The extractive sampling method
successfully recovered the required 1x106 CFU (minimum amount required to demonstrate 6 LR)
consistently from each material throughout the test series.
1.00E+08
1.00E+07
1.00E+06
1.00E+05
1.00E+04
& 1.00E+03
¦ Spore Recovery
gj 1.00E+02
1.00E+01
1.00E+00
Material
Figure 4-1. Average CFU recovery (± SD) from positive controls for each material
4.2.2 Efficacy of Individual Fogging Tests
Figure 4-2 shows the average efficacy results for each of the 25 chlorine-based fog decontamination
tests, organized by sporicide solution. The three tests providing the greatest average LR values in the
study were Test 19 (diluted bleach; 5.86 ± 0.80 LR), Test 15 (aqueous CIO2; 5.80 ± 1.08 LR), and Test 18
(dichlor; 5.76 ± 0.52 LR). It can be observed from Figure 4-2 that the lower numbered tests, which tended
to have less solution fogged and/or lower AIC values (refer to Table 4-1), generally resulted in lower LR
22
-------
values. As the test program proceeded, we endeavored to improve efficacy for a particular sporicide by
increasing AIC or liquid volume fogged. Thus maximizing the volume of solution fogged (maximum of
approximately 8 L) and the AIC (maximum of approximately 22,000 mg/L for pAB, diluted bleach, and
dichlor solution; tests performed with aqueous CIO2 had maximum CIO2 concentrations of approximately
5,900 mg/L) produced similar LR results for all sporicides. That is, average decontamination efficacies
associated with fogging relatively greater amounts of liquid and using higher AIC levels were generally > 5
LR for all four of the chlorinated sporicidal liquids. Note that because these LR values are averaged
across all materials, this resulted in standard deviation values greater than 2 LR for some tests; statistical
analyses comparing efficacy results and effects of test variables are further discussed below.
For additional details, refer to Appendix A, Table A-1, for the average LR values for each material for
each test. Refer to Appendix B for efficacy results graphically displayed for each test, indicating average
LR for each material at each location within the test chamber.
8.00
7.00
6.00
5.00
c
o
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1.00
0.00
i
Dichlor
T
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r
I
.QJ V) l/l V) VI
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4_, V) V)
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4—>
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oi
oi
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QJ
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QJ
H
h-
h-
H
H
1-
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1-
1-
H
Diluted bleach CI02 (aq)
pAB
Figure 4-2. Efficacy results (Avg. LR) grouped by sporicide (±SD)
4.2.3 Decontamination Results by Material
Figure 4-3 shows the average LR of viable spores for each material for all tests performed in this study
(analysis includes combined effects of sample location, sporicide, and test conditions). On average, non-
porous materials such as galvanized metal, glass, laminate, and PWB paper were easier to
decontaminate, with overall average efficacies of 6.08 ± 1.89, 6.54 ± 1.63, 6.08 ± 1.86, and 5.78 ± 1.86,
respectively. Conversely, porous materials proved more difficult to effectively decontaminate via the
fogging of chlorinated decontaminants, with average LR values for carpet, ceiling tile, wood, and concrete
being 2.69 ± 1.88, 1.89 ± 0.76, 3.49 ± 1.83, and 4.31 ± 1.82, respectively. Of the materials tested, ceiling
23
-------
tile was the most difficult to decontaminate; the highest LR achieved in the study for ceiling tile was 3.24 ±
0.20 (Test 19). Glass proved to be the least difficult to decontaminate overall with results showing > 6 LR
for 21 of the 25 treatments tested. Notably the three most difficult materials to decontaminate (carpet,
ceiling tile, and wood) are also comprised of organic based constituents. These general results related to
the effect of materials on decontamination efficacy when fogging chlorine-based decontaminants are
similar to results when pAB was applied as a spray or when materials were immersed. See for example,
US EPA (2006); Wood et al. (2011a); Wood et al. (2011 b); and Calfee et al. (2011).
Average LR for Materials
ii.iiiiiii
y y // & o* * ^ ^
Materia!
Figure 4-3. Average LR for materials (±SD)
Figure 4-4 displays efficacy results, but organized by material and sporicide. The average LR by material
was generally similar for each sporicide, although there were a few exceptions (e.g., the aqueous CIO2
solution much less effective on the galvanized metal compared to the other decontaminants). All four
chlorinated decontaminants were generally ineffective on carpet, concrete, wood, and ceiling tile.
24
-------
¦ Dichlor
¦ Diluted bleach
Material
Figure 4-4. Average efficacy for sporicidal solutions with respect to material
4.2.4 Decontamination Results for Each Location
The average LR (all tests, sporicides, and materials) for each mock office location and the average LR for
all locations combined are presented in Figure 4-5. Material coupons located on the desk show the
highest average LR, at 4.94 ±2.17. Coupons located under the desk (average LR 4.47 ± 2.30) and above
the ceiling (LR 4.42 ± 1.45) show effectively the same decontamination efficacy. An ANOVA showed no
significant difference between the average LR of coupons located under the desk and that of coupons in
other locations (p-value = 0.15). Findings were similar for coupons located above the ceiling (p-value =
0.28). Data analysis showed a significant difference (albeit small, i.e., ~ 0.5 LR difference) in the
decontamination efficacy of the desk location compared to the other locations (p-value = 0.012). Overall,
these minor differences in efficacy results as a function of test chamber location generally imply the fog
was fairly well distributed.
