EPA/600/R-17/156 | August 2017
www.epa.gov/homeland-security-
research
United States
Environmental Protection
Agency
&EPA
Evaluation of Commercially-Available
Equipment for the Decontamination
of Bacillus anthracis Spores in an
Urban Subway System
^^DANGER
Office of Research and Development
National Homeland Security Research Center

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EPA/600/R-17/156
August 2017
FINAL REPORT
Evaluation of Commercially-
Available Equipment for the
Decontamination of Bacillus
anthracis Spores in an Urban
Subway System
U.S. Environmental Protection Agency
Research Triangle Park, NC 27711

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Disclaimer
The U.S. Environmental Protection Agency (EPA), through its Office of Research and
Development's (ORD's) National Homeland Security Research Center (NHSRC), funded,
directed and managed this work through Contract Number EP-C-15-002, Task Order 007, with
Battelle. This report has been peer and administratively reviewed and has been approved for
publication as an EPA document. The views expressed in this report 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.
This study was funded through the Underground Transport Restoration Program by the U.S.
Department of Homeland Security Science and Technology Directorate under interagency
agreement (No. 7095866901).
Questions concerning this document or its application should be addressed to:
Dr. Worth Calfee
National Homeland Security Research Center
Office of Research and Development
U.S. Environmental Protection Agency
Mail Code E343-06
Research Triangle Park, NC 27711
919-541-7600
in

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Acknowledgments
Contributions of the following individuals and organization to this report are gratefully
acknowledged:
U.S. Environmental Protection Agency (EPA) Project Team
Worth Calfee (Principal Investigator, EPA, NHSRC)
Sang Don Lee (EPA NHSRC)
Lukas Oudejans (EPA NHSRC)
Shannon Serre (EPA CBRN Consequence Management Advisory Division (CMAD)
Leroy Mickelsen (EPA Office of Land and Emergency Management (OLEM) CMAD
Mike Nalipinski (EPA OLEM CMAD)
EPA Technical Review
Timothy Boe
Edward Bazenas
EPA Quality Assurance
Eletha Brady-Roberts
Ramona Sherman
EPA Editorial Review
Marti Sinclair
Battelle Memorial Institute
iv

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Executive Summary
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 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.
The Underground Transport Restoration (UTR) project is an inter-agency effort. This effort aims
to improve the capability for transit systems to quickly and efficiently recover from a biological
contamination incident by refining existing methods, tools and protocols for characterization,
clean-up, and clearance of contamination in physical structures (i.e., tunnels, stations) and rolling
stock (i.e., subway trains). The aim was to evaluate existing sampling, characterization, and
decontamination technologies through experimentation, table-top exercises and operational
demonstrations to develop guidance and decision frameworks and support tools through
interactions of local, state and federal partners.
In this investigation, a survey of commercially-available or fielded equipment was conducted
and resulted in three pieces of identified equipment that could be used or rapidly modified for
use in dispensing liquid chemicals to decontaminate surfaces following a biological
contamination incident. The equipment selected was the MM Sprayers, Air-O-Fan® (AOF), and
Dust Boss® sprayers. This equipment was selected based on rankings provided by a working
group comprised of EPA, EPA's technical support contractor for this effort, and stakeholders
representing the Transit Authority from across the US. This equipment was subjected to 100
hours of operation with pH-amended bleach (pAB) using smaller proxy equipment designed in
consultation with the vendors of the equipment to test for material compatibility with pAB.
Based on durability assessment, two pieces of equipment (AOF and Dust Boss sprayers) were
further down-selected to participate in a field-scale demonstration at a subway platform/tunnel at
Fort A.P. Hill (Bowling Green, VA). For purpose of demonstration, both pieces of equipment
were placed atop a flatbed railcar and used to spray water while the railcar was pulled through
the subway platform/tunnel at a speed of 1.2 miles per hours (mph). Video and leaf wetness data
(5 locations) were collected during this demonstration. The leaf wetness sensor measures the
percentage of the capacitive grid that is covered by moisture. Based on the leaf wetness data,
review of video, and observer input, a single piece of equipment was selected (AOF sprayer) to
perform field scale efficacy tests using Bacillus atrophaeus (E.g.) spores as a surrogate for B.
anthracis (B.a.), the causative organism of anthrax.
Efficacy testing was conducted within Battelle's ambient breeze tunnel (ABT) testing facility.
The ABT allowed full-scale implementation as the internal dimensions of this facility were
representative of many existing subway tunnels. Decontamination efficacy of operationally
sprayed pAB against surrogate B.g. was evaluated at target delivery speeds of 1.2 and 2.4 mph,
target temperature of 10 degrees Celsius (°C), uncontrolled relative humidity (RH) ranging from
59 to 98 percent (%), vertical and horizontal coupon orientations, and contact times ranging from
30 minutes (min) to 12 hours (overnight) for a total of 4 tests. Ceramic tile resulted > 6 log
reduction (LR) at each condition tested (Tests 1-3). Unpainted concrete resulted in LRs ranging
v

