EPA/600/R-16/321 I October 2016
www.epa.gov/homeland-security-research
United States
Environmental Protection
Agency
&EPA
Office of Research and Development
Homeland Security Research Program
Decontamination of Subway Railcar
and Related Materials Contaminated
with Bacillus anthracisSpores via the
Fogging of Peracetic Acid and
Aqueous Hydrogen Peroxide

-------
This page left Intentionally Blank

-------
EPA/600/R-16/321
October 2016
Decontamination of Subway
Railcar and Related Materials
Contaminated with Bacillus
anthracis Spores via the Fogging
of Peracetic Acid and Aqueous
Hydrogen Peroxide
U.S. Environmental Protection Agency
Research Triangle Park, NC 27711

-------
This page left Intentionally Blank

-------
Disclaimer
The U.S. Environmental Protection Agency (EPA), through its Office of Research and
Development's (ORD's) National Homeland Security Research Center (NHSRC), directed and
managed this work through Contract Number EP-C-11-038, Task Order 0017, with Battelle. This
study was funded by both the US EPA and through the Underground Transport Restoration
Program by the U.S. Department of Homeland Security Science and Technology Directorate
under interagency agreement (No. 7095866901).
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:
Mr. 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
l

-------
Acknowledgments
Contributions of the following individuals and organization to this report are gratefully
acknowledged:
U.S. Environmental Protection Agency (EPA) Project Team
Joseph Wood (Principal Investigator)
Worth Calfee
Lukas Oudejans
Office of Research and Development, National Homeland Security Research Center, Research
Triangle Park, NC 27711
Leroy Mickelsen and Shannon Serre
Office of Land and Emergency Management, U.S. EPA Research Triangle Park, NC 27711
Technical Reviewers of Report
Erin Silvestri, US EPA NHSRC
Christine Wagner, US EPA, Federal On-Scene Coordinator, Region 3
Mark Tucker, Sandia National Laboratories
US Department of Homeland Security
Donald Bansleben
US EPA Quality Assurance
Eletha Brady Roberts
Ramona Sherman
Battelle Memorial Institute
Other
We would also like to thank Lawrence Livermore National Laboratory for the collection of dirt
and grime from the undercarriage of a subway car, and Bay Area Rapid Transit (BART) for
providing subway railcar materials for testing.
li

-------
Executive Summary
The Department of Homeland Security's (DHS's) Underground Transport Restoration (UTR)
Program was established to identify potential methods for rapid characterization, cleanup, and
clearance of biological contamination in an underground transit system. This would include
physical structures (tunnels and stations) and rolling stock (railcars). The UTR Project is
expected to improve the capability for transit systems to recover rapidly from a biological release
event and thereby addresses a high-priority need expressed by the Transportation Security
Administration (TSA) and local transit systems. As part of this UTR Project, the U.S.
Environmental Protection Agency (EPA) is evaluating multiple methodologies for the
decontamination of subway and railcar materials contaminated by a biological agent.
This project supports the U.S. EPA Office of Research and Development's (ORD's) Homeland
Security Research Program (HSRP) mission of 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 technology and information that will help detect the intentional introduction of chemical
or biological contaminants in buildings or water systems; contain these contaminants;
decontaminate buildings, water systems, or other infrastructure; and facilitate the disposal of
material resulting from restoration activities.
In the event of a biological incident in a transportation hub such as a subway system, effective
remediation of railcars, subway tunnels and stations will require the use of various
decontamination approaches. One potential decontamination tool that could be used in such an
event is the fogging of sporicidal liquids. The study described in this report builds on previous
fogging decontamination research, but with a focus on decontaminating subway railcars and
related materials. More precisely, the purpose of this study was to evaluate the efficacy of
fogging to decontaminate a variety of subway railcar materials contaminated with Bacillus
anthracis (B.a.; Ames strain) spores. Multiple variables were investigated to assess their effect
on decontamination efficacy, including spore species (B.a. and Bacillus atrophaeus aka Bacillus
globigii, or B.g.), railcar or tunnel material, fogger types, air temperature, sporicidal liquid
(peracetic acid [PAA] or aqueous hydrogen peroxide [H2O2]), quantity of liquid fogged, and
location within the test chamber.
Summary of Major Findings
This evaluation focused on the decontamination of eleven types of subway railcar materials and a
common subway tunnel material (unpainted concrete). Decontamination efficacy tests were
conducted with spores of virulent B.a. Ames and non-virulent B.g., to assess the potential use of
B.g. as a surrogate for future studies with fogging equipment of sporicidal liquids. A summary of
the decontamination efficacy results, in terms of average logio reduction (LR) by material and
microorganism, is shown in Table ES-1. A decontaminant product is considered to be an
effective sporicide or sporicidal decontaminant if a 6 LR or greater is achieved based upon
appropriate laboratory testing.
111

-------
The data and statistical analyses generated from this evaluation suggest that B.g. may be a
suitable surrogate for B.a. Ames for future tests assessing the decontamination efficacy of PAA
or H2O2 using fogging equipment.
Many of the subway railcar materials were effectively decontaminated (achieved a 6LR or
greater) by fogging PAA. These materials include the rubber flooring, seat upholstery,
aluminum seat backing, Mylar® glass coating, and both new and used cabin air filters. Fogging
of PAA was ineffective for the carpet, concrete, and grease (with spores mixed in/encapsulated
into grease); and moderately effective (approximately 3-6 LR) for the interior fiberglass side
panel material, and the clean and dirty railcar grease (spores left on top of grease).
With respect to the effect of air temperature, while the higher temperature (20 °C) resulted in a
greater probability of complete spore population kill and greater LR values compared to the
results at 10 °C (an average of 1-2 LR better), many of these differences were not statistically
significant.
The two types of foggers yielded similar LR values when compared at 20 °C. Testing conducted
using the same parameters but at 10 °C generally yielded higher LR for the Sani-Tizer™ fogger
as compared to the Minncare equipment. Overall, however, statistical analysis using the logistic
regression model indicated that the type of fogger did not have a significant effect on LR.
There was minimal effect of location within the test chamber on decontamination efficacy.
However, as would be expected, coupons stationed horizontally on a cart facing upward, in the
center of the chamber, were more likely to show a complete kill compared to the other four
locations in the chamber (i.e.., vertical orientation on wall, in the duct, underneath the table, and
on the floor near the corner).
iv

-------
Table ES-1. Summary of B.a. Ames and B. atrophaeus Log Reductions by Material Type
Miilcriiil T\|H'
1 .«i*i Reduction Across All Tests
Minimum
Mitxiiniini
A\cr;ijic ± SI)
li. (iii/hnicis
li. (ilroi>li(icn.\
li. (inllinicis
li. tilrophucHs
li. (iiillirticis
li. (itrophtn-H.s
Rubber Flooring
".11
o.i:
8.0"
".31
" "(i 0 iS
0.92 0.4o
Seat Upholstery
7.12
6.23
8.08
7.56
7.79 ±0.45
6.96 ±0.57
Aluminum Seat
Back
7.38
7.06
8.01
7.65
7.81 ±0.29
7.30 ±0.25
Mylar® Glass
Window Coating
7.56
6.89
8.06
7.39
7.83 ±0.17
7.10 ±0.17
Fiberglass Side
Panel
3.39
3.21
7.58
6.84
5.82 ± 1.15
5.65 ± 1.06
Railcar Carpet
0.39
0.41
6.26
5.71
2.43 ± 1.64
1.91 ± 1.20
Unpainted
Concrete
0.60
0.30
2.70
2.21
1.62 ±0.60
1.36 ±0.65
New Grease
(Spores on Top
of Grease)
0.21
0.92
7.61
6.41
4.45 ± 2.62
4.70 ± 1.90
New Grease
(Spores mixed in
to grease aka
encapsulated)
0.33
0.80
2.77
4.83
1.59 ±0.85
2.24 ± 1.02
Used Grease
(Spores on Top
of Grease)
0.24
0.91
7.91
6.92
5.00 ±2.29
5.34 ± 1.58
Railcar Air Filter
(New)
5.85
6.38
7.99
6.64
6.77 ± 1.10
6.54 ±0.14
Railcar Air Filter
(Used)
2.10
2.63
7.92
7.20
7.10 ± 1.70
6.41 ± 1.30
New Industrial
Carpet
4.32
4.81
4.32
4.81
4.32 ±0.0
4.81 ±0.0
v

-------
Contents
Acknowledgments	ii
Executive Summary	iii
Abbreviations/Acronyms	x
1.0 Introduction	1
2.0 Procedures	2
2.1	Test Matrix	2
2.2	Biological Agents	3
2.3	Test Materials	3
2.4	Inoculation of Test Coupons	5
2.5	ARCA Test Chamber and Procedures	6
2.6	Fogging Equipment	8
2.7	Sporicidal Liquids	8
2.8	Fog Droplet Size Characterization	10
2.9	Coupon Extraction and Biological Agent Quantification	12
2.10Decontamination Efficacy	12
2.11	Statistical Analysis	14
2.12	Surface Damage	14
3.0 Quality Assurance/Quality Control	15
3.1	Equipment Calibration	15
3.2	QC Results	15
3.3	Operational Parameters	15
3.4	Audits	17
3.5	QA/QC Reporting	17
3.6	Data Review	17
4.0 Summary of Results and Discussion	18
4.1	Comparing Efficacy for the Different Species	18
4.2	Effects of Test Materials on PAA and H2O2 Efficacy for B.a. Ames	19
4.3	Effects of Temperature on Decontamination Efficacy	26
4.4	Effects of Fogging Equipment on Decontamination Efficacy	28
4.5	Effects of Sporicidal Liquid and Quantiity Fogged on Decontamination Efficacy	30
4.6	Effects of Test Location on Efficacy	31
4.7	Surface Damage to Materials	32
4.8	Summary	32
5.0 References	34
vi

-------
Figures
Figure 2-1. Coupon Types from Left to Right: Carpet, Mylar, Aluminum, Rubber Flooring,
New Filter, Used Filter, Fiberglass, Upholstery, Encapsulated New Grease, New
Grease SOT, Used Grease SOT, Unpainted Concrete, Industrial Carpet	4
Figure 2-2. Liquid Inoculation of Coupon Using a Micropipette	6
Figure 2-3. Schematic of ARCA Test Chamber	7
Figure 2-4. Representative Graph of Temperature, RH, and H2O2 Vapor Concentration (ppm)
During Fogging (Test 17)	8
Figure 2-5. ARCA during fog generation	9
Figure 2-6. Sani-Tizer fogger	9
Figure 2-7. Mini Dry Fog System	10
Figure 4-1. Summary of Sporicidal Liquid Efficacy Results on Rubber against B. anthracis
Ames and B. atrophaeus	20
Figure 4-2. Summary of Sporicidal Liquid Efficacy Results on Upholstery against B.
anthracis Ames and B. atrophaeus	20
Figure 4-3. Summary of Sporicidal Liquid Efficacy Results on Aluminum against B.
anthracis Ames and B. atrophaeus	21
Figure 4-4. Summary of Sporicidal Liquid Efficacy Results on Mylar against B. anthracis
Ames and B. atrophaeus	21
Figure 4-5. Summary of Sporicidal Liquid Efficacy Results on Fiberglass against B. anthracis
Ames and B. atrophaeus	22
Figure 4-6. Summary of Sporicidal Liquid Efficacy Results on Railcar Carpet against B.
anthracis Ames and B. atrophaeus	22
Figure 4-7. Summary of Sporicidal Liquid Efficacy Results on Concrete against B. anthracis
Ames and B. atrophaeus	23
Figure 4-8. Summary of Sporicidal Liquid Efficacy Results on New Grease SOT against B.
anthracis Ames and B. atrophaeus	23
Figure 4-9. Summary of Sporicidal Liquid Efficacy Results on Encapsulated NG against B.
anthracis Ames and B. atrophaeus	24
Figure 4-10. Summary of Sporicidal Liquid Efficacy Results on Used Grease SOT against B.
anthracis Ames and B. atrophaeus	24
Figure 4-11. Summary of Sporicidal Liquid Efficacy Results on New Filter against B.
anthracis Ames and B. atrophaeus	25
Figure 4-12. Summary of Sporicidal Liquid Efficacy Results on Used Filter against B.
anthracis Ames and B. atrophaeus	25
Figure 4-13. Effect of Temperature againsti?. anthracis Ames: Tests 1 and 11	277
Figure 4-14. Effect of Temperature against B. anthracis Ames: Tests 4 and 12	277
Figure 4-15. Effect of Temperature against B. anthracis Ames: Tests 7 and 13	277
Figure 4-16. Effect of Temperature againsti?. anthracis Ames: Tests 10 and 14	288
Figure 4-17. Effect of Temperature againsti?. anthracis Ames: Tests 17 and 20 	288
Figure 4-18. Effect of Fogger Equipment Type against B. anthracis Ames at 20°C	299
Figure 4-19. Effect of Fogger Equipment Type against B. anthracis Ames at 10°C	299
Figure 4-20. Effect of Sporicidal Liquid Type against B. anthracis Ames test 4 and 5	30
Figure 4-21. Effect of Sporicidal Liquid Type against B. anthracis Ames test 17 and 18	31
Figure 4-22. Effect of Sporicidal Liquid Type against B. anthracis Ames test 19 and 20	31
Vll

-------
Figure D-l.
Plot of Control Coupon Percent Recovery of Inoculum by Material
D-12
Tables
Table ES-1. Summary of B.a. Ames and B. atrophaeus Log Reductions by Material	vii
Table 2-1 Decontamination Test Matrix	2
Table 2-2. Test Materials	4
Table 2-3. Measured Droplet Size and Flux using the PDI	11
Table 3-1. Actual Fog Conditions for Tests	16
Table 3-2. Performance Evaluation Audits	17
Table 4-1. Summary of Average Differences in Efficacy between B.a. Ames and B.
atrophaeus*	19
Table 4-2. Summary of B.a. Ames and B. atrophaeus Log Reductions by Material
Type	266
Table A-l. Inactivation of B. anthracis Ames Spores Using Fogged Sporicidal Liquids51... A-l
Table A-l. Inactivation of B. anthracis Ames Using Fogged Sporicidal Liquids a
(Continued)	A-2
Table A-l. Inactivation of B. anthracis Ames Using Fogged Sporicidal Liquids a
(Continued)	A-3
Table A-2. Inactivation of B. atrophaeus Spores Using Fogged Sporicidal Liquids3	A-4
Table A-2. Inactivation of B. atrophaeus Spores Using Fogged Sporicidal Liquids a
(Continued)	A-5
Table A-2. Inactivation of B. atrophaeus Spores Using Fogged Sporicidal Liquids a
(Continued)	A-6
Table B-l. Difference in Efficacy Between B. anthracis Ames and B. atrophaeus*	B-l
Table B-2. Difference in Efficacy Between B. anthracis Ames and B. atrophaeus*	B-2
Table B-3. Difference in Efficacy Between B. anthracis Ames and B. atrophaeus*	B-4
Table C-l. Difference in efficacy Between B. anthracis Amesa at 10 °C and 20 °C	C-l
Table C-2. Difference in efficacy Between B. atrophaeousa at 10 °C and 20 °C	C-2
Table C-3. Difference in B. anthracis Amesa Efficacy Between Equipment Type	C-3
Table C-4. Difference in B. anthracis Amesa Efficacy Between Equipment Type	C-3
Table C-5. Difference in B. atrophaeusa Efficacy Between Equipment Type	C-4
Table C-6. Difference in B. atrophaeusa Efficacy Between Equipment Type	C-4
Table C-l. Difference in B. anthracis Ames& Efficacy Between Liquid Type (Tests 4/5) ... C-5
Table C-8. Difference in B. anthracis Amesa Efficacy Between Liquid Type (Tests
15/16)	C-5
Table C-9. Difference in B. anthracis Ames'A Efficacy Between Liquid Type (Tests
17/18)	C-5
Table C-10. Difference in B. anthracis AmesA Efficacy Between Liquid Type (Tests
19/20)	C-5
Table D-l. Mean Percent Recovery for Control Coupons for Each Agent and Material
with 95 Percent Confidence Intervals	D-4
Table D-2. Kruskal-Wallis Tests of Differences Among Materials for Each Agent	D-5
Table D-3. Kruskal-Wallis Tests of B.a. vs B.g. for Each Material	D-5
Table D-4. Proportion Success (Total Kill) for B.a. and B.g. with Exact 95 Percent
Confidence Intervals	D-6
viii

-------
Table D-5. Parameter Estimates for Final Selected Model Fit to More Balanced Data
Subset	D-10
Table D-6. Odds Ratio Estimates for Pairwise Material Comparisons	D-10
Table D-7. Odds Ratio Estimate for Comparisons of Locations within Chamber	D-l 1
Table D-8. Odds Ratio Estimates for Decontamination SL Comparisons	D-l 1
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 Sporicidal Liquid Efficacy....C-l
Appendix D. Detailed Statistical Analysis	D-l
IX

-------
Abbreviations/Acronyms
AO AC	Association of Official Analytical Chemists (now AO AC
International)
ARCA	Aerosol Research and Component Assessment Chamber
ASTM	American Society of Testing and Materials
B. anthracis Bacillus anthracis Ames
BART	Bay Area Rapid Transit
BBRC	Battelle Biomedical Research Center
B.g.	Bacillus globigii, aka Bacillus atrophaeus
BSC	biological safety cabinet
CFU	colony forming units
CI	confidence interval
cm	centimeter(s)
°C	degree(s) Celsius
DHS	Department of Homeland Security
DNA	Deoxyribonucleic Acid
E-beam	electron beam
EPA	U.S. Environmental Protection Agency
h	hour
HC1	hydrochloric acid
HSRP	Homeland Security Research Program
HVAC	Heating, ventilation, and air conditioning
kGy	kilogray(s)
L	liter(s)
LAL	Limulus Amebocyte Lysate
LPM	liters per minute
LR	logio reduction
l_ig	microgram(s)
|xL	microliter(s)
m	meter
mg	milligram(s)
mL	milliliter(s)
mil	thousandth of an inch
min	minute(s)
mm	millimeter(s)
l_im	micrometer(s)
MMD	Mass Median Diameter
NA	not applicable
NHSRC	National Homeland Security Research Center
ORD	Office of Research and Development
PAA	peracetic acid
x

-------
PBS	phosphate buffered saline
PBST	PBS + 0.1% Triton X-100
PCR	polymerase chain reaction
PE	Performance evaluation
PDI	phase Doppler interferometer
psi	Pounds per square inch
PVC	probe volume corrected
QA	quality assurance
QAPP	Quality Assurance Project Plan
QC	quality control
QMP	Quality Management Plan
RH	relative humidity
rpm	revolution(s) per minute
s	second(s)
SD	standard deviation
SE	standard error
SFW	sterile filtered water (cell-culture grade)
SOT	Spores on top
SSE	Sum of squares due to error
SSM	Sum of squares about the mean
STREAMS II Scientific, Technical, Research, Engineering, and Modeling
Support Contract
TO	time zero
TSA	technical systems audit
UTR	Underground Transport Restoration
V	Volt
VMD	Volume Median Diameter
xi

-------
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 technology and information that will
help detect the intentional introduction of chemical or biological contaminants into buildings or
water systems; contain these contaminants; decontaminate buildings, water systems, or other
infrastructure; and facilitate the disposal of material resulting from restoration activities.
In the event of a biological incident in a transportation hub such as a subway system, effective
remediation of railcars, subway tunnels and stations will require the use of various
decontamination approaches. One potential decontamination tool that could be used in such an
event is the fogging of sporicidal liquids. The study described in this report builds on previous
fogging decontamination research(1), but with a focus on decontaminating subway railcar and
related materials. More precisely, the purpose of this study was to evaluate the efficacy of
fogging to decontaminate a variety of subway railcar materials contaminated with Bacillus
anthracis (B.a.) spores. Over the course of 21 tests, multiple variables were investigated to assess
their effect on decontamination efficacy, including spore species, material, fogger type, air
temperature, sporicidal liquid, quantity of liquid fogged, and location within the test chamber.
Many of the materials used in the study originated from actual in-use subway railcars, and
include carpet, aluminum seat back, seat upholstery, rubber flooring, Mylar® coating (from a
glass window), fiberglass interior siding, railcar axle grease, new cabin air filter, and a used
cabin air filter. Unpainted concrete was also included in the majority of tests, as this is a
common subway tunnel material. Most of the decontamination efficacy tests were conducted
using peracetic acid (PAA) fog, based on its use in a previous fog decontamination study(1), and
PAA's relatively high efficacy against B.a. spores on many materials when applied as a spray.
However, a few tests were conducted with the fogging of aqueous hydrogen peroxide (H2O2)
solutions. Tests were conducted with spores of B.a. Ames and a potential surrogate, Bacillus
atrophaeus (aka Bacillus globigii, or B.g.). Testing was conducted in a pilot-scale chamber at
either 20 °C or 10 °C, the latter to better represent the temperature of underground subway
tunnels and stations. Finally, two different foggers were used in the test program: a relatively
expensive fogger comprised of primarily stainless steel parts, and a less expensive fogger
constructed of primarily plastic components.
Decontamination efficacy was determined based on the logio reduction (LR) in viable spores
recovered from the inoculated samples, with and without exposure to the sporicidal fog. A
decontaminant is considered to be an effective sporicide if a 6 LR or greater is achieved in
appropriate laboratory testing on the materials tested for a given set of conditions.
The results of this investigation provide decontamination stakeholders and decision-makers with
high quality, peer-reviewed data to evaluate the use of fogging equipment to disperse sporicidal
liquids in a subway railcar and related environment as a function of the spore type, the material
the spore is associated with, temperature, equipment type and sporicidal liquid used.
1