25
-------
8.00
7.00
6.00
£
.2 5.00
+-»
¦a 4.00
*
g> 3.00
2.00
1.00
n nn
II
ll
\i
Under the desk On the desk Above the ceiling All Locations
Location
Figure 4-5. Average LR for mock office locations (±SD)
4.3 Summary of Efficacious Test Conditions and Impact of Test Variables
Although no one test proved efficacious for all materials, the majority (24 of 25) of the decontamination
tests returned > 6 LR for one or more materials. Table 4-2 summarizes the tests in which each material
was effectively decontaminated (achieved an average of > 6 LR) and a summary of the associated
treatment conditions (effective ranges for volume disseminated, AIC in solution, and dwell time). Refer to
Table 4-1 for a detailed summary of conditions for individual tests. There were no tests in which carpet or
ceiling tile was effectively decontaminated, and just one test (Test 19) in which concrete was effectively
decontaminated, and so these materials are excluded from the table.
Efficacious decontamination conditions for galvanized metal were achieved with pAB volumes as low as
2,921 mL at 6,480 mg/L FAC. Similarly, effective decontamination with diluted bleach on galvanized metal
was achieved using 2,891 mL at 17,400 mg/L FAC. While smaller volumes of dichlor were sufficient for
effective decontamination on galvanized metal, all tests were performed with relatively high FAC
concentrations (at least 20,000 mg/L). Maximizing pAB and diluted bleach volume and FAC concentration
was not required to achieve > 6 LR for galvanized metal. Low volumes of dichlor at higher FAC
concentration proved efficacious, but information is not available for treatments at lower FAC
concentrations.
Similar to galvanized metal, efficacious fogging conditions were achieved for glass at lower pAB volumes
and FAC concentrations (2,921 mL pAB at 6,480 mg/L FAC). Also, fogging with low volumes of diluted
bleach and FAC concentrations (as low as 4,840 mL and 16,720 FAC) were effective with zero air
exchanges. Volumes of dichlor as low as 7,165 mL with FAC concentrations of 20,701 were proven
effective for glass with zero air exchanges. Aqueous CIO2 solution also proved effective for glass material
at low volumes (3,960 mL).
Small volumes of pAB at lower FAC concentrations (as low as 3,941 mL and 7840 mg/L FAC) were
effective for decontaminating laminate. Low volumes of diluted bleach were effective with increased levels
26
-------
of FAC (2,891 mL and 17,401 mg/L FAC). Aqueous CIO2 solution was effective at low volumes and CIO2
concentrations (3,960 mL and 4,763 mg/L). Larger volumes ofdichlorat higher concentrations (7,165 mL
and 20,701 mg/L) were required for full decontamination of laminate coupons.
Painted wallboard paper required smaller volumes of pAB at higher FAC concentrations for effective
decontamination (5,817 mL required 18,301 mg/L FAC). Relatively high volumes and FAC concentrations
(at least 7,642 mL and 19,001 mg/L FAC) of diluted bleach were required for effective decontamination.
Aqueous CIO2 solution proved effective at low volumes (3,960 mL). Similar to diluted bleach, high
volumes and FAC concentrations (at least 7,165 mL and 20,701 mg/L FAC) were required for successful
decontamination of painted wallboard paper with dichlor.
Wood material was successfully decontaminated with diluted bleach and aqueous CIO2. A relatively large
volume of diluted bleach at high FAC levels was required for effective decontamination. Aqueous CIO2
proved efficacious with relatively low volumes (3,960 mL).
In the case of nonporous materials, dichlor fogging proved effective at low volumes and 0.75 air
exchanges, but exceedingly high concentrations were required (3,141 mL and 32,502 mg/L FAC). It was
suspected that the high FAC levels of dichlor resulted in failure of the fogging equipment (one of two
foggers malfunctioned during testing). Further testing is required to assess equipment compatibility with
concentrated sporicide solutions.
Figure 4-6 is a distillation of the above information, and shows the minimum mass of AIC disseminated
(concentration of AIC X volume fogged) that resulted in effective decontamination, as a function of each
material and sporicide.
27
-------
Table 4-2. Summary of Efficacious (> 6 LR) Decontamination Conditions per Material
Coupon
Material
Efficacious Test Conditions and Test identification (ID) Numbers
pAB
Diluted bleach
Aqueous CI02
Dichlor
Galvanized
metal
Volume disseminated:
2,921-7,860 mL
AIC: 6,480-18,701 mg/L
Dwell: 0-20 hours
Tests: 2, 4, 9, 12, 16, 20
Volume disseminated: 2,891-
7,674 mL
AIC: 16,721-24,201 mg/L
Dwell: 16-23 hours
Tests: 3, 6, 14, 17, 19, 21, 26
-
Volume disseminated: 3,141-
7,915 mL
AIC: 20,701-32,502 mg/L
Dwell: 16-21 hours
Tests: 13, 18, 22-25
Glass
Volume disseminated:
2,921-7,860 mL
AIC: 6,480-18,701 mg/L
Dwell: 0-20 hours
Tests: 2, 4, 9, 12, 16, 20
Volume disseminated: 4,840-
7,674 mL
AIC: 16,721-24,201 mg/L
Dwell: 16-20 hours
Tests: 6, 14, 17, 19, 21, 26
Volume disseminated: 3,960-
7,738 mL
AIC: 4,763-5,907 mg/L
Dwell: 19 hours
Tests: 11, 15
Volume disseminated: 3,141-
7,915 mL
