&EPA
EPA/600/R-14/170 | August 2014 | www.epa.gov/nhsrc
United States
Environmental Protection
Agency
Methyl Bromide Decontamination
of Indoor and Outdoor Materials
Contaminated with
anthracis Spores
Office of Research and Development
National Homeland Security Research Center

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EPA/ 600/R-14/170
August 2014
Methyl Bromide Decontamination
of Indoor and Outdoor Materials
Contaminated with Bacillus
anthracis Spores
U.S. Environmental Protection Agency
Research Triangle Park, NC 27711
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Disclaimer
The U.S. Environmental Protection Agency (EPA), through its Office of Research and
Development's (ORD) National Homeland Security Research Center (NHSRC), funded, directed
and managed this work through Contract Number EP-C-10-001 with Battelle. This report has
been peer and administratively reviewed and has been approved for publication as an EPA
document. The views expressed in this report are those of the authors and do not necessarily
reflect the views or policies of the Agency. Mention of trade names or commercial products does
not constitute endorsement or recommendation for use of a specific product.
Questions concerning this document or its application should be addressed to:
Joseph Wood
National Homeland Security Research Center
Office of Research and Development
U.S. Environmental Protection Agency
Mail Code E343-06
Research Triangle Park, NC 27711
919-541-5029
li

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Acknowledgments
Contributions of the following individuals and organizations to this report are gratefully
acknowledged:
United States Environmental Protection Agency (EPA)
Leroy Mickelsen, CBRN Consequence Management Advisory Team
Richard Rupert, US EPA On-Scene Coordinator
Peer Reviewers
Shannon Serre, EPA National Homeland Security Research Center
Benjamin Franco, EPA On-Scene Coordinator
Steven Merritt, EPA On-Scene Coordinator
Battelle Memorial Institute
111

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Executive Summary
The U.S. Environmental Protection Agency (EPA), Office of Research and Development (ORD),
is striving to protect human health and the environment from adverse impacts resulting from the
intentional release of threat agents. As part of this mission, the EPA's Homeland Security
Research Program (HSRP) is investigating the effectiveness and applicability of technologies for
homeland security (HS)-related applications. This report provides the results of an assessment to
determine the decontamination efficacy of methyl bromide (MeBr) fumigant in inactivating
Bacillus anthracis (B.acausative agent for anthrax) spores on indoor and outdoor materials. In
particular, to facilitate future decontaminations employing MeBr, this investigation focused on
finding efficacious conditions when using MeBr at temperatures and relative humidity (RH)
levels lower than used in previous studies. Another objective of the study was to compare these
results with other spore-forming microorganisms to assess their potential as a representative
surrogate for B.a. Ames, for use in field studies and additional lab-based investigations.
This investigation focused on the decontamination of six types of common indoor and outdoor
materials: glass, ceiling tile, carpet, painted wallboard paper, bare pine wood, and unpainted
concrete. Decontamination efficacy tests were conducted with spores of virulent B.a. Ames and
the non-virulent strains Geobacillus stearothermophilus [G..s.], B.a. NNR1A1, and B.a. Sterne.
Decontamination efficacy was quantified in terms of log reduction (LR), based on the difference
in the number of bacterial spores recovered from positive control coupons and test coupons.
Tests were conducted at varying temperatures, RH levels, MeBr concentrations, and contact
times to assess the effect of these operational parameters on decontamination efficacy. Twenty
tests were conducted with MeBr, with target concentrations of either 212 or 300 milligrams per
liter (mg/L). Additionally, the target temperature during testing ranged from 22 to 32 °C, the
target RH was either 45 or 75 %, and contact times ranged from 18 to 72 hours.
Summary of Results
As seen in other similar fumigant evaluations1, the concentration, temperature, RH, and contact
time affect the efficacy of MeBr against B.a. Ames. Table E-l shows the contact time required to
achieve >6 LR (a decontaminant that achieves an LR value >6 is considered effective)2 on all
materials tested for a given set of fumigation conditions (MeBr level, temperature, and RH). For
example, a contact time of 36 hours was required to achieve > 6 LR of B.a. Ames on all
materials when fumigating at 212 mg/L, 22 °C, and 75 % RH. However, only 18 hours of contact
time were required to achieve > 6 LR of B.a. Ames on all materials when the MeBr
concentration was increased to 300 mg/L and temperature increased to 27 °C.
The test program originally began using two microorganisms and the six aforementioned test
materials in each experiment. But to evaluate three microorganisms at once, the number of
coupon materials for each experiment was reduced from six to four, due to the size of the MeBr
test chamber. Painted wallboard and unpainted concrete were removed from the latter part of the
study, as these two material types generally exhibited higher decontamination efficacy than the
other materials. In contrast, test results showed that glass and wood were the materials most
difficult to decontaminate (exhibited lower efficacy than the other four material types).
The data generated from this investigation show that G.s. is less resistant than B.a. Ames (G.s.
was inactivated at a higher average LR than B.a. Ames) in the two tests conducted with G.s. In
every test conducted with the NNR1A1 strain, the NNR1A1 strain was always more resistant
than the Ames strain. (In a few of the tests using the NNR1A1 strain, the average difference in
LR with the Ames strain was more than 6.0.) In the tests with the Sterne strain, Sterne was
iv

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always inactivated to a higher degree than the Ames strain when fumigating at 45 % RH. But
when fumigating at 75 % RH, the Sterne strain was more resistant than B.a. Ames.
This study shows the important role that RH plays when fumigating with MeBr. There were no
tests in which >6 LR of B.a. Ames was achieved on all materials when fumigating at 45 % RH.
When fumigating at 45 % RH, increasing the MeBr concentration, temperature, or contact time
generally did not improve decontamination efficacy. In contrast, when fumigating at 75 % RH,
increasing the MeBr concentration, temperature and contact time did generally improve efficacy
Table E-l. Contact Time Required to Achieve >6 LR on all Materials*
Target MeBr Target Target Time (hours) Required to Achieve >6 LR on All Materials0 Number
Concentration
(mg/L)
Temperature
(°C)
RH
(%)
B.a. Ames
G.s.
B.a. NNR1A1
B.a. Sterne
lest rxumuer
Reference3
212
22
45
>60
48
>60
>60
1,2, 11
212
22
75
36
b
>36
--
3,5
212
27
45
>48
--
>36
>48
4, 6,8
212
27
75
36
"
>36
36
7, 9
212
32
45
>72
--
>72
>72
13, 16, 19
212
32
75
24
"
>24
24
12,
300
22
45
>60
--
>60
>60
10, 15
300
22
75
24
"
>24
>24
14
300
27
45
>60
--
>60
>60
18
300
27
75
18
"
> 18
> 18
17
300
32
45
>60
"
>60
>60
20
* Materials tested were glass, ceiling tile, carpet, painted wallboard paper, bare pine wood and unpainted concrete.
a Detailed data from each test number can be referenced in Tables A-l through A-4 in Appendix A.
b" Not tested.
0 > indicates that >6 LR on all materials was not achieved at the contact time listed, and contact time was the longest tested.
Impact of Study
This research provides information on the efficacy of MeBr fumigation to decontaminate
materials that have been contaminated with B. anthracis spores. Such results may be useful in
the development of guidance to aid in deployment of MeBr fumigation after a wide-area release
of B. anthracis spores. In particular, these results will provide decision makers with information
for effectively using MeBr at temperatures and RH levels lower than has been recommended
previously, which will facilitate its use. This report also provides data to assist in selection of an
avirulent surrogate for B. anthracis Ames when using MeBr, for use in future field studies and
additional lab-based investigations.
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Contents
Disclaimer	ii
Acknowledgments	iii
Executive Summary	iv
Abbreviations/Acronyms	viii
1.0 Introduction.. 1
2.0 Technology Description and Test Matrices	2
2.1	Technology Description	2
2.2	Test Matrix	2
3.0 Test Procedures	4
3.1	Biological Agents	4
3.2	Test Materials	4
3.3	Preparation of Coupons	5
3.4	Coupon Extraction and Biological Agent Quantification	6
3.5	Decontamination Efficacy	7
3.6	Repeatability	8
3.7	Surface Damage	9
4.0 Fumigation Description and Procedures	10
5.0 Quality Assurance/Quality Control	13
5.1	Equipment Calibration	13
5.2	QC Results	13
5.2.1. Operational Parameters	13
5.3	Audits	14
5.3.1	Performance Evaluation Audit	14
5.3.2	Technical Systems Audit	15
5.3.3	Data Quality Audit	15
5.4	Test/Quality Assurance Plan Deviations	15
5.5	QA/QC Reporting	15
5.6	Data Re view	15
6.0 Summary of Results and Discussion	16
6.1	Comparing Efficacy for the Different Species	16
6.2	Effects of Test Materials on MeBr efficacy for B.a. Ames	17
6.3	Effect of Temperature on Efficacy of MeBr against B. anthracis Ames	20
6.4	Effect ofRelative Humidity on Efficacy of MeBr against B. anthracis Ames	21
6.5	Effect of MeBr Concentration on Efficacy against B. anthracis Ames	21
6.6	Effect of Contact Time on Efficacy of MeBr against B. anthracis Ames	22
6.7	Surface Damage to Materials	23
6.8	Summary and Conclusion	23
7.0 References	24
Figures
Figure 3-1. Coupon types from left to right: ceiling tile, carpet, glass, painted wallboard paper,
bare pine wood, and unpainted concrete	5
Figure 3-2. Liquid inoculation of coupon using a micropipette	6
Figure 4-1. Schematic of MeBr decontamination test chamber housed inside custom compact
glove box	11
Figure 6-1. Summary of MeBr efficacy (Tests 1-10) results, by material, for B. anthracis Ames	18
Figure 6-2. Summary of MeBr efficacy (Tests 11-20) results, by material, against B. anthracis
Ames	19
Figure 6-3. Effect of temperature on MeBr decontamination efficacy against B. anthracis Ames	20
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Figure 6-4. Effect of relative humidity on MeBr decontamination efficacy against B. anthracis
Ames	21
Figure 6-5. Summary of effect of increasing concentration on average MeBr decontamination
efficacy for B. a. Ames	22
Figure 6-6. Summary of the effect of contact time on average MeBr decontamination efficacy
against B. anthracis Ames	22
Figure C-l. Summary of MeBr efficacy against B. anthracis Ames on glass and bare pine wood... C-2
Figure C-2. Summary of MeBr efficacy against B. anthracis Ames on ceiling tile and carpet	C-2
Figure C-3. Summary of MeBr efficacy against B. anthracis Ames on painted wallboard paper
and unpainted concrete	C-3
Tables
Table E-l. Contact Time Required to Achieve >6 LR on all Materials*	v
Table 2-1. MeBr Test Matrix	3
Table 3 -1. Test Materials	5
Table 5-1. Actual Fumigation Conditions for MeBr Tests	14
Table 5-2. Performance Evaluation Audits	14
Table 6-1. Contact Time Required to Achieve >6 LR on all Materials*	16
Table 6-2. Summary of Average Differences in Efficacy between B.a. Ames and Avirulent
Strains*	17
Table 6-3. Summary of B.a Ames Average Log Reductions by Material Type	20
Table A-l. Inactivation of B. anthracis Ames Spores using Methyl Bromide3	A-l
Table A-2. Inactivation of G. stearothermophilus Spores using Methyl Bromide3	A-3
Table A-3. Inactivation of B. anthracis NNR1A1 Spores using Methyl Bromide3	A-4
Table A-4. Inactivation of B. anthracis Sterne Spores using Methyl Bromide3	A-6
Table B-l. Difference in MeBr Efficacy between B. anthracis Ames and G.
stearothermophilus*	B-l
Table B-2. Difference in MeBr Efficacy between B. anthracis Ames, B. anthracis NNR1 Al,
and B. anthracis Sterne*	B-2
Table C-l. Effect of Increasing Temperature at Low Relative Humidity on MeBr Efficacy*	C-4
Table C-2. Effect of Increasing Temperature at High Relative Humidity on MeBrEfficacy*	C-5
Table C-3. Effect of Increasing Relative Humidity at Low and High Temperatures on B.
anthracis Ames*	C-5
Table C-4. Effect of Increasing MeBr Concentration at Low Relative Humidity on B.
anthracis Ames*	C-6
Table C-5. Effect of Increasing MeBr Concentration at High Relative Humidity on B.
anthracis Ames*	C-6
Table C-6. Effect of Increasing Contact Time at Low Relative Humidity on B. anthracis
Ames*	C-7
Table C-7. Effect of Increasing Contact Time at High Relative Humidity on B. anthracis
Ames*	C-8
List of Appendices
Appendix A Detailed Test Results	A-l
Appendix B Comparing Efficacy for the Different Microorganisms	B-l
Appendix C Effects of Materials and Operational Parameters on MeBr Efficacy	C-l
vii

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Abbreviations/Acronyms
B. a.	Bacillus anthracis
BBRC	Battelle Biomedical Research Center
BOTE	Bio-Response Operational Testing and Evaluation
BSC	biological safety cabinet
CFU	colony-forming unit(s)
CI	confidence interval
cm	centimeter(s)
°C	degrees Celsius
DNA	deoxyribonucleic acid
EPA	U.S. Environmental Protection Agency
ft	foot/feet
G.s.	Geobacillus stearothermophilus
HC1	hydrochloric acid
HS	homeland security
HSRP	Homeland Security Research Program
kGy	kilogray
L	liter(s)
LAL	Limulus Amebocyte Lysate (assay)
LED	light emitting diode
LR	log reduction
MeBr	methyl bromide
min	minute(s)
mg	milligram(s)
mL	milliliter(s)
|_iL	microliter(s)
NA	not applicable
NHSRC	National Homeland Security Research Center
NT	not tested
ORD	EPA Office of Research and Development
oz	ounce(s)
PBST	phosphate-buffered saline + 0.1% Triton X-100
PCR	polymerase chain reaction
ppm	part(s) per million
QA	quality assurance
QC	quality control
QMP	Quality Management Plan
RH	relative humidity
rpm	revolution(s) per minute
SD	standard deviation
SE	standard error
SFW	sterile filtered water (cell-culture grade)
TSA	technical systems audit(s)
viii

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1.0 Introduction
The U.S. Environmental Protection Agency's (EPA's) 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 the intentional introduction of chemical or biological contaminants into buildings or water
systems; contain these contaminants; decontaminate buildings, outdoor environments, or water
systems; and facilitate the disposal of material resulting from restoration activities.
In this work, the efficacy of methyl bromide (MeBr) against Bacillus anthracis (B.a.) Ames and
candidate surrogate spores applied to indoor and outdoor materials (glass, ceiling tile, carpet,
painted wallboard paper, bare pine wood and unpainted concrete) was investigated. The study
builds on previous laboratory research conducted by EPA to assess decontamination efficacy of
MeBr for inactivating B. anthracis spores on different materials1"3. In addition, MeBr was used
in Phase 2 of the full-scale Bio-Response Operational Testing and Evaluation (BOTE) project.
At the BOTE Phase 2 demonstration, issues related to achieving the target fumigation conditions
were encountered, i.e., there were difficulties in achieving the relatively high target fumigation
temperature, humidity, and MeBr concentration in the facility. One objective of this study was,
therefore, to find effective fumigation conditions at relatively lower temperature and/or RH
conditions (i.e., conditions that would not require supplementary heating or humidification),
which would facilitate fumigation with MeBr.
Lastly, another objective of this work was to obtain side-by-side efficacy data for B.a. Ames and
other microbes that could be used to assess the suitability of candidate surrogates for B. a. Ames
when decontaminating with MeBr. Previous tests1"4 with B. atrophaeus or B. subtilis showed
these species to be overly resistant to MeBr compared with B.a. Ames. The Ames strain of B.a.
was chosen for testing because the Ames strain of B.a. was the strain identified in the Amerithrax
incident in 20017.
1