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from 1.62 to 2.34 and 1.32 to 3.02 at locations 1 (column) and 2 (floor), respectively. Since
concrete was more challenging, Test 4 utilized concrete only with repeat applications (2 and 3)
with 30 min contact times between applications. This resulted in increased LR ranging from 3.51
to 4.70 for 2 and 3 applications, respectively. A decontaminant or fumigant technology is
considered to be effective if a 6 LR or greater is achieved on the materials tested for a given set
of fumigation conditions [sporicidal liquid volume, temperature, and relative humidity (RH)](1).
Summary of Major Findings
Over the course of the study all testing conducted with ceramic tile resulted in >6 LR of B.g.,
while no conditions were found that resulted in >6 LR of B.g on unpainted concrete. It was
observed that neither decontamination delivery speeds of 1.2 or 2.4 mph, nor increased contact
times greater than 30 min resulted in a significant effect on LR. Repeat applications of pAB up to
3 each resulted in increased efficacy as shown in Figure ES-lthrough ES-3.
Contact Time Comparison
°	7
»n	r
ON	o
-H
C	5
o
r
3.	3
O	1
-I
~	30 Min Contact
~	Overnight Contact
Column	Floor	Column	Floor
Ceramic Tile	Ceramic Tile Unpainted Concrete Unpainted Concrete
Figure ES-1. 30 min (Test 1) vs overnight contact time (Test 2).
vi