-------
2.0 Procedures
This section provides an overview of the procedures used for the pilot-scale evaluation of
fogging sporicidal liquids to inactivate B.a. and B.g. spores on 13 different materials. Testing
was performed in accordance with the peer-reviewed and EPA-approved Quality Assurance
Project Plan (QAPP) for the Decontamination of Subway and Other Materials through the
Fogging of Sporicidal Liquids and associated amendments.(2) The QAPP provides additional
procedural details that are not included in this report.
2.1 Test Matrix
The test matrix for the study is shown in Table 2-1. Each of the 21 tests was performed using a
subset of six materials (chosen from a total of 13 materials) inoculated with spores of B.a. and
the same six materials inoculated with B.g. Operational parameters such as type of fogger, air
temperature, decontaminant chemical, decontaminant volume fogged, and contact time were
varied to assess effect on decontamination efficacy. Each of these test variables is further
described below.
Table 2-1 Decontamination Test Matrix


Open
ilioiiiil Pelr;imold's


losl
Nil in Ikt
r.(|iiii>iiK-ni
Air
Tcmpcr;iliiiv
(°C)
Sporicidiil
l.i(|iiid
Decon I ;i in i n;i t icin
Volume l-'niiiiod
(ml.)
( onliicl
TiiiK' (111
Miilcriiils
1


PAA
160
18
R, U, A, M, F, Ca
2


8% H202
2365
168
3
Sani-

PAA
160
1-5
Days
Ca
4
Tizer*



18
R, M, F, Ca, NGSOT, NF
5

20
22% H202
78
6




8
R, M, F, Co, NGSOT, UF
7





Ca, Co, NGSOT, NGM,
8



160

UGSOT, UF
9
MinnCare




R, U, A, M, F, Ca
10



500

F, Ca, Co, NGM, UGSOT, UF
11


PAA
160

R, U, A, M, F, Ca
12
Sani-


78

R, M, F, Ca, NG, NF
13
Tizer*
10

160

Ca, Co, NGSOT, NGM,
UGSOT, UF
14
MinnCare



18

15
Sani-


500

F, Ca, Co, NGM, UGSOT, UF
Tizer*





16
MinnCare
20
35% H202
500

F, Ca, Co, NGM, UGSOT, UF
17



F, Ca, Co, NGM, UGSOT, UF
18
Sani-
Tizer*

PAA
1000

F, Ca, Co, NGSOT, NGM,
UGSOT
19
10
PAA


F, Ca, Co, NGM, UGSOT, IC
20

35% H202


F, Ca, Co, NGM, UGSOT, UF
2

-------

Minncare

PAA


Ca, Co, NGSOT, NGM,
21
10
500






UGSOT, UF
Material Key: R=Rubber, U=Upholstery, A=Aluminum, M=Mylar, F=Fiberglass, Ca=Carpet, Co=Concrete, NGSOT=New Grease Spores on
Top, NGM=New Grease Spores Mixed (i.e., encapsulated), UGSOT=Used Grease Spores on Top, NF=New Filter, UF=Used Filter,
IC=Industrial Carpet (new).
2.2	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) using a
BioFlo 3000 fermenter (New Brunswick Scientific Co., Inc., Edison, NJ). The spore lot was
subject to a stringent characterization and qualification process required by the Battelle 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 can yield 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 confirmed
by an independent third party. The virulence of the spore lot was measured by challenging
guinea pigs intradermally with a dilution series of spore suspensions, and virulence was
expressed as the intradermal median lethal dose. (Note, the tests with guinea pigs were
conducted previously and not conducted under this study.) In addition, testing was conducted for
robustness of the spores via hydrochloric acid (HC1) resistance.
The B.g. spores (Lot 19076-03268) were supplied in powder form by the US EPA, and were
originally obtained from Dugway Proving Ground (Tooele County, UT). The B.g. stock spore
suspensions were prepared in sterile phosphate-buffered saline containing 0.1% Triton X-100
surfactant (PBST; Sigma, St. Louis, MO) at the same concentration as the B.a. stock and stored
at 2 to 8 degrees Celsius (°C). No further activities were performed to verify the identity of the
organism.
The B.a. stock spore suspension was prepared in sterile filtered water (SFW) at an approximate
concentration of 1 x io9 CFU/mL and stored at 2 to 8 °C. This buffer was chosen to be
consistent with previous work conducted with the same B.g. spores at EPA.
2.3	Test Materials
Decontamination testing was conducted using a number of materials removed from an actual
subway railcar and a common subway tunnel material. These materials are listed in table 2-2..
In one test (Test 19), we included new industrial carpet in order to compare with the used/dirty
railcar carpet, to assess whether the dirt and grime was a factor in decontamination efficacy of
the railcar carpet. In addition, both new and used railcar grease were used as a "coupon" when
applied to glass. The grease coupons were tested in two configurations: 1) spores dried on top of
the grease, and 2) dried spores mixed (encapsulated) into the grease. Information on all of these
3

-------
materials is presented in Table 2-2, and a picture of each is presented in Figure 2-1. Coupons
used for testing were cut to uniform length and width (Table 2-2) from the larger pieces of stock
material. Coupons materials were prepared for testing by either sterilization via electron beam
(E-beam) irradiation at -200 kilogray (kGy; E-beam Services Inc., Lebanon, OH) or autoclaved
at 121 °C for 15 minutes (min). E-beam-irradiated material coupons were sealed in 6 mil (0.006
inch) Uline Poly Tubing (Cat. No. S-2940, Uline, Chicago, IL), and autoclaved coupons were
sealed in sterilization pouches (Cat. No. 01-812-50, Fisher, Pittsburgh, PA) to preserve sterility
until the coupons were ready for use. Sterilization was intended to eliminate contamination by
endogenous microorganisms.
Figure 2-1. Coupon Materials from Left to Right: Railcar Carpet, Mylar, Aluminum,
Rubber Flooring, New Filter, Used Filter, Fiberglass, Upholstery, Encapsulated New
!
Grease, New Grease SOT, Used Grease SOT, Unpainted Concrete, Industrial Carpet
Table 2-2. Test Materials
Material
(abbrevia-tion)
Lot, Batch, or ASTM No.,
or Observation
Manufacturer/
Supplier Name
Location
Approximate Coupon Size, Width
x Length x Thickness
Material
Preparation
Used Carpet
(CA)
Received from Bay Area
Rapid Transit (BART)
U.S. EPA
1.9 cm x 3.8 cmx 0.2 cm
E-Beam
Aluminum seat
backing (A)
Received from BART
U.S. EPA
1.9 cm x 3.8 cmx 0.2 cm
Autoclave
Seat Upholstery
(U)
Received from BART
U.S. EPA
1.9 cmx 3.8 cm x0.2 cm
Autoclave
Rubber
Flooring (R)
Received from BART
U.S. EPA
1.9 cmx 3.8 cmx 0.2 cm
Autoclave
Mylar® glass
window coating
(M)
Received from BART
U.S. EPA
1.9 cmx 3.8 cmx0.2 cm
Autoclave
Fiberglass
interior siding
(F)
Received from BART
U.S. EPA
1.9 cmx 3.8 cmx0.2 cm
Autoclave
if 1• m
m1 -ti
4

-------
New Cabin Air
Filter (NF)
Received from BART
U.S. EPA
1.9 cm x 3.8 cm x 0.2 cm
Autoclave
Used Cabin Air
Filter (UF)
Received from BART
U.S. EPA
1.9 cm x 3.8 cm x 0.2 cm
Autoclave
New Grease
(NG)
Ultra-Duty EP, NLGI2
Chevron, San
Ramon, CA
1 mL of grease onto glass 1.9 cm x
7.6 cm x 0.2 cm
Autoclave
Grimy Used
Grease (UG)
Received from BART
U.S. EPA
1 mL of grease onto glass 1.9 cm x
7.6 cm x 0.2 cm
Autoclave
Glass (used
only with
grease samples)
C1036
Brooks Brothers,
Columbus, OH
1.9 cm x 7.6 cm x 0.2 cm
Autoclave
Unpainted
Concrete (Co)
ASTMC90 cinder block
Wellnitz
Columbus, OH
1.9 cm x 7.6 cm x 0.2 cm
Autoclave
Industrial carpet
(IC)
Shaw Swizzle EcoWorx,
Style: 10401 Color: Jacks
Shaw Industries,
Dalton, GA
1.9 cm x 7.5 cm x 0.3 cm
E-beam
2.4 Inoculation 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 108 CFU of viable B.a. Ames or surrogate
B.g. spores per coupon. A 100 microliter (|iL) aliquot of a stock suspension of approximately 1 x
109 CFU/mL was dispensed using a micropipette applied as 10 |iL droplets across the coupon
surface (see Figure 2-2). This approach provided a more uniform distribution of spores across the
coupon surface than would be obtained through a single drop of the suspension. Although
application of the inoculum onto each material was uniform, the behavior of the inoculum
droplets was not. Droplets beaded on the surface of the glass (nonporous material) while they
soaked into the other porous materials after producing a liquid bead for a short period of time.
The difference in the behavior of the inoculum droplets on each material could lead to a variance
in microorganism distribution across coupons; however, this effect was not studied in this
evaluation. 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).
The grease test materials were prepared by first applying 1 mL of grease using a 3 mL syringe at
one end of the glass material. The grease was then spread across the test material using a sterile
colony spreader, creating a thin film, and then the target organism was applied in a manner
identical to the other test materials. For the "coupon" where the spores were mixed
(encapsulated) into the clean grease, after the spore inoculum was dried, a sterile glass rod was
used to mix the dried spores into the grease using a circular motion across the glass.
5

-------
Figure 2-2. Liquid Inoculation of Coupon Using a Micropipette
The number and type of replicate coupons used for each combination of material, decontaminant,
concentration, foggertype, and environmental condition included:
•	Five test coupons (inoculated with B. ant.hracis or surrogate spores and exposed to
sporicidal fog)
•	Five positive controls (inoculated with B. anthracis or surrogate spores but not exposed
to sporicidal fog)
•	One laboratory blank (not inoculated and not exposed to sporicidal fog)
•	One procedural blank (not inoculated and exposed to sporicidal fog).
On the day following inoculation, coupons intended for decontamination (including blanks) were
transferred into the aerosol research and component assessment (ARCA) test chamber, placed in
one of five designated positions, and exposed to the sporicidal liquid fog using the apparatus and
application conditions specified in Section 2.5. Control coupons remained in the BSC III
chamber where they were dried and collected for processing at the conclusion of the test
chamber contact time.
2.5 ARCA Test Chamber and Procedures
Decontamination testing was conducted inside the ARCA test chamber with an approximate
internal volume of 16 cubic meters (m3). Figure 2-3 shows a schematic of the .ARCA test
chamber as well as fogging equipment and test coupon locations. This BSC III chamber is hard-
ducted to the facility exhaust system ventilation, but during each test, valves on both the exhaust
and supply were closed to create a sealed enclosure. Once test contact duration had concluded,
the exhaust and supply valves were opened to allow for any residual fumigant to be removed.
6

-------
Temp/RH probe
H202 probe
Position #3
Position #2
n under tab
Mixing Fan
Figure 2-3. Schematic of ARCA Test Chamber
For testing targeting 10 °C conditions, the temperature was controlled using a Krack HTSS-
0100MSD air cooled condensing unit and KR26A-089EB low profile evaporator (Krack,
Bolingbrook, IL) refrigerant system. Temperature and RH in the test chamber were measured
using an 1TMT368 temperature and humidity probe (Vaisala, Inc., Woburn, M A). The RH of the
chamber was not controlled during testing, and rapidly increased with the onset of fogging.
Since the PAA solution contains hydrogen peroxide, hydrogen peroxide vapor was measured as
an indicator of the fog process using an ATI B12 2-wire gas transmitter (Analytical Technology,
Inc., Collegeville, PA). All parameters were recorded every minute during the experimental
exposure time using a UX120-006M HOBO data logger and associated HOBOware software
(Onset, Bourne, MA).
Five test positions were selected within the ARCA chamber, including three horizontal positions
(1, 3, and 5), one vertical (4), and one inverted position (2). One replicate of each coupon
material was placed at each location. In addition, at each chamber location, wetness was
measured in terms of total percent coverage by using a HOBO S -LWA-M003 leaf wetness
sensor connected to a HOBO H21-002 micro station data logger that recorded wetness
measurements every minute for the duration of each test. Test locations 1-4 were all located
within the main chamber of the ARCA, while test position 5 was located approximately five feet
off the main chamber in a 2'x T duct. This location was selected to challenge the ability of the
fog to disseminate through a more complex area. A representative graph of the ARCA chamber
test conditions (from Test 17) data collection can be seen in Figure 2-4.
7

-------
280
Sani-Tizer Test #17
Time
200
180
•|= 160
ZD
ro 140
^ 120
<
100
80
Temp, *C
RH, %
PPM
Figure 2-4. Representative Graph of Temperature, RH, and H2O2 Vapor Concentration
(ppm) During Fogging
2.6 Fogging Equipment
Figure 2.5 is a photo of the ARCA chamber being fogged with a sporicidal liquid. Two fogger
technologies were tested for the ability to disseminate fogged sporicidal liquids throughout the
large test chamber. The first technology tested was the Sani-Tizer 3001-1 (Curtis Dyna-Fog Ltd.,
Jackson, GA). This fogger was constructed largely of plastic parts and required a 120 volt (V)
circuit to operate. Figure 2-6 shows the Curtis Dyna-Fog Sani-Tizer fogger. The unit was
equipped with a one gallon tank, three spray nozzles, and a rotary knob for control of liquid flow
rates that are listed as 0 to 4.5 gallons per hour. The median droplet size of 31 microns, generated
using PAA (method used to measure droplet size distribution (by volume) is discussed in Section
2.8), was within the published particle size distribution of 5-50 microns as listed in the product
manual(3). All testing conducted with this fogger used the low flow setting as indicated on the
rotary knob and resulted in flow rates ranging from 63-187 mL/min.
8

-------
Figure 2-5. ARCA During Fog Generation
Figure 2-6. Sani-Tizer
Fogger
The second fogger tested was the Mini Dry Fog System (Mar Cor Purification, Plymouth, MN).
Figure 2-7 shows the Mini Dry Fog system which was made entirely of stainless steel and
required a controlled outside air source as its means of generating the aerosol droplets using an
air atomizing nozzle. The unit was equipped with one spray nozzle, a 500 mL liquid reservoir,
and an in-line regulator to maintain pressure at the nozzle. The measured median droplet size (by
volume) of 12.4 microns for this evaluation, as described below in Section 2.8, was slightly
larger than the 7.5 microns listed in the product literature14'. This device required a controlled
9

-------
pressure of 75 pounds per square inch (psi) as well as minimum flow rate of 56 liters per minute
(LPM). Pressure was measured using a Dwyer DPG-205-NIST (Dwyer, Michigan City, IN).
Flow rate was measured using an Aalborg GFM47 flow meter (Aalborg Instruments and
Controls, Orangeburg, NY). Data from these devices were recorded every minute during
operation using a UX120-006M HOBO data logger.
Figure 2-7. Mini Dry Fog System
2.7	Sporicidal Liquids
Two types of sporicidal liquids were examined in this study (PAA and H2O2). PAA was used as
received (Minncare Cold Sterilant, Cat. No. 78325-150, Mar Cor Purification, Plymouth, MN)
and consisted of 22 % hydrogen peroxide, 9 % acetic acid, and 4.5 % peroxyacetic acid. Three
concentrations of aqueous H2O2 were used (35 %, 22 % and 8 %). The 35 % solution was used
as received (Cat. No. HPV-AQ, Horsham, PA) which consisted of 35 % w/w aqueous hydrogen
peroxide solution. This stock solution was diluted to target concentrations of 22 % and 8 %
using sterile water. These concentrations were verified by permanganate titration and resulted in
final concentrations of 22.4 % and 8.6 % respectively.
2.8	Fog Droplet Size Characterization
During this evaluation, droplet size measurements were made using an Artium Phase Doppler
Interferometer (PDI; Model 200MD, Sunnyvale, CA). These tests were conducted to confirm
droplet size information reported by the vendors. Fogger droplet size distribution is important,
since smaller droplets tend to remain aloft in the air longer and therefore are more easily and
widely distributed throughout the volume being decontaminated. The techniques used by the PDI
to measure droplet size have previously been described in literature(5 6:). The probe volume
corrected (PVC) fluxes were also recorded in order to accurately assess the overall spray plume
size distribution. The Artium PDI PVC flux measurements have already been shown to be in
good agreement with traditional mechanical patternation local volume flux measurements(7).
The two-dimensional Artium Technologies PDI - 200MD instrument was used to acquire droplet
size measurements across the spray plume. The PDI system was operated in a 1-D orientation for
10

-------
these measurements, resulting in a purely stream-wise velocity component. The transmitter and
receiver were mounted on a rail assembly with rotary plates at a 40 degree forward scattering
collection angle. The 500 millimeter (mm) lenses were used for both the transmitter and receiver,
which allowed for measurement of droplets in the range of 1.5 to 160 micrometers (|im)(8).
The spray nozzles were placed 21 centimeters (cm, 8.25 inches) from the PDI measurement
location. The nozzle was sprayed horizontally into a chemical fume hood while affixed to a
traversing system. The nozzle traversed both the x and y directions (always 21 cm from the PDI
measurement volume) to fully analyze the spray plume.
The nozzle was moved in both the positive and negative x- and y-directions, by 2 cm increments,
until the edge of the spray plume was reached. For each test configuration, the spray plume was
measured at -35 measurement locations.
On average, a total of 10,000 droplets were measured at each measurement location as they
passed through the PDI laser intersection. Towards the edge of the spray, the PDI was operated
for a total of 15 seconds and collected as many droplets as possible during that time. The PVC
distribution is used to provide the DV0.1, DV0.5, and DV0.9 diameters as well as the volume
flux. The DV0.1 diameter is the value where 10 % of the total volume fogged is made up of
drops with diameters less than or equal to the DV0.1. The DV0.5, or volume/mass median
diameter (VMD/MMD), is the diameter where 50 % of the total volume of liquid fogged is made
up of droplets with diameters smaller than the DV0.5. Finally, the DV0.9 is the value where 90%
of the total volume of liquid fogged is made up of droplets with diameters smaller than the
DV0.9. The volume flux (cm3/cm2/s) is a measurement of the liquid volume (cm3) that passes
through the probe volume (cm2) per unit time (s).
To determine the overall flux of the entire spray plume, a surface was fitted to the volume fluxes
measured at each x,y location. The surface was integrated over the measurement range to provide
the overall flux, which was then compared to the known liquid flow rate.
The DV0.1, DV0.5, and DV0.9 diameters measured at each x,y location were then multiplied by
the volume flux measured at the same measurement location. A surface was also fitted to the
flux*diameter values and integrated over the same range. The resulting values were then divided
by the calculated volume flux for the entire spray plume to provide the DV0.1, DV0.5, and
DV0.9 diameters for the entire spray. Refer to Table 2-3 for these results.
Table 2-3. Measured Droplet Size and Flux Using the PDI.
Test
Fogger
Device
Solution
PDI Measurements
Dvo.i,
in
Dvo.5,
^1 111
Dvo.9,
^1 111
Flux,
mL/min
1
Minncare
Water
8.1
15.7
26.2
13.2
2
Minncare
PAA
6.5
12.4
19.5
1.2
3
Sani-Tizer
Water
18.2
39.9
65.9
380.9
4
Sani-Tizer
PAA
13.4
31.0
58.3
246.8
11

-------
2.9	Coupon Extraction and Biological Agent Quantification
Spore extraction was achieved by placing test, positive control, and blank coupons in 50 mL
polypropylene conical tubes containing 10 mL of sterile PBST. The vials were capped, placed on
their side and agitated on an orbital shaker for 15 min at approximately 200 revolutions per
minute (rpm) at room temperature.
The amount of residual viable spores was determined using a dilution plating approach.
Following extraction, the extract was removed, and a series of tenfold dilutions was prepared in
SFW. An aliquot (0.1 mL) of either the undiluted extract and/or each serial dilution was plated
onto tryptic soy agar in triplicate and incubated for 18 to 24 h at 37 ± 2 °C. Colonies were
counted manually and CFU/mL was 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 target acceptance
criterion for extracts of laboratory or procedural blanks was zero CFU.
After each decontamination test, the ARCA and control chambers were thoroughly cleaned
(using separate steps involving bleach, ethanol, water, then drying). This involved, but was not
limited to, removal/bleaching of waste materials and test coupon racks. The immediate test area
was wiped with bleach followed by water rinse. Negative control samples (which were negative
for all tests) assured we were not getting any cross contamination within the chamber.
2.10	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 was known from enumeration of the stock spore suspension. One
aliquot of the stock suspension was 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 4 and Appendix A.
The performance or efficacy of the sporicidal liquid fog 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 organism 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 controls 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.:
12