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2.0 Technology Description and Test Matrices
2.1	Technology Description
MeBr (Chemtura, Philadelphia, PA) has been registered by the EPA for soil fumigation (injected
into the soil before a crop is planted to effectively sterilize the soil), commodity treatment (used
for post-harvest pest control), structural pest control (used to fumigate buildings for termites, and
warehouses and food processing facilities for insects and rodents), and quarantine uses (used to
treat imported commodities). Although MeBr has also been demonstrated to be an effective
biocide against B. anthracis on building materials and soil,1"3 the focus of this study was to
determine effective conditions at lower RH levels and/or temperatures, thereby making MeBr
fumigation for B.a. easier to implement. Furthermore, although MeBr use is being phased out
under the Montreal Protocol, MeBr is still currently and widely used via critical use exemptions
as a soil and commodity (quarantine) fumigant.6
2.2	Test Matrix
The test matrix for the MeBr fumigation tests is shown in Table 2-1. As testing proceeded,
adjustments were made to one of the fumigation parameters (contact time, temperature, RH,
concentration) to assess the effect of that parameter and to find efficacious conditions. The first
eight tests were conducted with all six materials, using B.a Ames and one other bacterium. In
Tests 9-20, two materials were eliminated from testing (unpainted concrete and painted
wallboard paper) to allow for the simultaneous testing of three microorganisms. In the latter 12
tests, testing focused on B.a. Ames, Sterne, and NNR1 Al, and (is. was no longer tested.
Unpainted concrete and wallboard paper were removed from the latter phase of testing as
decontamination efficacy was the highest for these materials. Tests 6 and 8 utilized the same
operational parameters to assess repeatability.
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Table 2-1. MeBr Test Matrix
Target Fumigation Parameters
Test
Number
Materials
Microorganisms
MeBr
Concentration
(mg/L)
Temperature
(°C)
RH
(%)
Time
(hours)
1

B. anthracis Ames
G. stearothermophilus
212
22
45
36


B. anthracis Ames
212
22
45
48
L

G. stearothermophilus


B. anthracis Ames
212
22
75
36

Glass
B. anthracis NNR1A1
4
Ceiling Tile
Carpet
B. anthracis Ames
B. anthracis NNR1A1
212
27
45
36
5
Painted Wallboard Paper
Bare Pine Wood
B. anthracis Ames
B. anthracis NNR1A1
212
22
75
24
6
Unpainted Concrete
B. anthracis Ames
B. anthracis Sterne
212
27
45
48
7

B. anthracis Ames
B. anthracis Sterne
212
27
75
24
8

B. anthracis Ames
212
27
45
48

B. anthracis Sterne


B. anthracis Ames




9

B. anthracis NNR1A1
B. anthracis Sterne
212
27
75
36


B. anthracis Ames




10

B. anthracis NNR1A1
B. anthracis Sterne
300
22
45
48


B. anthracis Ames




11

B. anthracis NNR1A1
B. anthracis Sterne
212
22
45
60


B. anthracis Ames




12

B. anthracis NNR1A1
B. anthracis Sterne
212
32
75
24


B. anthracis Ames




13

B. anthracis NNR1A1
B. anthracis Sterne
212
32
45
48


B. anthracis Ames




14
Glass
Ceiling Tile
B. anthracis NNR1A1
B. anthracis Sterne
300
22
75
24

Carpet
B. anthracis Ames




15
Bare Pine Wood
B. anthracis NNR1A1
B. anthracis Sterne
300
22
45
60


B. anthracis Ames




16

B. anthracis NNR1A1
B. anthracis Sterne
212
32
45
60


B. anthracis Ames




17

B. anthracis NNR1A1
B. anthracis Sterne
300
27
75
18


B. anthracis Ames




18

B. anthracis NNR1A1
B. anthracis Sterne
300
27
45
60


B. anthracis Ames




19

B. anthracis NNR1A1
B. anthracis Sterne
212
32
45
72


B. anthracis Ames




20

B. anthracis NNR1A1
B. anthracis Sterne
300
32
45
60
3

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3.0 Test Procedures
This section provides an overview of the procedures that were used for the bench-scale
evaluation of MeBr to inactivate B. anthracis Ames and potential surrogate spore species on six
different materials.
3.1	Biological Agents
The virulent B.a. spores used for this testing were prepared from a qualified stock of the Ames
strain at the Battelle Biomedical Research Center (BBRC, Lot B21, West Jefferson, OH). The
spore lot was subjected to a stringent characterization and qualification process required by
Battelle's standard operating procedure for spore production. Specifically, the spore lot was
characterized prior to use by observation of colony morphology, direct microscopic observation
of spore morphology, and size and determination of percent refractivity and percent
encapsulation. In addition, the number of viable spores was determined by colony count and
expressed as colony forming units per milliliter (CFU/mL). Theoretically, once plated onto
bacterial growth media, each viable spore germinates and yields one CFU. Variations in the
expected colony phenotypes were recorded. Endotoxin concentration of each spore preparation
was determined by the Limulus Amebocyte Lysate (LAL) assay to assess whether contamination
from gram-negative bacteria occurred during the propagation and purification process of the
spores. Genomic deoxyribonucleic acid (DNA) was extracted from the spores and DNA
fingerprinting by polymerase chain reaction (PCR) was done to confirm the genotype. This work
was performed by Dr. Paul Keim at Northern Arizona University. The virulence of the spore lot
was measured at Battelle by challenging guinea pigs intradermally with a dilution series of spore
suspensions, and virulence was expressed as the intradermal median lethal dose. In addition,
testing was conducted for robustness of the spores via hydrochloric acid (HC1) resistance.
Geobacillus stearothermophilus (G.s.; ATCC 12980), B.a. NNR1A1 (Received from Edgewood
Chemical and Biological Center, Edgewood, MD), and B.a. Sterne 34f2 (Colorado Serum
Company, Denver, CO) spores were tested alongside the virulent form of B.a. (Ames) to assess
their potential as a surrogate. Using growth from a stock culture, G.s., B.a. NNR1A1, or B. a.
Sterne was inoculated into 10 mL tubes of nutrient broth and incubated in a shaking incubator for
24 ± 2 hours at approximately 150 revolutions per minutes (rpm). The B.a. Ames strain was
prepared using a BioFlo 3000 fermenter (New Brunswick Scientific Co., Inc, Edison, NJ).
Incubation temperature for the B.a. strains was 37 degrees Celsius (°C), while for G.s., an
incubation temperature of 55 °C was used. This culture was used to inoculate amended nutrient
agar plates. Plates were inoculated with 500 microliters (|iL) of the culture and spread with a
sterile plate spreader. Plates were inverted (with no shaking) and incubated for 12-14 days.
Following incubation, plates were harvested by washing with 10 mL sterile water and scraped
into sterile tubes. The harvested spores were centrifuged at 5000 rpm and washed with water
three times and resuspended in sterile water. The prepared spores were examined via microscopy
and determined to have >95 % refractile spores with <5 % cellular debris. All stock spore
suspensions were prepared in sterile filtered water (SFW) at an approximate concentration of 1 x
109 CFU/mL and stored under refrigeration at 2 to 8 °C.
3.2	Test Materials
Decontamination testing was conducted on glass, ceiling tile, carpet, painted wallboard paper,
bare pine wood and unpainted concrete. Information on these materials is presented in Table 3-1,
and a picture of each is presented in Figure 3-1. Material coupons were cut to uniform length and
width from a larger piece of stock material. Materials were prepared for testing either by
sterilization via gamma irradiation at -40 kilogray (kGy; STERIS Isomedix Services,
4

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Libertyville, IL) or by autoclaving at 121 °C for 15 minutes. Gamma-irradiated material coupons
were sealed in 6 mil Uline Poly Tubing (Uline, Chicago, IL), and autoclaved coupons were
sealed in sterilization pouches (Fisher, Pittsburgh, PA) to preserve sterility until the coupons
were ready for use.
Table 3-1. Test Materials
Material
Lot, Batch, or ASTM No., or
Observation
Manufacturer/
Supplier Name
Approximate Coupon
Size,
width x length x
thickness
Material
Preparation
Glass
C1036
Brooks Brothers,
Columbus, OH
1.9 centimeter (cm) x 7.5 cm
x 0.3 cm
Autoclave
Ceiling
Tile
Armstrong® B513, classic fine textured
Armstrong,
Columbus, OH
1.9 cm X 7.5 cm x 0.3 cm
Gamma
Irradiation
Carpet
Shaw Swizzle EcoWorx, Style: 10401
Color: Jacks
Shaw Industries,
Dalton, GA
1.9 cm x 7.5 cm x 0.3 cm
Gamma
Irradiation
Painted
Wallboard
Paper
Roller painted on one side using Martin
Senour Paints. One primer (#71-1185)
and two finish (flat, #70-1001) coats
United States
Gypsum Company,
Chicago, IL
1.9 cm x 7.5 cm x 0.1 cm
Gamma
Irradiation
Bare Pine
Wood
Generic Molding
Lowes,
Columbus, OH
1.9 cm x 7.5 cm x 0.3 cm
Gamma
Irradiation
Unpainted
Concrete
ASTM C90 cinder block
Wellnitz
Columbus, OH
1.9 cm x 7.5 cm x: 0.6 cm
Autoclave
•V JL



Figure 3-1. Coupon types from left to right: ceiling tile, carpet, glass, painted wallboard
paper, bare pine wood, and unpainted concrete.
3.3 Preparation of Coupons
Test and positive control coupons were placed on a flat surface within a Class II biological safety
cabinet (BSC) and inoculated with approximately 1 x 10 CFU of viable B.a. Ames (or indicated
surrogate) spores per coupon. A 100 (iL aliquot of a stock suspension of approximately 1 x 1Q9
CFU/mL was dispensed using a micropipette applied as 10 jaL droplets across the coupon
surface (see Figure 3-2). This approach provided a more uniform distribution of spores across the
coupon surface than would be obtained through a single drop of the suspension. After
inoculation, the coupons were transferred to a Class III BSC and left undisturbed overnight to
dry under ambient conditions, approximately 22 °C and 40 % relative humidity (RH).
5

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Figure 3-2. Liquid inoculation of coupon using a micropipette.
The number and type of replicate coupons used for each combination of material, decontaminant,
concentration, and environmental conditions were:
•	five test coupons (inoculated with B. anthracis or surrogate spores and exposed to
decontaminant)
•	five positive controls (inoculated with B. anthracis or surrogate spores but not exposed
to decontaminant)
•	one laboratory blank (not inoculated and not exposed to the decontaminant)
•	one procedural blank (not inoculated and exposed to the decontaminant)
On the day following liquid spore inoculation, coupons intended for decontamination (including
blanks) were transferred into a test chamber and exposed to the MeBr fumigant using the
apparatus and application conditions specified in Section 4.0 of this report. Control coupons
were added to the control chamber as described in Section 4.0.
3.4 Coupon Extraction and Biological Agent Quantification
For sample extraction, test coupons, positive controls, and blanks were placed in 50 mL
polypropylene conical tubes containing 10 mL of sterile phosphate buffered saline + 0.1% Triton
X-100 (PBST). The vials were capped, placed on their sides and agitated on an orbital shaker for
15 minutes (min) at approximately 200 rpm at room temperature.
Residual viable spores were determined using a dilution plating approach. Following extraction,
the extract was removed and a series of 10-fold dilutions was prepared in sterile water. An
aliquot (0.1 mL) of either the undiluted extract and/or each serial dilution were plated onto
tryptic soy agar in triplicate and were incubated for 18-24 hours at 35-37 °C. Colonies were
counted manually and CFU/mL were determined by multiplying the average number of colonies
per plate by the reciprocal of the dilution. Dilution data representing the greatest number of
individually definable colonies were expressed as arithmetic mean ± standard deviation (SD) of
the numbers of CFU observed. Laboratory blanks controlled for sterility, and procedural blanks
controlled for viable spores inadvertently introduced to test coupons. The blanks were spiked
with an equivalent amount of 0.1 mL of SFW. The target acceptance criterion was that extracts
of laboratory or procedural blanks were to contain no CFU.
After each decontamination test, the BSC 111 and the MeBr test and control chambers were
thoroughly cleaned (using separate steps involving bleach, ethanol, water, then drying).
6

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3.5
Decontamination Efficacy
The mean percent spore recovery from each coupon was calculated using results from positive
control coupons (inoculated, not decontaminated), by means of the following equation:
Mean % Recovery = [Mean CFUpc/CFUspike] x 100	(1)
where Mean CFUpc is the mean number of CFU recovered from five replicate positive control
coupons of a single material, and CFUspike is the number of CFU spiked onto each of those
coupons. The value of CFUspike is known from enumeration of the stock spore suspension. One
aliquot of the stock suspension is plated and enumerated on each day of testing to confirm
CFUspike concentration. Spore recovery was calculated for B.a. Ames or surrogate on each
coupon, and the results are included in Section 5 and Appendix A.
The performance or efficacy of MeBr was assessed by determining the number of viable
organisms remaining on each test coupon after decontamination. Those numbers were compared
to the number of viable organisms extracted from the positive control coupons.
The number of viable spores of B.a. Ames or surrogate in extracts of test and positive control
coupons was determined to calculate efficacy of the decontaminant. Efficacy is defined as the
extent (as logio reduction or LR) to which viable spores extracted from test coupons after
decontamination were less numerous than the viable spores extracted from positive control
coupons. The logarithm of the CFU abundance from each coupon extract was determined, and
the mean of those logarithm values was then determined for each set of control and associated
test coupons, respectively. Efficacy of a decontaminant for a test organism/test condition on the
z'th coupon material was calculated as the difference between those mean log values, i.e.:
Efficacy (LR) = (log10 CFUci}) - (log10 CFUty)	(2)
where logio CFUcy refers to the j individual logarithm values obtained from the positive control
coupons, logio CFUty refers to the j individual logarithm values obtained from the corresponding
test coupons, and the overbar designates a mean value. In tests conducted under this plan, there
were five positive controls and five corresponding test coupons (i.e. J = 5) for each coupon. A
decontaminant that achieves a 6 LR or greater is considered effective.2
In the case where no viable spores were found in any of the five test coupon extracts after
decontamination, a CFU abundance of 1 was assigned, resulting in a logio CFU of zero for that
material. This situation occurred when the decontaminant was highly effective, and no viable
spores were found on the decontaminated test coupons. In such cases, the final efficacy on that
material was reported as greater than or equal to (>) the value calculated by Equation 2.
The variances (i.e., the square of the SD) of the logio CFUcy and logio CFUty values were also
calculated for both the control and test coupons (i.e., S2Cy and 5%), and were used to calculate
the pooled standard error (SE) for the efficacy value calculated in Equation 3, as follows:
p>
where the number 5 again represents the number j of coupons in both the control and test data
sets. Each efficacy result is reported as a LR value with an associated 95 % confidence interval
(CI), calculated as follows:
7