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u
5? 6
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C
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t3
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Contents
Acknowledgments	iv
Executive Summary	v
1.0 Introduction	1
2.0 Market Survey	3
3.0 Durability Testing	6
4.0 Field Scale Demonstration	7
5.0 Efficacy Testing	9
5.1	Test Matrix	9
5.2	Biological Organism	9
5.3	Test Materials	9
5.4	Inoculation of Coupons	10
5.5	Spraying Equipment	11
5.6	Ambient Breeze Tunnel and Procedures	12
5.7	Coupon Extraction and Biological Agent Quantification	14
5.8	Decontamination Efficacy	14
5.9	Surface Damage	16
6.0 Quality Assurance/Quality Control	17
6.1	Equipment Calibration	17
6.2	QC Results	17
6.3	Operational Parameters	17
6.4	Audits	18
6.5	QA/QC Reporting	19
6.6	Data Review	19
7.0 Results and Discussion	20
7.1	Durability Testing	20
7.2	Field Scale Demonstration	22
7.3	Efficacy Testing	24
7.4	Surface Damage to Materials	26
7.5	Summary	26
8.0 References	28
viii
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Figures
Figure ES-1. 30 min (Test 1) vs overnight contact time (Test 2)	vi
Figure ES-2. 1.2 mph (Test 2) vs 2.4 mph application rate (Test 3)	vii
Figure ES-3. Test 2, 4a, and 4b repeat application comparison	vii
Figure 2-2. Air-O-Fan D-40R radial type sprayer	4
Figure 2-1. MM Sprayers USA MM LG 400 radial fan sprayer	4
Figure 2-3. Dust Boss DB-30 sprayer	5
Figure 3-1. Durability test device	6
Figure 4-1. Demonstration equipment on railcar	7
Figure 4-2. Schematic of Fort A.P. Hill subway tunnel	8
Figure 5-1. Coupon types from left to right: unpainted concrete, ceramic tile on test fixture.
Orange tab indicates blank coupon	10
Figure 5-3. Customized AOF Model 2-36 sprayer	11
Figure 5-2 Liquid inoculation of coupon using a micropipette	11
Figure 5-3. Customized AOF Model 2-36	12
Figure 5-5. AOF and test cart configuration.Figure 5-3. Customized AOF Model 2-36	12
Figure 5-5. AOF sprayer and test cart configuration	13
Figure 5-4. Ambient breeze tunnel test facility	13
Figure 7-1. MM sprayer failure	20
Figure 7-2. Dust Boss nozzle failure. From left: control, test 1 test 2 and test 3 nozzles	21
Figure 7-3. Durability test flow rate	21
Figure 7-4. Durability test pressure	21
Figure 7-5. AOF nozzle failure. From left: control, test 1, test 2, and test 3 nozzles	22
Figure 7-6. AOF sprayer Fort A.P. Hill (FAPH) demonstration % wetness	23
Figure 7-7. Dust Boss sprayer Fort A.P. Hill (FAPH) demonstration % wetness	23
Figure 7-8. 30 min (Test 1) vs overnight contact time (Test 2)	24
Figure 7-9. 1.2 mph (Test 2) vs 2.4 mph application rate (Test 3)	25
Figure 7-10. Test 2, 4a, and 4b comparison	25
Figure 7-11. Test 2, 4a, 4b, and 4c comparison	26
Figure Dl. pH amended bleach (pAB) vs 2% dilute bleach at 2.4 mph	D-2
Figure D2. Efficacy of pH amended bleach (pAB) at varied application speeds	D-3
Figure D3. Mass deposition of water at various delivery speeds	D-3
Figure D4. Efficacy of multiple applications of pH amended bleach (pAB) and 2% bleach	D-4
Tables
Table 2-1. Equipment Scoring Criteria	3
Table 2-2. Final Down Selected Equipment for Subway Decontamination	4
Table 5-1 pH Amended Bleach Decontamination Test Matrix using the AOF at 10 °C	9
Table 5-2. Test Materials	10
Table 6-1. Actual Operational Conditions for Tests	18
Table 6-2. Performance Evaluation Audits	18
Table 7-1. Fort A.P. Hill Subway Percent Wetness	23
Table A-l. Inactivation of B. atrophaeus Spores using pH Amended Bleacha	A-l
Table B-l. Durability Testing Pressure	B-l
Table B-2. Durability Testing Flow	B-2
IX
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Table B-2. Durability Testing Flow (Continued)	B-2
Table C-l. Commercial Equipment for Subway Decontamination	C-l
Table C-2. Commercial Equipment for Subway Decontamination (Continued)	C-Error!
Bookmark not defined.
Table D-l. Add-on Inactivation of B. atrophaeus Spores using pH Amended and dilute 2%
Bleacha	D-l
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List of Appendices
Appendix A Detailed Test Results	A-l
Appendix B Detailed Durability Results	B-l
Appendix C Detailed Market Survey Results	C-l
Appendix D Mod 5 Add-on Test Results	D-l
Appendix E Technology Demonstration Feedback Results	E-l
XI
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Abbreviations/Acronyms
AO AC	Association of Analytical Communities
ABT	Ambient Breeze Tunnel
ATCC	American Type Culture Collection
B. a.	Bacillus anthracis
B.g.	Bacillus atrophaeus
BSC	biological safety cabinet
CFU	colony forming units
CI	confidence interval
cm	centimeter(s)
CMAD	Consequence Management Advisory Division
°C	degree(s) Celsius
Decon	decontamination
EPA	U.S. Environmental Protection Agency
FAPH	Fort A.P. Hill
HSRP	Homeland Security Research Program
kg	Kilogram
kPa	kilopascal
Kw	kilowatt
L	liter(s)
lpm	liters per minute
LR	log reduction
l_iL	microliter(s)
m	meter
mph	miles per hour
mL	milliliter(s)
min	minute(s)
NA	not applicable
NHSRC	National Homeland Security Research Center
NIST	National Institute of Standards and Technology
OLEM	Office of Land and Emergency Management
pAB	pH-amended bleach
PBS	phosphate buffered saline
PBST	PBS + 0.1% Triton X-100
PCR	polymerase chain reaction
ppm	parts per million
ppmv	parts per million by volume
psi	pounds per square inch
QA	quality assurance
QAPP	Quality Assurance Project Plan
xii
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QC	quality control
QMP	Quality Management Plan
RH	relative humidity
rpm	revolution(s) per minute
s	second(s)
SD	standard deviation
SE	standard error
UTR	underground transportation restoration
xiii
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1.0 Introduction
The U.S. Environmental Protection Agency's (EPA's) Homeland Security Research Program