-------
Efficacy (LR) = (log10 CFUct]) - (log10 CFUty)
(2)
where logio CFUcij refers to the j individual logarithm values obtained from the positive control
coupons and logio CFUtij refers to the j individual logarithm values obtained from the individual
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 or fumigant technology is considered to be an effective sporicide
via (AO AC International) AO AC method 966.04 if a 6 LR or greater is achieved.^
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 0 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 CFUtij values were also
calculated for both the control and test coupons (i.e., S2c,, and 5%), and were used to calculate
the pooled standard error (SE) for the efficacy value calculated in Equation 2, as follows:
=	(3)
where the number 5 again represents the number j of coupons in both the control and test data
sets. Each efficacy result is reported as an LR value with an associated 95 % confidence interval
(CI), calculated as follows:
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 % CI 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 that 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 an LR value. This difference in efficacy was calculated as follows:
n
LRa,2 - LRa, 1
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 surrogate microorganism (B.g.) for which results are being
compared. The letter n represents the number of materials tested. 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.
13

-------
In some instances, significant differences in average efficacy for a material between tests were
assessed with a t-test using Microsoft® Excel, according to the formula below:
17-57
*	v-
where and 5^ are the means of Tests 1 and 2, respectively. St-y: is the standard error of the
difference between Tests 1 and 2. This formula compares the averages of two tests to see if they
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.
2.11	Statistical Analysis
The mean and 95 percent confidence intervals on the percent recovery for the positive control
coupons were calculated by agent and material. For each agent separately, Kruskal-Wallis tests(9)
were performed to compare whether percent recovery differs by material. Kruskal-Wallis tests
also were performed to compare whether percent recovery differs by agent for each material. No
adjustment for multiple tests was applied.
All test results were transformed to binary measurement of either successful decontamination
(pass) or fail. A trial was recorded as a success if either: 1) the LR is greater than or equal to 6,
LR; or 2) the LR is equal to the average control recovery (e.g., no spores recovered from test
coupons, i.e., complete inactivation). The proportion of tests that pass (successful
decontamination) and 95 percent Clopper-Pearson(10) confidence intervals were computed by
agent, material, equipment, decontaminant, temperature, decontamination volume, and contact
time. A chi-squared test for association was performed to test whether the B.a. decontamination
success proportion was significantly different from the B.g. decontamination success proportion
across all test conditions.
For B.a., a logistic regression model with main effects for material, fog equipment,
decontaminant, temperature, decontaminant quantity, and contact time was fitted to the data to
compare the proportions of success. Statistically significant two-factor interactions were added to
the model. Models were fitted to the full data set and to a more balanced subset of the data.
All statistical analyses were performed using SAS (Version 9.4, Cary, NC). All results are
reported at the 0.05 level of significance.
2.12	Surface Damage
The physical effect of the sporicidal liquids as delivered by the fogging equipment to the
materials was qualitatively monitored during the evaluation. This approach provided a gross
visual assessment of whether the environmental state changed the appearance of the test
materials. The procedural blank was visually compared to a laboratory blank coupon.
14

-------
3 Quality Assurance/Quality Control
Quality assurance (QA)/quality control (QC) procedures were performed in accordance with the
Scientific, Technology, Research, Engineering, and Modeling Support (STREAMS II) Program
Quality Management Plan (QMP), Version 3 and the quality assurance project plan (QAPP)(2).
The QA/QC procedures and results are summarized below.
3.1	Equipment Calibration
All equipment (e.g., pipettes, incubators, pressure sensor, PDI, Vaisala, biological safety
cabinets) and monitoring devices (e.g., thermometer, hygrometer) used at the time of the
evaluation were verified as being certified, calibrated, or validated.
3.2	QC Results
QC efforts conducted during decontaminant testing included positive control samples, procedural
blanks, laboratory blanks, and inoculation control samples.
Most positive control results were within the target recovery range of 5 to 120 % of the
inoculated spores, except for a few instances. The average percent recoveries of both B.a. and
B.g. spores, by material, for positive controls are detailed in Table D-l and Figure D-l of the
Appendices. Recoveries of spores from positive controls were significantly higher for B.a. than
for B.g. (See Table D-2 and D-3). Generally lower recoveries of spores occurred with materials
such as unpainted concrete, fiberglass, and spores encapsulated in grease. Despite the low
recoveries of spores from some of these materials, in most cases recoveries were greater than 6
log CFU. 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, 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 io9 CFU/mL (±1 log) for all tests.
3.3	Operational Parameters
The temperature, RH, and H2O2 vapor concentration during each test were monitored as
described in Section 2.0. For select tests, the temperature was actively controlled by using the
Krack evaporator as described in Section 2.5. This device was set to the target conditions and
allowed to cool the ARC A chamber as needed to stay within target ranges of 10 °C ± 20 %
(when required). Readings were taken once every minute for the duration of the contact time.
The volume of liquid introduced into the test chamber via the fogger was measured after each
test by volumetric pipette to determine residual volume which was subtracted from total volume
added to the fogger. The actual operational parameters for each test are shown in Table 3-1 and
reported as the average value ± SD. Note that the RH for both the test chamber during fogging
and the control chamber for the positive controls were uncontrolled. The average RH for the
controls was left at laboratory ambient conditions, while the RH during fogging was typically
much higher due to the increase in water vapor released during the fog process.
15

-------
Table 3-1. Actual Fog Conditions for Tests
rest
Vumber
Sporicidal
Liquid Volume
Fogged (mL)
H2O2 Vapor
Concentration
(ppm)
Temperature (°C)
RH (%)
Target
Actual
Target
Fogging
Actual
Control
Actual
Fogging
Actual
Control
Actual
1
160
154
53.94 ±51.46
20
20.58 ±0.14
18.10 ±
0.13
80.73 ± 5.25
60.49 ±
0.52
2
2365
2627
12.37 ± 12.38
20
21.18 ±0.28
18.05 ±
0.37
94.83 ± 1.10
60.89 ±
0.22
3
160
162
4.96 ± 20.96
20
20.45 ± 0.24
18.48 ±
0.38
72.37 ±4.05
61.36 ±
0.21
4
78
82
16.26 ±38.25
20
20.45 ± 0.23
19.14 ±
0.25
69.86 ±4.94
59.11 ±
0.24
5
78
78
13.55 ±38.51
20
20.67 ±0.34
18.60 ±
0.15
68.49 ±4.20
57.57 ±
0.15
6
78
78
35.34 ±52.98
20
20.96 ±0.12
19.43 ±
0.05
70.08 ±7.10
54.20 ±
0.18
7
160
166
39.06 ±53.57
20
21.46 ±0.18
18.75 ±
0.35
78.46 ±5.17
59.57 ±
0.19
8
160
161
40.55 ± 72.09
20
20.96 ±0.27
20.44 ±
0.22
66.30 ± 11.61
58.20 ±
0.24
9
160
160
41.66 ±65.91
20
20.32 ±0.10
19.83 ±
0.03
67.24 ± 13.31
53.16 ±
0.16
10
500
497
68.72 ± 92.47
20
21.73 ±0.17
19.53 ±
0.36
76.09 ±9.52
21.31 ±
1.51
11
160
187
12.05 ± 10.52
10
9.70 ±0.71
19.67 ±
0.41
82.19 ±7.63
43.31 ±
1.17
12
78
104
4.86 ±4.97
10
9.88 ±0.54
19.87 ±
0.12
80.69 ±6.83
44.21 ±
0.16
13
160
166
7.69 ±8.38
10
9.87 ±0.57
19.78 ±
0.15
79.42 ±7.18
44.63 ±
0.31
14
500
497
14.47 ± 15.09
10
9.64 ±0.59
19.96 ±
0.74
84.57 ±8.98
48.62 ±
2.18
15
500
506
38.23 ± 12.52
20
20.18 ±0.21
19.7 ±
0.15
90.75 ±3.79
55.05±
0.31
16
500
500
16.51 ±42.08
20
20.04 ± 0.26
20.05 ±
0.13
48.32 ± 10.83
47.58 ±
0.36
17
1000
998
58.85 ±62.97
20
20.60 ±0.32
19.51 ±
0.25
94.83 ± 3.69
53.85 ±
0.27
18
1000
1001
24.97 ±20.30
20
21.80 ±0.22
19.86 ±
0.35
92.93 ±2.32
56.16 ±
1.32
19
1000
1001
15.69 ± 13.23
10
9.32 ±0.63
19.95 ±
0.26
78.87 ± 10.13
44.77 ±
1.96
20
1000
1000
23.42 ±25.37
10
9.64 ±0.57
18.65 ±
0.26
82.23 ± 10.37
59.62 ±
0.04
21
160
161
1.36 ±0.51
10
9.64 ±0.68
20.17 ±
0.49
57.08 ±5.66
56.52 ±
0.92
Data reported as average ± SD.
16

-------
3.4 Audits
3.4.1 Performance Evaluation Audit
Performance evaluation (PE) audits were conducted to assess the quality of the results obtained
during these experiments. Table 3-2 summarizes the PE audits that were performed.
No PE audits were performed for confirmation of the concentration and purity of B.a. or
surrogate spores because quantitative standards do not exist for these organisms. The titer
enumerations and the control and blank test coupons support the spore measurements.
Table 3-2. Performance Evaluation Audits
Measurement
Audil
Procedure
Allow iihle
Tolenince
Aelu;il
Tolenince
Volume of liquid from
micropipettes
Gravimetric evaluation
± 10 %
t 0.14% to 5.89%
Time
Compared to independent clock
± 2 seconds/hour
0 seconds/hour
Temperature
Compared to independent calibrated thermometer
± 2 °C
10.29 to 0.39 °C
Relative Humidity
Compare to independent calibrated hygrometer
± 10 %
t 3.52 to 3.63 %
3.4.2 Technical Systems Audit
Observations and findings from the technical system audit (TSA) were documented and
submitted to the laboratory technical lead for response. TSAs were conducted on July 13 and 14,
2015 to ensure that tests were being conducted in accordance with the appropriate QAPP and
QMP. As part of the audit, test procedures were compared to those specified in the QAPP and
data acquisition and handling procedures were reviewed. None of the findings of the TSA
required corrective action.
3.4.3 Data Quality Audit
At least 10 % of the data acquired during the evaluation were audited. Data were reviewed in
five separate batches from August 2015 through July 2016. 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
verified. Only minor issues were noted with the data, mostly data transcription errors that were
corrected.
3.5	QA/QC Reporting
Each assessment and audit was documented in accordance with the QAPP and QMP. For these
tests, findings were noted (none significant) in the data quality audit, and no follow-up corrective
action was necessary. The findings were mostly minor data transcription errors requiring some
recalculation of efficacy results, but none were gross errors in recording. QA/QC procedures
were performed in accordance with the QAPP.
3.6	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 this report.
17

-------
4 Summary of Results and Discussion
The decontamination efficacy of fogged PAA and three concentrations of H2C>2(8 %, 22 %, and
35 %) against virulent B.a. Ames and B.g. was evaluated at target delivery volumes of 78, 160,
500, 1000, and 2365 mL; target temperatures of 10 or 20 °C; and contact times ranging from 8 to
168 hours, for a total of 21 tests. Actual operational parameters as measured are detailed in
Section 3. The detailed decontamination efficacy results, showing average CFU recovery from
each material for both positive controls and test coupons, for all tests, are found in Appendix A.
This chapter of the report discusses decontamination efficacy results as a function of some of the
variables that were tested in the study. Some statistical results are also presented to indicate
whether test variables significantly affected efficacy.
4.1 Comparing Efficacy for the Different Species
The average difference in decontamination efficacy for each test for the two microorganisms is
shown in Table 4-1. These results indicate that B.g. had resistance similar to B.a. Ames, with
average differences ranging from -1.32 to 1.02 LR. (A positive difference in result indicates that
B.g. was inactivated to a higher degree, i.e., was less resistant, than B.a. Ames.) Overall, in 12
tests, B.g. was inactivated to a higher degree, and in nine tests, B.a. was inactivated to a higher
degree.
Estimates with exact 95 percent confidence intervals for the proportion of successes (as defined
in Section 2.10) are presented in Table D-4 of Appendix D. Estimates for B.a. and B.g. are
presented side-by-side for comparison. The chi-squared test of statistical dependence between
agent and success failed to reject the null hypothesis (p = 0.1119); thus, B.a. and B.g. are not
statistically significantly different with respect to the proportion of successes across all test
conditions. These results suggest B.g. may be a suitable surrogate for B.a. Ames when
conducting similar types of testing (i.e., fogging with PAA or H2O2). Detailed comparison results
are found in Appendix B.
18

-------
Table 4-1. Summary of Average Differences in Efficacy between B.a. Ames and B
atrophaeus*
Test
Number
Equipment
Sporicidal
Liquid
Target
Temperature
(°C)
Contact
Time
(hours)
Average Difference
in Efficacy
Between Species*
1
Sani-Tizer
PAA
20
18
0.12
2
Sani-Tizer
8% H2O2
20
168
0.22
3
Sani-Tizer
PAA
20
1-7 Days
0.56
4
Sani-Tizer
PAA
20
18
-0.45
5
Sani-Tizer
22% H2O2
20
18
0.26
6
Sani-Tizer
PAA
20
8
-0.03
7
Sani-Tizer
PAA
20
18
0.08
8
MinnCare
PAA
20
18
0.14
9
MinnCare
PAA
20
18
-0.73
10
MinnCare
PAA
20
18
-0.75
11
Sani-Tizer
PAA
10
18
-0.18
12
Sani-Tizer
PAA
10
18
0.15
13
Sani-Tizer
PAA
10
18
1.02
14
MinnCare
PAA
10
18
0.50
15
Sani-Tizer
PAA
20
18
-1.32
16
MinnCare
35%H202
20
18
0.37
17
Sani-Tizer
35%H202
20
18
-0.06
18
Sani-Tizer
PAA
20
18
-0.79
19
Sani-Tizer
PAA
10
18
0.27
20
Sani-Tizer
35%H202
10
18
-1.23
21
MinnCare
PAA
10
18
0.44
* Results shown as average difference in efficacy (log reduction). A positive result indicates th
inactivated to a higher degree (less resistant) than B.a. Ames
the aviralent microorganism (B.g.) was
4.2 Effects of Test Materials on PAA and H2O2 Efficacy for B.a. Ames
The LR results for each material, by test, are shown in Figures 4-1 through 4-12, in terms of the
average LR ± 95% CI. Differences in efficacy between two materials for the same test are
significant if the 95 % CIs of the two efficacy results do not overlap. Table 4-2 shows the
average LR for each material for the tests that used that material. Note that only six materials
were used during each test, and some of the materials tested initially in the study that were
relatively easy to decontaminate (readily achieved > 6 LR) were dropped from further testing.
Materials that were harder to decontaminate (e.g., railcar carpet) were included in more tests to
find conditions in which decontamination would be successful.
Materials such as such rubber flooring, seat upholstery, aluminum, Mylar, and both air filter
types exhibited >6 LR for B.a. in all or nearly all conditions tested. The carpet, concrete, and
encapsulated grease were the most difficult materials to decontaminate (lowest average LR
values), with the latter two materials having no test conditions resulting in >6 LR. In Test 19, in
which clean industrial carpet was tested to compare with the used railcar carpet, there was no
significant difference in decontamination efficacy results for the two materials. B.a. spores
inoculated onto the interior fiberglass siding, and the clean and dirty railcar grease (spores left on
top of grease) were moderately inactivated compared to the other materials.
Further details on the decontamination efficacy results and statistical analyses are found in
Appendix A.
19

-------
Material - Rubber
¦ B.anthracis ¦ B.atrophaeus
9.00
Test-1	Test-2	Test-4	Test-5	Test-6	Test-9	Test-11	Test-12
Figure 4-1. Summary of Decontamination Efficacy Results on Rubber against B. anthracis Ames and B. atrophaeus.
Material - Upholstery
¦ B.anthracis ¦ B.atrophaeus
9.00
8.00
7.00
§ 6.00 	
1 5.00
£ 4.00
00
o 3.00
2.00
1.00
0.00
Test-1
Test-2
Test-9
Test-11
Figure 4-2. Summary of Decontamination Efficacy Results on Upholstery against B. anthracis Ames and B. atrophaeus.
20

-------
Material - Aluminum
9.00
8.00
7.00
o 6.00
§ 5.00
£ 4.00
60
o 3.00
2.00
1.00
0.00
Figure 4-3. Summary of Decontamination Efficacy Results on Aluminum against B. anthracis Ames and B. atrophaeus.
Material - Mylar
¦ B.anthracis ¦ B.atrophaeus
9.00
8.00
7.00
| 6.00
= 5.00
"O
£ 4.00
60
o 3.00
2.00
1.00
0.00
Figure 4-4. Summary of Decontamination Efficacy Results on Mylar against B. anthracis Ames and B. atrophaeus.
¦ B.athracis ¦ B.atrophaeus
Test-1	Test-2	Test-9	Test-11
Test-1	Test-2	Test-4	Test-5	Test-6	Test-9	Test-11	Test-12
21

-------
Material - Fiberglass
¦	B.arvthratis I S.atropliaeus
9.00
8.00
7.00
o 6.00 -
3 5.00
SL 4-00
2 3-00
2.00
1.00
0.00
Test-1 Test-2 Test-4 Test-5 Test-6 Test-9 Test-10 Test-ll Test-12 Test-14 Test-lS Test-16 Test-17 Test-IB Test-19 Test-2Q
Figure 4-5. Summary of Decontamination Efficacy Results on Fiberglass against B. anthracis Ames and B. atrophaeus.
Material - Used Railcar Carpet
¦	B.anthracis ¦ B.atfophaeus
9.00
8.00
7.00
o 6.00
5 5.00
£ 4.00
OS
o 3.00
2.00
1.00
0.00
H
]
J
L
I 1
li li
il il ii ii
li
Test-1 Test-2 Test-3 Test-4 Test-5 Test-7 Test-8 Test-9 Test-10 Test-llTest-12 Test-13 Test-14 Test-15 Test-16 Test-17 Test-18 Test-19 Test-2QTest-21
Figure 4-6. Summary of Decontamination Efficacy Results on Railcar Carpet against B. anthracis Ames and B. atrophaeus.
22

-------
Material - Concrete
¦ B.anthracis ¦ B.atrophaeus
9.00
8.00
7.00
| 6.00 	
§ 5.00
£ 4.00
Gfl	T
I ii i. ii ii ii ii I. ii ii ii ii....
Test-6 Test-7 Test-8 Test-10 Test-13 Test-14 Test-15 Test-16 Test-17 Test-18 Test-19 Test-20 Test-21
Figure 4-7. Summary of Decontamination Efficacy Results on Concrete against B. anthracis Ames and B. atrophaeus.
Material - New Grease SOT
¦ B.anthracis ¦ B.atrophaeus
a nn
ft nn


o.UU
7 nn
""




/ .uu
c




ii



|





Q O.UU
^ ^ nn

1

--
¦








3 J«UU
"O
^ a nn

















1
1

ry- H.UU
CtO
o ^ nn













¦ 1
¦¦

II





J.UU
^ nn


il

















Z.UU
i nn


















T
i.UU
n nn


l















u.uu
Test-4
Test-5

Test-7
Test-8

Test-12
Test-13
Test-18
Test-21
Figure 4-8. Summary of Decontamination Efficacy Results on New Grease SOT against B. anthracis Ames and B. atrophaeus.
23

-------
Material - Encapsulated New Grease
¦	B.anthracis ¦ B.atrophaeus
9.00
8.00
7.00
o 6.00
§ 5.00
£ 4.00
60
o 3.00
2.00
1.00
0.00
Test-6 Test-7 Test-8 Test-10 Test-13 Test-14 Test-15 Test-16 Test-17 Test-18 Test-19
Figure 4-9. Summary of Efficacy Results on Encapsulated NG against B. anthracis Ames and B. atrophaeus.
Material - Used Grease SOT
¦	B.anthracis ¦ B.atrophaeus

¦¦ T


-i

1 .
H


n
i ii

i
i j
i i
o 6.00
P 3.00
II
Test-7 Test-8 Test-10 Test-13 Test-14 Test-15 Test-16 Test-17 Test-18 Test-19 Test-20
Figure 4-10. Summary of Efficacy Results on Used Grease SOT against B. anthracis Ames and B. atrophaeus.
Test-20
¦
Test-21
24