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95 % CI = Efficacy (LR) ± (1.96 x SE)
(4)
The significance of differences in efficacy across different test conditions and spore types was
assessed based on the 95 % confidence interval of each efficacy result. Differences in efficacy
were judged to be significant if the 95 % CIs of the two efficacy results did not overlap. Any
results based on this formula are hereafter noted as significantly different. Note this comparison
is not applicable when the two efficacy results being compared are both reported with LRs as >
some value.
The average difference in efficacy was determined when comparing the results of two tests and
reported as a LR value. This difference in efficacy was calculated as follows:
n
LRa,2 - LRa,\
Avg Difference in Efficacy (LR) = —		(5)
n
where the letters a through n represent the material types, the number 1 represents B.a. Ames,
and the number 2 represents the avirulent (potential surrogate) microorganism for which results
are being compared. The letter n represents the number of materials tested, with n = 6 for Tests
1-8, and equal to 4 for the remaining tests. When both values were > LR (indicating complete
inactivation), these were not included in the formula. A positive value indicates that the avirulent
organism was inactivated on average to a higher degree (i.e., it was less resistant) across the
materials tested compared to B.a. Ames.
In some instances, significant differences in average efficacy between tests were assessed with a
t-test using Microsoft Excel®, according to the formula below:
t =	(6)
X1—X2
where X1 and X2 are the means of Tests 1 and 2, respectively.	is the standard error of the
difference between Tests 1 and 2. Excel produces a p-value, a statistic calculated from the t-test,
used to assess whether the averages of the two tests are reliably different from each other. Using
this formula, a p-value was assigned where indicated. If the calculated p-value was <0.05, then
the two sets of data were considered to be significantly different.
3.6 Repeatability
It was desired to perform a statistical evaluation to formally test for repeatability between Test 6
and 8 within each of B.a. Ames and B.a. Sterne. The limited number of decontamination data
points in each test (n=6), as well as the underlying variability in decontamination results, make
this comparison a challenge. To address this challenge, a statistical hypothesis test approach was
developed. In this form of a statistical comparison, a statistical measure of interest is identified.
Then, a null hypothesis relative to the underlying comparison is proposed along with an
alternative hypothesis (usually what is desired to be shown). The statistical measure from the
observed data is compared to what could have been observed. If there is adequate probabilistic
evidence that the observed results are unusual, the null hypothesis can be rejected in favor of
concluding the alternative.
A statistical measure to show the repeatability of the testing is the square root of the sum of the
squared differences in average LR of bacteria between the first and second tests within each
material.
8

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		(6)
6
Test Statistic = X (LRm.Test 8 LRmjTest 6)
m= 1
where m is the material types (glass, ceiling tile, carpet, painted wallboard paper, bare pine
wood, and unpainted concrete) and LR„, /esli s the mean log reduction for material m in Test x.
Note: For results with a reduction ">" a value, the statistic was calculated using the subsequently
reported value (e.g., 7.34 for >7.34).
Since there is not adequate information regarding the true variability in LR at a population level,
a bootstrap approach was used where the observed data serve as the source population from
which sampling is done. The statistical analysis for this evaluation consisted of enumerating all
the 720 permutations of how the second set of six material log reduction results could have been
observed. As an example, consider for the Ames test that one possible permutation of Test 8
would have been for the observed log reductions to have been >6.94 for glass, 2.03 for ceiling
tile, >7.60 for carpet, 4.31 for painted wallboard paper, 3.29 for bare pine wood, and 2.29 for
painted concrete. For each of the 720 permutations, a test statistic is calculated. In the Ames
example just noted, a value of 6.979 would be calculated. The actual observed test statistic
(2.701 for Ames, 3.194 for Sterne) was then compared to a ranking of the entire set of 720
permutations within each agent. If the observed statistic was below the 5th percentile of the
ranking, it provided at least 95 % confidence that the test results were repeatable.
3.7 Surface Damage
The physical effect of MeBr on the materials was also qualitatively monitored during the
evaluation. This approach provided a gross visual assessment of whether the decontaminants
altered the appearance of the test materials. The procedural blank (coupon that is
decontaminated, but has no spores applied) was visually compared to a laboratory blank coupon
(a coupon not exposed to the decontaminant and that has no spores applied). Obvious visible
damage might include structural damage, surface degradation, discoloration, or other aesthetic
impacts.
9

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4.0 Fumigation Description and Procedures
Methyl bromide is a colorless and odorless volatile gas. Chloropicrin was added to the MeBr
source gas (0.5 % chloropicrin, 99.5 % MeBr) as a warning irritant (lacrimator) for the safety of
laboratory staff. The gas mixture was used at full strength and injected into the test chamber at
the indicated target concentrations.
Figure 4-1 shows a schematic drawing of the MeBr test chamber and containment system.
Decontamination testing was conducted inside an approximately 38 liter (L) stainless steel
chamber. The chamber was insulated to prevent condensation on the inside chamber walls. As a
means of secondary containment and laboratory personnel safety, this test chamber was housed
inside a custom acrylic compact glove box (Plas Labs, Inc., Lansing, MI) that was hard-ducted to
the facility exhaust system.
Temperature was controlled using a heated/cooled water bath, and relative humidity (RH) was
elevated using a Nafion tube pervaporation system (controlled using a water bath). Temperature
and RH in the test chamber was measured using an HMT368 temperature and humidity probe
(Vaisala, Inc., Woburn, MA). Temperature, RH, and MeBr concentration were controlled with a
CNI-822 controller (Omega Engineering, Stamford, CT) and were data-recorded every minute
during the contact time using the associated iLOG software.
The MeBr concentration in the test chamber was measured continuously during the contact
period using a Fumiscope™ Version 5.0 (Key Chemical and Equipment Company, Clearwater,
FL). MeBr was added to the chamber, as necessary, to maintain the specified concentration
within ±10 %. The Fumiscope meter was calibrated by the manufacturer for MeBr, displaying
the concentration on a digital light-emitting diode (LED) display in ounces (oz) of MeBr per
3	3
1000 cubic feet (ft ). One oz per 1000 ft is approximately 257 parts per million (ppm) at 25 °C
and is approximately 1 mg/L (independent of temperature). The Fumiscope meter included an air
pump that pulled a gas sample from the test chamber through the thermal conductivity meter at a
controlled rate and exhausted the gas back into the test chamber. Moisture was removed from the
gas sample using a small paper filter before it was measured in the Fumiscope to eliminate
interference from water.
10

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%RH
T ernp
Iiilet Valve
Regulator
nn
RH WB
Outlet V;
WB = water bath
~~ Indicates RH lines
Indicates Temperature Lines
Indicates MeBr loop from chamber to fiimiscope
~~ Indicates electrical lines to and from RH, temperature, and MeBr concentration controllers
Figure 4-1. Schematic of MeBr decontamination test chamber housed inside custom compact glove box.
11

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A 9L Lock & Lock® airtight container (Lock & Lock, Farmers Branch, TX) served as the
positive control chamber. Fixed humidity point salts5 were added as a slurry to a separate
container placed in the bottom of the MeBr positive control chamber. Sodium chloride was used
to control the RH at 75 % and potassium carbonate to control the RH at 45 %. The control
chamber was placed in an incubator (Thermo Scientific, Waltham, MA) for all tests and set to
the appropriate temperature (i.e., 22, 27 or 32 °C). The temperature and RH of the positive
control chamber were measured and datalogged using a HOBO® data logger model U12-11
(Onset Computer Corporation, Cape Cod, MA).
As in previous studies with MeBr1, multiple coupons of each material were inoculated with the
biological agent and placed on a wire rack inside the test chamber. Blank (i.e., uninoculated) and
positive control (i.e., inoculated but not decontaminated) samples were also prepared for each
material and were utilized with data from the test samples (inoculated and decontaminated) to
determine decontamination efficacy.
The 20 MeBr tests were conducted at concentrations of either 212 or 300 mg/L, as shown in
Table 2-1. Target contact times ranged from 18 to 72 hours, temperature from 22 to 32 °C and
RH from 45 to 75 %. During each test run, inoculated test samples were placed inside the MeBr
test chamber, and the chamber was sealed. The chamber was allowed sufficient time to
equilibrate to the target temperature and RH prior to start of the run. Once the temperature and
RH were stable, MeBr was slowly injected into the chamber until the target concentration was
reached. The test chamber remained sealed until the end of the required contact time. At this
time, the MeBr was turned off and the seal of the test chamber broken by removing the lid. The
test chamber and BSC III were allowed to off-gas until the MeBr levels in the chamber reach 0
mg/L, which happened within minutes of lid removal. At this time, the samples were removed
and processed as stated in Section 3.4.
12

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5.0 Quality Assurance/Quality Control
Quality assurance/quality control (QC) procedures were performed in accordance with the
Quality Management Plan (QMP) and the test/QA Plan. The QA/QC procedures and results are
summarized below.
5.1	Equipment Calibration
All equipment (e.g., pipettes, incubators, biological safety cabinets) and monitoring devices (e.g.,
thermometer, hygrometer) used at the time of evaluation were verified as being certified,
calibrated, or validated.
5.2	QC Results
Quality control efforts conducted during decontaminant testing included positive control samples
(inoculated, not decontaminated), procedural blanks (not inoculated, decontaminated), laboratory
blank (not inoculated, not decontaminated), and inoculation control samples (analysis of the
stock spore suspension).
All positive control results were within the target recovery range of 1 to 150 % of the inoculated
spores, and all procedural and laboratory blanks met the criterion of no observed CFU for both
organisms.
Inoculation control samples were taken from the spore suspension on the day of testing and
serially diluted, nutrient plated, and counted to establish the spore density used to inoculate the
samples. The spore density levels met the QA target criterion of 1 x 109 CFU/mL (±1 log) for all
tests.
5.2.1. Operational Parameters
The temperature, RH, and MeBr concentration during each test were controlled using Omega
controllers, as described in Section 4.1. These controllers were set to the target conditions and
allowed heat, RH, or MeBr to be injected as needed to stay within target ranges of ±2 °C, ±20 %
RH and ±10 % MeBr. Readings were taken once every minute for the duration of the contact
time. The actual operational parameters for each test are shown in Table 5-1 and reported as the
average value ± SD.
13

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Table 5-1. Actual Fumigation Conditions for MeBr Tests
Test
Number
MeBr Concentration
(mg/L)
Target Actual*
Temperature (°C)
Target Actual*
Target
RH (%)
Actual*
Contact
Time
(hours)
1
212
213.39 ±5.50
22
22.47 ±0.31
45
45.03 ±0.59
36
2
212
211.16 ± 2.95
22
22.20 ±0.38
45
46.45 ± 1.87
48
3
212
211.74 ±3.29
22
22.41 ±0.16
75
75.29 ±0.27
36
4
212
213.01 ±4.23
27
27.58 ±0.51
45
48.66 ±8.90
36
5
212
212.38 ±2.87
22
22.13 ±0.13
75
74.90 ±0.35
24
6
212
212.16 ±2.96
27
27.14 ±0.16
45
45.75 ± 1.63
48
7
212
212.17 ±2.79
27
27.14 ±0.23
75
75.98 ± 1.61
24
8
212
212.55 ±3.14
27
27.13 ±0.10
45
45.39 ± 1.52
48
9
212
211.97 ±2.67
27
27.24 ±0.14
75
75.72 ± 1.56
36
10
300
301.29 ±3.07
22
22.32 ±0.13
45
45.81 ± 1.30
48
11
212
210.81 ±2.72
22
21.99 ±0.19
45
46.07 ± 1.60
60
12
212
212.78 ±3.21
32
32.14 ±0.23
75
75.28 ± 1.64
24
13
212
212.16 ±2.98
32
32.16 ±0.16
45
45.99 ± 1.15
48
14
300
302.62 ±3.49
22
22.20 ± 0.28
75
76.30 ±2.36
24
15
300
302.87 ±3.74
22
22.23 ±0.11
45
46.10 ± 1.19
60
16
212
211.38 ±2.93
32
32.17 ±0.29
45
45.79 ±0.84
60
17
300
300.87 ±2.76
27
25.56 ±0.58
75
81.16 ±2.99
18
18
300
301.18 ±2.86
27
27.28 ±0.21
45
45.95 ± 1.03
60
19
212
212.10 ±3.82
32
32.16 ±0.28
45
46.23 ± 1.18
72
20
300
301.53 ±4.41
32
32.14 ±0.18
45
45.87 ±0.89
60
* Data reported as average ± SD.
^ Contact time did not deviate from target during any test.
5.3 Audits
5.3.1 Performance Evaluation Audit
Performance evaluation audits were conducted to assess the quality of the results obtained during
these experiments. Table 5-2 summarizes the performance evaluation audits that were
performed.
No performance evaluation audits were performed to confirm the concentration and purity of
B.a. or surrogate spores because quantitative standards do not exist for these organisms. The
control coupons and blanks support the spore measurements.
Table 5-2. Performance Evaluation Audits
Measurement
Audit
Allowable
Actual
Procedure
Tolerance
Tolerance
Volume of liquid from
micropipettes
Gravimetric evaluation
± 10 %
±0.15% to 2.5%
Time
Compared to independent clock
± 2 seconds/hour
0 seconds/hour
Temperature
Compared to independent calibrated
thermometer
±2 °C
± 1.12 °C
Relative Humidity
Compare to independent calibrated
hygrometer
± 20 %
± 1.52%
14

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5.3.2	Technical Systems Audit
Observations and findings from the technical systems audit (TSA) were documented and
submitted to the laboratory staff lead for response. TSAs were conducted on August 9 and
August 14, 2013, to ensure that the tests were being conducted in accordance with the
appropriate test/QA plan and QMP. As part of the audit, test procedures were compared to those
specified in the test/QA plan and data acquisition and handling procedures were reviewed. None
of the findings of the TSA required corrective action.
5.3.3	Data Quality Audit
At least 10 % of the data acquired during the evaluation were audited. A QA auditor traced the
data from the initial acquisition, through reduction and statistical analysis, to final reporting to
ensure the integrity of the reported results. All calculations performed on the data undergoing the
audit were checked.
5.4	Test/Quality Assurance Plan Deviations
Section 3.2 of the test/QA plan states "The temperature and RH of the control and test chambers
(excluding the MeBr test chamber) will be measured with a thermometer/hygrometer (Fisher
Scientific Cat. No. S66283, Pittsburgh, PA), and the data will be recorded using a data logger
(Onset Part No. U12-001, Bourne, MA)". For Test #9 started on 9/3/13, the HOBO® was
inadvertently not launched inside the control chamber, resulting in no temperature or RH data at
the end of the contact period. The parameters for Test #9 were as follows: 212 mg/L MeBr; 27
°C; 75 % RH; 36 hour contact time using B.a. Ames, Sterne and NNR1A1 against glass, ceiling
tile, carpet and bare pine wood. The data for the decontamination samples were not affected.
5.5	QA/QC Reporting
Each assessment and audit was documented in accordance with the test/QA plan and QMP. For
these tests, findings were noted (none significant) in the data quality audit, but no followup
corrective action was necessary. The findings were mostly minor data transcription errors
requiring some recalculation of efficacy results, but none were gross errors in recording. Copies
of the assessment reports were distributed to the EPA QA Manager and laboratory staff. QA/QC
procedures were performed in accordance with the test/QA plan.
5.6	Data Review
Records and data generated in the evaluation received a QC/technical review before they were
utilized in calculating or evaluating results and prior to incorporation in reports.
15

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6.0 Summary of Results and Discussion
The decontamination efficacy of MeBr against virulent B.a. Ames and surrogates was evaluated
at target concentrations of 212 and 300 mg/L, at a target temperatures of 22 to 32 °C, target RH
of 45 and 75 %, and contact times ranging from 18 to 72 hours for a total of twenty tests. Table
6-1 shows the contact time required to achieve >6 LR (the level considered effective) on all
material types tested (glass, ceiling tile, carpet, painted wallboard paper, bare pine wood, and
unpainted concrete) and at all target operational parameters. Actual operational parameters as
measured were well within acceptable ranges and are detailed in Section 5. The detailed
decontamination efficacy results are found in Appendix A. As seen in the table, a contact time of
36 hours was required to achieve > 6 LR of B.a. Ames when fumigating at 212 mg/L, 22 °C, and
75 % RH. Only 18 hours were required to achieve > 6 LR when the MeBr concentration was
increased to 300 mg/L and temperature increased to 27 °C.
Table 6-1. Contact Time Required to Achieve >6 LR on all Materials*
Target MeBr
Target
Target
Time (hours) Required to Achieve >6 LR on All Materials0
- Test Number
Reference3
Concentration
(mg/L)
Temperature
(° C)
RH
(%)
B.a. Ames
G.s.
B.a. NNR1A1
B.a. Sterne
212
22
45
>60
48
>60
>60
1,2, 11
212
22
75
36
b
>36
--
3, 5
212
27
45
>48
--
>36
>48
4, 6,8
212
27
75
36
"
>36
36
7, 9
212
32
45
>72
--
>72
>72
13, 16, 19
212
32
75
24
"
>24
24
12,
300
22
45
>60
--
>60
>60
10, 15
300
22
75
24
"
>24
>24
14
300
27
45
>60
--
>60
>60
18
300
27
75
18
"
> 18
> 18
17
300
32
45
>60
	