(HSRP) is helping protect human health and the environment from adverse impacts resulting
from the release of chemical, biological, or radiological agents. The program's emphasis is on
decontamination and consequence management, water infrastructure protection, and threat and
consequence assessment. The HSRP is working to develop technology and information that will
detect the 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.
Contamination of an underground transportation system (i.e., subway tunnel or platform)
following a biological terror incident would have a crippling effect on a city's economy and
stability. Rapid decontamination following such an incident is paramount for returning to
normalcy. To facilitate a rapid mobilization following an incident and to shorten the remediation
process, utilization of commercially available equipment is desirable. Equipment that can rapidly
be obtained and utilized to dispense liquid sporicides to subway infrastructure would be very
useful for underground transportation system decontamination.
In this investigation, a survey of currently fielded equipment or other readily-adaptable
commercially available equipment that could dispense liquid sporicides was conducted and
compiled into a spreadsheet containing applicable operational specifications. This spreadsheet
was evaluated by a working group comprised of EPA, EPA's support contractor for this for
effort (Battelle), and transit authority staff. The groups provided ranking scores in one of three
categories (Commercial Readiness, Ease of Deployment, and Decontamination ("Decon")
Application Rate). The scores across all categories were then aggregated and used to select the
top three technologies. A durability assessment was conducted for each piece of equipment to
identify potential material compatibility issues with the selected sporicidal liquid, pH-amended
bleach (pAB). Due to the large scale of the equipment being investigated, a series of proxy
equipment were designed, with input from the equipment manufacturers, using parts from the
larger equipment and were tested in triplicate for up to 100 hours of operation. pAB or water
(control) flow rate and pressure were recorded at the beginning and end of each day of testing.
Any observable changes to operational pressure or flow rate were investigated as potential
failure and documented with photographs.
A field demonstration was conducted at Fort A.P. Hill (Bowling Green, VA) facility using the
top two performing pieces of equipment as measured by the durability assessment. This facility
houses a subway training facility with an approximate 84 meter (m) subway platform and 113 m
subway tunnel. Each piece of equipment was operated by spraying water and was pulled through
the subway tunnel/platform at a fixed speed of 1.2 miles per hour (mph). Leaf wetness sensors
and video were utilized to determine the amount and efficiency of each equipment to deposit
liquid onto several complex surfaces and orientations. The leaf wetness sensor measures the
percentage of the capacitive grid that is covered by moisture.
Finally, field-scale efficacy tests were conducted to evaluate the performance of the
demonstrated equipment for decontamination of materials found in subway tunnels or stations
that could become contaminated with biological agents such as Bacillus anthracis (B.a.) spores.
(B. anthracis is the bacterial pathogen that causes anthrax.) The efficacy of pAB was evaluated
1
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on both unpainted concrete and ceramic tile contaminated with Bacillus atrophaeus (B.g.) as a
surrogate spore for B.a. Decontamination efficacy was determined based on the log reduction
(LR) in viable spores recovered from the inoculated samples (with and without exposure to the
sporicidal liquids). A decontaminant or fumigant technology is considered to be effective via
Association of Analytical Communities (AOAC) (test method 966.04) if a 6 LR or greater is
achieved on the materials tested (AOAC material types not used for present study) for a given set
of fumigation conditions [sporicidal liquid volume, temperature, and relative humidity (RH)](1).
The results of this investigation provide decontamination stakeholders and decision makers with
high quality, peer-reviewed data on the use of equipment to disperse sporicidal liquids in a
subway environment, as a function of the spore type, the material the spore is associated with,
temperature, equipment type and sporicidal liquid used.
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2.0 Market Survey
A thorough search was conducted for equipment currently fielded or equipment readily capable
of disseminating large amounts of liquids in a quick and efficient manner and capable of
targeting the multiple surface orientations that exist in a complex subway system. Information
such as operational pressure, liquid flow rate, mode of liquid delivery, power requirements, tank
size, material of construction, weight, and dimensions were collected and compiled into a
spreadsheet that can be found in Appendix C in its entirety. The information was collected using
internet search engines, literature review and by leveraging connections within the working
group for recommendations based on practical industrial use and knowledge. Members of each
part of the working group reviewed the developed spreadsheet and provided ranked scores in one
of three categories: Battelle ranked Commercial Readiness, Transit Authorities ranked Ease of
Deployment, and EPA ranked Decon Application Rate (Table 2-1). Each category had a possible
ranking from 1 to 5, with the highest possible total aggregate score of 15. The summed total of
the three categories were aggregated for all groups.
Table 2-1. Equipment Scoring
Brief
Score Description
Long Description