-------
Material - New HVAC Filter
¦	B.anthraeis ¦ B.atrophaeus
9.00
8.00
7.00
§ 6.00 	
| 5.00
£ 4.00
o 3.00
2.00
1.00
0.00
Test-4	Test-5	Test-12
Figure 4-11. Summary of Decontamination Efficacy Results on New Filter against B. anthracis Ames and B. atrophaeus.
Material - Used HVAC Filter
¦	B,anthracis ¦ B.atrophaeus

9.00

8.00

7.00
c

o
6.00
6
3
5.00
T3

V
DC
4.00
o5
3.00
_>


2.00

LOO

0.00
ii
Test-6	Test-7	Test-8	Test-10 Test-13 Test-14 Test-lS Test-16 Test-17 Test-20 Test-21
Figure 4-12. Summary of Decontamination Efficacy Results on Used Filter against B. anthracis Ames and B. atrophaeus.
25

-------
Table 4-2. Summary of B.a. Ames and B.g. Log Reductions by Material Type
Miiloriiil
Number of
losls
A\er;iiie />'.
-------
Sani-Tizer B.anthracis (Tests 1 & 11)
9.00
8.00
7.00
0	6.00
1	5.00
1 4.00
of 3.00
2.00
1.00
0.00
Rubber Flooring Upholstery
0 Indicates complete inactivation
¦—
• 20*C
H0°C
Aluminum	Mylar
Material Type
Fiberglass Interior Used Railcar
Siding	Carpet
Figure 4-13. Effect of Temperature Against B. anthracis Ames: Tests 1 and 11. Results are
shown in terms of average LR ± 95% CI.
Sani-Tizer B.anthracis (Tests 4 & 12}
9.00
8.00
7.00
tins
0	6.00 -|	1
1	5.00
I 4.00
J* 3.00
2.00
1.00
0.00
Rubber Flooring New Grease SOT New HVAC Filter Mylar
0 Indicates complete inactivation	Material Type
2CTC
110*C
Fiberglass Interior Used Railcar
Siding	Carpet
Figure 4-14. Effect of Temperature Against B. anthracis Ames: Tests 4 and 12. Results are
shown in terms of average LR ± 95% CI.
Sani-Tizer B.anthracis (Tests 7 & 13)
9.00
8.00
7.00
S G.00
1 5.00
1 4.00
o1 3.00
—I
2.00
1.00
0.00
0 0
R

¦	20*C
¦	10°C
Used Railcar Unpainted
Carpet	Concrete
0 Indicates complete inactivation
Used HVAC Filter New Grease SOT Encapsulated New Used Grease SOT
Grease
Material Type
Figure 4-15. Effect of Temperature Against & anthracis Ames: Tests 7 and 13. Results are
shown in terms of average LR ± 95% CI.
27
-------
Minncare B.anthracis (Tests 10 & 14)
5 5.00
i 2CTC
110*C
0 indicates complete inaetivation
Used Railear Encapsulated New Used HVAC Filter Fiberglass Interior Unpainted Used Grease SOT
Carpet	Grease	Siding	Concrete
Material Type
Figure 4-16. Effect of Temperature Against B. anthracis Ames: Tests 10 and 14. Results are
shown in terms of average LR ± 95% CI.
Sani-Tizer B.anthracis (Tests 17 & 20)
9.00
| 6.00
a 5.00
^ 3.00
0.00
I 20°C
ncrc
0 Indicates complete inactivatian
Used Railcar Encapsulated New Used HVAC Filter Fiberglass Interior Unpainted Used Grease SOT
Carpet	Grease	Siding	Concrete
Material Type
Figure 4-17. Effect of Temperature Against B. anthracis Ames: Tests 17 and 20. Results are
shown in terms of average LR ± 95% CI.
4.4 Effect of Fogging Equipment on Decontamination Efficacy
The decontamination efficacy of fogging PAA or aqueous H2O2 against B.a. and B.g. was
evaluated using two types of fog generating equipment as previously described in Section 2.6.
The Minncare cold fogger generated a mean droplet size of 12.4 |im, while the Sani-Tizer
generated a larger mean droplet size of 31.0 when spraying PAA solution. The two types of
equipment yielded similar LR values when compared under identical test conditions at 20 °C
(Figure 4-18). Testing conducted using the same parameters but at 10 °C generally yielded
higher LR for the Sani-Tizer as compared to the Minncare equipment (Figure 4-19). That a
somewhat higher LR is associated with the fogger producing larger size droplets is an
unexpected result, since the larger droplets would tend to settle out sooner. Overall, however,
statistical analysis using the logistic regression model indicated that the type of fogger did not
have a significant effect on LR. Additional analyses of the effect of equipment type are included in
Appendices A and D.
28
-------
10,00
9.00
8.00
7.00
6.00
5.00
, 4.00
3.00
2.00
1,00
0.00
Equipment Comparision Tests 7 and 8
i Minncare ¦ Sani-Tizer
ii -¦
Used Railcar Carpet
Unpainted Concrete
Used HVAC Filter	New Grease (SOT)

Encapsulated New
Grease
Used Grease (SOT)
Figure 4-18. Effect of Fogger Equipment Type Against B. anthracis Ames at 20°C. Results
are shown in terms of average LR ± 95% CI.
9.00
8.00
7.00
o 6.00
§ 5.00
£ 4.00
o 3.00
2.00
1.00
0.00
Equipment Comparision Tests 13 and 21
¦ Minneare ¦ Sani-Tizer
I
I
Used Railcar Carpet
Unpalnted Concrete
Used HVAC Filter
New Grease (SOT) Encapsulated New Grease Used Grease (SOT)
Figure 4-19. Effect of Fogger Equipment Type Against B. anthracis Ames at 10°C. Results
are shown in terms of average LR ± 95% CI.
29
-------
4.5 Effect of Sporicidal Liquid and Quantity Fogged on Decontamination Efficacy
The decontamination efficacy of fogging PAA was evaluated for 16 tests, while the fogging of
aqueous H2O2 was evaluated for five tests. Three of the H2O2 tests were conducted under the
same operational conditions as three PAA tests, and thus allow us to compare results. For
example, in Tests 4 and 5, both tests were conducted with the same fogger, temperature, and
volume of sporicidal liquid. Similarly, in Tests 17 and 18, both tests were conducted at the same
temperature (20 °C), using the same fogger, and using the same quantity of sporicidal liquid.
The results of the comparisons are shown in Figures 4-20 to 4-23, and indicate that while the
aqueous H2O2 solutions were in most cases less effective than the PAA, there were only a few
cases in which there was a significant difference in efficacy. In addition, the lower concentration
H2O2 solution (22 %) appears to be somewhat less effective than the higher concentration (35 %)
H2O2 solution.
With respect to the quantity of sporicidal liquid fogged, within the parameters assessed in the
statistical analyses, the probability of a complete kill increases as a function of the logio of the
volume of the sporicidal liquid (further discussed in Appendix D).
Sporicidal Liquid Type Comparision Tests 4 and 5
¦ PAA ¦ 22% H202
10.00
9.00
1II I. IIII h
Rubber Flooring	New Grease (SOT)	New HVAC Filter	Mylar	Fiberglass Siding	Carpet
Figure 4-20. Effect of Sporicidal Liquid Type Against B. anthracis Ames Tests 4 and 5.
Results are shown in terms of average LR ± 95 % CI.
30
-------
Sporicidal Liquid Type Comparision Tests 17 and 18
I PAA ¦ 35% H202
ii ii
Carpet
Encapsulated New Grease Used HVAC Filter
Fiberglass Siding
ii
Unpainted Concrete
Used Grease (SOT)
Figure 4-21. Effect of Sporicidal Liquid Type Against B. anthracis Ames Tests 17 and 18.
Results are shown in terms of average LR ± 95 % CI.
Sporicidal Liquid Type Comparision Tests 19 and 20
I PAA ¦ 35% H202
9.00
8.00
7.00
o 6.00
§ 5.00
£ 4.00
oo
O 3.00
2.00
1.00
0.00
i
¦_
Carpet (Subway) Encapsulated New Grease
Carpet (New)
Fiberglass Siding
Unpainted Concrete Used Grease (SOT)
Figure 4-22. Effect of Sporicidal Liquid Type Against B. anthracis Ames Tests 19 and 20.
Results are shown in terms of average LR ± 95 % CI.
4.6 Effects of Test Location on Efficacy
The decontamination efficacy of fogging was evaluated at five locations within the test chamber
(refer to Figure 2-3), as previously described in Section 2.5. Log reductions for B.a. Ames were
averaged across all 21 tests per location (Figure 4-23). These results showed minimal difference
in LR by location. However, the logistic model indicates that the probability of complete kill is
significantly different for each location compared to location 3 (coupons placed on the cart,
horizontally facing upward, in the center of the chamber), with all locations less likely to result
in a complete kill compared to location 3. See Table D7 in the Appendix.
When average percent wetness per location was examined (Figure 4-24), apparent differences
existed between horizontal upward facing locations (location 1 and 3, more wet) and inverted,
vertical, or offset locations (location 2, 4, and 5, less wet, respectively).
31
-------
Avgerage Log Reduction By Location B. othracis Ames
£ 3.00
00
o
>
<
1-00
ARCA Location
Figure 4-23. Summary of Effect of Location Against B. anthracis Ames as Average of All
Tests. Results are shown in terms of average LR ± 95% CI.
Avgerage Wetness By Location
5 6.00
ARCA Location
Figure 4-24. Summary of Wetness per Location in Chamber as Average of All Tests
4.7 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 the PAA or H2O2 fog 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 thirteen material surfaces after being exposed to the sporicidal
fogs. While not a test material, copper tubing installed in the test chamber as part of the cooling
equipment exhibited severe corrosion when exposed to the PAA sporicidal liquid.
4.8 Summary
This evaluation focused on the decontamination of eleven types of subway railcar materials
(carpet, aluminum, upholstery, rubber flooring, Mylar® coating, fiberglass, new cabin filter, used
cabin filter, new grease with spores mixed (encapsulated) into the grease, new grease with spores
32
-------
left on top of the grease, and used grease (spores left on top of the grease) and a common subway
tunnel material (unpainted concrete). Decontamination efficacy tests were conducted with
spores of virulent B.a. Ames and non-virulent B.g., to assess the potential use of B.g. as a
surrogate for future studies with the fogging of sporicidal liquids. Other fogger operational and
environmental variables were evaluated for their effect on decontamination efficacy, such as air
temperature, location, sporicidal liquid chemical and quantity fogged, and fogging equipment.
The data generated from this evaluation suggest that B.g. may be a suitable surrogate for B.a.
Ames for future tests assessing the decontamination efficacy of PAA or H2O2 using fogging
equipment.
Many of the subway railcar materials were effectively decontaminated with fogging PAA. These
materials include the rubber flooring, seat upholstery, aluminum seat backing, Mylar glass
coating, and both new and used cabin air filters. Fogging of PAA was ineffective for the carpet
(both the dirty railcar carpet and the new, clean industrial carpet), concrete, and grease (with
spores mixed in/encapsulated into the grease); and moderately effective for the interior fiberglass
siding, and the clean and dirty railcar grease (spores left on top of grease).
With respect to the effect of air temperature, while the higher temperature (20 °C) resulted in a
greater probability of complete spore population kill and greater LR values compared to the
results at 10 °C (an average of 1-2 LR better), many of these differences were not statistically
significant.
The two types of foggers yielded similar LR values when compared at 20°C. Testing conducted
using the same parameters but at 10°C generally yielded higher LR for the Sani-Tizer as
compared to the Minncare equipment. Overall, however, statistical analysis using the logistic
regression model indicated that the type of fogger did not have a significant effect on LR.
In terms of the effect of chamber location on efficacy, there was minimal difference in average
LR by location within the test chamber. However, as would be expected, coupons stationed at
location 3 (coupons placed horizontally on a cart facing upward, in the center of the chamber),
were more likely to result in a complete kill compared to the other four locations in the chamber.
33
-------
5 References
1.	Wood, J.P., Calfee, M.W., et al. Evaluation of peracetic acid fogging for the inactivation of
Bacillus spores. J. Haz. Matls. 2013, Vol. 250-251, 61-67.
2.	US EPA. 2014. Quality Assurance Project Plan for the Decontamination of Subway and
Other Materials through the Fogging of Sporicidal Liquids, Version Final. April 2015.
(Available upon request by contacting EPA).
3.	Curtis Dynafog Sani-Tizer™ Operation and Maintenance Manual Model 3001-1 and 3001-2
Rev. 2-17-2014. Available from the WWW (accessed on 8/30/16) at
http://www.dvnafog.com/wp-content/uploads/2015/06/SANI-TIZER-MANUAL-MASTER-
2-17-2014.pdf
4.	MarCor Minncare® Mini Fog System Technical Sheet Rev C P/N: 3024402. Available from
the WWW (accessed on 8/30/16) at http://www.mcpur.com/disinfection/dryfogmini
5.	Bachalo, W. D. (1980). A Method for Measuring the Size and Velocity of Spheres By Dual
Beam Light Scatter Interferometry, Applied Optics 19 (3): 363-370.
6.	Bachalo, W. D. and Houser, M. J. (1984). Phase Doppler Spray Analyzer for Simultaneous
Measurements of Drop Size and Velocity Distributions, Optical Engineering 23 (5): 583-
590.
7.	Bade, K. M. and Schick, R. J. (2011). Phase Doppler Interferometry Volume Flux Sensitivity
to Parametric Settings and Droplet Trajectory, Atomization and Sprays 21 (7): 537-551.
8.	Artium Technologies, Inc. (2012). PDI-200 MD User Manual, Sunnyvale, CA 94086.
Available from the WWW (accessed on 8/30/16) at http://www.artium.com/cgi-
bin/DJgallery.cgi?T=products.html&ZONE=PDl
9.	Kruskal; Wallis (1952). "Use of ranks in one-criterion variance analysis". Journal of the
American Statistical Association. 47 (260): 583-621. doi: 10.1080/0162 1459.1952.10483441
10.	Clopper, C.; Pearson, E. S. (1934). "The use of confidence or fiducial limits illustrated in the
case of the binomial". Biometrika. 26: 404-413. doi: 10.1093/biomet/26.4.404
34
-------
Appendix A
Detailed Test Results
Efficacy Results
The detailed decontamination efficacy results for sporicidal liquids fogged against B.a. Ames andB.
atrophaeus on up to thirteen material types are shown in Tables A-l through A-3. Zero CFU were
observed on all laboratory and procedural blanks.
Table A-l. Inactivation of B. anthracis Ames Spores using Fogged Sporicidal Liquids3
Test
Number
Decon
Solution
(mL)
Equipment
Contact
Time
(hour)
Temp
(°C)
Material
Inoculum
(CFU/coupon)
Mean Recovered B. anthracis (CFU/coupon)
Positive Controlb Test Coupon0
Efficacy ± Cl"





Rubber Flooring

8.95 ± 2.14 xlO7
0.00 ± 0.00
>7.94 ± 0.10





Upholstery

1.20 ± 0.04 x10s
0.00 ± 0.00
>8.08 ± 0.01
1
PAA
(160)
Sani-Tizer
18
20
Aluminum
Mylar
Fiberglass Siding
Railcar Carpet
1.35E+08
7.65 ± 0.85 xlO7
1.17 ± 0.31 xlO8
3.90 ± 1.08 xlO7
4.60 ± 2.19 xlO7
0.00 ± 0.00
0.00 ± 0.00
0.00 ± 0.00
1.06 ± 1.71 x 106
>7.88 ± 0.04
>8.06 ±0.10
>7.58 ± 0.10
2.37 ± 1.30





Rubber Flooring

9.71 ± 1.40 x 107
0.00 ± 0.00
>7.98 ± 0.05





Upholstery

9.87 ± 1.70 x 107
0.00 ± 0.00
>7.99 ±0.06
2
8%
H2O2
(2635)
Sani-Tizer
168
20
Aluminum
Mylar
Fiberglass Siding
Railcar Carpet
1.26E+08
1.02 ± 0.12 x10s
8.61 ± 1.05 x 107
4.05 ± 2.08 xlO7
5.63±2.17xl07
0.00 ± 0.00
0.00 ± 0.00
9.95 ± 22.2 xlO2
1.95 ± 2.60 xlO5
>8.01 ± 0.04
>7.93 ± 0.05
6.77 ± 1.49
4.51 ± 2.59



24hr



2.49 ± 1.00 xlO7
9.90 ± 12.1 x 105
1.71 ± 0.67

PAA
(160)

48 hr



2.49 ± 1.00 xlO7
1.53 ± 1.64 x 105
2.69 ±0.94
3
Sani-Tizer
120 hr
20
Railcar Carpet
1.17E+08
2.49 ± 1.00 xlO7
4.27 ± 2.77 xlO5
1.84 ± 0.36


144 hr



2.49 ± 1.00 xlO7
1.74 ± 3.08 x 106
1.66 ±0.68



168 hr



2.49 ± 1.00 xlO7
5.13 ±6.71 xlO5
2.84 ± 1.47





Rubber Flooring

1.18 ± 0.06 x10s
0.00 ± 0.00
>8.07 ± 0.02





New Grease
(SOT)

1.06 ± 0.10 x10s
1.27 ± 2.85 x 106
5.93 ± 2.70
4
PAA
(78)
Sani-Tizer
18
20
New HVAC Filter
1.72E+08
9.83 ± 1.02 xlO7
0.00 ± 0.00
>7.99 ±0.04




Mylar
Fiberglass Siding
Railcar Carpet

8.39 ± 1.24 x 107
3.99 ± 0.86 xlO7
6.42 ± 3.09 xlO7
1.41 ±2.94x10
3.80 ± 5.54 xlO2
4.85 ±3.24x10®
7.56 ±0.72
6.03 ± 1.30
1.24 ± 0.58





Rubber Flooring

8.52 ± 1.05 xlO7
0.00 ± 0.00
>7.93 ± 0.05
5
22%
H2O2
(78)
Sani-Tizer
18
20
New Grease
(SOT)
New HVAC Filter
Mylar
Fiberglass Siding
Railcar Carpet
1.09E+08
1.07 ± 0.09 x10s
9.79 ± 1.20 xlO7
8.51 ± 1.09 x 107
4.01 ± 0.95 x 107
4.17 ± 2.52 x 107
3.98 ±4.53 x 106
9.04 ± 12.6 xlO4
0.00 ± 0.00
2.40 ± 3.96 xlO4
1.90 ± 1.80 xlO7
1.70 ±0.50
5.85 ± 2.57
>7.93 ± 0.05
3.58 ± 0.50
0.39 ±0.42
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 Coupons = samples inoculated, decontaminated.
11 CI = confidence interval (± 1.96 SE).
A-l
-------
Table A-l. Inactivation of B. anthracis Ames using Fogged Sporicidal Liquids a
(Continued)
Test
Number
Decon
Solution
(mL)
Equipment
Contact ~
Temp
Time 1
(hour) '
Material
Inoculum
(CFU/coupon)
Mean Recovered B. anthracis (CFU/coupon)
Positive Controlb Test Coupon0
Decontamination
Efficacy ± CId




Rubber Flooring

7.13 ± 0.16 x
07
8.74 ± 19.3 x 106
7.33 ± 1.03




Encapsulated New
Grease

7.87 ± 4.33 x
06
1.88 ± 1.45 x 106
0.85 ±0.68
6
PAA
(78)
Sani-
Tizer
8 20
Used HVAC Filter
Mylar
Fiberglass Siding
Unpainted Concrete
1.09E+08
7.75 ± 0.70 x
6.73 ± 0.45 x
2.98 ± 1.27 x
6.13 ± 4.81 x
07
07
07
06
0.00 ± 0.00
0.00 ± 0.00
1.34 ± 2.20 xlO4
1.72 ± 1.26 x 10s
>7.89 ±0.04
>7.83 ±0.03
4.93 ± 2.03
1.57 ±0.49




Railcar Carpet

2.10± 1.81 x
07
3.53 ±4.11 x 10s
2.16 ±0.85




Unpainted Concrete

2.81 ± 2.38 x
06
5.97 ± 4.82 x 104
1.66 ±0.68
7
PAA
(160)
Sani-
Tizer
18 20
Used HVAC Filter
New Grease (SOT)
1.03E+08
6.27 ± 0.58 x
5.06 ± 0.58 x
07
06
0.00 ± 0.00
1.31 ± 2.86 x 103
>7.80 ±0.04
5.97 ± 1.19



Encapsulated New
Grease
Used Grease (SOT)

6.95 ± 6.34 x
5.35 ± 0.85 x
06
07
2.16 ± 3.55 x10s
0.00 ± 0.00
1.78 ± 1.14
>7.72 ±0.06




Railcar Carpet

2.08 ± 1.32 x
07
2.42 ± 4.36 x 10s
2.44 ±0.71




Unpainted Concrete

3.00 ± 2.96 x
o6
2.00 ± 2.13 x10s
1.27 ±0.72
8
PAA
(160)
Minncare
18 20
Used HVAC Filter
New Grease (SOT)
1.04E+08
7.17 ± 0.72 x
8.60 ± 0.80 x
o7
o7
0.00 ± 0.00
9.46 ± 21.2 xlO3
>7.85 ±0.04
7.00 ± 1.83