>60
>60
20
* Materials tested were glass, ceiling tile, carpet, painted wallboard paper, bare pine wood and unpainted concrete.
a Detailed data from each test number can be referenced in Tables A-l through A-4 in Appendix A.
bNot tested.
c > dictates that >6 LR on all materials was not achieved at the contact time listed, and contact time was the longest tested.
6.1 Comparing Efficacy for the Different Species
A summary of the results comparing the average difference in decontamination efficacy for the
microorganisms that were compared is shown in Table 6-2. Testing was first conducted using
G.s. as a potential surrogate for B.a. Ames (Tests 1 & 2). The results showed that G.s. is less
resistant than B.a. Ames to MeBr exposure. Therefore G.s. was eliminated from further testing,
since a surrogate should be at least as resistant as the virulent strain. Thus additional potential
surrogates, B.a. NNR1A1 and B.a. Sterne, were subsequently tested. In an attempt to evaluate all
three organisms simultaneously, the number of materials tested was reduced due to the limited
size of the MeBr test chamber. Painted wallboard paper and unpainted concrete were eliminated
from testing after Test 8 as these materials were the easiest to decontaminate (highest LR values
obtained). Refer to Appendix B for detailed efficacy results (e.g., results for each material)
comparisons among microorganisms.
The results in Table 6-2 show that B.a. Sterne was more resistant (lower decontamination
efficacy) to MeBr compared to B.a. Ames at the high RH condition (75 %), with the average
16

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difference in efficacy ranging from -0.22 to -5.35 LR. In contrast, at 45 % RH, B.a. Sterne was
always less resistant to MeBr than B.a. Ames.
The avirulent B.a. NNR1A1 was also tested alongside B.a. Ames in Tests 3-5 and 9-20. This
organism was more resistant to MeBr than B.a. Ames in all tests performed, regardless of the
temperature, RH, MeBr concentration, and contact time. However, the difference between B.a.
Ames and the NNR1A1 strain was generally greater than the difference between the Ames and
Sterne strains. In some tests (Tests 3 and 14), the difference in efficacy between B.a. Ames and
the NNR1A1 strain was quite high (-6.33 and -6.18 LR, respectively).
Table 6-2. Summary of Average Differences in Efficacy between B.a. Ames and Avirulent
Strains
Test
Number
Target MeBr
Concentration
(mg/L)
Target
Temperature
(°C)
Target RH
(%)
Contact
Time (hour)
Average Difference
B.a.
NNR1A1
in Efficacy
B.a. Sterne
1
212
22
45
36
2.26*
a
--
2
212
22
45
48
1.10
--
--
11
212
22
45
60
--
-1.63
0.72
10
300
22
45
48
--
-1.55
1.86
15
300
22
45
60
--
-1.03
1.61
4
212
27
45
36
--
-1.29
--
6
212
27
45
48
--
--
1.74
8
212
27
45
48
--
--
1.54
18
300
27
45
60
--
-1.66
1.28
13
212
32
45
48
--
-1.59
1.31
16
212
32
45
60
--
-1.92
1.43
19
212
32
45
72
--
-2.06
1.63
20
300
32
45
60
--
-1.51
0.98
5
212
22
75
24
--
-2.20*
--
3
212
22
75
36
--
-6.33*
--
14
300
22
75
24
--
-6.16*
-5.35*
7
212
27
75
24
--
--
-3.75*
9
212
27
75
36
--
-1.83
-0.39
17
300
27
75
18
--
-4.96*
-3.67*
12
212
32
75
24
—
-2.54
-0.22
Results shown as average difference in efficacy (log reduction). A positive result indicates that the avirulent microorganism was inactivated to a
higher degree (less resistant) than B.a. Ames. * An asterisk denotes a significant difference in efficacy.
a " Not tested at that condition.
6.2 Effects of Test Materials on MeBr efficacy for B.a. Ames
The LR results by material, for each test, are shown in the bar graphs in Figures 6-1 and 6-2.
Differences in efficacy between two materials are significant if the 95 % CIs of the two efficacy
results do not overlap. As discussed previously, testing was originally conducted (Tests 1
through 8) using six test materials. But in an attempt to evaluate three organisms at once {B.a.
Ames, B.a. NNR1A1, and B.a. Sterne), the number of coupon materials tested was reduced due
to the size of the MeBr test chamber. Painted wallboard and unpainted concrete were removed
from testing in Tests 9 through 20, as these two material types generally exhibited higher
efficacy than the other material types. Table 6-3 shows the average LR for each of the six
materials in Tests 1-8, and the average LR for the four materials tested in Tests 9-20. In general,
glass and wood were the materials most difficult to decontaminate (exhibited lower efficacy than
17

-------
the other four material types). Further details on the decontamination efficacy results are found
in Appendices A through C.
Test #1:212 mg/L MeBr. 22 °C, 45% RH, if. hours
I I
*
ii.i
Ceiling
Tile
Caipet
Painted
Wallboard
Faper
Bare	Unpainted
Pine	C^crele
Wood
Test #3: 212 mg/L MeBr, 22 °C\ 75% RH, 36 hours
Carpet
Painted
Wallboard
Paper
Bare	Unpainted
Fine	Concrete
Wood
Test#?: 212 mg/L MeBr, 22"C, 75% RH, 24 hours
Ceiling
Tile
Caipet
Painted
Wallboard
Paper
Bare	Unpainted
Pine	Concrete
Wo-od
Test #7: 212 mg/L MeBr, 27 °C, 75% RH, 24hours
.Mill
Carpet	Painted	Bare	Unpainted
Wallboard	Puie	Cosicrete
Paper	Wood
i:
Test #9: 212 mg/L MeBr, 27 °C, 75% RH, 36 hours
1 *
III)
Ceiling
Tile
Bare
Puie
Wood
I
Test #2: 212 mg/L MeBr, 22 »C, 45% RH. 48 hours
Lii.i
Ceiling
Tile
Carpet	Pawled	Bare	Unpainted
Wallboard	Pine	concrete
Paper	Wood
Test #4: 212 mg/L MeBr, 27 °C\ 45% RH, 36 hours
* *
_	_	*	8	^	*	*
II I III
Glass	Ceiling
Tile
Carpet
Painted
Wallboard
Paper
Bare	Unpainted
Pine	Concrete
Wood
Test #6: 212 mg/L MeBr. 27 »C, 45% RH. 48 hours
r	IM
Stf 5 1	£ J -
ii I I I I I I
II.I
Ceiling
Tile
Carpet
Painted
Wallboard
Paper
Bare	Unpainted
Pine	Concrete
Wood
Test #8: 212 mg/L MeBr, 27 °C, 45% RH, 48 hours
•= 4
i' i
i I
i I	*
Carpet	Painted	Bare
Wallboard	Pine
Paper	Wood
lest #111: 300 mg/L MeBr. 22 «C. 45% RH. 4» hours
£• J
I
i
Ceiling
M
Bare
Pme
Wood
Figure 6-1. Summary of MeBr efficacy (Tests 1-10) results, by material, for B. anthracis
Ames. Results shown are average log reduction ± CI.
18

-------
Complete inactivation achieved
Te.vr #11: 212 mg/I. MeBr, 22•('. 45% RH. 60 hours
* *
£ "
I
i i
Carpet
Bare
Pine
Wood
Test #13: 212 mg/L MeBr, 32 °C\ 45% RH, 48 hours
|	6
as	?
M
*	4
i3
W	1
i
i i
Ceiling
Tile
Carpel
i
Bare
Pine
Wood
Test #15: 3(H) mg/L MeBr, 22 °C, 45% RH, 60 hours
t- 3
I
i i
Ceiling
Tile
Carpet
Bare
Pine
Wood
Test #17: 300 mg/L MeBr, 27 °C, 75% RH, 18hours
lill
Ceiling
Carpet
Test #19: 212 mg/L MeBr. 32°C\ 45% RH. 72 hours
b 3
\
i i
Ceiling
Carpel
Bare
Pine
Wood
Test #12: 212 mg/L MeBr, 32 °C\ 75% RH. 24 hours
| *
ill i
Ceiling
Tile
Carpet
Bare
Pine
Wood
S
I 6
ai 5
u
£ 4
3
£ 1
Test#14: 3(H) mg/L MeBr, 22 °C, 75% RH, 24 hours
III
Ceiling
Tile
Carpet
Pine
Wood
Test #16: 212 mg/L MeBr. 32 °C, 45% RH, 60 hours
j 4
i I
i I
Ceiling
Tile
Carpet
i
Bare
Pine
Wood
Test #18: 300 mg/L MeBr, 27 °C\ 45% RH, 60 hours
sc	5
M
p2	4
t-	3
S
S	1
i
i I
Ceiling
Tile
Carpet
i
Bare
Pine
Wood
Test #20: 300 mg/L MeBr, 32 °C, 45% RH. 60 hours
| 6
£ 5
ri I
Ceiling
Tile
Carpet
Figure 6-2. Summary of MeBr efficacy (Tests 11-20) results, by material, against B.
anthracis Ames. Results shown in average log reduction ± CI.
* Complete inactivation achieved
19

-------
Table 6-3. Summary of B.a Ames Average Log Reductions by Material Type
Material Type
Average (±SD) LR
for Tests 1-8
Material Type
Average LR (±SD)
for Tests 9-20
Glass
3.96 ± 1.83
Glass
4.56 ±2.30
Ceiling Tile
5.11 ± 1.91
Ceiling Tile
5.69 ± 1.38
Carpet
6.00 ± 1.83
Carpet
6.04 ± 1.61
Painted Wallboard Paper
6.87 ± 1.71


Bare Pine Wood
3.89 ±2.15
Bare Pine Wood
4.02 ±2.18
Unpainted Concrete
6.52 ± 1.32


6.3 Effect of Temperature on Efficacy of MeBr against B. anthracis Ames
The decontamination efficacy of MeBr against virulent B.a. Ames was evaluated at target
temperatures of 22, 27, or 32 °C. These temperatures were tested at various combinations of RH,
MeBr concentration, and contact time; the results are organized by test condition in Figure 6-3 to
visualize the effect of temperature. Additional analyses of the effect of temperature, including LR
data for each specific material, are included in Tables C-l and C-2 of Appendix C.
12
3 9
11
13
16
15
18
20
n 5
ll
¦	22 °C
¦	27 °C
~ 32 °C
212.45.36 212.45.48 212.45.48 212.45.60 300.45.60 212.75.24 212.75.24 212.75.36
MeBr Concentration (mg/L), %RH, Contact Time
Figure 6-3. Effect of temperature on MeBr decontamination efficacy against B. anthracis
Ames (Test numbers shown above each bar). Bars are the LR values averaged across the
materials tested. In comparing with Test 13, which included only four materials, the second
bar for Test 6 is the average of the same four materials. Similarly for the second bar shown
for Test 7.
In general, increasing temperature (while holding all other test variables constant) either
increased decontamination efficacy or had no significant effect on efficacy. At 45 % RH, there
was only one test condition in which efficacy improved with increasing temperature; see
comparison between Tests 1 and 4 in Figure 6-3. The remainder of the comparisons made for 45
% RH appear to show slightly reduced efficacy with increasing temperature, although these
differences in efficacy are not statistically significant. At 75 % RH, there was just one test
condition (out of three) in which increasing temperature resulted in a statistically significant
20

-------
increase in efficacy; see comparison for Tests 5 and 7. The other two test conditions being
compared did not show any significant increase in efficacy.
6.4 Effect of Relative Humidity on Efficacy of MeBr against B. anthracis Ames
The decontamination efficacy of MeBr against B.a. Ames was evaluated at target relative
humidities of 45 or 75 %. The actual %RH conditions for each test are shown in Section 5. These
RH levels were tested at various temperatures, MeBr concentrations, and contact times. The
comparisons are shown in Figure 6-4 and detailed tabulated results to assess the effect of RH are
summarized in Table C-3 of Appendix C.
212, 22, 36	212, 27, 36
Concentration (mg/L), Temperature (°C), Contact Time (hours)
¦ 45% RH
~ 75% RH
Figure 6-4. Effect of relative humidity on MeBr decontamination efficacy against B.
anthracis Ames (Test numbers shown in each bar). Results shown as average log reduction
± standard deviation and corresponding test numbers are at the bottom of each bar.
The effect of increasing the %RH from 45 % to 75 % at low temperature (22 °C; compare Tests
1 and 3) and also at high temperature (27 °C; compare tests 4 and 9) was evaluated while
keeping all other parameters constant (MeBr concentration and contact time). The average
decontamination efficacy across all materials increased with increasing RH in both instances.
Overall, no test conducted at 45 % RH resulted in >6 LR of B.a. Ames for all materials tested.
6.5 Effect of MeBr Concentration on Efficacy against B. anthracis Ames
The decontamination efficacy of MeBr against virulent B.a. Ames was also evaluated at target
concentrations of 212 and 300 mg/L. Refer to Section 5 for the actual MeBr concentrations
achieved for each test. These concentrations were tested at various combinations of temperature
and RH. Four test conditions showed results that could be compared to assess the effect of
increasing MeBr concentration. These comparisons are shown in Figure 6-5, below, with
detailed results for each material presented in Tables C-4 and C-5 of Appendix C.
For the three test conditions conducted at 45 % RH, there was no significant change in efficacy
when increasing the MeBr concentration. Although only assessed once (between Tests 5 and 14),
increasing the MeBr concentration from 212 to 300 mg/L in the presence of 75 % RH resulted in
a significant increase in decontamination efficacy (4.47 LR), with complete inactivation or >6
LR on all materials tested.
21

-------
22, 45, 48	22, 45, 60	32, 45, 60	22, 75, 24
Temperature (°C), %RH, Contact Time (hours)
¦ 212 mg/L MeBr
~ 300 mg/L MeBr
Figure 6-5. Summary of effect of increasing concentration on average MeBr
decontamination efficacy for B.a. Ames. Results shown as average log reduction ± standard
deviation and corresponding test numbers are at the bottom of each bar.
6.6 Effect of Contact Time on Efficacy of MeBr against B. anthracis Ames
The effect of increasing the contact time on the efficacy against B.a. Ames was also assessed.
The contact times tested ranged from 18 to 72 hours; six sets of test conditions could be
compared to assess the effect of increasing contact time. These comparisons are summarized in
Figure 6-6 and presented in full detail in Tables C-6 and C-7 of Appendix C.
w
m

1 £ JJL
n1
5


lir
i


3 5






















j
¦

JL
9
¦


1












1







1


1
~	24 hours
¦ 36 hours
~	48 hours
~	60 hours
~	72 hours
212,22,45 212,27,45 212,32,45 300,22,45 212,22,75 212,27,75
MeBr Concentration (mg/L), Temperature (°C), %RH
Figure 6-6. Summary of the effect of contact time on average MeBr decontamination
efficacy against B. anthracis Ames. Corresponding test numbers are shown above each bar.
Similar to what we saw with the effect of increasing MeBr concentration (and to some extent,
increasing temperature), there was no significant effect of increasing contact time on
decontamination efficacy when fumigating at 45 % RH. At 75 % RH, there were two test
conditions that could be compared to assess the effect of contact time. When increasing the
contact time from 24 to 36 hours at 212 mg/L MeBr and 22 °C, there was a significant increase
22