AIC: 20,701-32,502 mg/L
Dwell: 16-21 hours
Tests: 13, 18, 22-25
Laminate
Volume disseminated:
3,941-7,860 mL
AIC: 7,840-18,701 mg/L
Dwell: 19-21 hours
Tests: 4, 12, 16, 20
Volume disseminated: 2,891-
7,766 mL
AIC: 17,401-24,201 mg/L
Dwell: 16-23 hours
Tests: 3, 14, 17, 19
Volume disseminated: 3,960-
7,738 mL
AIC: 4,763-5,907 mg/L
Dwell: 19 hours
Tests: 11, 15
Volume disseminated: 3,141-
7,915 mL
AIC: 20,701-32,502 mg/L
Dwell: 16-21 hours
Tests: 13, 18, 22-25
PWB paper
Volume disseminated:
5,817-7,229 mL
AIC: 17,401-18,301 mg/L
Dwell: 19 hours
Tests: 12, 16
Volume disseminated: 7,642-
7,766 mL
AIC: 19,001-24,201 mg/L
Dwell; 16-19 hours
Tests: 14, 17, 19
Volume disseminated: 3,960-
7,738 mL
AIC: 4,763-5,907 mg/L
Dwell: 19 hours
Tests: 11, 15
Volume disseminated: 3,141-
7,915 mL
AIC: 20,701-32,502 mg/L
Dwell: 16-21 hours
Tests: 13, 18, 22-25
Wood
-
Volume disseminated: 7,766
mL
AIC: 23,701 mg/L
Dwell: 19 hours
Tests: 19
Volume disseminated: 3,
960-7,738 mL
AIC: 4,763-5,907 mg/L
Dwell: 19 hours
Tests: 11, 15
-
PAB
diluted bleach
aqueous CI02
dichlor
galv. metal glass laminate PWB wood concrete
Figure 4-6. Minimum mass of active ingredient needed to achieve effective decontamination
28
-------
4.3.1 Effect of Disseminated Volume on Fogging Efficacy
The effect of liquid volume fogged on decontamination efficacy was examined by comparing tests
subjected to similar conditions (solution type, AIC, dwell time, pH, RH and T) but with dissimilar volumes
of solution (mL) fogged. Tests paired for comparative analysis include Tests 7 and 13, 1 and 2, and 10
and 15. Table 4-1 summarizes the test conditions for each set of tests evaluated (AIC, volume of solution
disseminated, and dwell time) as well as the average LR and statistical analysis findings (ANOVA).
Tests 7 and 13 used dichlor solution; Test 7 conditions included an FAC concentration of 20,601 mg/L, a
disseminated volume of 5,873 mL, and 17 hours of dwell. Test 13 conditions included an FAC concentration
of 20,701 mg/L, a disseminated volume of 7,165 mL, and 17 hours of dwell. Statistical analysis of these data
indicated that varying the volume of diluted bleach solution disseminated has a statistically significant effect
on decontamination efficacy at the prescribed test conditions (0.00122 p-value).
Tests 1 and 2 were performed with pAB solution. Test 1 conditions included an FAC concentration of
6,440 mg/L, a disseminated volume of 6,480 mL, and 20 hours of dwell. Test 2 conditions included an
FAC concentration of 6,480 mg/L, a disseminated volume of 2,921 mL, and 24 hours of dwell. Statistical
analysis of these data indicated that varying the volume of pAB solution disseminated has a statistically
significant effect on decontamination efficacy at the prescribed test conditions (1.22E-05 p-value).
Tests 10 and 15 used aqueous CIO2 solution. Test 10 conditions included a CIO2 concentration of 5,906
mg/L, a disseminated volume of 5,907 mL, and 19 hours of dwell. Test 15 conditions included a CI02(aq)
concentration of 5,907 mg/L, a disseminated volume of 1,910 mL, and 19 hours of dwell. Statistical
analysis of these data indicated that varying the volume of aqueous CIO2 solution disseminated has a
statistically significant effect on decontamination efficacy at the prescribed test conditions (1.03E-07 p-
value).
The disseminated volume of solution proved to have a significant effect on efficacy for each of the three
comparisons evaluated, i.e., increasing the volume fogged increased the efficacy. This effect seemingly
persisted regardless of the AIC (relatively high or low).
Table 4-3. Decontamination Efficacy Comparison of Similar Tests with Different Volumes of
Disseminated Solution
Test
ID
Sporicidal
Solution
AIC in Solution
(mg/L)
Volume of
Solution
Disseminated
(mL)
Dwell
Time (h)
Avg. LR
p-Value
(a = 0.05)
7
Dichlor
20601
5873
17
3.71 ± 1.58
0.00122
13
20701
7165
17
5.53 ± 1.90
1
pAB
6440
887
20
0.90 ± 0.32
1.22E-05
2
6480
2921
24
3.28 ± 0.91
10
Aqueous
CIO2
5906
1910
19
2.66 ± 0.52
1.03E-07
15
5907
7738
19
5.80 ± 1.13
29
-------
4.3.2 Effect of Air Exchange on Decontamination Efficacy
Two air exchange rates were used for this study: 0 and 0.75 air exchanges per hour. The majority of fog
tests used no air exchange, while there were four tests that used an air exchange of 0.75. Statistical
analyses were performed to assess the impact of air exchange on decontamination efficacy using paired
tests with similar conditions, but with and without air exchange. Sets compared included Tests 22 and 23,
7 and 25, and 26 and 21. Table 4-3 summarizes test conditions (the AIC for the sporicidal solution, the
volume of solution disseminated, and the total air exchanges [fraction of chamber volume replaced each
hour]) as well as the comparative analysis (ANOVA) findings. The results show lower average efficacy in
all three comparisons where air exchange was used, however the effect was significant only for the
comparison between Tests 7 and 25 and therefore the lower values for air exchange in the other two
comparisons were due to natural variation.