Encapsulated New
Grease
Used Grease (SOT)

4.32 ± 3.80 x
9.04 ± 1.47 x
06
07
9.27 ± 13.1 x 10s
3.95 ± 8.58 x 10s
1.00 ±0.93
5.76 ±2.67




Rubber Flooring

6.98 ± 0.59 x
o7
0.00 ± 0.00
>7.84 ±0.03




Upholstery

9.44 ± 0.69 x
o7
0.00 ± 0.00
>7.97 ±0.03
9
PAA
(160)
Minncare
18 20
Aluminum
Mylar
Fiberglass Siding
Railcar Carpet
8.40E+07
8.91 ± 1.18 x
7.97 ± 1.06 x
3.74 ± 0.42 x
5.31 ± 2.37 x
o7
o7
o7
o7
0.00 ± 0.00
0.00 ± 0.00
1.34 ± 1.90 x 102
4.39 ± 7.19 x 10s
>7.95 ± 0.05
>7.90 ±0.05
5.97 ±0.87
3.85 ±2.17




Railcar Carpet

4.84 ± 1.94 x
o7
2.82 ± 3.25 xlO3
4.90 ± 1.39




Encapsulated New
Grease

1.78 ± 1.63 x
o6
2.11 ± 4.27 x 10s
2.40 ± 1.65
10
PAA
(500)
Minncare
18 20
Used HVAC Filter
1.01E+08
8.39 ± 0.89 x
o7
0.00 ± 0.00
>7.92 ±0.04



Fiberglass Siding
Unpainted Concrete
Used Grease (SOT)

3.25 ± 0.90 x
2.04 ± 1.55 x
8.62 ± 0.63 x
o7
o7
o7
1.21 ± 2.68 x 102
1.01 ± 1.07 x 10s
1.33 ± 2.98 x 103
6.94 ± 1.10
2.41 ± 0.55
7.17 ± 1.50




Rubber Flooring

7.85 ± 0.57 x
o7
0.00 ± 0.00
>7.89 ±0.03




Upholstery

8.83 ± 1.22 x
o7
2.54 ± 5.68 x 103
7.12 ± 1.61
11
PAA
(160)
Sani-
Tizer
18 10
Aluminum
Mylar
Fiberglass Siding
Railcar Carpet
9.67E+07
8.50 ± 0.85 x
6.13 ± 1.44 x
4.40 ± 0.76 x
3.39 ± 1.64 x
o7
o7
o7
o7
1.07	± 2.38 x 102
0.00 ± 0.00
7.08	± 15.4 x 102
6.43 ±6.96x10®
7.38 ± 1.07
>7.78 ±0.10
6.56 ± 1.40
0.99 ±0.61




Rubber Flooring

7.14 ± 0.65 x
o7
1.07 ± 2.40 xlO3
7.11 ± 1.46




New Grease (SOT)

1.07 ± 0.16 x
o8
2.04 ± 2.95 x 106
3.40 ± 2.47
12
PAA
(78)
Sani-
Tizer
18 10
New HVAC Filter
Mylar
Fiberglass Siding
Railcar Carpet
1.04E+08
1.04 ± 0.09 x
8.41 ± 0.72 x
3.39 ± 0.38 x
2.80 ± 2.33 x
o8
o7
o7
o7
3.06 ± 6.84 x10s
7.46 ± 14.4
5.63 ± 7.14 x 104
9.70 ± 7.79 x 106
6.47 ±2.35
7.62 ±0.60
3.39 ±0.87
0.54 ±0.58




Railcar Carpet

4.04 ± 1.73 x
o7
7.90 ±5.97x10®
0.73 ± 0.32




Unpainted Concrete

5.66 ± 3.12 x
o6
4.85 ± 3.97 x 10s
1.24 ±0.58
13
PAA
(160)
Sani-
Tizer
18 10
Used HVAC Filter
New Grease (SOT)
1.01E+08
4.87 ±0.71 x
7.30 ± 0.67 x
o7
o7
0.00 ± 0.00
4.27 ± 7.72 x 10s
>7.68 ± 0.06
3.81 ±2.16


Encapsulated New
Grease
Used Grease (SOT)

1.32 ± 1.41 x
7.67 ± 0.24 x
06
07
3.96 ± 3.87 x10s
2.32 ± 4.32 x 106
0.77 ± 1.26
3.31 ±2.41
" 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 Coupons = samples inoculated, decontaminated.
11 CI = confidence interval (± 1.96 SE).
A-2
-------
Table A-l. Inactivation of B. anthracis Ames using Fogged Sporicidal Liquids a
(Continued)
Test
Number
Decon
Solution
(mL)
Equipment
Contact ~
—. Temp
Tmie '
(hour) ^ '
Material
Inoculum
(CFU/coupon)
Mean Recovered B. anthracis (CFU/coupon)
Positive Controlb Test Coupon0
Decontamination
Efficacy ± CId




Railcar Carpet

4.75 ± 2.57 x
07
1.89 ± 1.46 x 106
1.43 ±0.41




Encapsulated New

1.38 ± 0.40 x
07
5.81 ± 7.65 x 105
1.65 ±0.51
14
PAA
(500)
Minncare
18 10
Used HVAC Filter
1.13E+08
8.11 ± 1.04 x
07
9.42 ±20.8x10
7.37 ± 1.05



Fiberglass Siding
Unpainted Concrete
Used Grease (SOT)

3.08 ± 1.05 x
1.23 ± 0.54 x
6.65 ± 2.24 x
000
1.12 ± 2.23 xlO4
9.76 ± 2.70 xlO4
4.89 ± 10.1 x 105
5.38 ± 1.87
2.06 ±0.29
3.87 ±2.11




Railcar Carpet

2.09 ± 1.31 x
07
2.22 ± 4.96 x 104
6.26 ± 1.99




Encapsulated New

4.75 ± 0.83 x
o6
3.41 ±4.95 x 105
2.77 ±2.19
15
PAA
Sani-
18 20
Used HVAC Filter
1.00E+08
6.72 ± 2.14 x
o7
0.00 ± 0.00
>7.80 ±0.15
(500)
Tizer
Fiberglass Siding
Unpainted Concrete
Used Grease (SOT)
3.12 ± 2.08 x
1.48 ± 1.15 x
8.74 ± 1.20 x
000
1.27 ± 2.83 x 102
7.27 ± 8.64 x 104
1.16 ±2.59x105
6.86 ± 1.13
2.70 ±0.86
6.22 ±2.25




Railcar Carpet

2.98 ± 1.76 x
o7
3.62 ±3.59x10®
1.01 ±0.49

35%


Encapsulated New
Grease

5.05 ± 2.17 x
o6
5.83 ± 4.84 xlO5
1.15 ±0.61
16
H2O2
Minncare
18 20
Used HVAC Filter
9.03E+07
6.98 ± 0.76 x
0'
3.39 ± 4.66 x 10
7.08 ± 0.92

(500)


Fiberglass Siding
Unpainted Concrete
Used Grease (SOT)

3.54 ± 2.10 x
7.10 ± 12.8 x
6.49 ± 3.63 x
000
3.06 ± 5.59 xlO3
5.37 ± 4.89 x 105
1.02 ± 1.56 x 106
5.30 ± 1.50
1.87 ± 0.72
2.34 ± 1.43




Railcar Carpet

2.92 ± 0.79 x
o7
1.73 ± 0.79 xlO5
2.29 ±0.31

35%
Sani-
Tizer

Encapsulated New
Grease

8.55 ± 0.60 x
o7
4.24 ± 5.96 xlO5
2.69 ±0.59
17
H2O2
18 20
Used HVAC Filter
1.03E+08
7.20 ± 0.39 x
o7
1.16 ± 2.55 xlO3
6.74 ± 1.47

(1000)

Fiberglass Siding
Unpainted Concrete
Used Grease (SOT)

5.37 ± 4.39 x
6.87 ± 1.46 x
7.92 ± 0.46 x
000
4.74 ± 1.02 x 102
3.00 ± 2.78 xlO5
8.14 ± 8.30 x 102
6.61 ± 1.35
1.57 ±0.49
5.73 ± 1.23




Railcar Carpet

5.28 ± 1.85 x
07
3.43 ± 5.65 x 105
3.31 ± 1.45




Encapsulated New

1.22 ± 6.53 x
06
1.46 ± 3.15 x 105
1.99 ±0.97
18
PAA
Sani-
8 20
New Grease (SOT)
6.60E+07
8.27 ± 1.40 x
07
7.46 ± 1.44
7.61 ± 0.60
(1000)
Tizer
Fiberglass Siding
4.18 ± 0.85 x
07
5.67 ± 12.6 x 105
5.56 ±2.60




Unpainted Concrete

1.38 ± 0.70 x
07
1.95 ± 1.79 x 105
1.91 ±0.36




Used Grease (SOT)

8.39 ± 2.09 x
07
0.00 ±0.00
>7.91 ±0.10




Carpet (Subway)

2.46 ± 2.56 x
07
6.07 ±6.71 x 105
3.69 ±2.88




Encapsulated New

4.18 ± 3.01 x
06
9.35 ± 6.76 x 105
0.67 ±0.52




Grease





19
PAA
(1000)
Sani- . 0
Tizer 18
10
Industrial Carpet
Fiberglass Siding
Unpainted Concrete
Used Grease (SOT)
7.17E+07
7.01 ± 0.50 x
3.47 ± 0.35 x
4.38 ± 4.77 x
8.50 ± 0.55 x
07
07
06
07
1.30 ±2.21 x 104
1.16 ± 1.82 x 103
1.85 ± 2.38 x 105
8.50 ± 5.45 x 107
4.32 ±0.74
6.17 ± 1.65
1.50 ±0.70
5.29 ±2.65




Railcar Carpet

4.70 ± 3.75 x
07
2.28 ±2.42 106
3.23 ± 2.75




Encapsulated New

3.86 ± 2.23 x
08
1.26 ± 1.73 x 106
2.64 ±0.51
20
35%
H2O2
(1000)
®am" 18
lizer
10
Grease
Used HVAC Filter
Fiberglass Siding
Unpainted Concrete
Used Grease (SOT)
9.60E+07
7.58 ± 1.39 x
3.47 ± 1.18 x
4.90 ± 3.98 x
7.62 ± 1.01 x
07
07
06
07
0.00 ±0.00
2.12 ± 3.80 x 103
8.11 ±3.07x 105
2.17 ± 4.77 x 105
>7.87 ±0.07
5.56 ± 1.62
0.70 ±0.34
4.44 ± 1.51




Railcar Carpet

7.45 ± 0.64 x
07
2.78 ± 1.64 x 107
0.49 ±0.23




Unpainted Concrete

4.64 ± 3.48 x
06
1.00 ± 0.32 x 106
0.60 ±0.29
21
PAA
(160)
Minncare 18
10
Used HVAC Filter
New Grease (SOT)
Encapsulated New
Grease
8.03E+07
6.47 ± 6.25 x
8.07 ± 0.97 x
2.59 ± 2.34 x
07
07
06
5.16 ± 10.9 x 106
5.01 ± 1.06 x 106
9.03 ± 5.57 x 105
2.10 ±0.92
0.21 ± 0.09
0.33 ±0.51




Used Grease (SOT)

6.95 ±0.57x107
4.49 ±2.17 x 107
0.24 ±0.23
' 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 Coupons = samples inoculated, decontaminated.
11 CI = confidence interval (± 1.96 SE).
A-3
-------
Table A-2. Inactivation of B. atrophaeus Spores using Fogged Sporicidal Liquids3
Test
Number
Decon
Solution
(mL)
Equipment
Contact
Time
(hour)
Temp
(°C)
Material
Inoculum
(CFU/coupon)
Mean Recovered B. atrophaeus
(CFU/coupon)
Positive Control1* Test Couponc
Efficacy ± CId





Rubber Flooring

2.30 ± 2.57 x 107
0.00 ±0.00
>7.21 ± 0.33





Upholstery

7.61 ± 2.49 x 106
0.00 ±0.00
>6.86 ±0.12
1
PAA
(160)
Sani-Tizer
18
20
Aluminum
Mylar
Fiberglass Siding
Railcar Carpet
8.50E+07
1.20 ± 0.39 x 106
8.38 ± 3.62 x 106
3.11 ± 1.31 x 106
1.75 ± 1.27 x 107
0.00 ±0.00
0.00 ±0.00
0.00 ±0.00
6.05 ± 7.00 x
105
>7.06 ±0.13
>6.89 ±0.17
>6.47 ±0.14
2.49 ±2.30





Rubber Flooring

6.89 ± 2.77 x 106
0.00 ±0.00
>6.81 ±0.16





Upholstery

1.82 ± 0.75 x 106
0.00 ±0.00
>6.23 ±0.14

8%



Aluminum

1.68 ± 1.02 x 107
0.00 ±0.00
>7.18 ±0.18
2
H2O2
(2635)
Sani-Tizer
168
20
Mylar
Fiberglass Siding
Railcar Carpet
1.11E+08
1.35	± 1.56 x 107
2.37 ±0.62 xlO6
4.36	± 1.03 x 106
0.00 ±0.00
1.41 ±2.94x10
7.86 ± 17.6 x
103
>6.96 ±0.33
6.00 ±0.72
5.71 ± 1.80



24hr



1.31 ± 0.96 x 107
1.36 ± 1.63 x
105
2.54 ± 1.07



48 hr



1.31 ± 0.96 x 107
1.67 ± 2.83 x
105
2.68 ± 1.35
3
PAA
(160)
Sani-Tizer
120 hr
20
Railcar Carpet
1.19E+08
1.31 ± 0.96 x 107
1.62 ± 2.64 x
105
2.71 ± 1.46



144 hr



1.31 ± 0.96 x 107
4.99 ± 1.54 x
104
2.34 ±0.31



168 hr



1.31 ± 0.96 x 107
6.19 ± 6.51 x
104
3.27 ± 1.91





Rubber Flooring

2.13 ±0.81 x 107
0.00 ±0.00
>7.30 ±0.16





New Grease
(SOT)

6.30 ±2.81 x 106
1.41 ±2.94x10
6.39 ±0.74
4
PAA
(78)
Sani-Tizer
18
20
New HVAC Filter
Mylar
Fiberglass Siding
Railcar Carpet
1.06E+08
3.97 ± 1.04 x 106
9.75 ± 1.60 x 106
3.12 ± 1.50 x 106
1.26 ± 0.88 x 107
0.00 ±0.00
0.00 ±0.00
1.39 ± 1.77x10
2.96 ± 2.43 x
106
6.59 ±0.10
6.98 ± 0.06
5.84 ±0.76
0.70 ±0.40





Rubber Flooring

1.38 ± 0.93 x 107
9.94 ± 2.22 x
104
6.12 ± 1.86

22%
H2O2
(78)



New Grease
(SOT)

6.60 ± 4.97 x 106
6.31 ± 12.2 x
104
3.74 ± 1.90
5
Sani-Tizer
18
20
New HVAC Filter
Mylar
Fiberglass Siding
Railcar Carpet
1.09E+08
2.44 ±0.51 x 103
1.02	± 0.33 x 107
2.08 ± 0.93 x 106
1.03	±0.61 x 107
0.00 ±0.00
0.00 ±0.00
1.11 ± 1.45 x
103
3.69 ± 1.29 x
106
>6.38 ± 0.08
>6.99 ±0.13
4.07 ± 1.28
0.41 ± 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 Coupons = samples inoculated, decontaminated.
11 CI = confidence interval (± 1.96 SE).
A-4
-------
Table A-2. Inactivation of B. atrophaeus Spores using Fogged Sporicidal Liquids a
(Continued)
Test
Number
Decon
Solution
(mL)
Equipment
Contact
Time
(hour)
Temp
PC)
Material
Inoculum
(CFU/coupon)
Mean Recovered B. trophaeus
(CFU/coupon)
Positive Controlb
Test Couponc
Decontamination
Efficacy ± CId
PAA
(78)
Sani-
Tizer
20
Rubber Flooring
Encapsulated New
Grease
Used HVAC Filter
Mylar
Fiberglass Siding
Unpainted Concrete
2.74 ± 1.29 x
9.89 ± 8.05 x
1.02E+08
5.89 ± 1
1.52 ± 0
4.77 ± 3
2.50 ± 1
99 x
94 x
01 x
42 x
4.03 ± 5.43 x 10
3.08 ± 3.88 x 104
0.00 ±
0.00 ±
1.41 ±
2.53 ±
0.00
0.00
2.08 x 102
1.49 x 104
6.35 ±0.89
1.57 ±0.94
>6.75 ±0.14
>7.13 ±0.20
5.61 ± 1.24
1.03 ±0.38
PAA
(160)
Sani-
Tizer
20
Railcar Carpet
Unpainted Concrete
Used HVAC Filter
New Grease (SOT)
Encapsulated New
Grease
Used Grease (SOT)
2.31 ± 1
2.39 ±2
5.31 ±0
1.74 ± 1
42 x
05 x
59 x
74 x
1.12 ± 1.37 x
1.40 ± 1.96 x
5.55 ±
1.12 ±
0.00 ±
4.35 ±
1.54 ±
1.50 ±
6.55 x 105
1.07 x 105
0.00
9.70 x 102
2.10 x 102
3.35 x 104
2.23 ± 1.14
0.30 ±0.46
>6.72 ±0.04
6.38 ± 1.37
4.83 ± 1.29
5.94 ± 1.95
PAA
(160)
Railcar Carpet
Unpainted Concrete
Used HVAC Filter
New Grease (SOT)
Encapsulated New
Grease
Used Grease (SOT)
9.32 ±3.
7.92 ± 7.
2.04 ± 1.
2.90 ± 1.
42 x
66 x
32 x
70 x
1.08	± 1.17 x
1.09	± 1.64 x
7.32 ±
6.82 ±
0.00 ±
0.00 ±
6.64 x 105
8.71 x 103
0.00
0.00
3.59 ± 3.37 x 103
4.73 ± 8.65 x 10
2.46 ±0.85
2.07 ±0.63
>7.20 ±0.33
>6.41 ± 0.22
1.57 ± 1.21
5.67 ± 1.22
(160)
Rubber Flooring
Upholstery
Aluminum
Mylar
Fiberglass Siding
Railcar Carpet
1.11 ±0.
5.23 ±4.
4.50 ±0.
4.75 ±5.
4.95 ±2.
1.09 ±0.
24 x
09 x
94 x
32 x
34 x
46 x
0.00 ±0.00
0.00 ±0.00
0.00 ±0.00
0.00 ±0.00
0.00 ±0.00
3.54 ± 3.95 x 105
>7.04 ±0.08
>7.56 ±0.40
>7.65 ± 0.08
>7.39 ±0.50
>6.66 ±0.18
1.70 ±0.49
PAA
(500)
Railcar Carpet
Encapsulated New
Grease
Used HVAC Filter
Fiberglass Siding
Unpainted Concrete
Used Grease (SOT)
2.58 ± 1.59 x
5.69 ± 5.35 x
8.60 ±0
1.20 ±0
3.73 ± 1
1.60 ±0
82 x
84 x
73 x
16 x
9.26 ± 14.8 x 104
7.15 ± 15.7 x 104
2.92 ±0.79
2.47 ± 1.33
0.00 ±0.00	>6.93 ±0.04
7.46 ±14.4	6.71 ±0.64
4.16 ± 3.21 x 103	2.04 ±0.36
1.14 ± 1.57 x 102	5.94 ± 1.06
PAA
(160)
Sani-
Tizer
10
Rubber Flooring
Upholstery
Aluminum
Mylar
Fiberglass Siding
Railcar Carpet
9.13E+07
2.11 ± 1
1.62 ±0
2.35 ±0
1.59 ± 0
5.44 ±2
2.35 ± 1
15 x
29 x
73 x
37 x
00 x
13 x
0.00 ±0.00
0.00 ±0.00
0.00 ±0.00
0.00 ±0.00
2.20 ± 3.52 x 102
9.36 ±5.01 x 105
>7.28 ±0.17
>7.20 ±0.07
>7.35 ±0.12
>7.19 ±0.08
5.30 ± 1.19
1.48 ±0.44
12
PAA
(78)
Sani-
Tizer
10
Rubber Flooring
New Grease (SOT)
New HVAC Filter
Mylar
Fiberglass Siding
Railcar Carpet
1.22E+08
2.19 ± 0
6.41 ±0
4.57 ± 1
1.90 ±0
5.11 ±3
1.09 ± 1
83	x
84	x
86 x
73 x
35 x
13 x
0.00 ±0.00
7.02 ± 15.5 x 104
0.00 ±0.00
0.00 ±0.00
1.35 ± 1.78 x 104
3.41 ± 1.71 x 105
>7.31 ±0.16
3.58 ± 1.29
>6.64 ±0.13
>7.26 ±0.14
3.21 ± 0.94
1.43 ±0.36
PAA
(160)
Sani-
Tizer
10
Railcar Carpet
Unpainted Concrete
Used HVAC Filter
New Grease (SOT)
Encapsulated New
Grease
Used Grease (SOT)
2.21 ±2
3.52 ±2
1.08 ± 0
1.05 ±0
2.60 ±2
7.40 ± 5
76 x
45 x
64 x
12 x
96 x
30 x
1.52 ± 0.94 x 105
2.93 ± 4.50 x 104
0.00 ±0.00
2.86 ± 4.69 x 103
8.39 ± 9.23 x 103
7.47 ± 11.2 x 102
0.99 ±0.48
1.36 ±0.64
>6.96 ±0.25
4.97 ± 1.69
2.16 ± 1.59
5.50 ± 1.58
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 Coupons = samples inoculated, decontaminated.
d CI = confidence interval (± 1.96 x SE).
A-5
-------
Table A-2. Inactivation of B. atrophaeus Spores using Fogged Sporicidal Liquids a
(Continued)
Test
Number
Decon
Solution
(mL)
Equipment
Contact
Time
(hour)
Temp
(°C)
Material
Inoculum
(CFU/coupon)
Mean Recovered B. atrophaeus (CFU/coupon)
Positive Controlb Test Couponc
Decontamination
Efficacy ± CId