-------
in efficacy. These results show that when RH is low, RH is the predominant factor controlling
efficacy.
6.7	Surface Damage to Materials
At the end of each decontamination test, the procedural blanks were visually compared to the
laboratory blanks, and test coupons were visually compared to positive controls, to assess any
impact MeBr may have had on each material type. Based on the visual appearance of the
decontaminated coupons, there were no apparent changes in the color, reflectivity, or roughness
of the six material surfaces after being exposed to MeBr.
6.8	Summary and Conclusion
This investigation focused on finding efficacious conditions when fumigating with MeBr at
temperatures and RH levels lower than used in previous studies. Eliminating or reducing the
need to humidify and/or heat a building would greatly facilitate MeBr fumigation when used to
decontaminate a building contaminated with B. anthracis spores. Another objective of the study
was to compare the decontamination results for B. anthracis (Ames) with avirulent spore-
forming microorganisms, to assess their potential as surrogates for use in future studies with
MeBr.
This study shows the important role that RH plays when fumigating with MeBr. There were no
tests in which >6 LR of B.a. Ames was achieved on all materials when fumigating at 45 % RH.
When fumigating at 45 % RH, increasing the MeBr concentration, temperature, or contact time
generally did not improve decontamination efficacy. In contrast, when fumigating at 75 % RH,
increasing the MeBr concentration, temperature and contact time did generally improve efficacy.
For example, a contact time of 36 hours was required to achieve > 6 LR of B.a. Ames on all
materials when fumigating at 212 mg/L, 22 °C, and 75 % RH. However, only 18 hours of contact
time were required to achieve > 6 LR of B.a. Ames on all materials when the MeBr
concentration was increased to 300 mg/L and temperature increased to 27 °C.
From two of the initial tests, the study showed that G.s. spores are less resistant to MeBr than
B.a. Ames. Therefore G.s. was eliminated from further testing, since a surrogate should be at
least as resistant as the virulent strain. In every test conducted with the NNR1A1 strain, the
NNR1A1 strain was always more resistant than the Ames strain, and in a few tests, the average
difference in LR with the Ames strain was more than 6.0. In the tests with the Sterne strain,
Sterne was always inactivated to a higher degree than the Ames strain when fumigating at 45 %
RH. But when fumigating at 75 % RH, the Sterne strain was more resistant than B.a. Ames.
Impact of Study
This work provides information on the efficacy of MeBr fumigation to decontaminate materials
that have been contaminated with B. anthracis spores. Such results may be useful in the
development of guidance to aid in deployment of MeBr fumigation after a wide-area release of
B. anthracis spores. In particular, these results will provide decision makers with information for
effectively using MeBr at temperatures and RH levels lower than has been recommended
previously, which will facilitate its use. This report also provides data to assist in selection of an
avirulent surrogate for B. anthracis Ames, for use in future field studies and additional lab-based
investigations utilizing MeBr.
23

-------
7.0 References
1.	Decontamination of Soil Contaminated with Bacillus anthracis Spores. US EPA Report
600/R-13/110, July 2013
(http://cfpub.epa.gov/si/si public record report.cfm?dirEntryId=258007&fed org id=12
53&address=nhsrc/&view=desc&sortBv=pubDateYear&showCriteria=l&count=25&sea
rchall=).
2.	Determining the Efficacy of Liquids and Fumigants in Systematic Decontamination
Studies for Bacillus anthracis Using Multiple Test Methods. US EPA Report 600/R-
10/088, December 2010.
(http://cfpub.epa.gov/si/si public record report.cfm?dirEntryId=227175&fed org id=12
53&address=nhsrc/si/&view=desc&sortBv=pubDateYear&showCriteria=l&count=25&s
earchall='indoor%20outdoor%20decontamination').
3.	Systematic Investigation of Liquid and Fumigant Decontamination Efficacy against
Biological Agents Deposited on Test Coupons of Common Indoor Materials. US EPA
Report 600/R-l 1/076, August 2011.
(http://cfpub.epa.gov/si/si public record report.cfm?dirEntryId=235044).
4.	Evaluation of Ethylene Oxide for the Inactivation o/'Bacillus anthracis. EPA Technology
Evaluation Report. December 2013. EPA/13/R-13-220.
(http://cfpub.epa.gov/si/si public record report.cfm?dirEntryId=262949&fed org id=12
53&address=nhsrc%2Fsi%2F&view=desc&sortBv=pubDateYear&count=25&showCrite
ria=l&searchall=EPA%2F600%2FR+13%2F220+&submit=Search).
5.	Standard Practice for Maintaining Constant Relative Humidity by Means of Aqueous
Solutions, AS I'M El 04-02. ASTM International, October, 2006.
6.	The Phaseout of Methyl Bromide. (http://www. epa. eov/ozone/mbr/index.html) Accessed
February 15, 2014.
7.	Amerithrax Investigative Summary. United States Department of Justice.
(http://www.iustice.gov/archive/amerithrax/docs/amx-investigative-summarv.pdf) Accessed
June 2, 2014.
24

-------
Appendix A
Detailed Test Results
Efficacy Results
The detailed decontamination efficacy results for methyl bromide against B.a. Ames, G.s., B.a.
NNR1A1, and B.a. Sterne on six material types (glass, ceiling tile, carpet, painted wallboard paper,
bare pine wood and unpainted concrete) are shown in Tables A-l through A-4. Zero CFU were
observed on all laboratory and procedural blanks.
Table A-l. Inactivation of B. anthracis Ames Spores using Methyl Bromide3
Test
Number
Target Parameters

Inoculum
(CFU/coupon)
Mean Recovered B. a. Ames
Decontamination
Efficacy ± CId
Concentration (mg/L) / Temp (°C) /
Material
(CFU/coupon)

Contact Time (hr) RH (%)

Positive Control1*
Test Coupon0


Glass

3.13 ±0.69x10'
2.84 ± 2.24 xlO4
3.14 ± 0.30


Ceiling Tile

7.67 ± 1.66 x10s
2.55 ± 0.39 xlO3
3.47 ±0.11
1
212/36 22/45
Carpet
Painted Wallboard Paper
5.97x10'
4.30 ±0.94x10'
1.11 ±0.24x10'
0.70 ± 1.10 xlO4
0.00 ± 0.00
4.18 ±0.60
>7.04 ±0.08


Bare Pine Wood

3.23 ± 0.44 x 10s
2.99 ± 2.39 xlO4
2.16 ±0.34


Unpainted Concrete

1.76 ± 1.11 xl0s
0.00 ± 0.00
>6.17 ±0.25


Glass

3.67 ±0.30x10'
4.01 ± 1.76 xlO4
3.00 ±0.20


Ceiling Tile

5.90 ± 1.34 x 10s
0.00 ± 0.00
>6.76 ± 0.09
2
212/48 22/45
Carpet
Painted Wallboard Paper
7.00x10'
4.45 ±0.56x10'
1.72 ±0.63x10'
0.71 ± 1.03 xlO3
0.00 ± 0.00
6.35 ± 1.55
>7.21 ±0.12


Bare Pine Wood

5.70 ± 1.59 x 10s
1.20 ± 0.83 xlO4
2.82 ± 0.44


Unpainted Concrete

8.03 ± 2.34 x 10s
0.00 ± 0.00
>6.89± 0.11


Glass

8.36 ±0.78x10'
0.00 ± 0.00
>7.92 ± 0.04


Ceiling Tile

1.18 ±0.05x10'
0.00 ± 0.00
>7.07 ±0.02
3
212/36 22/75
Carpet
Painted Wallboard Paper
1.07x10s
8.55 ±0.79x10'
5.84 ±0.59x10'
0.00 ± 0.00
0.00 ± 0.00
>7.93 ± 0.04
>7.76 ± 0.04


Bare Pine Wood

1.07 ± 0.18 x 10'
0.00 ± 0.00
>7.02 ± 0.06


Unpainted Concrete

3.45 ± 1.19 x 10s
0.00 ± 0.00
>7.51± 0.15


Glass

6.57 ± 1.64x10'
3.07 ± 3.21 xlO3
4.86 ± 0.90


Ceiling Tile

1.13 ±0.15x10'
3.35 ± 7.46 xlO2
6.41 ± 1.26
4
212/36 27/45
Carpet
Painted Wallboard Paper
8.80x10'
8.98 ± 0.61 x 10'
3.18 ±0.43x10'
0.00 ± 0.00
0.00 ± 0.00
>7.95 ± 0.03
>7.50 ± 0.05


Bare Pine Wood

9.77 ± 3.27 x 10s
0.48 ± 1.02 xlO3
5.93 ± 1.34


Unpainted Concrete

2.01 ± 1.31 x 10'
0.00 ± 0.00
>7.23± 0.24


Glass

5.03 ± 1.01 x 10'
1.60 ± 0.91 xlO5
2.54 ±0.22


Ceiling Tile

1.10 ± 0.12 x 10'
3.69 ± 3.56 xlO4
2.70 ± 0.48
5
212/24 22/75
Carpet
Painted Wallboard Paper
1.02 x10s
7.51 ± 1.33x10'
4.38 ±0.66x10'
9.85 ± 8.62 xlO5
1.08 ± 0.62 xlO5
3.31 ±0.78
2.69 ±0.30


Bare Pine Wood

5.63 ± 1.40 x 10s
1.35 ± 2.30 xlO4
2.99 ±0.50


Unpainted Concrete

2.10 ±0.93x10'
1.65 ± 2.40 xlO4
3.41± 0.56


Glass

6.28± 1.24x10'
6.20 ± 7.55 xlO4
3.49 ± 0.74


Ceiling Tile

1.09 ±0.10x10'
2.41 ±2.31 xlO3
4.00 ± 0.65
6
212/48 27/45
Carpet
Painted Wallboard Paper
1.19 x10 s
9.08 ±0.49x10'
2.31 ±0.79x10'
1.59 ± 3.09 xlO3
0.00 ± 0.00
6.60 ± 1.66
>7.34 ±0.14


Bare Pine Wood

6.15 ± 0.82 x 10s
9.06 ± 3.64 xlO4
1.86 ±0.19


Unpainted Concrete

7.39 ± 3.26 x10s
0.00 ± 0.00
>6.84± 0.15


Glass

6.15 ±0.95x10'
6.97 ± 6.21 xlO3
4.42 ± 0.96


Ceiling Tile

1.40 ±0.20x10'
0.00 ± 0.00
>7.14 ±0.06
7
212/24 27/75
Carpet
Painted Wallboard Paper
1.04 x10s
1.09 ± 0.13 x10s
6.15 ±0.39x10'
2.95 ± 6.57 xlO2
0.00 ± 0.00
7.40 ± 1.24
>7.79 ± 0.02


Bare Pine Wood

6.78 ± 3.74 x 10s
0.61 ± 1.34 xlO2
6.29 ±0.99


Unpainted Concrete

1.65 ±0.95x10'
0.00 ± 0.00
>7.17± 0.20


Glass

6.91 ± 1.22 x 10'
3.54 ± 0.67 xlO5
2.29 ±0.10


Ceiling Tile

1.33 ±0.26x10'
7.78 ± 4.57 xlO3
3.29 ±0.26
8
212/48 27/45
Carpet
Painted Wallboard Paper
1.35 x10 s
1.26 ± 0.25 x10s
4.03 ±0.52x10'
9.61 ±6.63 xlO3
0.00 ± 0.00
4.31 ±0.55
>7.60 ± 0.05


Bare Pine Wood

8.76 ± 4.18 x10s
8.11 ±5.02 xlO4
2.03 ±0.32


Unpainted Concrete

9.16 ± 3.03 x10s
0.00 ± 0.00
>6.94± 0.12
a Data are expressed as the mean (± SD) of the logs of the number of spores (CFU) observed on five individual samples, and decontamination
efficacy (log reduction).
b Positive Controls = samples inoculated, not decontaminated.
c Test Samples = samples inoculated, decontaminated. 11 CI = confidence interval (± 1.96 SE).
A-l

-------
Table A-l. Inactivation of B. anthracis Ames Spores using Methyl Bromide3 (Continued)
Test
Number
Target Parameters
Concentration (mg/L) /
Contact Time (hr)
Temp (°C) /
RH (%)
Inoculum
(CFU/coupon)
Mean Recovered B. a. Ames
(CFU/coupon)
Positive Control
Test Coupon0
Decontamination
Efficacy ± Cl"
Glass
Ceiling Tile
Carpet
Bare Pine Wood
5.65 ± 1.58 xlO7
1.20 ± 0.26 xlO7
1.11 ±0.09 xlO8
8.53 ± 1.69 xlO6
0.00 ± 0.00
0.00 ± 0.00
0.00 ± 0.00
0.00 ± 0.00
>7.74 ±0.10
>7.07 ±0.08
>8.04 ±0.03
>6.92 ±0.08



Glass

3.96 ± 0.61 xlO7
4.62 ± 4.96 xlO4
3.11 ±0.38
10
300/48
22/45
Ceiling Tile
Carpet
Bare Pine Wood
1.15 x10s
1.71 ±0.19xl07
9.23 ± 1.42 xlO7
9.67 ± 5.48 x 10s
0.00 ±0.00
6.95 ±6.08x10-'
1.41 ± 1.29 xlO4
>7.23 ±0.04
4.60 ±0.89
2.95 ±0.42



Glass

5.61 ± 1.42 xlO7
8.15 ±5.43xl05
3.90 ±0.27
11
212/60
22/45
Ceiling Tile
Carpet
Bare Pine Wood
9.67x10'
1.47 ± 0.24 x 107
8.98 ± 2.83 xlO7
8.13 ± 2.76 x10s
0.00 ±0.00
5.26 ± 4.77 xlO4
1.52 ± 2.23 xlO4
>7.16 ±0.06
3.41 ±0.47
3.03 ±0.52



Glass

4.08 ± 0.77 xlO7
0.00 ±0.00
>7.61 ±0.07
12
212/24
32/75
Ceiling Tile
Carpet
Bare Pine Wood
1.12 x10 s
1.10 ± 0.41 x 107
8.13 ± 1.20 x 107
8.01 ±2.53 x 10s
0.00 ±0.00
0.00 ±0.00
0.00 ±0.00
>7.04 ±0.05
>7.91 ±0.06
>6.89 ±0.10



Glass

5.31 ± 1.03 xlO7
5.57 ±4.95x10=
2.28 ±0.61
13
212/48
32/45
Ceiling Tile
Carpet
Bare Pine Wood
1.14x10 s
1.29 ± 0.31 xlO7
1.07 ± 0.06 x 10s
1.05 ± 0.50 x 10s
2.36 ± 1.71 x 10'
4.01 ±3.11 x 10'
5.31 ±4.45 xlO4
3.93 ±0.54
4.65 ± 0.52
2.47 ±0.53



Glass

4.22 ±0.71 xlO7
0.00 ±0.00
>7.62 ±0.07
14
300/24
22/75
Ceiling Tile
Carpet
Bare Pine Wood
1.31 x 10 s
2.02 ± 0.84 xlO7
1.05 ± 0.07 x 10s
1.11 ± 0.28 x 107
1.34 ± 2.98 x 102
0.00 ±0.00
0.00 ±0.00
6.72 ± 1.12
>8.02 ±0.03
>7.04 ±0.09



Glass

4.63 ± 1.16 xlO7
3.41 ±2.82 xlO4
3.25 ±0.37
15
300/60
22/45
Ceiling Tile
Carpet
Bare Pine Wood
1.27x10s
1.46 ± 0.24 x 107
7.98 ± 2.60 xlO7
8.61 ± 1.76 x10s
1.06 ± 1.55x10'
7.14 ± 6.82 x 102
5.76 ± 4.40 xlO4
5.36 ± 1.50
5.30 ± 0.59
2.38 ±0.53