1
Not at all
not commercially available

2
Poor
very limited commercial availability
Commercial
Readiness
3
Moderate
available, but in limited quantities or select locations

4
Good
generally available

5
Excellent
readily available, multiple vendors, any region in US

1
Not at all
unable to deploy equipment in subway environments

2
Poor
major technical hurdles to deploy equipment in subways

3
Moderate
some modifications needed for deployment in subways
Deployment
4
Good
minor modifications or logistical challenges for deployment in
subways (i.e., needs flatbed railcar to transport)

5
Excellent
easily deployed into subways with no modification (i.e., can be
directly deployed to subways with no modification or additional
equipment required)

1
Not at all
unable to dispense decontaminants, due to incompatibility or
technical issues

2
Poor
very limited ability to dispense decontaminants, application rate or
spray reach is insufficient for subway application
Decon
Application
Rate
3
Moderate
moderate ability to dispense decontaminants, application rate or spray
reach is acceptable for some subway applications

4
Good
good ability to dispense decontaminants, application rate or spray
reach is acceptable for most/many subway applications

5
Excellent
superior ability to dispense decontaminants, application rate or spray
reach is acceptable for all subway applications
3
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A total of 22 pieces of equipment were identified from several industrial sectors such as
agricultural sprayers, roadway de-icing equipment, construction dust suppression, crowd cooling,
firefighting, and insect control, which were divided into six categories (Radial Fan Sprayers, Air
Directed Sprayers, Electrostatic Sprayers, Dust Suppression Equipment, Foggers and De-icing
Equipment). In some cases, multiple versions or brands of a similar technology existed. To
make the final evaluation spreadsheet as concise as possible, one to two representative
technologies were selected where market availability and lower cost was preferred. The final
raking was achieved by aggregating all scoring across functional working groups per piece of
equipment. The top three pieces of equipment were chosen to be carried through for durability
testing (Table 2-2). These top three pieces of equipment were all found to be: readily available;
able to be deployed in a subway environment with minimal modifications; and capable of
dispensing liquid decontaminants into a subway system environment.
Table 2-2. Final Down-Selected Equipment for Subway Decontamination
SCORE Company Category Model # Web link to sprayer
12.3
MM
Sprayers
USA
Radial Fan
Sprayer
MM LG
400
http://www.mmspraversusa.com/product/mm-le-
400-eas-trailer/
11.1
Air-O-fan
Radial Fan
Sprayer
D-40R
1,000
Gallon
http: //airofan .com/OrchardSpravers/Engine Drive/
D40RModel/D40Rl OOOGal.aspx
10.8 Dust Boss „ Dust	DB-30 http://www.dustboss.com/Droducts/db-30/
Suppression
The top scoring piece of equipment was the MM Sprayers
USA MM LG 400 (MM Sprayers USA, Lynden, WA) radial
fan sprayer (Figure 2-1). This sprayer is typically used in the
vineyard or berry-growing industry. It has a 400 liter (L)
polyethylene tank, 0.64 m diameter fan, 10 Braglia brass
swivel rollover anti-drip nozzle bodies with TeeJet" stainless
steel hollow cone
D4 spray tips Figure 2-1. MM Sprayers USA
( ee et	MM LG 400 radial fan sprayer.
Technologies,
Glendale Heights, IL). It uses a A.R. 403 diaphragm
pump, powered by a 13 horse power Honda engine.
Dimensions of the equipment are 1.2 m width, 1.4 m
height, 2.5 m length with a dry weight of 264
kilograms (kg). Typical cost of this unit is
approximately $10,000.
Figure 2-2. Air-G-Fan D-40R
radial type sprayer.
The second ranked piece of equipment was the Air-
O-Fan (AOF) D-40R 3800 L (Air-O-Fan Products
Corp, Reedley, CA) radial type sprayer (Figure 2-2).
This sprayer is typically used in the orchard and nut-
4
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growing industry. Several models of this type of
sprayer are available. It has a 3800 L type 304
stainless steel tank and is expandable through use
of rapid fill cam lock fittings. The sprayer is
powered with 156 John Deere® (Deere & Co.,
Moline, IL) diesel engine and powers a 2-stage
200 pounds per square inch (psi) centrifugal pump
and twin steel co-axial fans capable of throwing
liquid droplets from 43 m to 82 m. Composite
nylon adjustable air vanes house up to two nozzles
per vane (total 36). Liquid flow rates are
controllable between 1 and 380 liters per minute
(lpm). Dimensions of the equipment are 2.7 m
width, 1.5-1.8 m height, 6.2 m length and a dry
weight of 3838 kg. Equipment costs range from Figure 2-3. Dust Boss DB-30 sprayer.
$60,000 to $100,000 based on model type and
options selected.
The third ranked piece of equipment was the Dust Boss® DB-30 (Figure 2-3) sprayer (BossTek,
Peoria, IL). This equipment is used in demolition and other industrial settings as a dust
suppression cannon. Several models of this sprayer are available. This sprayer is powered by an
electric direct drive motor and is capable of throwing liquid droplets up to 30 m with liquid
delivery rates from 5.3 to 10.6 lpm. Standard unit oscillates through a 70 degree pattern and has a
manual 0 to 50 degree vertical angle adjustment. Unlike the first two pieces of equipment, this
device requires external input for liquid at approximately 50 psi and electric power (60 kilowatt
[kw] generator). Equipment costs range from $22,000 to $60,000 based on model type but it is
worth noting this does not include power generation or pressurized liquid delivery.
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3.0 Durability Testing