Railcar Carpet
Encapsulated New

2.18 ±2.01 x 107
7.28 ± 7.55 x 10s
3.03 ±3.52x10®
2.88 ± 6.22 xlO4
0.90 ±0.59
2.55 ± 1.86
14
PAA
(500)
Minncare
18
10
Used HVAC Filter
Fiberglass Siding
Unpainted Concrete
Used Grease (SOT)
8.53E+07
7.23 ±2.48x10®
4.63 ±2.02x10®
7.10 ± 3.88 xlO5
1.01 ± 0.30 x 107
0.00 ± 0.00
0.00 ± 0.00
1.00 ± 0.68 xlO4
1.29 ± 2.86 xlO3
>6.84 ±0.13
>6.63 ±0.16
1.90 ±0.35
5.92 ± 1.47





Railcar Carpet
Encapsulated New

1.05 ± 0.50 xlO7
4.04 ± 5.50 x10s
6.04 ±4.18 x10s
3.18 ± 4.33 xlO4
1.36 ±0.47
2.33 ± 1.83
15
PAA
(500)
Sani-
Tizer
18
20
Used HVAC Filter
Fiberglass Siding
Unpainted Concrete
Used Grease (SOT)
1.00E+08
8.26 ± 1.21 x 106
7.99 ±5.09x10®
5.32 ± 7.14 x10s
8.17 ± 3.77 x 106
0.00 ± 0.00
0.00 ± 0.00
4.71 ± 2.51 x 104
0.00 ± 0.00
>6.91 ± 0.06
>6.84 ±0.21
0.78 ± 0.72
>6.88 ±0.14
16
35%
H2O2
(500)
Minncare
18
20
Railcar Carpet
Encapsulated New
Grease
Used HVAC Filter
Fiberglass Siding
Unpainted Concrete
Used Grease (SOT)
1.03E+08
8.22 ±1.76x10®
6.29 ± 6.68 x10s
9.25 ±1.70x10®
2.37± 1.65 x 106
9.54 ± 4.46 x10s
1.20 ± 0.55 xlO7
1.32 ±0.84x10®
1.89 ± 2.49 x10s
0.00 ± 0.00
5.01 ± 11.2 x 102
1.38 ± 1.25 x 10s
6.51 ± 12.5 x 103
1.05 ±0.69
1.25 ± 1.28
>6.96 ±0.06
5.63 ± 1.35
1.10 ±0.75
4.98 ± 1.79
17
35%
H2O2
(1000)
Sani-
Tizer
18
20
Railcar Carpet
Encapsulated New
Grease
Used HVAC Filter
Fiberglass Siding
Unpainted Concrete
Used Grease (SOT)
1.11E+08
1.08 ± 0.69 xlO7
8.01 ± 1.99 x 106
6.85 ±1.89x10®
4.77 ±2.37x10®
2.10 ± 1.02 x10s
8.93 ±4.44 x 106
1.71 ± 1.83 x 10s
1.87 ± 3.05 x 10s
0.00 ± 0.00
3.33 ± 7.20 xlO3
8.14 ±4.21 x 103
0.00 ± 0.00
2.27 ±0.881
2.59 ± 1.27
>6.82 ±0.09
5.26 ± 1.73
1.41 ±0.27
>6.92 ±0.16





Railcar Carpet
Encapsulated New

1.25 ± 0.93 xlO7
4.66 ± 4.65 x10s
3.28 ± 6.00 x10s
1.32 ± 1.74 x 103
2.09 ±0.75
2.83 ±0.93
18
PAA
(1000)
Sani-
Tizer
18
20
New Grease (SOT)
Fiberglass Siding
Unpainted Concrete
Used Grease (SOT)
1.08E+08
2.40 ±2.23x10®
7.50 ±3.02x10®
1.18 ±0.81 x 10®
6.11 ± 1.48 x 10®
5.88 ± 12.8 x 102
1.88 ± 4.20 xlO4
2.54 ± 3.23 xlO4
2.02 ± 4.46 x 103
5.21 ± 1.40
5.24 ±1.79
2.21 ± 1.01
5.58 ± 1.57
19
PAA
(1000)
Sani-
Tizer
18
10
Carpet (Subway)
Encapsulated New
Grease
Industrial Carpet
Fiberglass Siding
Unpainted Concrete
Used Grease (SOT)
9.33E+07
1.73 ± 1.58 x 107
1.16 ±6.55x106
3.65 ± 1.13 x 107
4.87 ±2.93x106
5.87 ± 4.74 x 105
8.09 ±7.22x106
1.07 ± 0.94 106
6.26 ± 10.80x104
7.85 ±6.09x102
0.00 ± 0.00
5.34 ± 7.97 x 103
3.70 ± 8.27 x 104
1.15 ±0.48
2.82 ±2.03
4.81 ±0.43
>6.61 ± 0.26
2.13 ±0.71
5.75 ± 2.08
20
35%
H2O2
(1000)
Sani-
Tizer
18
10
Railcar Carpet
Encapsulated New
Grease
Used HVAC Filter
Fiberglass Siding
Unpainted Concrete
Used Grease (SOT)
1.29E+08
1.36 ±0.99x107
5.26 ±5.93x105
4.08	± 1.14x 106
3.35 ±0.99x106
5.07 ±3.41 x 105
6.09	±1.99x106
1.68 ± 1.43 x 106
5.88 ± 5.74 x 104
8.07 ± 16.1 x 10
1.15 ± 1.66 x 103
9.22 ±3.39x104
9.31 ± 20.0 x 104
1.62 ± 1.65
0.80 ±0.70
5.78 ± 1.04
4.32 ± 1.27
0.67 ±0.32
4.08 ± 2.24
21
PAA
(160)
Minncare
18
10
Railcar Carpet
Unpainted Concrete
Used HVAC Filter
New Grease (SOT)
Encapsulated New
Grease
Used Grease (SOT)
9.83E+07
1.65	±1.39x107
6.66	±3.42x106
3.49 ±0.78x106
6.22 ±5.17x106
1.60 ±1.64x106
6.90 ±4.80x106
7.71 ± 0.79 x 107
1.33 ±0.56x105
4.48 ± 8.91 x 104
8.16	±5.90x105
1.17	± 1.74x 105
1.55 ± 2.17x 106
-0.04 ±0.37
0.70 ±0.31
2.63 ± 0.75
0.92 ±0.46
1.36 ±0.89
0.91 ±0.59
" 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 Coupons = samples inoculated, decontaminated.
11 CI = confidence interval (± 1.96 SE).
A-6
-------
Appendix B
Comparing Efficacy for the Different Microorganisms
All 21 tests were conducted using B. cmthracis Ames andB. atrophaeus (B.g.). The results
showed that B. atrophaeus has resistance similar to B.a. Ames when exposed to PAA and H2O2
fog at both the ambient (20°C) and lower simulated subway (10°C) conditions. 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 Efficacy between B. anthracis Ames and B. atrophaeus*
Test
Number
Decon
Solution
(mL)
Equipment
Contact
Time
(hour)
Temp
(°C)
Material
B.a Ames
Efficacy
B.g. Efficacy
Average
Difference in
Efficacy





Rubber Flooring
>
7.94
>
7.21






Upholstery
>
8.08
>
6.86

1
PAA (160)
Sani-Tizer
18
20
Aluminum
Mylar
Fiberglass Siding
Railcar Carpet
>
>
>
7.88
8.06
7.58
2.37
>
>
>
7.06
6.89
6.47
2.49
0.12





Rubber Flooring
>
7.98
>
6.81






Upholstery
>
7.99
>
6.23

2
8° 0 H2O2
(2635)
Sani-Tizer
168
20
Aluminum
Mylar
Fiberglass Siding
Railcar Carpet
>
>
8.01
7.93
6.77
4.51
>
>
7.18
6.96
6.00
5.71
0.22



168+



1.71

2.54




24hr,



2.69

2.68

3
PAA (160)
Sani-Tizer
48hr,
120hr,
144hr,
20
Railcar Carpet

1.84

2.71
0.56






1.66

2.34




168hr)



2.84

3.27






Rubber Flooring
>
8.07
>
7.30






New Grease (SOT)

5.93

6.39

4
PAA (78)
Sani-Tizer
18
20
New HVAC Filter
Mylar
Fiberglass Siding
Railcar Carpet
>
7.99
7.56
6.03
1.24

6.59
6.98
5.84
0.70
-0.45





Rubber Flooring
>
7.93

6.12






New Grease (SOT)

1.70

3.75

5
22° 0 H0O0
(78)
Sani-Tizer
18
20
New HVAC Filter
Mylar
Fiberglass Siding
Railcar Carpet
>
5.85
7.93
3.58
0.39
>
>
6.38
6.99
4.07
0.41
0.26





Rubber Flooring

7.33

6.35






Encapsulated New

0.85

1.57






Grease



6
PAA (78)
Sani-Tizer
8
20
Used HVAC Filter
Mylar
Fiberglass Siding
Unpainted Concrete
>
>
7.89
7.83
4.93
1.57
>
>
6.75
7.13
5.61
1.03
-0.03





Railcar Carpet

2.16

2.23






Unpainted Concrete

1.66

0.3






Used HVAC Filter
>
7.80
>
6.72

7
PAA (160)
Sani-Tizer
18
20
New Grease (SOT)

5.97

6.38
0.08





Encapsulated New

1.78

4.83






Grease








Used Grease (SOT)
>
7.72

5.94

* Results shown as average difference in efficacy (log reduction). A positive result indicates that the avirulent microorganism (B.g.) was
inactivated to a higher degree (less resistant) than B.a. Ames
B-l
-------
Table B-2. Difference in Efficacy between B. anthracis Ames and B. atrophaeus*
Test
Number
Decon
Solution
(mL)
Equipment
Contact
Time
(hour)
Temp
CO
Material
B.a. Ames
Efficacy
B.g. Efficacy
Average
Difference
in Efficacy





Railcar Carpet

2.44

2.46






Unpainted Concrete

1.27

2.07


PAA
(160)



Used HVAC Filter
>
7.85
>
7.20

8
Mimicare
18
20
New Grease (SOT)

7.00

6.41
0.14




Encapsulated New
Grease
Used Grease (SOT)

1.00
5.76

1.57
5.67






Rubber Flooring
>
7.84
>
7.04






Upholstery
>
7.97
>
7.56

9
PAA
(160)
Minncare
18
20
Aluminum
Mylar
Fiberglass Siding
Railcar Carpet
>
>
7.95
7.90
5.97
3.85
>
>
7.65
7.39
6.66
1.70
-0.73





Railcar Carpet

4.90

2.92


PAA
(500)



Encapsulated New
Grease

2.40

2.47

10
Minncare
18
20
Used HVAC Filter
>
7.92
>
6.93
-0.75




Fiberglass Siding
Unpainted Concrete
Used Grease (SOT)

6.94
2.41
7.17

6.71
2.04
5.94






Rubber Flooring
>
7.89
>
7.28






Upholstery

7.12
>
7.20

11
PAA
(160)
Sani-Tizer
18
10
Aluminum
Mylar
Fiberglass Siding
Railcar Carpet
>
7.38
7.78
6.56
0.99
>
>
7.35
7.19
5.30
1.48
-0.18





Rubber Flooring

7.11
>
7.31






New Grease (SOT)

3.40

3.58

12
PAA
(78)
Sani-Tizer
18
10
New HVAC Filter
Mylar
Fiberglass Siding
Railcar Carpet

6.47
7.62
3.39
0.54
>
>
6.64
7.26
3.21
1.43
0.15





Railcar Carpet

0.73

0.99






Unpainted Concrete

1.24

1.36


PAA
(160)



Used HVAC Filter
>
7.68
>
6.96

13
Sani-Tizer
18
10
New Grease (SOT)

3.81

4.97
1.02




Encapsulated New
Grease
Used Grease (SOT)

0.77
3.31

2.16
5.50






Railcar Carpet

1.43

0.90


PAA
(500)



Encapsulated New
Grease

1.65

2.55

14
Minncare
18
10
Used HVAC Filter

7.37
>
6.84
0.50




Fiberglass Siding
Unpainted Concrete
Used Grease (SOT)

5.38
2.06
3.87
>
6.63
1.90
5.92






Railcar Carpet

6.26

1.36


PAA
(500)



Encapsulated New
Grease

2.77

2.33

15
Sani-Tizer
18
20
Used HVAC Filter
>
7.80
>
6.91
-1.32




Fiberglass Siding
Unpainted Concrete
Used Grease (SOT)

6.86
2.70
6.22
>
>
6.84
0.78
6.88

B-2
-------
* Results shown as average difference in efficacy (log reduction). A positive result indicates that the avirulent microorganism (B.g.) was
inactivated to a higher degree (less resistant) than B.a. Ames
B-3
-------
Table B-3. Difference in Efficacy between B. anthracis Ames and B. atrophaeus*
Test
Number
Decon
Solution
(mL)
Equipment
Contact
Time
(hour)
Temp
(°Q
Material
B.a. Ames
Efficacy
B.g. Efficacy
Average
Difference
in Efficacy





Railcar Carpet

1.01

1.05

16
35%
H,a
(500)
Minncare
18
20
Encapsulated New
Grease
Used HVAC Filter
Fiberglass Siding
Unpainted Concrete
Used Grease (SOT)

1.15
7.08
5.30
1.87
2.34
>
1.25
6.96
5.63
1.10
4.98
0.37





Railcar Carpet

2.29

2.27

17
35%
h2o2
(1000)
Sani-Tizer
18
20
Encapsulated New
Grease
Used HVAC Filter
Fiberglass Siding
Unpainted Concrete
Used Grease (SOT)

2.69
6.74
6.61
1.57
5.73
>
>
2.59
6.82
5.26
1.41
6.92
-0.06





Railcar Carpet

3.31

2.09

18
PAA
(1000)
Sani-Tizer
18
20
Encapsulated New
Grease
New Grease (SOT)
Fiberglass Siding
Unpainted Concrete
Used Grease (SOT)
>
1.99
7.61
5.56
1.91
7.91

2.83
5.21
5.24
2.21
5.58
-0.79





Carpet (Subway)

3.69

1.15

19
PAA
(1000)
Sani-Tizer
18
10
Encapsulated New
Grease
Industrial Carpet
Fiberglass Siding
Unpainted Concrete
Used Grease (SOT)

0.67
4.32
6.17
1.5
5.29
>
2.82
4.81
6.61
2.13
5.75
0.27





Railcar Carpet

3.23

1.62

20
35%
h2o2
(1000)
Sani-Tizer
18
10
Encapsulated New
Grease
Used HVAC Filter
Fiberglass Siding
Unpainted Concrete
Used Grease (SOT)
>
2.64
7.87
5.56
0.7
4.44

0.8
5.78
4.32
0.67
4.08
-1.23





Railcar Carpet

0.49

-0.04






Unpainted Concrete

0.6

0.7

21
PAA
(160)
Sani-Tizer
18
10
Used HVAC Filter
New Grease (SOT)
Encapsulated New
Grease
Used Grease (SOT)

2.1
0.21
0.33
0.24

2.63
0.92
1.36
0.91
0.44
* Results shown as average difference in efficacy (log reduction). A positive result indicates that the aviralent microorganism (B.g.) was
inactivated to a higher degree (less resistant) than B.a. Ames
B-4
-------
Appendix C
Effects of Materials and Operational Parameters on Decontamination
Efficacy
Effects of Temperature on Efficacy
The decontamination efficacy of PAA and H2O2 fog against B.a. Ames and B. g. was evaluated at
target temperatures of 10 or 20 °C. These temperatures were tested at uncontrolled RH and volumes
of sporicidal liquid ranging from 78 to 500 mL PAA and 500 mL H2O2. Results are summarized in
Table C-l and C-2. The comparisons are made for two test conditions that share the same fogging
parameters except temperature. A negative result for the average difference in efficacy indicates a
higher efficacy at the higher temperature.
Table C-l. Difference in Efficacy Between B. anthracis Ames3 at 10°C and 20°C

Test 1
Test 11
Average
Material Type
PAA 160 mL; Sani-Tizer; 20 °C;
PAA 160 mL
Sani-Tizer; 10 °C;
Difference in

18 hr

18 hr

Efficacy
Rubber Flooring

> 7.94
>
7.89

Upholstery

> 8.08

7.12

Aluminum

> 7.88

7.38
-0.97
Mylar

> 8.06
>
7.83
Fiberglass Interior Siding

> 7.58

6.55

Railcar Carpet

2.37

0.99


Test 4
Test 12
Average
Material Type
PAA 78 mL; Sani-Tizer; 20 °C;
PAA 78 mL;
Sani-Tizer; 10 °C;
Difference in

18 hr

18 hr

Efficacy
Rubber Flooring

> 8.07

7.11

New Grease SOT

5.93

3.40

New Filter

> 7.99

6.47
-1.38
Mylar

7.56

7.62
Fiberglass Interior Siding

6.03

3.39

Railcar Carpet

1.24

0.54


Test 7
Test 13
Average
Material Type
PAA 160 mL; Sani-Tizer; 20 °C;
PAA 160 mL
Sani-Tizer; 10 °C;
Difference in

18 hr

18 hr

Efficacy
Railcar Carpet

2.16

0.73

Unpainted Concrete

1.66

1.24

Used Filter

> 7.80
>
7.68
-1.89
New Grease SOT

5.97

3.81
Encapsulated New Grease

1.78

0.77

Used Grease SOT

7.72

3.31


Test 10
Test 14
Average
Material Type
PAA 500 mL; Minncare; 20 °C;
PAA 500 mL
Minncare; 10 °C;
Difference in

18 hr

18 hr

Efficacy
Railcar Carpet

4.90

1.43

Encapsulated New Grease

2.40

1.65

Used Filter

> 7.92

7.37
-1.66
Fiberglass Interior Siding

6.94

5.38
Unpainted Concrete

2.41

2.06

Used Grease SOT

7.17

3.87


Test 17
Test 20
Average
Material Type
35% H202 500 mL; Sani-Tizer;
35% H202 500 mL; Sani-Tizer;
Difference in

20 °C; 18 hr
10 °C; 18 hr

Efficacy
Railcar Carpet

2.29

3.23

Encapsulated New Grease

2.69

2.64

Used HVAC Filter

6.74
>
7.87
-0.20
Fiberglass Interior Siding

6.61

5.56
Unpainted Concrete

1.57

0.70

Used Grease SOT

5.73

4.44

a Data are expressed as decontamination efficacy (log reduction).
C-l
-------
Table C-2. Difference in Efficacy Between B. atrophaeousa at 10°C and 20°C

Test 1
Test 11
Average
Material Type
PAA 160 mL; Sani-Tizer; 20 °C;
PAA 160 mL
Sani-Tizer; 10 °C;
Difference

18 hr

18 hr

in Efficacy
Rubber Flooring

> 7.21
>
7.25

Upholstery

> 6.86
>
7.20

Aluminum

> 7.06
>
7.29
-1.09
Mylar

> 6.89
>
7.20
Fiberglass Interior Siding

> 6.47

5.30

Railcar Carpet

2.49

1.48


Test 4
Test 12
Average
Material Type
PAA 78 mL; Sani-Tizer; 20 °C;
PAA 78 mL;
Sani-Tizer; 10 °C;
Difference

18 hr

18 hr

in Efficacy
Rubber Flooring

> 7.30
>
7.31

New Grease SOT

6.39

3.58

New Filter

> 6.59
>
6.64
-1.57
Mylar

> 6.98
>
7.26
Fiberglass Interior Siding

5.84

3.21

Railcar Carpet

0.70

1.43


Test 7
Test 13
Average
Material Type
PAA 160 mL; Sani-Tizer; 20 °C;
PAA 160 mL
Sani-Tizer; 10 °C;
Difference