Glass

7.23 ± 2.32 xlO7
1.27 ± 1.45x10=
3.03 ±0.59
16
212/60
32/45
Ceiling Tile
Carpet
Bare Pine Wood
9.23 xlO7
1.09 ± 0.16 xlO7
1.04 ± 0.11 x 10s
6.32 ± 0.60 x10s
1.78 ± 1.02 x 10'
1.31 ± 1.39x10'
6.23 ± 7.31 xlO4
4.36 ± 1.31
5.29 ±0.71
2.40 ±0.68



Glass

7.77 ± 0.80 xlO7
0.75 ± 1.44x10'
7.58 ± 0.60
17
300/18
27/75
Ceiling Tile
Carpet
Bare Pine Wood
1.08x10s
1.46 ± 0.37 x 107
1.30 ± 0.13 x10s
8.42 ± 1.30 x10s
1.34 ± 2.79 x 102
0.00 ±0.00
0.00 ±0.00
6.29 ±1.12
>8.11 ±0.04
>6.92 ±0.06



Glass

7.78 ± 1.42 xlO7
1.02 ±0.78x10=
3.01 ±0.38
18
300/60
27/45
Ceiling Tile
Carpet
Bare Pine Wood
1.13 x10 s
1.90 ± 0.98 xlO7
1.39 ± 0.23 x10s
1.59 ± 1.04 x 107
1.77 ± 1.81 x 103
2.11 ±3.60x10'
3.68 ± 2.86 xlO4
4.36 ± 0.75
5.28 ±0.62
2.71 ±0.44



Glass

7.10 ± 2.40 xlO7
1.62 ± 1.35x10=
2.72 ±0.31
19
212/72
32/45
Ceiling Tile
Carpet
Bare Pine Wood
1.21 x 10 s
1.58 ± 0.24 x 107
1.15 ± 0.23 x 0s
8.41 ± 1.02 x 10s
2.37 ±3.57x10'
4.73 ± 4.26 xlO2
5.59 ± 2.60 xlO4
5.20	± 1.65
6.02 ± 1.13
2.21	±0.16



Glass

4.27 ± 0.79 xlO7
7.98 ± 4.70 xlO4
2.84 ±0.37
20
300/60
32/45
Ceiling Tile
Carpet
Bare Pine Wood
1.27x10s
1.22 ± 0.29 xlO7
9.33 ± 0.37 xlO7
8.39 ± 1.25 x10s
4.52 ±4.08x10'
7.07 ± 8.41 xlO2
0.79 ± 1.08x10=
3.60 ±0.41
5.82 ± 1.15
2.27 ±0.42
3 Data are expressed as the mean (± SD) of the logs of the number of spores (CFU) observed on five individual samples, and decontamination
efficacy (log reduction).
b Positive Controls = samples inoculated, not decontaminated.
c Test Samples = samples inoculated, decontaminated.
d CI = confidence interval (± 1.96 x SE).
A-2

-------
Table A-2. Inactivation of (7. stearothermophilus Spores using Methyl Bromide3
Test
Number
Target Parameters

Inoculum
(CFU/coupon)
Mean Recovered G.s.
Decontamination
Efficacy ± CId
Concentration (mg/L) / Temp (°C) /
Material
(CFU/coupon)

Contact Time (hr) RH (%)

Positive Control15
Test Couponc


Glass

5.37 ± 0.91 xlO7
0.00 ± 0.00
>7.72 ± 0.07


Ceiling Tile

4.22 ±0.94x10=
3.39 ± 5.72 xlO1
4.89 ± 0.90
1
212/36 22/45
Carpet
Painted Wallboard Paper
6.80 xlO7
1.60 ± 1.10 x 107
6.69 ± 0.43 xlO7
1.41 ±2.94 xlO1
0.00 ± 0.00
6.77 ±0.75
>7.82 ± 0.02


Bare Pine Wood

7.87 ±6.67x10=
0.00 ± 0.00
>5.75 ± 0.36


Unpainted Concrete

1.03 ± 0.64 x 107
0.00 ± 0.00
>6.78 ± 0.62


Glass

5.81 ±0.94 xlO7
0.00 ± 0.00
>7.76 ± 0.06


Ceiling Tile

1.32 ± 1.37x10=
0.00 ± 0.00
>5.00 ± 0.29
2
212/48 22/45
Carpet
Painted Wallboard Paper
7.77x10'
1.65 ± 0.66 xlO7
2.89 ± 1.30 xlO7
0.00 ± 0.00
0.00 ± 0.00
>7.19 ±0.14
>7.43 ±0.17


Bare Pine Wood

1.59 ± 3.34 x 10s
0.00 ± 0.00
>5.37 ±0.75


Unpainted Concrete

7.53 ± 3.50 x 10s
0.00 ± 0.00
>6.85± 0.15
a Data are expressed as the mean (± SD) of the logs of the number of spores (CFU) observed on five individual samples, and decontamination
efficacy (log reduction).
b Positive Controls = samples inoculated, not decontaminated.
c Test Samples = samples inoculated, decontaminated.
11 CI = confidence interval (± 1.96 SE).
A-3

-------
Table A-3. Inactivation of B. anthracis NNR1A1 Spores using Methyl Bromide3
Test
Number
Target Parameters
Concentration (mg/L) /
Contact Time (hr)
Temp (°C) /
RH (%)
Inoculum
(CFU/coupon)
Mean Recovered B. a. NNR1A1
(CFU/coupon)
Positive Controlb
Test Coupon0
Decontamination
Efficacy ± CId



Glass

2.21 ± 1.09 xlO6
5.35 ± 1.47 xlO5
0.60 ±0.20



Ceiling Tile

1.28 ± 0.41 xlO6
1.19 ± 1.12 x 105
1.12 ± 0.31
3
212/36
22/75
Carpet
Painted Wallboard Paper
Bare Pine Wood
Unpainted Concrete
9.33 xlO7
4.83 ± 0.86 xlO7
1.40 ± 0.42 xlO7
1.28 ± 0.45 xlO7
2.00 ± 1.10 xlO7
1.38 ± 0.44 xlO6
1.81 ±0.30 xlO6
2.15 ± 1.32 xlO5
1.17 ± 0.64 x 106
1.56 ±0.14
0.88 ±0.15
1.80 ±0.24
1.26 ±0.34



Glass

8.05 ± 1.36 xlO7
9.49 ± 5.72 xlO5
1.97 ± 0.21



Ceiling Tile

4.30 ± 1.43x106
1.74 ± 2.55 xlO2
5.57 ± 1.26
4
212/36
27/45
Carpet
Painted Wallboard Paper
Bare Pine Wood
Unpainted Concrete
1.01 xlO8
8.49 ± 0.81 xlO7
5.99 ± 1.26 xlO7
2.43 ± 1.32x107
8.13 ± 3.32 xlO6
1.85 ± 1.83 xlO3
0.00 ± 0.00
8.26 ± 9.71 xlO2
0.00 ± 0.00
4.89 ±0.51
>7.77 ±0.08
5.08 ± 1.19
>6.88 ±0.18



Glass

8.24 ± 0.71 xlO7
3.72 ± 0.50 xlO7
0.35 ±0.06



Ceiling Tile

6.52 ± 2.04 xlO6
1.07 ± 0.40 xlO6
0.80 ±0.21
5
212/24
22/75
Carpet
Painted Wallboard Paper
Bare Pine Wood
Unpainted Concrete
1.12 x 108
7.42 ± 1.38x107
5.85 ± 0.20 xlO7
7.51 ± 1.80 xlO6
1.20 ± 0.38 xlO7
1.57 ± 0.34 xlO7
1.44 ± 0.16 xlO7
1.12 ± 0.43 xlO6
1.20 ± 0.88 xlO6
0.68 ±0.12
0.61 ±0.04
0.84 ± 0.19
1.16 ±0.47



Glass

6.00 ± 2.85 xlO7
2.20 ± 2.87 xlO5
2.79 ± 0.68
9
212/36
27/75
Ceiling Tile
Carpet
Bare Pine Wood
8.13 xlO7
3.89 ± 3.07 xlO6
8.29 ± 1.81 xlO7
3.83 ± 2.63 xlO6
0.00 ± 0.00
0.00 ± 0.00
5.20 ± 7.73 xlO2
>6.48 ± 0.30
>7.91 ±0.08
5.26 ± 1.51



Glass

5.82 ± 0.69 xlO7
1.39 ± 0.10 xlO7
0.62 ±0.06
10
300/48
22/45
Ceiling Tile
Carpet
Bare Pine Wood
1.68 xlO8
1.01 ±0.20 xlO7
8.82 ± 0.58 xlO7
7.30 ± 0.76 xlO6
0.73 ± 1.62 xlO2
2.26 ± 1.31 xlO6
9.64 ± 8.96 xlO3
6.29 ± 1.40
1.65 ±0.24
3.13 ±0.52



Glass

8.28 ± 1.30 xlO7
1.14 ± 0.26 x 107
0.87 ±0.11
11
212/60
22/45
Ceiling Tile
Carpet
Bare Pine Wood
1.31 xlO8
5.30 ± 1.18 xlO6
8.68 ± 1.29 xlO7
7.75 ± 2.96 xlO6
1.60 ± 1.96 xlO2
4.45 ± 1.29x106
3.59 ± 3.17 xlO3
5.33 ± 1.14
1.30 ±0.13
3.48 ± 0.48



Glass

4.80 ± 1.14 xlO7
9.24 ± 3.46 xlO5
1.73 ±0.19
12
212/24
32/75
Ceiling Tile
Carpet
Bare Pine Wood
1.27 x 108
4.47 ± 0.82 xlO6
5.23 ± 1.25 xlO7
5.17 ± 1.85 x 106
0.00 ± 0.00
4.08 ± 8.90 xlO1
1.57 ± 1.93 xlO3
>6.64 ±0.07
7.25 ±0.91
3.69 ± 0.40



Glass

8.09 ± 0.71 xlO7
2.08 ± 0.65 xlO7
0.60 ±0.11
13
212/48
32/45
Ceiling Tile
Carpet
Bare Pine Wood
1.02 xlO8
1.22 ± 0.45 xlO6
7.98 ± 1.20x107
1.32 ± 0.95 xlO6
3.15 ± 2.50 xlO3
1.95 ± 0.59 xlO6
9.90 ± 3.50 xlO3
2.67 ±0.36
1.63 ±0.16
2.06 ±0.29
a Data are expressed as the mean (± SD) of the logs of the number of spores (CFU) observed on five individual samples, and decontamination
efficacy (log reduction).
b Positive Controls = samples inoculated, not decontaminated.
c Test Samples = samples inoculated, decontaminated.
d CI = confidence interval (± 1.96 x SE).
A-4

-------
Table A-3. Inactivation of B. anthracis NNR1A1 Spores using Methyl Bromide3
(Continued)
Target Parameters	Mean Recovered II. a. NNR1A1
Test
Number
Concentration (mg/L) /
Temp (°C) /
Material
Inoculum
(CFU/coupon) .
(CFU/coupon)
Decontamination
Efficacy ± CId
Contact Time (hr)
RH (%)

Positive Controlb
Test Coupon0



Glass

4.38 ± 0.89 xlO7
4.15 ± 1.04 x10s
1.03 ±0.13
14
300/24
22/75
Ceiling Tile
Carpet
Bare Pine Wood
1.22 xlO8
3.72 ± 0.43 x 10s
4.62 ± 1.55x10'
1.08 ±0.50x10'
1.89 ± 1.15x10=
8.12 ± 2.29 x10s
2.77 ± 1.09 x 105
1.36 ±0.24
0.75 ±0.17
1.63 ±0.28



Glass

7.33 ±2.44x10'
9.57 ± 2.43 x10s
0.87 ± 0.19
15
300/60
22/45
Ceiling Tile
Carpet
Bare Pine Wood
1.12 x 108
4.31 ± 1.19 x 10s
6.98 ± 1.63x10'
4.08 ± 2.02 x10s
4.07 ±7.20x10'
1.21 ±0.74xl0s
1.01 ±0.55xl05
5.87 ±0.93
1.85 ±0.33
3.60 ±0.33



Glass

4.68 ± 1.58x10'
1.64 ± 0.86 x 10'
0.48 ± 0.22
16
212/60
32/45
Ceiling Tile
Carpet
Bare Pine Wood
1.23 xlO8
9.91 ±5.32 xlO5
7.66 ± 1.28x10'
1.43 ± 0.73 x 10s
4.01 ±4.25x10-'
0.94 ± 1.23 x10s
8.31 ±4.34x10'
2.53 ± 0.43
2.14 ±0.42
2.24 ±0.30



Glass

6.12 ± 2.11 x 10'
2.88 ±4.67x10=
2.68 ±0.53
17
300/18
28/75
Ceiling Tile
Carpet
Bare Pine Wood
1.13 x 108
3.10 ± 2.29 x 10s
9.60 ± 1.88x10'
7.35 ± 6.29 x10s
1.78 ± 1.49 xlO4
1.91 ± 0.98 x10s
3.34 ± 2.59 xlO4
2.33 ± 0.45
1.74 ±0.21
2.30 ± 0.48



Glass

8.68 ±0.90x10'
1.62 ±0.32x10'
0.73 ± 0.08
18
300/60
27/45
Ceiling Tile
Carpet
Bare Pine Wood
1.10x10s
3.98 ± 0.72 x10s
7.88 ±0.59x10'
0.96 ± 1.33x10'
2.15 ± 1.72 x 10'
1.53 ± 0.55 x10s
8.28 ±4.66x10'
3.38 ±0.33
1.73 ±0.14
2.88 ± 0.42



Glass

7.32 ± 1.20x10'
1.18 ± 0.31 x 10'
0.80 ±0.12
19
212/72
32/45
Ceiling Tile
Carpet
Bare Pine Wood
1.02 xlO8
2.97 ± 1.30 x10s
9.06 ±2.70x10'
5.81 ± 3.66 x10s
1.64 ± 1.68x10'
2.50 ± 1.65 x10s
8.72 ± 8.43 xlO4
3.46 ± 0.50
1.67 ±0.40
1.98 ±0.63



Glass

4.51 ± 1.50x10'
1.56 ±0.08x10'
0.44 ±0.14
20
300/60
32/45
Ceiling Tile
Carpet
Bare Pine Wood
1.03 xlO8
3.33 ± 2.15 x10s
1.16 ± 0.12 x10s
3.64 ± 2.90 x10s
2.40 ±2.16x10'
2.50 ± 3.07 x10s
3.62 ± 1.87x10'
3.19 ±0.43
1.89 ±0.42
2.96 ±0.34
a Data are expressed as the mean (± SD) of the logs of the number of spores (CFU) observed on five individual samples, and decontamination
efficacy (log reduction).
b Positive Controls = samples inoculated, not decontaminated.
c Test Samples = samples inoculated, decontaminated.
11 CI = confidence interval (± 1.96 SE).
A-5

-------
Table A-4. Inactivation of B. anthracis Sterne Spores using Methyl Bromide3
Test
Number
Target Parameters

Inoculum
(CFU/coupon)
Mean Recovered B. a. Sterne
Decontamination
Efficacy ± CId
Concentration (mg/L) / Temp (°C) /
Material
(CFU/coupon)

Contact Time (hr) RH (%)

Positive Controlb
Test Couponc


Glass

9.82 ± 3.80 x 10s
0.00 ± 0.00
>6.97 ±0.15


Ceiling Tile

1.01 ±0.15 xlO7
0.00 ± 0.00
>7.00 ± 0.06
6
212/48 27/45
Carpet
Painted Wallboard Paper
9.63 xlO7
7.46 ± 0.39 xlO7
3.89 ± 0.82 xlO7
0.00 ± 0.00
0.00 ± 0.00
>7.87 ±0.02
>7.58 ± 0.07