The ability to use commercially available equipment provides advantages in terms of reduced
implementation time, less operators per surface/length, and potential lower overall cost.
However, a potential drawback is the equipment may not be fully compatible with many of the
sporicidal liquids considered for field-scale remediation. Many of these chemicals are corrosive,
such as pAB, and can cause premature material degradation and equipment failure.
To assess this potential in advance of field-scale testing, a laboratory-scale durability test was
conducted for the three down-selected pieces of equipment to identify potential material
compatibility issues with the selected sporicidal liquid, pAB. Due to the large scale of the
equipment being investigated, a series of smaller proxy equipment was designed using parts or
representative parts from the larger equipment and were tested in triplicate for up to 100 hours of
operation (Figure 3-1). Each vendor was consulted and asked to provide a list of the wetted
components within their larger equipment
to aid in design. During testing, pAB
(n=3) or water (control, n I) flow rate
and pressure were recorded at the
beginning and end of each day using a
timed collection into a graduated cylinder
and a Wika® Model 2135325 National
Institute of Standards and Technology
(NIST) traceable pressure gauge (Wika
Instrument, Lawrenceville, GA). The
system was designed such that the liquid
exiting the spray nozzles was collected
into flexible tubing and recirculated back
to the liquid holding tank. Any observable
changes were investigated as potential
failures and documented with
photographs.
During a typical test run, the pAB
solution was prepared fresh each day as
described in the EPA crisis exemption
requirements for use against B.a. spores'2'. The solution was prepared by combining one part
Germicidal Clorox® Bleach (Clorox Corp., Oakland, CA, USA) with eight parts deionized water
and one part 5% (v/v) Heinz1' distilled white vinegar (Kraft Heinz Company, Mendota Heights,
MN, USA). The pH was adjusted to 6.5-7.0 with additional vinegar, and the free available
chlorine content was measured and acceptable if > 8000 parts per million by volume (ppmv).
Pressure measurements were collected by briefly starting each sprayer and recording pressure
from NIST traceable pressure gauges. Flow rate was measured by a timed collection of fluid
exiting each individual spray nozzle. The equipment was then operated for a defined amount of
time per test day. After each test, pressure, flow rate, free available chlorine, and pH
measurements were collected and recorded.
Figure 3-1. Durability test device.
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4.0 Field Scale Demonstration
A field-scale operational demonstration was performed on October 13, 2016 at Fort A.P. Hill
(FAPH) in Virginia. The FAPH maintains a subway platform and a tunnel with an approximate
84 m subway platform and 113 m subway tunnel that was used for this testing. Based on
performance from the durability testing, the AOF and Dust Boss systems were selected by the
EPA working group for use.
Due to weight limitations of the cart intended for use initially (Xinxiang Hundred Percent
Electrical and Mechanical Co., Ltd, Model: KPX40T, He'nan Provice, China), a few
modifications to the AOF equipment were necessary. At the time of the demonstration, a full-
size flatbed railcar became available and was utilized for the demonstration. A smaller D2-36
model was selected and modifications included removal of the pneumatic wheels (unit to be
mounted on rail car), exchange of diesel engine for VI0 aluminum block gasoline engine,
smaller 250-gallon stainless steel tank, and an added 12 foot downward facing spray bar. The
AOF and Dust Boss equipment (including 60 kw generator) were placed atop a flatbed rail car
(Figure 4-1) and pulled through the subway tunnel and platform at a fixed speed of 1.2 mph
using a Maxi railcar mover (Railquip Inc, Atlanta, GA). Each piece of equipment was operated
(one at a time) while the railcar was pulled through the entire length of the tunnel. Multiple runs
Figure 4-1. Demonstration equipment on railcar.
were conducted for each piece of equipment.
Leaf wetness sensors were placed at 5 locations within the platform or tunnel sections to assess
the distribution of liquid droplets as total percent coverage over a range of orientations as noted
in Figure 4-2. In addition, high definition video of each equipment test run was collected from
various angles and was provided as a deliverable in addition to the report.
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Sensor #3 —	— Sensor 01
(Railing)	airs	(Back Column)
Tunnel Entrance
Sensor 85 —
(Tunnel Wall)
	i
Sensor #4	Sensor #2
(Tracks) q	A <«"'> 0	Column
Figure 4-2. Schematic of Fort A.P. Hill subway tunnel.
8
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5.0 Efficacy Testing
This section provides an overview of the procedures used for the field-scale evaluation of
commercially-available equipment for spraying sporicidal liquids to inactivate B.g. on up to two
material types. Testing was performed in accordance with EPA ORD's QA program and
Battelle's QA program.
5.1 Test Matrix
The test matrix for the decontamination tests is shown in Table 5-1. Tests 1-3 were performed
using two materials commonly found in a subway environment (ceramic tile and unpainted
concrete) while Test 4 used unpainted concrete only. Operational parameters were chosen to
assess conditions representative of field environments and equipment in the case of a wide area
subway contamination event. Testing was conducted at a target of 10 °C, but varied due to the
field-scale testing environment.
Table 5-1 pH Amended Bleach Decontamination Test Matrix using the AOF Sprayer at
10 °C