Table 4-4. Efficacy Comparison of Similar Tests with Different Rates of Air Exchange
Test
ID
Sporicidal
solution
AIC in
Solution
(mg/L)
Volume of
Solution
Disseminated
(mL)
Air Exchange
(fraction of
chamber volume
replaced per
hour)
Avg. LR (±
SD)
p-Value
(a = 0.05)
22
Dichlor
21901
7778
0.75
5.44 ± 0.67
0.89
23
20701
7396
0
5.52 ± 0.48
7
Dichlor
20601
5873
0.75
3.72 ± 0.99
0.027
25
20801
5406
0
5.12 ± 0.78
26
Diluted bleach
22201
7674
0.75
4.55 ± 0.93
0.91
21
23001
7780
0
4.63 ± 0.65
4.3.3 Effect of Dwell on Decontamination Efficacy
For this study, the dwell period (the post fogging time period during which the chamber environment was
allowed to remain undisturbed prior to starting aeration) was typically overnight. However, two tests were
performed with low dwell times (0 and 2 hours) to assess the impact of dwell on decontamination efficacy.
Comparative analysis was performed using two sets of paired tests with similar conditions but different
dwell times. Test sets included Tests 8 and 6 and 9 and 16. Table 4-4 summarizes test conditions (AIC
for the sporicidal solution, volume of solution disseminated, and dwell time) as well as the average LR
and statistical analysis findings (ANOVA).
Table 4-5. Efficacy Comparison of Similar Tests with Different Dwell Times
Test
ID
Sporicidal
Solution
AIC in Solution
(mg/L)
Volume
Disseminated
(mL)
Dwell
Time (h)
Average LR (±
SD)
p-Value
(a = 0.05)
8
Diluted
Bleach
15920
5300
2
3.5 ± 1.25
0.059
6
16721
4840
20
4.59 ± 0.79
9
pAB
15701
5891
0
4.56 ±1.11
0.96
16
17401
7229
19
4.53 ± 1.17
Tests 8 and 6 using diluted bleach were paired for comparison. Test 8 conditions included 2 hours of
dwell, an FAC concentration of 15,920 mg/L, and a disseminated volume of 5,300 mL. Test 6 conditions
30
-------
included 20 hours of dwell, an FAC concentration of 16,721 mg/L, and a disseminated volume of 4,840.
Analysis of these data indicated dwell time did not have a statistically significant effect on
decontamination efficacy at the time prescribed test conditions (0.059 p-value).
Tests 9 and 16 using pAB were paired for comparison. Test 9 conditions included 0 hours of dwell, an
FAC concentration of 15,701 mg/L, and a disseminated volume of 5,891 mL. Test 16 conditions included
19 hours of dwell, an FAC concentration of 17,401 mg/L, and a disseminated volume of 7,229 mL.
Statistical analysis indicated dwell time has no statistically significant effect on decontamination efficacy
at the prescribed test conditions (0.96 p-value).
4.3.4 Effect of Active Ingredient Concentration on Decontamination Efficacy
The effect of AIC on decontamination efficacy was assessed by comparing paired tests that had similar
test conditions but different AlCs. Test sets examined were Test 14 and 17 and Tests 2 and 4. Table 4-5
summarizes the tests conditions (AIC for the sporicidal solution, volume of solution disseminated, and
dwell time) as well as the average LR and statistical analysis findings (ANOVA).
Table 4-6. Efficacy Comparison for Tests with Varying AlCs
Test
ID
Sporicidal
Solution
AIC in Solution
(mg/L)
Volume
Disseminated
(mL)
Dwell
Time (h)
Average LR (±
SD)
p-Value
(a = 0.05)
14
Diluted
19001
7642
19
5.73 ± 0.74
0.52
17
bleach
24201
7776
16
5.32 ± 0.66
2
pAB
6480
2921
24
3.28 ±0.91
0.29
4
7840
3941
21
4.03 ± 1.03
While statistical analysis for both diluted bleach and pAB tests did not indicate any significant effects of
FAC levels on decontamination efficacy, this is most likely because the difference in AIC was not large for
the relationship to be apparent.
31
-------
4.4 Biological Indicator Results
Although previous tests (Rastogi et al., 2010) have shown that spore populations on Bis are typically
much easier to inactivate than spores associated with coupons from building materials or actual
environmental surfaces, Bis were included in fogging tests to demonstrate the general concept and utility
of fogging technology.
Figure 4-7 presents the Bl results for tests that included Bis, in terms of the percent of Bis inactivated
(typically nine were used in each test). All positive control Bis (those not exposed to fogging conditions)
from every experiment tested positive for growth. In experiments with air exchange (Tests 22, 24, and
25), a portion of the Bis showed positive growth. In two instances (Tests 10 and 24), all Bis tested
positive. Other tests that had less than a 100% inactivation rate of Bis (Tests 2, 4, and 7) used low
disseminated volumes of sporicide. In general, relatively low dissemination volumes and the presence of
air exchange appear to challenge Bl decontamination efficacy (similar to B. atrophaeus spore inactivation
results discussed above) to a higher extent than other parameters tested in this study.
120
100
£
O
4= 80
60
£
CD
U
&_
CD
Q.
40
20
2 3 4 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25
Test ID
Figure 4-7. Percent of Bis inactivated for each test with Bis (n = 9)
Figure 4-8 shows the percentage of inactivated Bis as a function of location. Bis located on the desk
showed the highest inactivation percentage of 84.1%. Bis under the desk were inactivated at a rate of
81.9%, and Bis located above the ceiling had the lowest inactivation percentage of 69.6%.