Bare Pine Wood

6.95 ± 1.85 x 10s
4.01 ±4.85x103
4.28 ± 1.53


Unpainted Concrete

8.55 ± 6.09 x10s
0.00 ± 0.00
>6.85 ± 0.26


Glass

1.33 ± 0.36 xlO7
0.75 ± 1.08x104
4.17 ± 1.52


Ceiling Tile

9.25 ± 1.15 x 10s
1.85 ± 1.61 xlO4
3.53 ± 1.70
7
212/24 27/75
Carpet
Painted Wallboard Paper
1.00 x10 s
6.75 ± 0.44 xlO7
4.15 ± 0.48 xlO7
2.87 ± 1.70 xlO5
6.73 ± 3.44 xlO5
2.47 ±0.33
1.86 ±0.27


Bare Pine Wood

9.79 ± 3.55 x 10s
1.72 ± 1.83 xlO5
1.88 ±0.34


Unpainted Concrete

5.48 ± 2.64 x10s
0.94 ± 1.66 xlO4
3.83 ± 1.54


Glass

1.28 ± 0.28 xlO7
1.07 ± 2.20 xlO2
6.25 ± 1.08


Ceiling Tile

7.43 ± 1.18 x10s
2.52 ± 4.31 xlO3
3.99 ±0.69
8
212/48 27/45
Carpet
Painted Wallboard Paper
1.10 x10 s
6.18± 1.11 xlO7
3.73 ± 0.78 xlO7
0.00 ± 0.00
0.00 ± 0.00
>7.78 ± 0.07
>7.56 ± 0.08


Bare Pine Wood

9.37 ± 1.67 x10s
1.88 ± 3.21 xlO4
3.56 ±0.98


Unpainted Concrete

3.87 ± 2.08 x10s
0.00 ± 0.00
>6.54 ±0.21


Glass

3.86 ± 2.05 xlO7
0.00 ± 0.00
>7.53 ± 0.23
9
212/36 27/75
Ceiling Tile
Carpet
9.63 xlO7
1.15 ± 0.16 xlO7
6.07 ± 0.70 xlO7
0.00 ± 0.00
2.06 ± 2.93 xlO1
>7.06 ± 0.06
7.11 ±0.81


Bare Pine Wood

6.45 ± 0.59 x10s
0.75 ± 1.44 xlO1
6.50 ±0.60


Glass

1.27 ± 0.41 x 107
1.41 ±2.94 xlO1
6.72 ± 0.73
10
300/48 22/45
Ceiling Tile
Carpet
1.05 x10 s
1.37 ± 0.25 x 107
7.49 ± 1.70 xlO7
0.00 ± 0.00
1.07 ± 2.38 xlO2
>7.13 ±0.07
7.32 ± 1.07


Bare Pine Wood

1.15 ± 0.23 xlO7
9.23 ± 8.79 xlO3
4.14 ± 1.65


Glass

1.62 ± 0.77 x 107
2.91 ±6.20 xlO3
5.01 ± 1.31
11
212/60 22/45
Ceiling Tile
Carpet
1.00 x10 s
1.35 ± 0.38 xlO7
9.01 ±2.84xl07
1.41 ±2.94 xlO1
3.56 ± 5.27 xlO3
6.75 ± 0.72
5.25 ± 1.40


Bare Pine Wood

1.11 ±0.32 xlO7
1.18 ± 1.74 x 104
3.35 ±0.59


Glass

8.00 ± 2.03 x10s
0.00 ± 0.00
>6.89 ± 0.09
12
212/24 32/75
Ceiling Tile
Carpet
1.16 x10 s
1.21 ±0.36 xlO7
6.24 ± 0.87 xlO7
0.00 ± 0.00
0.00 ± 0.00
>7.07 ±0.10
>7.79 ± 0.06


Bare Pine Wood

7.16 ± 2.60 x10s
0.00 ± 0.00
>6.84 ±0.12


Glass

1.15 ± 0.62 xlO7
4.70 ± 2.63 xlO3
3.40 ± 0.34
13
212/48 32/45
Ceiling Tile
Carpet
9.30 X 107
9.76 ± 2.00 x10s
6.61 ±0.47 xlO7
1.02 ± 0.97 xlO3
2.73 ± 3.60 xlO1
4.13 ±0.38
7.09 ± 0.88


Bare Pine Wood

5.28 ± 1.07 x10s
1.11 ±0.92 xlO3
3.95 ±0.59
a Data are expressed as the mean (± SD) of the logs of the number of spores (CFU) observed on five individual samples, and decontamination
efficacy (log reduction).
b Positive Controls = samples inoculated, not decontaminated.
c Test Samples = samples inoculated, decontaminated.
11 CI = confidence interval (± 1.96 SE).
A-6

-------
Table A-4. Inactivation of B. anthracis Sterne Spores using Methyl Bromide3 (Continued)
Target Parameters	Mean Recovered B. a. Sterne
Test
Number
Concentration (mg/L) /
Temp (°C) /
Material
Inoculum
(CFU/coupon)
(CFU/coupon)
Decontamination
Efficacy ± CId
Contact Time (hr)
RH (%)

Positive Control1*
Test Coupon0



Glass

9.60 ± 3.37 x 10s
3.02 ± 2.11 xlO4
2.57 ±0.30
14
300/24
22/75
Ceiling Tile
Carpet
Bare Pine Wood
1.06 x10 s
2.59 ±0.74x10'
7.57 ± 1.30x10'
1.14 ± 0.18 x 10'
7.10 ±4.42x10=
7.56 ± 5.91 x 10=
2.54 ± 1.75x10=
1.61 ±0.25
2.10 ±0.30
1.72 ±0.25



Glass

1.04 ± 0.49 x 10'
0.00 ±0.00
>6.98 ±0.18
15
300/60
22/45
Ceiling Tile
Carpet
Bare Pine Wood
1.18 x10 s
2.74 ±0.99x10'
8.69 ±0.79x10'
1.53 ±0.62x10'
1.82 ± 1.49 x10s
2.06 ±2.93x10'
3.86 ± 2.41 x 10s
4.79 ± 1.32
7.27 ±0.81
3.67 ±0.34



Glass

1.01 ± 1.07x10'
2.85 ± 5.75 xlO4
4.21 ± 1.78
16
212/60
32/45
Ceiling Tile
Carpet
Bare Pine Wood
1.22 x10s
1.02 ±0.09x10'
6.40 ±0.66x10'
9.53 ± 2.46 x10s
1.50 ±2.15xlOs
0.00 ±0.00
3.95 ± 4.22 x10s
4.63 ± 1.26
>7.80 ±0.04
4.15 ± 1.43



Glass

2.15 ± 1.17 x 10'
7.73 ± 3.59 xl0s
4.41 ±0.32
17
300/18
27/75
Ceiling Tile
Carpet
Bare Pine Wood
9.97x10'
1.33 ±0.32x10'
1.07 ± 0.19 x 10s
1.63 ± 1.06x10'
6.82 ± 4.56 xlO4
1.62 ± 1.65 xlO4
2.33 ± 2.24 xlO4
2.40 ± 0.37
4.38 ± 1.09
3.04 ±0.58



Glass

7.55 ± 2.61 x 10s
2.71 ±2.74x10'
5.88 ±0.80
18
300/60
27/45
Ceiling Tile
Carpet
Bare Pine Wood
1.07x10s
1.09 ±0.22x10'
5.81 ± 1.03x10'
1.07 ± 0.27 x 10'
3.58 ± 2.86 x10s
2.74 ±5.90x10'
4.31 ±9.38 xlO4
3.60 ±0.33
7.33 ± 0.84
3.67 ± 1.04



Glass

1.13 ±0.32x10'
1.01 ± 1.60 x10s
6.10 ± 1.14
19
212/72
32/45
Ceiling Tile
Carpet
Bare Pine Wood
1.01 x 10 s
9.74 ± 2.51 x 10s
7.69 ± 1.08x10'
6.40 ± 1.44 x 10s
0.89 ± 1.43 x10s
0.00 ±0.00
4.36 ± 2.93 x10s
5.27 ± 1.45
>7.88 ± 0.05
3.41 ±0.64



Glass

1.71 ± 1.52 x 10'
3.41 ± 5.06 x10s
3.95 ±0.58
20
300/60
32/45
Ceiling Tile
Carpet
Bare Pine Wood
1.06 x10 s
1.07 ± 0.27 x 10'
5.78 ±0.82x10'
5.93 ± 0.90 x 10s
1.91 ± 2.60 x10s
0.75 ± 1.44x10'
7.08 ± 4.55 x10s
4.02 ± 0.47
7.45 ± 0.60
3.01 ±0.31
a Data are expressed as the mean (± SD) of the logs of the number of spores (CFU) observed on five individual samples, and decontamination
efficacy (log reduction).
b Positive Controls = samples inoculated, not decontaminated.
c Test Samples = samples inoculated, decontaminated.
11 CI = confidence interval (± 1.96 SE).
A-7

-------
Appendix B
Comparing Efficacy for the Different Microorganisms
Testing was first conducted using G.s. as a potential surrogate for B.a. Ames (Tests 1 & 2). The
results showed that G.s. is less resistant than B.a. Ames to MeBr exposure; therefore, the
additional potential surrogates, B.a. NNR1A1 and B.a. Sterne, were tested. In an attempt to
evaluate all three organisms at once, the number of coupon materials tested was reduced due to
the size of the MeBr test chamber. Painted wallboard and unpainted concrete were removed from
testing after Test 8 as these materials were the easiest to decontaminate. The detailed differences
in efficacy by material type and test number are shown in Tables B-l and B-2.
Table B-l. Difference in MeBr Efficacy between B. anthracis Ames and G.
stearothermophilus *
Test Target MeBr
,T . Concentration
Number , ,T.
(mg/L)
Target
Temperature
<°C)
Target
RH
(%)
Contact
Time
(hour)
Material Type
B.a,
Ames
Efficacy
G.s.
Efficacy
Average
Difference
in Efficacy




Glass
3.14
>
7.72





Ceiling Tile
3.47

4.89

1 212
22
45
36
Carpet
Painted Wallboard Paper
Pine Wood
Unpainted Concrete
4.18
>	7.04
2.16
>	6.17
>
>
6.77
7.82
5.75
6.78
2.26




Glass
3.00
>
7.76





Ceiling Tile
> 6.76
>
5.00

2 212
22
45
48
Carpet
Painted Wallboard Paper
Pine Wood
Unpainted Concrete
6.35
>	7.21
2.82
>	6.89
>
>
>
>
7.19
7.43
5.37
6.85
1.10
* Results shown as efficacy (log reduction).
B-l

-------
Table B-2. Difference in MeBr Efficacy between B. anthracis Ames, B. anthracis NNR1A1,
and B. anthracis Sterne*
Test
Number
Target MeBr
Concentration
(mg/L)
Target
Temperature
(°C)
Target
RH
(%)
Contact
Time
(hour)
Material Type
B.a.
Ames
Efficacy
B.a.
NNR1A1
Efficacy
Average
Difference
in Efficacy
B.a.
Sterne
Efficacy
Average
Difference
in Efficacy





Glass
>
7.92
0.60









Ceiling Tile
>
7.07
1.12




3
212
22
75
36
Carpet
Painted Wallboard Paper
Pine Wood
Unpainted Concrete
>
>
>
>
7.93
7.76
7.02
7.51
1.56
0.88
1.80
1.26
-6.33

NT
NT





Glass

4.86
1.97









Ceiling Tile

6.41
5.57




4
212
27
45
36
Carpet
Painted Wallboard Paper
Pine Wood
Unpainted Concrete
>
>
>
7.95
7.50
5.93
7.23
4.89
>	7.77
5.08
>	6.88
-1.29

NT
NT





Glass

2.54
0.35









Ceiling Tile

2.70
0.80




5
212
22
75
24
Carpet
Painted Wallboard Paper
Pine Wood
Unpainted Concrete

3.31
2.69
2.99
3.41
0.68
0.61
0.84
1.16
-2.20

NT
NT





Glass

3.49


>
6.97






Ceiling Tile

4.00


>
7.00

6
212
27
45
48
Carpet
Painted Wallboard Paper
Pine Wood
Unpainted Concrete
>
>
6.60
7.34
1.86
6.84
NT
NT
>
>
>
7.87
7.58
4.28
6.85
1.74





Glass

4.42



4.17






Ceiling Tile
>
7.14



3.53

7
212
27
75
24
Carpet
Painted Wallboard Paper
Pine Wood
Unpainted Concrete
>
>
7.40
7.79
6.29
7.17
NT
NT

2.47
1.86
1.88
3.83
-3.75





Glass

2.29



6.25






Ceiling Tile

3.29



3.99

8
212
27
45
48
Carpet
Painted Wallboard Paper
Pine Wood
Unpainted Concrete
>
>
4.31
7.60
2.03
6.94
NT
NT
>
>
>
7.78
7.56
3.56
6.54
1.54
* Results shown as efficacy (log reduction).
NT = Not Tested
B-2

-------
Table B-2. Difference in MeBr Efficacy between B. anthracis Ames, B. anthracis NNR1A1,
and B. anthracis Sterne* (Continued)
Test
Number
Target MeBr
Concentration
(mg/L)
Target
T emperature
(°C)
Target
RH
(%)
Contact
Time
(hour)
Material Type
B.a.
Ames
Efficacy
B.a,
NNR1A1
Efficacy
Average
Difference
in Efficacy
B.a.
Sterne
Efficacy
Average
Difference
in Efficacy





Glass
>
7.74
2.79

>
7.53

9
212
27
75
36
Ceiling Tile
Carpet
Bare Pine Wood
>
>
>
7.07
8.04
6.92
>	6.48
>	7.91
5.26
-1.83
>
7.06
7.11
6.50
-0.39





Glass

3.11
0.62


6.72

10
300
22
45
48
Ceiling Tile
Carpet
Bare Pine Wood
>
7.23
4.60
2.95
6.29
1.65
3.13
-1.55
>
7.13
7.32
4.14
1.86





Glass

3.90
0.87


5.01

11
212
22
45
60
Ceiling Tile
Carpet
Bare Pine Wood
>
7.16
3.41
3.03
5.33
1.30
3.48
-1.63

6.75
5.25
3.35
0.72





Glass
>
7.61
1.73

>
6.89

12
212
32
75
24
Ceiling Tile
Carpet
Bare Pine Wood
>
>
>
7.04
7.91
6.89
> 6.64
7.25
3.69
-2.54
>
>
>
7.07
7.79
6.84
-0.22





Glass

2.28
0.60


3.40

13
212
32
45
48
Ceiling Tile
Carpet
Bare Pine Wood

3.93
4.65
2.47
2.67
1.63
2.06
-1.59

4.13
7.09
3.95
1.31





Glass
>
7.62
1.03


2.57

14
300
22
75
24
Ceiling Tile
Carpet
Bare Pine Wood
>
>
6.72
8.02
7.04
1.36
0.75
1.63
-6.16

1.61
2.10
1.72
-5.35





Glass

3.25
0.87

>
6.98

15
300
22
45
60
Ceiling Tile
Carpet
Bare Pine Wood

5.36
5.30
2.38
5.87
1.85
3.60
-1.03

4.79
7.27
3.67
1.61





Glass

3.03
0.48


4.21

16
212
32
45
60
Ceiling Tile
Carpet
Bare Pine Wood

4.36
5.29
2.40
2.53
2.14
2.24
-1.92
>
4.63
7.80
4.15
1.43





Glass

7.58
2.68


4.41

17
300
27
75
18
Ceiling Tile
Carpet
Bare Pine Wood
>
>
6.29
8.11
6.92
2.33
1.74
2.30
-4.96