18 hi-

18 hr

in Efficacy
Railcar Carpet

2.23

0.99

Unpainted Concrete

0.30

1.36

Used Filter

> 6.72
>
6.96
-0.94
New Grease SOT

6.38

4.97
Encapsulated New Grease

4.83

2.16

Used Grease SOT

5.94

5.50


Test 10
Test 14
Average
Material Type
PAA 500 mL; Minncare; 20 °C;
PAA 500 mL
Minncare; 10 °C;
Difference

18 hi-

18 hr

in Efficacy
Railcar Carpet

2.92

0.90

Encapsulated New Grease

2.47

2.55

Used Filter

> 6.93
>
6.84
-0.44
Fiberglass Interior Siding

6.71
>
6.63
Unpainted Concrete

2.04

1.90

Used Grease SOT

5.94

5.92


Test 17
Test 20
Average
Material Type
35% H202 500 mL; Sani-Tizer;
35% H202 500 mL; Sani-Tizer;
Difference

20 °C; 18 hr
10 °C; 18 hi-

in Efficacy
Railcar Carpet

2.27

1.62

Encapsulated New Grease

2.59

0.80

Used HVAC Filter

> 6.82
>
5.78
-1.39
Fiberglass Interior Siding

5.26

4.32
Unpainted Concrete

1.41

0.67

Used Grease SOT

> 6.92

4.08

a Data are expressed as decontamination efficacy (log reduction).
C-2
-------
Effects of Fogger Equipment Type on Sporicidal Liquid Efficacy
The decontamination efficacy of PAA and H2O2 against B. a. Ames and B. g. was evaluated using
two types of fogging equipment (Minncare Mini Dry Fogger and Curtis Dynafogger Sani-Tizer).
These pieces of equipment were tested at uncontrolled RH and volumes of sporicidal liquid ranging
from 78 to 500 mL PAA and 500 mL H2O2. Results are summarized in Table C-3 and C-6. The
comparisons are made for two test conditions that share the same fumigation parameters except
equipment.
Table C-3. Difference in B. anthracis Ames3 Efficacy Between Equipment Type
Material Type3
Sani-Tizer (Tests 1 and
2)
Minncare (Tests 8 and
9)
Average
Difference
in
Efficacy
PAA 160 mL;
20 °C; 18 hr


Rubber Flooring

>
7.94
>
7.84

Upholstery

>
8.08
>
7.97

Aluminum

>
7.88
>
7.95

Mylar

>
8.06
>
7.90

Fiberglass Interior Siding

>
7.58

5.97

Railcar Carpet


2.37

2.44
-0.28
Railcar Carpet (other test)


2.16

3.85

Unpainted Concrete


1.66

1.27

Used HVAC Filter

>
7.80
>
7.85

New Grease (SOT)


5.97

7.00

Encapsulated New Grease


1.78

1.00

Used Grease (SOT)

>
7.72

5.76

" Data are expressed as decontamination efficacy (log reduction).
Table C-4. Difference in B. anthracis Ames3 Efficacy Between Equipment Type
Material Type3
Sani-Tizer (Test 13)
Minncare (Test 21)
Average
Difference
in
Efficacy
PAA 160 mL; 10 °C; 18 hr
Railcar Carpet
Unpainted Concrete
Used HVAC Filter
New Grease (SOT)
Encapsulated New Grease
Used Grease (SOT)
0.73 0.49
1.24 0.6
> 7.68 2.1
3.81 0.21
0.77 0.33
3.31 0.24
-2.26
" Data are expressed as decontamination efficacy (log reduction).
C-3
-------
Table C-5. Difference in B. atrophaeusa Efficacy Between Equipment Type
Material Type8
Sani-Tizer (Tests 1 and
7)
Minncare (Tests 8 and
9)
Average
Difference
in Efficacy
PAA 160 mL; 20 °C; 18 hr



Rubber Flooring
Upholstery
Aluminum
>	7.21
>	6.86
>	7.06

>
>
>
7.04
7.56
7.65

Mylar
Fiberglass Interior Siding
Railcar Carpet
Carpet
Unpainted Concrete
>	6.89
>	6.47
2.49
2.23
0.3

>
7.39
6.66
1.7
2.46
2.07
-0.30
UsedHVAC Filter
> 6.72

>
7.2

New Grease (SOT)
6.38


6.41

Encapsulated New Grease
Used Grease (SOT)
4.83
5.94


1.57
5.67

a Data are expressed as decontamination efficacy (log reduction).




Table C-6. Difference in B. atrophaeusa Efficacy Between Equipment Type

Sani-Tizer
Minncare
Average
Material Type3
PAA 160 mL; 10 °C; 18 hr
Difference
in Efficacy
Railcar Carpet
0.99