32
-------
¦ No growth
Above ceiling
Under desk
Mock office location
Figure 4-8. Bl inactivation percentages in each mock office location (n = 69, all tests combined)
4.5 Impact of Dichlor Residue on Spore Recovery
During the process of collecting samples exposed to dichlor fog, residue was observed on the coupon
surfaces and in COMMANDER itself. Subsequent tests confirmed that a dichloroisocyanurate salt was
present in the residue. Because of this residue, a test was conducted to determine the impact of the
dichlor residue on the recovery of spores subsequently inoculated onto coupons.
The results of this test are shown in Figure 4-9, and show a considerable effect of the residue, i.e., a 7 log
difference between viable spores recovered from surfaces containing dichlor residue (4.67E+01) and
those recovered from clean surfaces (5.83E+07). In response to these findings, additional neutralization
tests were performed to identify potential biases in data gathered from fogging tests with dichlor solution.
33
-------
1.00E+08
1.00E+07
1.00E+06
CL
ro 1.00E+05
ll 1.00E+04
0)
ro 1.00E+03
-------
1.00E+06
1.00E+05
£¦ 1.00E+04
o
£ 1.00E+03
cc
3
5 1.00E+02
1.00E+01
l.OOE+OO
Extraction Procedure
Figure 4-10. Effect of neutralizer during extraction on average CFU recovery (± SD)
Table 4-7. Average CFU Recoveries for Extraction Procedures
Material
Extraction
Solution
Floor
p-Value
Desk
p-Value
Ceiling
p-Value
Carpet
PBST
2.57E+04 ± 9.66E+03
0.087
5.00E+00 ± 0.00E+00
0.31
2.31 E+04 ± 9.60E+03
0.47
PBST +
STS
5.97E+04 ± 2.42E+04
7.06E+02 ± 1.05E+03
4.97E+04 ± 5.69E+04
Galvanized
Metal
PBST
5.00E+00 ± 0.00E+00
0.37
5.00E+00 ± 0.00E+00
n/a
5.00E+00 ± 0.00E+00
n/a
PBST +
STS
7.13E+01 ± 1.15E+02
5.00E+00 ± 0.00E+00
5.00E+00 ± 0.00E+00
Laminate
PBST
5.00E+00 ± 0.00E+00
0.37
8.62E+02 ± 1.38E+03
0.36
9.76E+02 ± 1.29E+03
0.90
PBST +
STS
2.91 E+04 ± 5.00E+04
4.87E+01 ± 7.56E+01
1.14E+03 ± 1.63E+03
Concrete
PBST
3.28E+03 ± 4.69E+03
0.73
2.60E+01 ± 3.64E+01
0.30
7.13E+01 ± 1.15E+02
0.051
PBST +
STS
4.77E+03 ± 5.05E+03
1.84E+03 ± 2.66E+03
5.14E+03 ± 3.17E+03
4.5.2 Coupon Storage Neutralization Test
The purpose of this second neutralization test (Test 28) was to identify the need to neutralize the coupons
(after fogging of dichlor) during the 72-hour period over the weekend when coupons were stored in a
refrigerator until they could be processed and extracted the following week. Figure 4-11 shows the results
for this test (Test 28) and Table 4-8 provides a summary. An ANOVA of the three sample hold procedures
(dry, in 10 mL PBST, or in 10 mL PBST+STS) for samples with dichlor residue was performed for each of
the three materials studied. For laminate and concrete, the analysis resulted in p-values greater than 0.05
for each material, indicating no statistically significant difference between the three coupon hold
procedures. For these materials, neither storing samples in 10 mL of PBST nor adding neutralizer to the
PBST appeared to improve average spore recoveries significantly compared to storing the samples dry.
However, in the case of carpet, an ANOVA comparison of the three storage procedures showed a
iLiilll
PBST PBST + PBST PBST + PBST PBST + PBST PBST +
STS STS STS STS
Carpet Gal. Metal Laminate Concrete
I Floor
I Desk
Ceiling
35
-------
statistical difference within the set, i.e., spore recoveries for carpet samples appeared to increase
significantly for samples stored in PBST with neutralizer (ANOVA p-value = 3.05E-09).
Dry -UsT PBST + DrJ PBST + Dry -t>BST PBST +
STS STS STS
Carpet Laminate Concrete
Storage Method
Figure 4-11. Effect of neutralizer during coupon storage (± SD)
Table 4-8. Average CFU Recoveries for Alternative Storage Procedures
Material
Storage
Procedure
CFU Recovery (± SD)
p-Value
Floor
Desk
Ceiling
Carpet
Dry
5.42E+00 ± 5.14E+00
7.73E-01 ±1.37E-02
7.77E-01 ± 1.83E-02
3.29E-08
PBST
1.02E+00 ± 4.27E-01
7.59E-01 ± 4.06E-02
7.54E-01 ± 6.53E-03
PBST + STS
4.10E+01 ± 2.22E+01
4.13E+01 ± 1.07E+01
2.35E+01 ± 9.74E+00
Laminate
Dry
2.19E+02 ± 3.07E+02
4.28E+01 ± 4.41 E+01
9.46E-01 ± 4.18E-01
0.26
PBST
1.02E+00 ± 4.27E-01
6.73E-01 ± 1.37E-02
1.38E+00 ± 1.21E+00
PBST + STS
3.29E+02 ± 2.37E+02
2.28E+01 ± 1.99E+01
1.00E+01 ± 8.08E+00
Concrete
Dry
8.00E-01 ± 5.09E-02
7.54E-01 ± 1.72E-02
8.34E-01 ± 2.45E-02
0.35
PBST
1.02E+00 ± 4.27E-01
3.30E+00 ± 4.36E+00
7.51 E-01 ± 2.86E-02
PBST + STS
8.67E-01 ± 8.29E-02
7.78E-01 ± 2.74E-02
8.38E-01 ± 2.16E-02
The aforementioned results are caveated by the fact that Test 28 coupons were inoculated using an MDI
manufactured by a different vendor (Research International) from what was used in all the previous tests.