Operational Parameters

Test Number
Equipment
Speed
Contact Time
Number of
Applications
Materials
1

30 Min
1
Ceramic Tile, Unpainted Concrete
2

Overnight
1
Ceramic Tile, Unpainted Concrete
3
1.2 mph
Overnight
1
Ceramic Tile, Unpainted Concrete
4a
Overnight
2*
Unpainted Concrete
4b

Overnight
3*
Unpainted Concrete
4c

Overnight
4*
Unpainted Concrete
*30 min contact time observed between applications.
5.2	Biological Organism
The B.g. spores (Lot DJS-BG-004) were supplied in powder form originally obtained from
Dugway Proving Ground (Tooele County, UT). The B.g. stock spore suspensions were prepared
in sterile phosphate-buffered saline (PBS) at an approximate concentration of 1 x 109 colony
forming units per milliliter (CFU/mL) and stored at 2 to 8 degrees Celsius (°C). Genomic DNA
was extracted from the spores and DNA fingerprinting by polymerase chain reaction (PCR) to
confirm the genotype (matches ATCC® 9372™, Manassas, VA). In addition, the number of viable
spores was determined by colony count and expressed as CFU/mL. Theoretically, once plated onto
bacterial growth media, each viable spore germinates and can yield one CFU although the
possibility does exist that multiple spores, if co-located when plating, can also result in one CFU.
5.3	Test Materials
Decontamination efficacy testing was conducted using common subway tunnel materials
(ceramic tile and unpainted concrete). Information on these materials is presented in Table 5-2,
and a picture of each is presented in Figure 5-1. Material coupons were cut to uniform length and
width (Table 5-2) from larger pieces of stock material. Materials were prepared for testing by
sterilization via autoclave at 121 °C, 103 kPa for 15 min. Autoclaved coupons were sealed in
sterilization pouches (Cat. No. 01-812-50, Fisher, Pittsburgh, PA) to preserve sterility until the
9
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coupons were ready for use. Sterilization was intended to eliminate contamination by
endogenous microorganisms.
e © @ —
Figure 5-1. Coupon types from left to right: unpainted concrete, ceramic tile on test
fixture. Orange tab indicates blank coupon.
Table 5-2. Test Materials
Material
Lot, Batch, or ASTM No.,
or Observation
Manufacturer/
Supplier Name
Location
Approximate Coupon Size,
Width x Length x Thickness
Material
Preparation
Ceramic
Tile
Model: PWHITW91L01
Lowes,
Milliard. OH
1.9 cm x 3.8 cm x 0.2 cm
Autoclave
Unpainted
Concrete
ASTM C90 cinder block
Wellnitz,
Columbus, OH
1.9 cm x 7.6 cm x 0.2 cm
Autoclave
5.4 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 i 10s CFU of viable B.g. spores per coupon.
A 100 microliter ( |_i L) aliquot of a stock suspension of approximately 1 * 109 CFU/mL was
dispensed using a micropipette applied as 10 |iL droplets across the coupon surface (Figure 5-2).
This approach provided a more uniform distribution of spores across the coupon surface than
10
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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 ceramic tile (nonporous material) while they soaked into the unpainted
concrete materials after
producing a liquid bead
for a short period. 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 left undisturbed
overnight to dry under
ambient conditions,
approximately 22 °C and
40% RH.
The number and type of replicate coupons used for each combination of material, decontaminant,
concentration and environmental condition included:
•	Three test coupons (inoculated with B. cttrophaeus spores and sprayed with sporicidal
liquid)
•	Three positive controls (inoculated with B. atrophaeus spores and sprayed with water)
•	One laboratory blank (not inoculated and not sprayed with sporicidal liquid)
•	One procedural blank (not inoculated and sprayed with sporicidal liquid).
On the day following inoculation, coupons intended for decontamination (including blanks) were
transferred to the ambient breeze tunnel (ABT) test facility, placed in one of two designated
positions, and exposed to the pAB using the AOF apparatus and application conditions specified
in Section 2.5. Details of coupon handling can be found in Section 5.6.
5.5 Spraying Equipment
Figure 5-3 is a photo of the modified AOF 2-36 orchard sprayer used for both the field scale
demonstration and efficacy testing. Although a larger D-40R model was selected in the market
suivey portion of the study, due to weight and size limitations for the field scale dem onstration
cart, a few modifications were necessary along with selecting the smaller sized D2-36 model.
Modifications included removal of the pneumatic wheels (unit to be mounted on rail car),
exchange of diesel engine for VI0 aluminum block gasoline engine, smaller 250-gallon stainless
Figure 5-2 Liquid inoculation of coupon using a micropipette.
11
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steel tank, and an added 3.7 m
downward facing spray bar.
The orchard sprayer fluidics
were controlled by an AgOtter
controller (AgOtter, Tempe,
AZ) which uses global
positioning systems and axle
mounted monitors to
determine speed and location
of the equipment. This device
continuously adjusts a series
of valves to ensure the target
surfaces are getting an equal
amount of liquid deposition
regardless of ground speed.
The 2-stage 200 pounds per Figure 5-3. Customized AOF Model 2-36
square inch (psi) centrifugal
myers pump supplied the water or pAB to 85 Teejet ceramic conejet (TeeJet Technologies,
Glendale Heights, IL Model# TXR800013) nozzles including 12 that were downward facing.
5.6 Ambient Breeze Tunnel and Procedures
Decontamination testing was conducted inside Battelle's ABT test facility located in West
Jefferson, Ohio, which has height and width dimensions of similar scale to many subway tunnels
(20 feet W x 20 feet H x 135 feet L). Figure 5-4 shows the exterior of the ABT test facility. The
ABT has an upstream and downstream blower exhaust system that was operated during each test
to achieve an approximately 1.5 mph cross wind. This not only replicated wind that would be
generated by traversing through the subway at 1.2 mph, it also minimized the amount of
wraparound from the pAB to the engine and operator area of the test equipment. Once testing
had concluded each day, all exhaust blowers were turned off to minimize any enhanced drying
effect from the excess air movement.
12
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Figure 5-4. Ambient breeze tunnel test facility.
measurements every ten
seconds for the duration
of each spray application.
Two test positions were
selected that exhibited the
lowest percent wetness
from an average of three
tests. These positions
included one vertical right
side of column position
(location 1), and one
horizontal floor (location
2).
Testing targeted 10 °C to replicate conditions commonly found in underground subway tunnels
and platforms. Due to the scale of testing (field scale), temperature was not controllable and
therefore tests were planned for days that met temperature requirements, which resulted in higher
Due to the large size and weight of the AOF sprayer, it was determined that controlling
movement of the test coupons would be more efficient than controlling the AOF sprayer. A
custom cart was designed and fabricated that would allow test coupons to be held at 5 different
orientations to the spray plume (Figure 5-5). These locations would be representative of the
tracks on the floor and all four sides of a pillar commonly found in a terminal platform location.
At each location, wetness was measured in terms of total percent coverage using a HOBO® S-
LWA-M003 leaf wetness
sensor connected to a
FIOBO H21-002 micro
station data logger (Onset,
Bourne, MA), which
recorded wetness
Figure 5-5. AOF sprayer and test cart configuration.
13
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than normal variability. Temperature and RH in the ABT were measured every minute during
experimental exposure using an HOBO MX temp/RH data logger.
Each test consisted of coupon inoculation the afternoon before testing was to occur. The
following day, those test coupons were transferred to the ABT testing facility and the control
coupons and blanks were loaded into test positions and held in place by metal clips. Once
operational test temperatures were observed the sprayer was operated with water and the test cart
was moved through the spray plume at the desired speed using a 1 horse power variable speed
DC motor (Dayton Manufacturing Co., Niles, IL) over a 15.2 m aluminum track. Control
coupons and laboratory blanks were then collected and placed into clean 50 mL conical tubes
and kept in the same orientation (vertical/horizontal) as they were sprayed. The caps of the
conical tubes were left open during the exposure to maintain evaporative effects. Each coupon
platform was then washed with bleach followed by ethanol wipe to minimize any carry over of
viable control organism to test coupons. The test coupons and procedural blanks were then