-0.04

Unpainted Concrete
1.36


0.7

UsedHVAC Filter
> 6.96


2.63
-2.58
New Grease (SOT)
4.97


0.92
Encapsulated New Grease
2.16


1.36

Used Grease (SOT)
5.5


0.91

" Data are expressed as decontamination efficacy (log reduction).
-------
Effects of Sporicidal Liquid on B.a. Ames Efficacy
The decontamination efficacy of PAA and H2O2 against B. a. Ames and B. g. was evaluated using
two types of sporicidal chemicals. These sporicidal liquids were tested at 10°C and 20°C,
uncontrolled RH, and volumes of sporicidal liquid ranging from 78 to 100 mL. Results are
summarized in Table C-7 and C-10. The comparisons are made for two test conditions that share the
same fogging operational parameters except sporicidal liquid type.
Table C-7. Difference in B. anthracis Ames3 Efficacy Between Liquid Type (Tests 4/5)
Material Type8
PAA 78 mL; Sani-
Tizer; 20 °C; 18 hr
22%H202 78 mL; Sani-
Tizer; 20 °C; 18 hr
Average
Difference
in
Efficacy
Rubber Flooring
New Grease (SOT)
New HVAC Filter
Mylar
Fiberglass Siding
Railcar Carpet
>	8.07 > 7.93
5.93 1.70
>	7.99 5.85
7.56 > 7.93
6.03 3.58
1.24 0.39
-1.86
" Data are expressed as decontamination efficacy (log reduction).
Table C-8. Difference in B. anthracis Ames3 Efficacy Between Liquid Type (Tests 15/16)
Material Type3
PAA 500 mL; Sani-
Tizer; 20 °C; 18 hr
35%H202 500 mL;
Sani-Tizer; 20 °C; 18
hr
Average
Difference
in
Efficacy
Railcar Carpet
Encapsulated New Grease
Used HVAC Filter
Fiberglass Siding
Unpainted Concrete
Used Grease (SOT)
6.26 1.01
2.77 1.15
> 7.80 7.08
6.86 5.30
2.70 1.87
6.22 2.34
-2.31
" Data are expressed as decontamination efficacy (log reduction).
Table C-9. Difference in B. anthracis Ames3 Efficacy Between Liquid Type (Tests 17/18)
Material Type3
PAA 1000 mL; Sani-
Tizer; 20 °C; 18 hr
35%H202 1000 mL;
Sani-Tizer; 20 °C; 18
hr
Average
Difference
in
Efficacy
Railcar Carpet
Encapsulated New Grease
Used HVAC Filter
Fiberglass Siding
Unpainted Concrete
Used Grease (SOT)
3.31 2.29
1.99 2.69
7.61 6.74
5.56 6.61
1.91 1.57
> 7.91 5.73
-0.44
a Data are expressed as decontamination efficacy (log reduction).
C-5
-------
Table C-10. Difference in B. anthracis Ames3 Efficacy Between Liquid Type (Tests 19/20)
Material Type8
PAA 1000 mL; Sani-
Tizer; 10 °C; 18 hr
35%H202 1000 mL;
Sani-Tizer; 10 °C; 18
hr
Average
Difference
in Efficacy
Carpet (Subway)
Encapsulated New Grease
Industrial Carpet (New)
Fiberglass Siding
Unpainted Concrete
Used Grease (SOT)
3.69 3.43
0.67 2.64
4.32 > 7.87
6.17 5.56
1.5 0.7
5.29 4.44
0.50
a Data are expressed as decontamination efficacy (log reduction).
C-6
-------
Appendix D
Detailed Statistical Analysis
Introduction
This report contains the statistical analysis of B. anthracis (B.a.) and B. atrophaeus (B.g.)
decontamination data for different decontamination methods on a variety of materials and location
of the materials in the decontamination chamber.
Results
Positive controls. Table D1 contains the mean percent recoveries for the positive controls for
each spore species and material with 95 percent confidence intervals on the means; percent
recoveries for each positive control coupon are plotted in Figure D-l. The Kruskal-Wallis tests to
compare materials by agent were statistically significant for both B.a. (p < 0.001) and B.g. (p <
0.001) (Table D-2). The p-values for each Kruskal-Wallis test to compare B.a. vs B.g. for each
material are presented in Table D-3. The percent recovery for B.a. is statistically significantly
different from the percent recovery for B.g. for all materials.
Comparing decontamination efficacy for Ba and Bg. Estimates with exact 95 percent
confidence intervals for the proportion of successes (complete inactivation or > 6 LR) are
presented in D-4. Estimates for B.a. and B.g. are presented side-by-side for comparison. The chi-
squared test of statistical dependence between agent and success failed to reject the null
hypothesis (p = 0.1119); thus, we conclude that B.a. and B.g. are not statistically significantly
different with respect to the proportion of successes across all test conditions.
Assessing the effect of parameters on efficacy. The main effects logistic regression model
could not be fitted to the complete data set as planned due to quasi-complete separation of the
data. Three materials that had successes for all tests or failures for all tests were removed from
the data set to allow the model to be fitted: clean carpet (no successes), Mylar (all successes),
and unpainted concrete (no successes). In addition, a more balanced data set was constructed by
removing the following records:
•	Materials: new HVAC filter, aluminum, and upholstery (in addition to clean carpet,
Mylar, and unpainted concrete already removed)
•	Decontaminant liquids: 22% H2O2 and 8% H2O2
•	Decontamination Volume: 2635 mL
•	Time: 8, 168, and 1-5 days
The main effects logistic regression model was fitted to the full dataset with the three materials
removed and the more balanced subset of the data. Conclusions from the two models were
equivalent, and time was not found to be statistically significant in the full data model.
Therefore, two-factor interactions were considered for the model of the more balanced subset of
data. Two of the two-factor interactions were found to be statistically significant and were added
D-l
-------
to the model: Temperature x logio Decontamination Volume and Equipment x Temperature.
Parameter estimates for the final logistic regression model are presented in Table D-5.
Odds ratios for all pairwise material comparisons, comparisons of all locations with location 3
(center of room), and decontamination sporicidal liquid comparison are presented in Tables D-6,
D-7, and D-8.
Based on the parameters of the logistic model, materials can be grouped by decontamination
effectiveness. The following groups are suggested:
•	Rubber Flooring, Used HVAC Filter - Highly effective decontamination
•	Clean Grease SOT, Fiberglass Interior Siding, Used Grease SOT - Moderately effective
decontamination
•	Used Carpet, Encapsulated Clean Grease - Ineffective decontamination
Though not included in the model due to quasi-complete separation, decontamination is highly
effective for Mylar (100% success) and highly ineffective for Clean Carpet and Unpainted
Concrete (0% complete kills). New HVAC filter, aluminum, and upholstery were also not
included in the logistic model and were not perfectly separated. However, all but one test was
successful for aluminum and upholstery and all but three were successful for new HVAC filter.
These limited number of results suggest that aluminum and upholstery group in the highly
effective decontamination category, and new HVAC filter group in the moderately or highly
effective decontamination category.
The logistic model indicates that the probability of a complete kill is different for each location
compared to location 3, with all locations less likely to result in a complete kill compared to
location 3.
The two decontamination sporicidal liquids are shown to be statistically significantly different,
with PAA more likely to result in a complete kill.
Temperatures are also statistically significant, with higher temperature having a greater
probability of a complete kill.
Finally, the probability of a complete kill increases as a function of the logio of the volume of the
decontaminant liquid.
Conclusions
Analysis of the percent recovery showed statistically significant differences in percent recovery
for different materials for each agent and for different agents for each material. We conclude that
B.a. and B.g. are not statistically significantly different with respect to the proportion of
successes (complete kills) across all test conditions. For B.a., materials can be grouped with
respect to effectiveness as follows:
•	Rubber Flooring, Used HVAC Filter, Mylar - Highly effective decontamination
•	Clean Grease SOT, Fiberglass Interior Siding, Used Grease SOT - Moderately effective
decontamination
D-2
-------
• Used Carpet, Encapsulated Clean Grease, Clean Carpet, Unpainted Concrete -
Ineffective decontamination
Decontamination time is not statistically significant, nor is equipment. Location 3 has the highest
probability of observing a complete kill. Higher temperature and greater volume of
decontamination SL both increase the probability of a complete kill. Use of PAA increases the
probability of complete kill compared to 35 % H2O2.
D-3
-------
Table D-l. Mean Percent Recovery for Control Coupons for Each Agent and Material
with 95 Percent Confidence Intervals
Agent
Material
N
Mean Percent Recovery
(95% Confidence Interval)
B. anthracis
New HVAC Filter
15
82.30 (70.94,93.67)
B. anthracis
Aluminum
20
82.93 (73.42,92.45)
B. anthracis
Clean Carpet
5
97.82 (89.17,100.0)*
B. anthracis
Clean Grease SOT
40
86.52 (78.30,94.75)
B. anthracis
Encapsulated Clean Grease
65
41.50 (11.35,71.65)
B. anthracis
Fiberglass Interior Siding
80
37.55 (33.63,41.47)
B. anthracis
Mylar
40
72.88 (67.11,78.66)
B. anthracis
Rubber Flooring
40
73.58 (70.28,76.88)
B. anthracis
Unpainted Concrete
65
14.30 (4.23,24.36)
B. anthracis
Upholstery
20
92.77 (85.39,100.0)*
B. anthracis
Used Carpet
100
40.47 (35.15,45.79)
B. anthracis
Used Grease SOT
60
83.89 (77.17,90.61)
B. anthracis
Used HVAC Filter
55
70.73 (67.14,74.32)
B. atrophaeus
New HVAC Filter
15
3.14(2.40, 3.88)
B. atrophaeus
Aluminum
20
24.45 (17.95,30.94)
B. atrophaeus
Clean Carpet
5
39.14 (24.15,54.14)
B. atrophaeus
Clean Grease SOT
40
6.07 (4.26, 7.88)
B. atrophaeus
Encapsulated Clean Grease
65
1.18(0.69, 1.67)
B. atrophaeus
Fiberglass Interior Siding
80
4.71 (3.91, 5.51)
B. atrophaeus
Mylar
40
16.54 (9.90,23.18)
B. atrophaeus
Rubber Flooring
40
17.84 (13.32,22.36)
B. atrophaeus
Un painted Concrete
65
0.53 (0.41, 0.65)
B. atrophaeus
Upholstery
20
19.53 (7.34,31.71)
B. atrophaeus
Used Carpet
100
17.47 (13.54,21.40)
B. atrophaeus
Used Grease SOT
60
8.81 (7.04,10.59)
B. atrophaeus
Used HVAC Filter
55
7.46 (6.03, 8.89)
* Confidence limits less than 0 or greater than 100 truncated to 0 or 100 to reflect valid range of
percent recovery values.
D-4
-------
Table D-2. Kruskal-Wallis Tests of Differences among Materials for Each Agent
Agent
DF
p value
B. anthracis
12
< 0.001
B. atrophaeus
12
< 0.001
Table D-3. Kruskal-Wallis Tests of B.a. vs B.g. for Each Material
Agent
DF
p value
New HVAC Filter
1
< 0.001
Aluminum
1
< 0.001
Clean Carpet
1
0.0088
Clean Grease SOT
1
< 0.001
Encapsulated Clean
Grease
1
< 0.001
Fiberglass Interior
Siding
1
< 0.001
Mylar
1
< 0.001
Rubber Flooring
1
< 0.001
Unpainted Concrete
1
< 0.001
Upholstery
1
< 0.001
Used Carpet
1
< 0.001
Used Grease SOT
1
< 0.001
Used HVAC Filter
1
< 0.001
D-5
-------
1 Table D-4. Proportion Success (> 6 LR or Total Kill) for B.a. and B.g. with Exact 95 Percent Confidence Intervals
Material
Equipment
Decon
liquid
Temp °C
Decon
Volume
(mL)
Time
(Hours)
B.a.
B.g.
Number
Success/N
Proportion
Success
(Exact 95%
Confidence
Interval)
Number
Success/
N
Proportion
Success
(Exact 95%
Confidence
Interval)
New HAVC Filter
Sani-Tizer
22% H2O2
20
78
18
3/5
0.60 (0.15, 0.95)
5/5
1.00 (0.48, 1.00)
New HVAC Filter
Sani-Tizer
PAA
10
78
18
4/5
0.80 (0.28, 0.99)
5/5
1.00 (0.48, 1.00)
New HVAC Filter
Sani-Tizer
PAA
20
78
18
5/5
1.00 (0.48, 1.00)
5/5
1.00 (0.48, 1.00)
Aluminum
MinnCare
PAA
20
160
18
5/5
1.00 (0.48, 1.00)
5/5
1.00 (0.48, 1.00)
Aluminum
Sani-Tizer
8% H2O2
20
2635
168
5/5
1.00 (0.48, 1.00)
5/5
1.00 (0.48, 1.00)
Aluminum
Sani-Tizer
PAA
10
160
18
4/5
0.80 (0.28, 0.99)
5/5
1.00 (0.48, 1.00)
Aluminum
Sani-Tizer
PAA
20
160
18
5/5
1.00 (0.48, 1.00)
5/5
1.00 (0.48, 1.00)
Clean Carpet
Sani-Tizer
PAA
10
1000
18
0/5
0.00 (0.00, 0.52)
0/5
0.00 (0.00, 0.52)
Clean Grease SOT
MinnCare
PAA
10
160
18
0/5
0.00 (0.00, 0.52)
0/5
0.00 (0.00, 0.52)
Clean Grease SOT
MinnCare
PAA
20
160
18
4/5
0.80 (0.28, 0.99)
5/5
1.00 (0.48, 1.00)
Clean Grease SOT
Sani-Tizer
22% H2O2
20
78
18
0/5
0.00 (0.00, 0.52)
1/5
0.20 (0.01, 0.72)
Clean Grease SOT
Sani-Tizer
PAA
10
78
18
1/5
0.20 (0.01, 0.72)
0/5
0.00 (0.00, 0.52)
Clean Grease SOT
Sani-Tizer
PAA
10
160
18
1/5
0.20 (0.01, 0.72)
2/5
0.40 (0.05, 0.85)
Clean Grease SOT
Sani-Tizer
PAA
20
78
18
3/5
0.60 (0.15, 0.95)
4/5
0.80 (0.28, 0.99)
Clean Grease SOT
Sani-Tizer
PAA
20
160
18
3/5
0.60 (0.15, 0.95)
4/5
0.80 (0.28, 0.99)
Clean Grease SOT
Sani-Tizer
PAA
20
1000
18
5/5
1.00 (0.48, 1.00)
3/5
0.60 (0.15, 0.95)
Encapsulated Clean Grease
MinnCare
35% H2O2
20
500
18
0/5
0.00 (0.00, 0.52)
0/5
0.00 (0.00, 0.52)
Encapsulated Clean Grease
MinnCare
PAA
10
160
18
0/5
0.00 (0.00, 0.52)
0/5
0.00 (0.00, 0.52)
Encapsulated Clean Grease
MinnCare
PAA
10
500
18
0/5
0.00 (0.00, 0.52)
1/5
0.20 (0.01, 0.72)
Encapsulated Clean Grease
MinnCare
PAA
20
160
18
0/5
0.00 (0.00, 0.52)
0/5
0.00 (0.00, 0.52)
Encapsulated Clean Grease
MinnCare
PAA
20
500
18
0/5
0.00 (0.00, 0.52)
0/5
0.00 (0.00, 0.52)
Encapsulated Clean Grease
Sani-Tizer
35% H2O2
10
1000
18
0/5
0.00 (0.00, 0.52)
0/5
0.00 (0.00, 0.52)
Encapsulated Clean Grease
Sani-Tizer
35% H2O2
20
1000
18
0/5
0.00 (0.00, 0.52)
0/5
0.00 (0.00, 0.52)
Encapsulated Clean Grease
Sani-Tizer
PAA
10
160
18
0/5
0.00 (0.00, 0.52)
1/5
0.20 (0.01, 0.72)
Encapsulated Clean Grease
Sani-Tizer
PAA
10
1000
18
0/5
0.00 (0.00, 0.52)
1/5
0.20 (0.01, 0.72)
Encapsulated Clean Grease
Sani-Tizer
PAA
20
78
8
0/5
0.00 (0.00, 0.52)
0/5
0.00 (0.00, 0.52)
D-6
-------
Material
Equipment
Decon
liquid
Temp °C
Decon
Volume
(mL)
Time
(Hours)
B.a.
B.g.
Number
Success/N
Proportion
Success
(Exact 95%
Confidence
Interval)
Number
Success/
N
Proportion
Success
(Exact 95%
Confidence
Interval)
Encapsulated Clean Grease
Sani-Tizer
PAA
20
160
18
0/5
0.00 (0.00, 0.52)
3/5
0.60 (0.15, 0.95)
Encapsulated Clean Grease
Sani-Tizer
PAA
20
500
18
1/5
0.20 (0.01, 0.72)
1/5
0.20 (0.01, 0.72)
Encapsulated Clean Grease
Sani-Tizer
PAA
20
1000
18
0/5
0.00 (0.00, 0.52)
0/5
0.00 (0.00, 0.52)
F
berglass Interior Siding
MinnCare
35% H2O2
20
500
18
1/5
0.20 (0.01, 0.72)
4/5
0.80 (0.28, 0.99)
F
berglass Interior Siding
MinnCare
PAA
10
500
18
2/5
0.40 (0.05, 0.85)
5/5
1.00 (0.48, 1.00)
F
berglass Interior Siding
MinnCare
PAA
20
160
18
2/5
0.40 (0.05, 0.85)
5/5
1.00 (0.48, 1.00)
F
berglass Interior Siding
MinnCare
PAA
20
500
18
4/5
0.80 (0.28, 0.99)
4/5
0.80 (0.28, 0.99)
F
berglass Interior Siding
San
-Tizer
22% H2O2
20
78
18
0/5
0.00 (0.00, 0.52)
1/5
0.20 (0.01, 0.72)
F
berglass Interior Siding
San
-Tizer
35% H2O2
10
1000
18
2/5
0.40 (0.05, 0.85)
1/5
0.20 (0.01, 0.72)
F
berglass Interior Siding
San
-Tizer
35% H2O2
20
1000
18
3/5
0.60 (0.15, 0.95)
3/5
0.60 (0.15, 0.95)
F
berglass Interior Siding
San
-Tizer
8% H2O2
20
2635
168
4/5
0.80 (0.28, 0.99)
4/5
0.80 (0.28, 0.99)
F
berglass Interior Siding
San
-Tizer
PAA
10
78
18
0/5
0.00 (0.00, 0.52)
0/5
0.00 (0.00, 0.52)
F
berglass Interior Siding
San
-Tizer
PAA
10
160
18
3/5
0.60 (0.15, 0.95)
2/5
0.40 (0.05, 0.85)
F
berglass Interior Siding
San
-Tizer
PAA
10
1000
18
3/5
0.60 (0.15, 0.95)
5/5
1.00 (0.48, 1.00)
F
berglass Interior Siding
San
-Tizer
PAA
20
78
8
2/5
0.40 (0.05, 0.85)
3/5
0.60 (0.15, 0.95)
F
berglass Interior Siding
San
-Tizer
PAA
20
78
18
2/5
0.40 (0.05, 0.85)
3/5
0.60 (0.15, 0.95)
F
berglass Interior Siding
San
-Tizer
PAA
20
160
18
5/5
1.00 (0.48, 1.00)
5/5
1.00 (0.48, 1.00)
F
berglass Interior Siding
San
-Tizer
PAA
20
500
18
4/5
0.80 (0.28, 0.99)
5/5
1.00 (0.48, 1.00)
F
berglass Interior Siding
San
-Tizer
PAA
20
1000
18
3/5
0.60 (0.15, 0.95)
2/5
0.40 (0.05, 0.85)
Mylar
MinnCare
PAA
20
160
18
5/5
1.00 (0.48, 1.00)
5/5
1.00 (0.48, 1.00)
Mylar
Sani-Tizer
22% H2O2
20
78
18
5/5
1.00 (0.48, 1.00)
5/5
1.00 (0.48, 1.00)
Mylar
Sani-Tizer
8% H2O2
20
2635
168
5/5
1.00 (0.48, 1.00)
5/5
1.00 (0.48, 1.00)
Mylar
Sani-Tizer
PAA
10
78
18
5/5
1.00 (0.48, 1.00)
5/5
1.00 (0.48, 1.00)
Mylar
Sani-Tizer
PAA
10
160
18
5/5
1.00 (0.48, 1.00)
5/5
1.00 (0.48, 1.00)
Mylar
Sani-Tizer
PAA
20
78
8
5/5
1.00 (0.48, 1.00)
5/5
1.00 (0.48, 1.00)
Mylar
Sani-Tizer
PAA
20
78
18
5/5
1.00 (0.48, 1.00)
5/5
1.00 (0.48, 1.00)
Mylar
Sani-Tizer
PAA
20
160
18
5/5
1.00 (0.48, 1.00)
5/5
1.00 (0.48, 1.00)
Rubber Flooring
MinnCare
PAA
20
160
18
5/5
1.00 (0.48, 1.00)
5/5
1.00 (0.48, 1.00)
D-7
-------
Material
Equipment
Decon
liquid
Temp °C
Decon
Volume
(mL)
Time
(Hours)
B.a.
B.g.
Number
Success/N
Proportion
Success
(Exact 95%
Confidence
Interval)
Number
Success/
N
Proportion
Success
(Exact 95%
Confidence
Interval)
Rubber Flooring
Sani-Tizer
22% H2O2
20
78
18
5/5
1.00 (0.48, 1.00)
4/5
0.80 (0.28, 0.99)
Rubber Flooring
Sani-Tizer
8% H2O2
20
2635
168
5/5
1.00 (0.48, 1.00)
5/5
1.00 (0.48, 1.00)
Rubber Flooring
Sani-Tizer
PAA
10
78
18
4/5
0.80 (0.28, 0.99)
5/5
1.00 (0.48, 1.00)
Rubber Flooring
Sani-Tizer
PAA
10
160
18
5/5
1.00 (0.48, 1.00)
5/5
1.00 (0.48, 1.00)
Rubber Flooring
Sani-Tizer
PAA
20
78
8
4/5
0.80 (0.28, 0.99)
2/5
0.40 (0.05, 0.85)
Rubber Flooring
Sani-Tizer
PAA
20
78
18
5/5
1.00 (0.48, 1.00)
5/5
1.00 (0.48, 1.00)
Rubber Flooring
Sani-Tizer
PAA
20
160
18
5/5
1.00 (0.48, 1.00)
5/5
1.00 (0.48, 1.00)
Unpainted Concrete
MinnCare
35% H2O2
20
500
18
0/5
0.00 (0.00, 0.52)
0/5
0.00 (0.00, 0.52)
Unpainted Concrete
MinnCare
PAA
10
160
18
0/5
0.00 (0.00, 0.52)
0/5
0.00 (0.00, 0.52)
Un painted Concrete
MinnCare
PAA
10
500
18
0/5
0.00 (0.00, 0.52)
0/5
0.00 (0.00, 0.52)
Un painted Concrete
MinnCare
PAA
20
160
18
0/5
0.00 (0.00, 0.52)
0/5
0.00 (0.00, 0.52)
Un painted Concrete
MinnCare
PAA
20
500
18
0/5
0.00 (0.00, 0.52)
0/5
0.00 (0.00, 0.52)
Unpainted Concrete
Sani-Tizer
35% H2O2
10
1000
18
0/5
0.00 (0.00, 0.52)
0/5
0.00 (0.00, 0.52)
Un painted Concrete
Sani-Tizer
35% H2O2
20
1000
18
0/5
0.00 (0.00, 0.52)
0/5
0.00 (0.00, 0.52)
Unpainted Concrete
Sani-Tizer
PAA
10
160
18
0/5
0.00 (0.00, 0.52)
0/5
0.00 (0.00, 0.52)
Unpainted Concrete
Sani-Tizer
PAA
10
1000
18
0/5
0.00 (0.00, 0.52)
0/5
0.00 (0.00, 0.52)
Unpainted Concrete
Sani-Tizer
PAA
20
78
8
0/5
0.00 (0.00, 0.52)
0/5
0.00 (0.00, 0.52)
Unpainted Concrete
Sani-Tizer
PAA
20
160
18
0/5
0.00 (0.00, 0.52)
0/5
0.00 (0.00, 0.52)
Unpainted Concrete
Sani-Tizer
PAA
20
500
18
0/5
0.00 (0.00, 0.52)
0/5
0.00 (0.00, 0.52)
Unpainted Concrete
Sani-Tizer
PAA
20
1000
18
0/5
0.00 (0.00, 0.52)
1/5
0.20 (0.01, 0.72)
Upholstery
MinnCare
PAA
20
160
18
5/5
1.00 (0.48, 1.00)
5/5
1.00 (0.48, 1.00)
Upholstery
Sani-Tizer
8% H2O2
20
2635
168
5/5
1.00 (0.48, 1.00)
5/5
1.00 (0.48, 1.00)
Upholstery
Sani-Tizer
PAA
10
160
18
4/5
0.80 (0.28, 0.99)
5/5
1.00 (0.48, 1.00)
Upholstery
Sani-Tizer
PAA
20
160
18
5/5
1.00 (0.48, 1.00)
5/5
1.00 (0.48, 1.00)
Used Carpet
MinnCare
35% H2O2
20
500
18
0/5
0.00 (0.00, 0.52)
0/5
0.00 (0.00, 0.52)
Used Carpet
MinnCare
PAA
10
160
18
0/5
0.00 (0.00, 0.52)
0/5
0.00 (0.00, 0.52)
Used Carpet
MinnCare
PAA
10
500
18
0/5
0.00 (0.00, 0.52)
0/5
0.00 (0.00, 0.52)
Used Carpet
MinnCare
PAA
20
160
18
1/10
0.10 (0.00, 0.45)
0/10
0.00 (0.00, 0.31)
D-8
-------
Material
Equipment
Decon
liquid
Temp °C
Decon
Volume
(mL)
Time
(Hours)
B.a.
B.g.
Number
Success/N
Proportion
Success
(Exact 95%
Confidence
Interval)
Number
Success/
N
Proportion
Success
(Exact 95%
Confidence
Interval)
Used Carpet
MinnCare
PAA
20
500
18
1/5
0.20 (0.01, 0.72)
0/5
0.00 (0.00, 0.52)
Used Carpet
San
-Tizer
22% H2O2
20
78
18
0/5
0.00 (0.00, 0.52)
0/5
0.00 (0.00, 0.52)
Used Carpet
San
-Tizer
35% H2O2
10
1000
18
1/5
0.20 (0.01, 0.72)
0/5
0.00 (0.00, 0.52)
Used Carpet
San
-Tizer
35% H2O2
20
1000
18
0/5
0.00 (0.00, 0.52)
0/5
0.00 (0.00, 0.52)
Used Carpet
San
-Tizer
8% H2O2
20
2635
168
2/5
0.40 (0.05, 0.85)
4/5
0.80 (0.28, 0.99)
Used Carpet
San
-Tizer
PAA
10
78
18
0/5
0.00 (0.00, 0.52)
0/5
0.00 (0.00, 0.52)
Used Carpet
San
-Tizer
PAA
10
160
18
0/10
0.00 (0.00, 0.31)
0/10
0.00 (0.00, 0.31)
Used Carpet
San
-Tizer
PAA
10
1000
18
2/5
0.40 (0.05, 0.85)
0/5
0.00 (0.00, 0.52)
Used Carpet
San
-Tizer
PAA
20
78
18
0/5
0.00 (0.00, 0.52)
0/5
0.00 (0.00, 0.52)
Used Carpet
San
-Tizer
PAA
20
160
18
0/10
0.00 (0.00, 0.31)
1/10
0.10 (0.00, 0.45)
Used Carpet
San
-Tizer
PAA
20
160
24
0/5
0.00 (0.00, 0.52)
0/5
0.00 (0.00, 0.52)
Used Carpet
San
-Tizer
PAA
20
160
48
0/5
0.00 (0.00, 0.52)
0/5
0.00 (0.00, 0.52)
Used Carpet
San
-Tizer
PAA
20
160
120
0/5
0.00 (0.00, 0.52)
0/5
0.00 (0.00, 0.52)
Used Carpet
San
-Tizer
PAA
20
160
144
0/5
0.00 (0.00, 0.52)
0/5
0.00 (0.00, 0.52)
Used Carpet
San
-Tizer
PAA
20
160
168
0/5
0.00 (0.00, 0.52)
1/5
0.20 (0.01, 0.72)
Used Carpet
San
-Tizer
PAA
20
500
18
4/5
0.80 (0.28, 0.99)
0/5
0.00 (0.00, 0.52)
Used Carpet
San
-Tizer
PAA
20
1000
18
0/5
0.00 (0.00, 0.52)
0/5
0.00 (0.00, 0.52)
Used Grease SOT
MinnCare
35% H2O2
20
500
18
0/5
0.00 (0.00, 0.52)
2/5
0.40 (0.05, 0.85)
Used Grease SOT
MinnCare
PAA
10
160
18
0/5
0.00 (0.00, 0.52)
0/5
0.00 (0.00, 0.52)
Used Grease SOT
MinnCare
PAA
10
500
18
1/5
0.20 (0.01, 0.72)
3/5
0.60 (0.15, 0.95)
Used Grease SOT
MinnCare
PAA
20
160
18
3/5
0.60 (0.15, 0.95)
3/5
0.60 (0.15, 0.95)
Used Grease SOT
MinnCare
PAA
20
500
18
4/5
0.80 (0.28, 0.99)
2/5
0.40 (0.05, 0.85)
Used Grease SOT
Sani-Tizer
35% H2O2
10
1000
18
1/5
0.20 (0.01, 0.72)
2/5
0.40 (0.05, 0.85)
Used Grease SOT
Sani-Tizer
35% H2O2
20
1000
18
2/5
0.40 (0.05, 0.85)
5/5
1.00 (0.48, 1.00)
Used Grease SOT
Sani-Tizer
PAA
10
160
18
1/5
0.20 (0.01, 0.72)
3/5
0.60 (0.15, 0.95)
Used Grease SOT
Sani-Tizer
PAA
10
1000
18
3/5
0.60 (0.15, 0.95)
4/5
0.80 (0.28, 0.99)
Used Grease SOT
Sani-Tizer
PAA
20
160
18
5/5
1.00 (0.48, 1.00)
4/5
0.80 (0.28, 0.99)
Used Grease SOT
Sani-Tizer
PAA
20
500
18
3/5
0.60 (0.15, 0.95)
5/5
1.00 (0.48, 1.00)
D-9
-------
Material
Equipment
Decon
liquid
Temp °C
Decon
Volume
(mL)
Time
(Hours)
B.a.
B.g.
Number
Success/N
Proportion
Success
(Exact 95%
Confidence
Interval)
Number
Success/
N
Proportion
Success
(Exact 95%
Confidence
Interval)
Used Grease Sot
Sani-Tizer
PAA
20
1000
18
5/5
1.00 (0.48, 1.00)
3/5
0.60 (0.15, 0.95)
Used HVAC F
Iter
MinnCare
35% H2O2
20
500
18
4/5
0.80 (0.28, 0.99)
5/5
1.00 (0.48, 1.00)
Used HVAC F
Iter
MinnCare
PAA
10
160
18
0/5
0.00 (0.00, 0.52)
0/5
0.00 (0.00, 0.52)
Used HVAC F
Iter
MinnCare
PAA
10
500
18
4/5
0.80 (0.28, 0.99)
5/5
1.00 (0.48, 1.00)
Used HVAC F
Iter
MinnCare
PAA
20
160
18
5/5
1.00 (0.48, 1.00)
5/5
1.00 (0.48, 1.00)
Used HVAC F
Iter
MinnCare
PAA
20
500
18
5/5
1.00 (0.48, 1.00)
5/5
1.00 (0.48, 1.00)
Used HVAC F
Iter
Sani-Tizer
35% H2O2
10
1000
18
5/5
1.00 (0.48, 1.00)
3/5
0.60 (0.15, 0.95)
Used HVAC F
Iter
Sani-Tizer
35% H2O2
20
1000
18
4/5
0.80 (0.28, 0.99)
5/5
1.00 (0.48, 1.00)
Used HVAC F
Iter
Sani-Tizer
PAA
10
160
18
5/5
1.00 (0.48, 1.00)
5/5
1.00 (0.48, 1.00)
Used HVAC F
Iter
Sani-Tizer
PAA
20
78
8
5/5
1.00 (0.48, 1.00)
5/5
1.00 (0.48, 1.00)
Used HVAC F
Iter
Sani-Tizer
PAA
20
160
18
5/5
1.00 (0.48, 1.00)
5/5
1.00 (0.48, 1.00)
Used HVAC F
Iter
Sani-Tizer
PAA
20
500
18
5/5
1.00 (0.48, 1.00)
5/5
1.00 (0.48, 1.00)
D-10
-------
Table D-5. Parameter Estimates for Final Selected Model Fit to More Balanced Data
Subset.
Variable
Variable Level
DF
Estimate
Standard
Error
Wald
Statistic
p value
Intercept
--
1
3.0269
1.9476
2.4156
0.1201
MATERIAL
Clean Grease
SOT
1
-4.1773
1.2164
11.7930
0.0006*
MATERIAL
Encapsulated
Clean Grease
1
-9.6562
1.6205
35.5091
0.0000*
MATERIAL
Fiberglass Interior
Siding
1
-4.3792
1.2067
13.1705
0.0003*
MATERIAL
Used Carpet
1
-7.3149
1.2727
33.0323
0.0000*
MATERIAL
Used Grease SOT
1
-4.6608
1.2267
14.4358
0.0001*
MATERIAL
Used HVAC Filter
1
-1.4842
1.2654
1.3756
0.2409
EQUIPMENT
MinnCare
1
-0.5114
0.4247
1.4501
0.2285
DECON
35% H2O2
1
-2.5871
0.5243
24.3506
0.0000*
TEMP
10
1
-7.2662
2.3292
9.7323
0.0018*
Log DeconVol
--
1
1.5852
0.6384
6.1657
0.0130*
LOCATION
1
1
-1.0337
0.5177
3.9871
0.0458*
LOCATION
2
1
-1.1647
0.5205
5.0075
0.0252*
LOCATION
4
1
-1.6939
0.5342
10.0558
0.0015*
LOCATION
5
1
-1.9625
0.5425
13.0848
0.0003*
logDeconVol*TEMP
10
1
2.3514
0.8896
6.9859
0.0082*
EQUIPMENTTEMP
MinnCare /10
1
-2.1316
0.8192
6.7701
0.0093*
There is no variable level for intercept of continuous variables.
Statistically significant at a = 0.05 level.
Table D-6. Odds Ratio Estimates for Pairwise Material Comparisons.
Material
Rubber
Flooring
Clean
Grease SOT
Encapsulated
Clean Grease
Fiberglass
Interior
Siding
Used
Carpet
Used Grease
SOT

Odds Ratio Estimate (p-value)#
Clean Grease
0.02





SOT
(0.0006*)





Encapsulated
Clean Grease
0.00
(<0.0001 *)
239.59
(<0.0001A)




Fiberglass
Interior Siding
0.01
(0.0003*)
1.22
(0.7235)
0.01
(<0.0001')



Used Carpet
0.00
(<0.0001 *)
23.05
(<0.0001 *)
0.10
(0.0347*)
18.84
(<0.0001 *)


Used Grease
0.01
1.62
0.01
1.33
0.07

SOT
(0.0001*)
(0.4203)
(<0.0001 *)
(0.5345)
(<0.0001')

Used HVAC
0.23
0.07
0.00
0.06
0.00
0.04
Filter
(0.2409)
(0.0003*)
(<0.0001 *)
(<0.0001 *)
(<0.0001 *)
(<0.0001 *)
#	Odds ratios greater (less) than one indicate that the odds of a success for row label material are
greater (less) than for the column label material.
*	Statistically significant at a = 0.05 level.
D-10
-------
Table D-7. Odds Ratio Estimate for Comparisons of Locations within Chamber.
Contrast
Estimate#
(p-value)
Location 1 vs. Location 3
0.36
(0.0458*)
Location 2 vs. Location 3
0.31
(0.0252*)
Location 4 vs. Location 3
0.18
(0.0015*)
Location 5 vs. Location 3
0.14
(0.0003*)
#	Odds ratios greater (less) than one indicate that the odds of a success for first location are
greater (less) than for Location 3.
*	Statistically significant at a = 0.05 level.
Table D-8. Odds Ratio Estimates for Decontamination SL Comparisons.
Contrast
Estimate
(p-value)
35 % H2O2 vs. PAA
0.08
(0.0000*)
#	Odds ratios less than one indicate that the odds of a success for first sporicidal liquid are greater
(less) than for second SL.
*	Statistically significant at a = 0.05 level.
D-ll
-------
AB. anthracis
IIJB. atrophaeus
Material
Figure D-l. Plot of Control Coupon Percent Recovery of Inoculum by Material and
Agent. Note That Percent Recovery Values Greater than 200 % Are Not Included in the
Plot.
D-l 2
-------
vvEPA
United States
Environmental Protection
Agency
PRESORTED STANDARD
POSTAGE & FEES PAID
EPA
PERMIT NO. G-35
Office of Research and Development (8101R)
Washington, DC 20460
Official Business
Penalty for Private Use
$300
-------