The use of the different MDI may have led to notably lower overall CFU recoveries from the Test 28
control set samples (the set of coupons stored dry; the same storage conditions at Tests 1-26) compared
to Test 27 control set samples (the set of samples extracted without addition of neutralizer; same
1.00E+03
1.00E+02
O
(J
(D
al
<
1.00E+01
1.00E+00
1.00E-01
36
-------
extraction conditions as Tests 1-26), despite having comparable fog test conditions (see Table 4-1). This
is because investigation revealed that when subjected to heat shock, samples inoculated with the newer
Research International MDI yielded approximately 50% less CFU than the samples not subjected to heat
shock. These results suggest a higher population of vegetative cells were loaded onto Test 28 coupons
compared to Test 27. Thus it is possible that results for Test 28 were not representative of previous tests
because of the different spore mixture and should be cautiously considered in reference to this study.
However, any additional vegetative cells on the coupons in Test 28 would have most likely been
inactivated during the fogging, yielding only spores on the coupons that were extracted. And thus the
comparison of coupon storage methods (dry, PBST, or PBST + STS) would still be valid.
37
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5 Summary and Conclusions
In this investigation, a series of 27 tests were conducted to assess the efficacy of fogging chlorine-based
sporicidal solutions for the decontamination of common materials found in indoor and outdoor
environments contaminated with a B. anthracis spore surrogate. While previous studies have shown that
chlorine-based sporicides applied as a spray are effective in inactivating bacterial spores on a number of
materials, commercially available fogging technologies have not been evaluated for their use with
chlorine-based sporicides.
The following summarizes some of the more important findings from the study:
• Maximizing the fogged solution volume (approximately 8 L total; or in terms of volume to be
decontaminated, 336 mL/m3) and the AIC (maximum of approximately 22,000 mg/L free available
chlorine for pAB, diluted bleach, and dichlor solution; and approximately 5000 mg/L aqueous
CIO2) generally produced similar results for all sporicides. That is, decontamination efficacy
averaged for all materials in these tests at these more optimal conditions was generally above 5
LR, independent of the sporicide fogged.
• While no individual test achieved an average decontamination efficacy of > 6 LR for all materials,
fogging methods were proven most effective (typically achieved > 6 LR) for the nonporous
materials: galvanized metal, glass, painted wallboard paper, and laminate. Fogging of the
chlorinated decontaminants was moderately effective for concrete. Ceiling tile, wood, and carpet
(porous and organic-based materials) were the most difficult materials to decontaminate. These
general trends in efficacy by material using chlorine-based sporicides are consistent with the
literature.
• Relatively high volumes of sporicidal solution at high concentrations appeared to cancel any
significant effects of increased air exchange rates on decontamination efficacy.
• Increasing the disseminated volume of solution proved to significantly increase decontamination
efficacy. This effect persisted regardless of the solution's AIC (high or low).
• Data analysis showed a significant yet minor average improvement (~ 0.5 LR) in the
decontamination efficacy of the coupons placed on the desk location compared to the other
locations (under the desk and above the ceiling tiles). Coupons located under the desk and above
the ceiling showed the same average decontamination efficacy. Overall, these minor differences
in efficacy results as a function of test chamber location generally imply the sporicidal fog was
fairly well distributed.
• In an experiment to evaluate the neutralization requirements for coupon samples containing
dichlor residue during the extraction process, it was determined that there were statistically
insignificant differences between samples extracted with PBST plus neutralizer and those
extracted with just PBST. This was shown to be the case for all materials. This result is caveated
by the fact that only one test was conducted, and that further research to investigate more fully
this issue is warranted.
• The recovery of spores inoculated onto coupons already having a dichlor residue was
significantly diminished.
This study has demonstrated the potential of using chlorine-based decontaminants applied with a
commercially available fogging technology for the decontamination of surfaces typical of indoor
38
-------
environments contaminated by Bacillus spores. However, this decontamination approach may be better
suited for areas that do not contain significant quantities of porous or organic materials such as carpet,
ceiling tile, or wood.
39
-------
6 References
Brown, G.S., Betty, R.G., Brockmann, J.E., Lucero, D.A., Souza, C.A., Walsh, K.A., Boucher, R.M.,
Tezak, M., Wilson, M.C., and Rudolph, T. (2007a) Evaluation of a wipe surface sample method for
collection of Bacillus spores from nonporous surfaces. Appl Environ Microbiol 73(3):706-710.
Calfee, M. W., Choi, Y., Rogers, J., Kelly, T., Willenberg, Z., & Riggs, K. (2011). Lab-scale assessment to
support remediation of outdoor surfaces contaminated with Bacillus anthracis spores. Journal of
Bioterrorism and Biodefense, 2(3).
Canter, D.A., Gunning, D., Rodgers, P., O'Connor, L., Traunero, C., Kempter, C.J. (2005) Remediation of
Bacillus anthracis contamination in the U.S. Department of Justice mail facility. Biosecur Bioterror 3:
119-127.
Lee, S.D., Ryan, S.P. and Snyder, E.G. (2011) Development of an aerosol surface inoculation method for
Bacillus spores. Appl Environ Microbiol 77, 1638-1645.