2.40
4.38
3.04
-3.67





Glass

3.01
0.73


5.88

18
300
27
45
60
Ceiling Tile
Carpet
Bare Pine Wood

4.36
5.28
2.71
3.38
1.73
2.88
-1.66

3.60
7.33
3.67
1.28





Glass

2.72
0.80


6.10

19
212
32
45
72
Ceiling Tile
Carpet
Bare Pine Wood

5.20
6.02
2.21
3.46
1.67
1.98
-2.06
>
5.27
7.88
3.41
1.63





Glass

2.84
0.44


3.95

20
300
32
45
60
Ceiling Tile
Carpet
Bare Pine Wood

3.60
5.82
2.27
3.19
1.89
2.96
-1.51

4.02
7.45
3.01
0.98
* Results shown as efficacy (log reduction).
B-3

-------
Appendix C
Effects of Materials and Operational Parameters on MeBr Efficacy
Effects of Test Materials on MeBr efficacy
Testing was originally conducted using six test materials (Tests 1 through 8): ceiling tile, carpet,
glass, painted wallboard paper, bare pine wood, and unpainted concrete. In an attempt to evaluate
three organisms at once {B.a. Ames, B.a. NNR1A1, and B.a. Sterne), the number of coupon
materials tested was reduced due to the size of the MeBr test chamber. Painted wallboard and
unpainted concrete were removed from testing in Tests 9 through 20, as these two material types
generally exhibited higher efficacy than the other material types. A summary of the results in
terms of LR are organized by operational parameters and can be seen in Figures C-l through C-3
below.
C-l

-------
Glass
Bare Pine Wood
d
t-
M
iiiiiii
2i L
22 °C
45% RH
27 °C
45% RH
32 °C
45% RH
22 °C
75% RH
27 °C
75% RH
32 °C
75% RH
mg L MeBr/Contact Time (hours)
t 6
Oh
& 3
11111111111111
CO CO
rvj rv|
rJL
22 °C
45% RH
27 °C
45% RH
32 °C
45% RH
22 °C
75% RH

27 °C
75%RH
32 °C
75%RH
ing L MeBr/Contact Time (hours)
Figure C-l. Summary of MeBr efficacy against B. anthracis Ames on glass and bare pine wood. Results shown in average log
reduction ± CI.
* Complete inactivation achieved
d
&
Ceiling Tile
75% RH 75% RH
d 4
Carpet
I

L

75% RH 75% RH
ing L MeBr Contact Tune (hours)
mgL MeBr/Contact Tune (hours)
Figure C-2. Summary of MeBr efficacy against B. anthracis Ames on ceiling tile and carpet. Results shown in average log
reduction ± CI.
: Complete Inactivation achieved
C-2

-------
Painted Wallboard Paper	Unpainted Concrete
22 °C	27 °C	22 °C	27 °C	22 °C	27 °C	22 °C	27 °C
45% RH	45% RH	75%RH	75%RH	45% RH	45% RH	75% RH	75%RH
mg/L MeBr Contact Time (hours)	mg/L MeBr Contact Tiine (hours)
Figure C-3. Summary of MeBr efficacy against B. anthracis Ames on painted wallboard paper and unpainted concrete.
Results shown in average log reduction ± CI.
* Complete inactivation achieved
C-3

-------
Effects of Temperature on MeBr Efficacy against B.a. Ames
The decontamination efficacy of MeBr against B.a. Ames was evaluated at target temperatures of 22,
27, or 32 °C. These temperatures were tested at various combinations of %RH, MeBr concentration,
and contact time and results are summarized in Tables C-l and C-2. The comparisons are made for
two test conditions which share the same fumigation parameters except temperature.
Table C-l. Effect of Increasing Temperature at Low Relative Humidity on MeBr
Efficacy*
Material Type
Test 1
212 mg/L; 22 °C;
45 %; 36 hr
Test 4
212 mg/L; 27 °C;
45 %; 36 hr
Average
Difference
in Efficacy
Test 2
212 mg/L; 22 °C;
45 %; 48 hr
Test 6
212 mg/L; 27 °C;
45 %; 48 hr
Average
Difference in
Efficacy
Glass
3.14
4.86

3.00
3.49

Ceiling Tile
3.47
6.41

> 6.76
4.00

Carpet
Painted Wallboard Paper
4.18
> 7.04
>	7.95
>	7.50
2.29
6.35
> 7.21
6.60
7.34
-0.48
Bare Pine Wood
2.16
5.93

2.82
1.86

Unpainted Concrete
> 6.17
> 7.23

> 6.89
6.84

Material Type
Test 6
212 mg/L; 27 °C;
45 %; 48 hr
Test 13
212 mg/L
32 °C
45 %
48 hr
Average
Difference
in Efficacy
Test 11
212 mg/L; 22 °C;
45 %; 60 hr
Test 16
212 mg/L; 32 °C;
45 %; 60 hr
Average
Difference in
Efficacy
Glass
3.49
2.28

3.90
3.03

Ceiling Tile
4.00
3.93

> 7.16
4.36

Carpet
Painted Wallboard Paper
6.60
> 7.34
4.65
	b
-0.66
3.41
5.29
-0.61
Bare Pine Wood
1.86
2.47

3.03
2.40

Unpainted Concrete
> 6.84





Material Type
T est 15
300 mg/L; 22 °C;
45 %; 60 hr
Test 18
300 mg/L
27 °C
45 %
60 hr
Average
Difference
in Efficacy
Test 18
300 mg/L; 27 °C;
45 %; 60 hr
Test 20
300 mg/L; 32 °C;
45 %; 60 hr
Average
Difference in
Efficacy
Glass
3.25
3.01

3.01
2.84

Ceiling Tile
5.36
4.36

4.36
3.60

Carpet
Painted Wallboard Paper
5.30
5.28
-0.23
5.28
5.82
-0.21
Bare Pine Wood
2.38
2.71

2.71
2.27

Unpainted Concrete
* Data are expressed as decontamination efficacy (log reduction).
a Parameters of each test listed in order of MeBr concentration (mg/L), temperature (°C), %RH, and contact time (hrs).
b Not tested.
C-4

-------
Table C-2. Effect of Increasing Temperature at High Relative Humidity on MeBr
Efficacy*

Test 5
Test 7
Average

Test 3

Test 9
Average
Material Type


Difference




Difference in

212 mg/L; 22 °C;
212 mg/L; 27 °C;
in Efficacy
212 mg/L; 22 °C;
212 mg/L; 27 °C;
Efficacy

75%; 24 hr
75 %; 24 hr

75 %; 36 hr
75 %; 36 hr

Glass
2.54
4.42

>
7.92
>
7.74

Ceiling Tile
2.70
> 7.14

>
7.07
>
7.07

Carpet
3.31
7.40

>
7.93
>
8.04



3.76




0.00
Painted Wallboard Paper
2.69
> 7.79

>
7.76

~

Bare Pine Wood
2.99
6.29

>
7.02
>
6.92

Unpainted Concrete
3.41
> 7.17

>
7.51



Material Type
Test 7
212 mg/L; 27 °C;
75 %; 24 hr
Test 12
212 mg/L; 32 °C;
75 %; 36 hr
Average
Difference
in Efficacy
Glass
4.42
> 7.61

Ceiling Tile
> 7.14
> 7.04

Carpet
Painted Wallboard Paper
7.40
> 7.79
> 7.91
1.05
Bare Pine Wood
6.29
> 6.89

Unpainted Concrete
> 7.17


* Data are expressed as decontamination efficacy (log reduction).
a Parameters of each test listed in order of MeBr concentration (mg/L), temperature (°C), %RH, and contact time (hrs).
b Not tested.
Effect of Relative Humidity on Efficacy of MeBr against B. anthracis Ames
The decontamination efficacy of MeBr against B. a. Ames was evaluated at target relative humidities
of 45 or 75 %. The actual %RH conditions for each test are shown in Appendix A. These RH levels
were tested at various temperatures, MeBr concentrations, and contact times and results are
summarized in Table 6-5 below and discussed in Section 6.4. The comparisons are made for two test
conditions which share the same fumigation parameters except RH.
Table C-3. Effect of Increasing Relative Humidity at Low and High Temperatures on B.
anthracis Ames*

Test l3

Test 3

Test 4
Test 9





Average


Average
Material Type



Difference


Difference in

212 mg/L; 22 °C;
212 mg/L; 22 °C;
in Efficacy
212 mg/L; 27 °C;
212 mg/L; 27 °C;
Efficacy

45%; 36 hr
75%; 36 hr

45%; 36 hr
75%; 36 hr

Glass
3.14
>
7.92

4.86
> 7.74

Ceiling Tile
3.47
>
7.07

6.41
> 7.07

Carpet
4.18
>
7.93

> 7.95
> 8.04




3.18

.
1.16
Painted Wallboard Paper
> 7.04
>
7.76

> 7.50
~

Bare Pine Wood
2.16
>
7.02

5.93
> 6.92

Unpainted Concrete
> 6.17
>
7.51

> 7.23


* Data are expressed as decontamination efficacy (log reduction).
a Parameters of each test listed in order of MeBr concentration (mg/L), temperature (°C), %RH, and contact time (hrs).
b Not tested.
C-5

-------
Effects of MeBr Concentration on Efficacy against B. anthracis Ames
The decontamination efficacy of MeBr against virulent B.a. was also evaluated at target
concentrations of 212 and 300 mg/L. These concentrations were tested in various combinations of
temperature and RH. The results are summarized in Tables C-4 and C-5, below. The comparisons are
made for two test conditions that share the same fumigation parameters except MeBr concentration.
Table C-4. Effect of Increasing MeBr Concentration at Low Relative Humidity on B.
anthracis Ames*

Test 2a
Test 10
Average
Test 11
Test 15
Average
Material Type


Difference


Difference in

212 mg/L; 22 °C;
212 mg/L; 22 °C;
in Efficacy
212 mg/L; 22 °C;
300 mg/L; 22 °C;
Efficacy

45%; 48 hr
45%; 48 hr

45%; 60 hr
45%; 60 hr

Glass
3.00
3.11

3.90
3.25

Ceiling Tile
> 6.76
> 7.23

> 7.16
5.36

Carpet
Painted Wallboard Paper
6.35
> 7.21
4.60
	b
-0.26
3.41
5.30
-0.30
Bare Pine Wood
2.82
2.95

3.03
2.38

Unpainted Concrete
> 6.89













Test 16
Test 20
Average



Material Type
212 mg/L; 32 °C;
45%; 60 hr
300 mg/L; 32 °C;
45%; 60 hr
Difference
in Efficacy



Glass
3.03
2.84




Ceiling Tile
4.36
3.60




Carpet
Painted Wallboard Paper
5.29
5.82
-0.14



Bare Pine Wood
2.40
2.27




Unpainted Concrete






* Data are expressed as decontamination efficacy (log reduction).
a Parameters of each test listed in order of MeBr concentration (mg/L), temperature (°C), %RH, and contact time (hrs).
b Not tested.
Table C-5. Effect of Increasing MeBr Concentration at High Relative Humidity on B.
anthracis Ames*
Material Type
Test 53
212 mg/L; 22 °C;
75 %; 24 hr
Test 14
300 mg/L; 22 °C;
75 %; 24 hr
Average
Difference
in Efficacy
Glass
2.54
> 7.62

Ceiling Tile
2.70
6.72

Carpet
3.31
> 8.02


.
4.47
Painted Wallboard Paper
2.69
—

Bare Pine Wood
2.99
> 7.04

Unpainted Concrete
3.41


* Data are expressed as decontamination efficacy (log reduction).
a Parameters of each test listed in order of MeBr concentration (mg/L), temperature (°C), %RH, and contact time (hrs).
b Not tested.
C-6

-------
Effects of Contact Time on Efficacy of MeBr against B. anthracis Ames
The effect of increasing the contact times to MeBr at low and high %RH on the efficacy against
B.a. Ames was also assessed. The contact times tested ranged from 18 to 72 hours and actual
contact times did not deviate from these targets. The results are summarized in Tables C-6 and
C-7 and Figure 6-6. The comparisons are made for two test conditions that share the same
fumigation parameters except contact time.
Table C-6. Effect of Increasing Contact Time at Low Relative Humidity on B. anthracis
Ames*

Test l3
Test 2

Test 2
Test 11

Material Type
212 mg/L; 22 °C;
45 %; 36 hr
212 mg/L; 22 °C;
45 %; 48 hr
Average
Difference
in Efficacy
212 mg/L; 22 °C;
45 %; 48 hr
212 mg/L; 22 °C;
45 %; 60 hr
Average
Difference in
Efficacy
Glass
3.14
3.00

3.00
3.90

Ceiling Tile
3.47
> 6.76

> 6.76
> 7.16

Carpet
Painted Wallboard Paper
4.18
> 7.04
6.35
> 7.21
1.15
6.35
> 7.21
3.41
	b
-0.36
Bare Pine Wood
2.16
2.82

2.82
3.03

Unpainted Concrete
> 6.17
> 6.89

> 6.89



Test 4
Test 6

Test 13
Test 16

Material Type
212 mg/L; 27 °C;
45 %; 36 hr
212 mg/L; 27 °C;
45 %; 48 hr
Average
Difference
in Efficacy
212 mg/L; 32 °C;
45 %; 48 hr
212 mg/L; 32 °C; 4
5%; 60 hr
Average
Difference in
Efficacy
Glass
4.86
3.49

2.28
3.03

Ceiling Tile
6.41
4.00

3.93
4.36

Carpet
Painted Wallboard Paper
>	7.95
>	7.50
6.60
> 7.34
-1.63
4.65
5.29
0.44
Bare Pine Wood
5.93
1.86

2.47
2.40

Unpainted Concrete
> 7.23
> 6.84





Test 16
Test 19

Test 10
Test 15

Material Type
212 mg/L; 32 °C;
45 %; 60 hr
212 mg/L; 32 °C;
45 %; 72 hr
Average
Difference
in Efficacy
300 mg/L; 22 °C;
45 %; 48 hr
300 mg/L; 22 °C;
45 %; 60 hr
Average
Difference in
Efficacy
Glass
3.03
2.72

3.11
3.25

Ceiling Tile
4.36
5.20

> 7.23
5.36

Carpet
Painted Wallboard Paper
5.29
6.02
0.27
4.60
5.30
-0.40
Bare Pine Wood
2.40
2.21

2.95
2.38

Unpainted Concrete






* Data are expressed as decontamination efficacy (log reduction).
a Parameters of each test listed in order of MeBr concentration (mg/L), temperature (°C), %RH, and contact time (hrs).
b Not tested.
C-7

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Table C-7. Effect of Increasing Contact Time at High Relative Humidity on B. anthracis
Ames*

Test 5

Test 3
Average
Test 7

Test 9
Average
Material Type



Difference



Difference in

212 mg/L; 22 °C;
212 mg/L; 22 °C;
in Efficacy
212 mg/L; 27 °C;
212 mg/L; 27 °C;
Efficacy

75 %; 24 hr
75 %; 36 hr

75 %; 24 hr
75 %; 36 hr

Glass
2.54
>
7.92

4.42
>
7.74

Ceiling Tile
2.70
>
7.07

> 7.14
>
7.07

Carpet
3.31
>
7.93

7.40
>
8.04




4.60



1.13
Painted Wallboard Paper
2.69
>
7.76

> 7.79

—

Bare Pine Wood
2.99
>
7.02

6.29
>
6.92

Unpainted Concrete
3.41
>
7.51

> 7.17



* Data are expressed as decontamination efficacy (log reduction).
a Parameters of each test listed in order of MeBr concentration (mg/L), temperature (°C), %RH, and contact time (hrs).
b Not tested.
C-8

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United States
Environmental Protection
Agency
PRESORTED STANDARD
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