National Council of the Paper Industry for Air and Stream Improvement, Inc. (NCASI). (1997) Methods
Manual, Determination of Chlorine and Chlorine Dioxide in Pulp Mill Bleach Plant Vents. Research
Triangle Park, N.C.
Rastogi, V.K., Ryan, S.P., Wallace, L., Smith, L.S., Shah, S.S., Martin, G.B. (2010) Systematic evaluation
of the efficacy of chlorine dioxide in decontamination of building interior surfaces contaminated with
anthrax spores. Appl Environ Microbiol 76: 3343-3351.
Ryan, S.P., Lee, S.D., Calfee, M.W., Wood, J.P., McDonald, S., Clayton, M., Griffin-Gatchalian, N. (2014)
Effect of inoculation method on the determination of decontamination efficacy against Bacillus spores.
World J Microbiol Biotechnol, DOI 10.1007/s11274-014-1684-2.
Schmitt, K., N.A. Zacchia, N.A. (2012) Total decontamination cost of the anthrax letter attacks. Biosecur
Bioterror 10:98-107.
Standard Methods Online (2005). Standard Methods 21st Edition, Standard Methods for the Examination
of Water and Wastewater, http://standardmethods.org/ (last accessed November 28, 2016).
U.S. EPA (2010) Determining the Efficacy of Liquids and Fumigants in Systematic Decontamination
Studies for Bacillus anthracis Using Multiple Test Methods, EPA/600/R-10/088. Washington, DC.
U.S. EPA (2013). Bio-response Operational Testing and Evaluation (BOTE) Project - Phase 1:
Decontamination Assessment, EPA/600/R-13/168. U.S. Environmental Protection Agency,
Washington, DC.
U.S. EPA (2012). Assessment of Liquid and Physical Decontamination Methods for Environmental
Surfaces Contaminated with Bacterial Spores: Evaluation of Spray Method Parameters and Impact of
Surface Grime, EPA/600/R/12/591. U.S. Environmental Protection Agency, Washington, DC.
40
-------
U.S. EPA (2015) Surface Decontamination Methodologies for a Wide-Area B. Anthracis Incident,
EPA/600/S-15/172, 2015. U.S. Environmental Protection Agency, Washington, DC.
U.S. EPA. (2006) Evaluation of Spray-Applied Sporicidal Decontamination Technologies, EPA/600/R-
06/146. U.S. Environmental Protection Agency, Washington, DC.
Wood, J.P., Calfee, M.W., Clayton, M., Griffin-Gatchalian, N., Touati, A., Ryan, S., Mickelsen, L., Smith,
L., Rastogi, V. (2016) A simple decontamination approach using hydrogen peroxide vapor for Bacillus
anthracis spore inactivation. J Appl Microbiol. Accepted Author Manuscript, doi: 10.1111/jam.13284.
Wood, J.P., Calfee, M.W., Clayton, M., Griffin-Gatchalian. N., Touati, A., Egler, K. (2013) Evaluation of
peracetic acid fogging for the inactivation of Bacillus spores. J Hazard Mater 250-251:61-67.
Wood, J.P., Calfee, M.W., Clayton, M., Griffin-Gatchalian, N., and Touati A. Optimizing acidified bleach
solutions to improve sporicidal efficacy on building materials. Article first published online: 27 OCT
2011 | DOI: 10.1111/j.1472-765X.2011.03162.x
Wood, J.P., Choi, Y., and Rogers, J.V. Efficacy of liquid spray decontaminants for inactivation of Bacillus
anthracis spores on building and outdoor materials. J. Appl Microbiol. 2011,110, 1262-1273.
41
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Appendix A:
Detailed Decontamination Results
42
-------
Table A-1. Detailed LR Summary
Test ID
Carpet
Ceiling Tile
Concrete
Galvanized Metal
Glass
Laminate
PWB Paper
Wood
Avg LR
SD
Avg LR
SD
Avg LR
SD
Avg LR
SD
Avg LR
SD
Avg LR
SD
Avg LR
SD
Avg LR
SD
1
0.33
0.12
0.44
0.10
0.59
0.11
1.58
0.46
1.26
0.58
1.45
0.30
1.11
0.31
0.45
0.22
2
0.84
0.18
1.04
0.14
1.94
0.21
6.70
0.94
6.30
1.20
4.50
1.49
3.46
1.27
1.50
0.57
3
0.49
0.19
1.11
0.23
4.16
1.67
6.84
1.21
5.90
2.45
6.00
1.70
5.53
1.62
2.42
0.65
4
0.89
0.21
1.57
0.30
3.55
1.07
6.88
1.59
6.76
1.43
6.31
1.22
4.51
1.07
1.73
0.15
6
1.92
0.21
1.86
0.26
4.77
0.87
7.28
0.73
7.47
0.01
5.24
0.47
5.60
1.78
2.54
0.41
7
2.25
0.72
1.55
0.32
2.93
0.48
4.59
1.38
5.48
1.87
4.51
0.82
5.78
0.70
2.64
0.66
8
1.36
0.19
1.37
0.11
3.57
1.34
4.78
1.09
5.45
2.17
4.62
1.95
4.52
0.96
2.30
0.42
9
2.09
2.04
1.81
0.20
4.90
0.84
7.15
0.37
7.37
0.14
5.58
1.21
5.57
1.78
2.05
0.38
10
1.80
0.63
1.21
0.12
2.09
0.34
1.98
0.62
5.18
0.50
3.21
1.20
3.01
0.57
2.78
0.52
11
4.74
1.67
2.40
0.40
3.95
0.74
2.27
0.36
7.13
0.29
6.56
1.42
6.50
1.20
6.25
0.89
12
2.78
0.46
2.43
0.24
5.74
1.08
7.19
0.01
7.16
0.09
7.10
0.57
6.70
1.12
3.81
0.98
13
3.57
1.15
2.20
0.14
5.33
0.98
6.88
0.05
6.97
0.06
7.19
0.30
7.29
0.03
4.84
0.35
14
3.18
1.59
2.23
0.33
5.96
0.52
7.40
0.06
7.17
0.13
7.51
0.53
7.12
0.80
5.25
0.72
15
5.96
1.15
3.20
0.57
4.91
1.18
5.75
0.37
6.63
0.17
6.88
1.52
6.41
1.58
6.66
1.13
16
1.49
0.30
1.37
0.29
3.47
1.81
7.12
0.02
6.87
0.44
6.50
1.95
6.39
1.77
3.00
0.51
17
2.01
0.85
2.09
0.31
5.66
0.95
7.20
0.05
7.26
0.14
7.33
0.96
7.09
0.38
3.90
0.83
18
4.64
1.19
2.48
0.20
5.35
0.48
6.99
0.18
6.96
0.01
7.33
0.44
7.50
0.01
4.83
0.52
19
3.93
0.79
3.24
0.70
6.23
0.54
6.75
0.05
6.83
0.10
6.86
0.62
6.62
1.28
6.41
1.30
20
2.67
0.34
2.21
0.88
5.77
0.99
6.35
0.54
6.81
0.10
7.09
0.02
5.54
0.95
3.64
0.37
21
2.57
0.55
2.04
0.32
4.46
1.13
5.81
0.92
6.60
0.81
5.63
0.23
5.79
0.19
4.12
0.35
22
5.06
1.36
1.82
0.28
5.35
0.97
6.86
0.04
6.91
0.01
7.35
0.03
6.64
0.80
3.50
0.22
23
3.56
0.42
2.15
0.43
5.46
0.87
7.02
0.22
7.50
0.01
7.35
0.62
6.99
0.02
4.09
0.52
24
3.91
0.94
1.89
0.21
4.97
0.66
6.63
0.37
7.21
0.00
6.91
0.35
6.03
0.94
1.75
0.22
25
3.12
0.98
1.50
0.16
3.55
0.76
6.84
1.37
7.20
0.07
7.00
0.59
6.60
0.67
N/A
N/A
26
2.12
1.04
2.00
0.35
3.11
0.95
7.26
0.06
7.22
0.36
5.97
1.71
5.49
1.30
3.25
0.34
Note: Test 5 not included in table since this test was used for method development, in which peracetic acid (a non-chlorine based decontaminant) was fogged. Bold numbers indicate
complete inactivation of spore population and no spores were detected.
43
-------
Appendix B:
Efficacy Charts for Individual Tests
44
-------
7.00
6.00
5.00
4.00
3.00
Figure B-1. Test 1 LR Summary (±SD)
9.00
8.00
Figure B-2. Test 2 LR Summary (±SD)
Material
Material
45
-------
¦ Ceiling
Material
Figure B-3. Test 3 LR Summary (±SD)
¦ Desk
¦ Ceiling
Material
Figure B-4. Test 4 LR Summary (±SD)
46
-------
¦ Ceiling
Material
Figure B-5. Test 8 LR Summary (±SD)
¦ Desk
¦ Ceiling
Material
Figure B-6. Test 7 LR Summary (±SD)
47
-------
Ceiling
Material
Figure B-7. Test 8 LR Summary (±SD)
¦ Desk
¦ Ceiling
Material
Figure B-8. Test 9 LR Summary (±SD)
48
-------
8.00
Material
Figure B-9. Test 10 LR Summary (±SD)
8.00
7.00
6.00
5.00 | |
4,00 ¦
"° ! ii i
I11
-------
Material
Figure B-11. Test 12 LR Summary (±SD)
Material
Figure B-12. Test 13 LR Summary (±SD)
50
-------
¦ Ceiling
Material
Figure B-13. Test 14 LR Summary (±SD)
¦ Desk
¦ Ceiling
Material
Figure B-14. Test 15 LR Summary (±SD)
51
-------
9.00
8.00 T T"
r il I II T
6.00
T
¦ ¦ II ¦ T aF|oor
I I II ¦ ]jIt ii
Eiill II111
i<^ d;J^ (-$• &
-------
¦ Ceiling
Material
Figure B-17. Test 18 LR Summary (±SD)
¦ Desk
¦ Ceiling
Material
Figure B-18. Test 19 LR Summary (±SD)
53
-------
¦ Ceiling
Material
Figure B-19. Test 20 LR Summary (±SD)
¦ Desk
¦ Ceiling
Material
Figure B-20. Test 21 LR Summary (±SD)
54
-------
9.00
8.00
=1 iniii
= ! f Ifl
jf' ^ 0Ob ,<£-
or . <& # & 9 ^ ^
c# c? ^ s/ c/
Material
Figure B-21. Test 22 LR Summary (±SD)
..mid
-------
¦ Ceiling
Material
Figure B-23. Test 24 LR Summary (±SD)
¦ Desk
¦ Ceiling
Material
Figure B-24. Test 25 LR Summary (±SD)
56
-------
¦ Ceiling
Material
Figure B-25. Test 26 LR Summary (±SD)
57
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United States
Environmental Protection
Agency
PRESORTED STANDARD
POSTAGE & FEES PAID
EPA
PERMIT NO. G-35
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