placed onto the test cart and the pAB was prepared within the AOF equipment. Aliquots were
removed to ensure proper concentration and pH of the prepared pAB. The pH was measured
using a handheld Thermo Seven-Go pH meter (Thermo Scientific, Waltham, MA). pAB ppm
was measured an iodometric determination of chlorine dioxide and chlorite using a HACH® test
kit (HACH, Loveland, CO). The test cart was then traversed through the spray plume and
coupons collected in an identical manner as the controls. The test and control coupons were held
in the ABT for the defined contact time per test.
5.7	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 phosphate buffered saline with 0.1%
Triton™ x-100 [Sigma-Aldrich, St. Louis, MO (PBST)]. The vials were capped, placed on their
side and agitated on an orbital shaker for 15 minutes (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
sterile filtered water. 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 hours 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 that could have been inadvertently introduced to
test coupons. The target acceptance criterion for extracts of laboratory or procedural blanks was
zero CFU.
After each decontamination test, the test cart was thoroughly cleaned (using separate steps
involving bleach, ethanol, then drying).
5.8	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)
14
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where Mean CFUpc is the mean number of CFU recovered from three 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.g. on each coupon, and the results
are included in Section 7 and Appendix A.
The performance or efficacy of the sporicidal liquids was assessed by determining the number of
viable spores (CFU) 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.g. in extracts of test and positive control coupons was
determined to calculate efficacy of the decontaminant. Efficacy is defined as the extent (as logio
reduction or LR) to which viable spores extracted from test coupons after decontamination were
less numerous than the viable spores extracted from positive control coupons. The logarithm of
the CFU abundance from each coupon extract was determined, and the mean of those logarithm
values was then determined for each set of control and associated test coupons, respectively.
Efficacy of a decontaminant for a test organism/test condition on the z'th coupon material was
calculated as the difference between those mean log values, i.e.:
where logio CFUcy refers to the j individual logarithm values obtained from the positive control
coupons and logio CFUty 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 three positive controls and three corresponding test coupons (i.e. J = 3) for
each coupon. A decontaminant or fumigant technology is considered to be effective via AO AC
test method 966.04 if a 6 LR or greater is achieved (1).
In the case where no viable spores were found in any of the three 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 CFUctj and logio CFUtij values were also
calculated for both the control and test coupons (i.e., S2Cij and S2ty), and were used to calculate
the pooled standard error (SE) for the efficacy value calculated in Equation 2, as follows:
where the number 3 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:
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
Efficacy (LR) = (log10 CFUcy) - (log10 CFUti})
(2)
(3)
95 % CI = Efficacy (LR) ± (1.96 x SE)
(4)
15
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significant if the 95% CIs of the two efficacy results did not overlap. Any results based on this
formula are hereafter noted as significantly different. Note this comparison is not applicable
when the two efficacy results being compared are both reported with LRs as > some value.
5.9 Surface Damage
The physical effect of the sporicidal liquids as delivered by the AOF equipment on 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.
16
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6.0 Quality Assurance/Quality Control
Quality assurance (QA) and quality control (QC) procedures were performed in accordance with
with EPA ORD's QA program and Battelle's QA program. The QA/QC procedures and results
are summarized below.
6.1	Equipment Calibration
All equipment (e.g., pipettes, incubators, wetness sensor, 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.
6.2	QC Results
QC efforts conducted during decontaminant testing included positive control samples procedural
blanks, laboratory blanks, and inoculation control samples.
Positive control results were in many cases lower than the target recovery range of 5 to 120% of
the inoculated spores. Recoveries ranged from 3.55% to 15.3%, and 0.13% to 3.24% from
ceramic tile and unpainted concrete, respectively. Low recoveries from unpainted concrete are
not uncommon due to the porosity of the materials; however, the low recoveries from the
ceramic tile were most likely due to mechanical removal of spores from the coupons as observed
by re-deposition and enumeration of target bacterial colonies from blank materials of both
unpainted concrete and ceramic tile. LRs of >6 were achievable in most instances, even with the
low recoveries.
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.
6.3	Operational Parameters
The temperature, RH, and wetness for each test was monitored as described in Section 5.0. For
all tests, the temperature and relative humidity was uncontrolled but monitored as described in
Section 5.6. Testing was scheduled based on weather forecasting and not initiated until minimum
of 7.2 °C was achieved. Readings were taken once every minute for the duration of the spray and
contact time. The percent wetness was measured for all 5 locations (only target locations
reported) every 10 seconds for the duration of the spray and up to 15 min post application. The
actual operational parameters for each test are shown in Table 6-1 and reported as the average
value ± SD.
17
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Table 6-1. Actual Operational Conditions for Tests
Test
Number
Avg. Control Wetness
Avg. pAB Wetness
Temperature (°C)
RH (%)
Contact
Time
(hours)
Location #1
Location #2
Location #1
Location #2
Target
Actual*
Target
Actual*
1
30.2 ±3.81
98.0 ± 10.9
43.4 ±0.83
98.3 ±3.33
10
15.5 ±1.17
None
59.1 ±3.93
0.5
2
27.1 ±5.23
72.5 ± 10.2
45.4 ±4.58
84.8 ±7.87
10
12.7 ± 1.41
None
79.7 ±7.74
12T
3
37.1 ±2.40
54.1 ±4.12
43.3 ± 11.0
57.1 ±20.5
4a
27.5 ±2.85
62.3 ±14.5
30.0 ± 1.38
48.5 ±1.80
10
11.2 ± 1.26
None
98.0 ±4.94
10t
4b
26.9 ±5.66
47.5 ±4.39
55.2 ±3.82
72.7 ±3.37
4c
36.7 ±4.36
60.1 ±5.92
63.9 ±4.71
66.5 ±5.70
* Data reported as average ± SD.
t Overnight contact time, samples were not staggered and thus value is approximate.
6.4 Audits
6.3.1 Performance Evaluation Audit
Performance evaluation (PE) audits were conducted to assess the quality of the results obtained
during these experiments. Table 6-2 summarizes the PE audits that were performed.
No PE audits were performed for confirmation of the concentration and purity of B.g. spores
because quantitative standards do not exist for this organism. The titer enumerations and the
control and blank test coupons support the spore measurements.
Table 6-2. Performance Evaluation Audits
Measurement
Audit
Procedure
Allowable
Tolerance
Actual
Tolerance
Volume of liquid from
micropipettes
Gravimetric evaluation
± 10 %
±0.8%to 3.0%
Time
Compared to independent clock
± 2 seconds/hour
0 seconds/hour
Temperature
Compared to independent calibrated
thermometer
± 2 °C
±0.58 to 1.73 °C
Relative Humidity
Compare to independent calibrated
hygrometer
± 10%
± 0.04 to 0.67 %
6.3.2 Technical Systems Audit
Observations and findings from the technical system audit were documented and submitted to
the laboratory technical lead for response. The audit was conducted on November 29, 2016 to
ensure that tests were conducted in accordance with the QAPP. 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 audit required corrective action.
6.3.3 Data Quality Audit
At least 10 % of the data acquired during the evaluation were audited. Data was reviewed in one
batch February 2017. 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
18
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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.
6.5	QA/QC Reporting
Each assessment and audit was documented in accordance with EPA ORD's QA program and
Battelle's QA program. 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 EPA ORD's QA program and
Battelle's QA program.
6.6	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.
19
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7.0 Results and Discussion
A survey of commercially available or fielded equipment was conducted in order to inform
remediation efforts following a large-scale contamination incident involving an underground
transportation system. A working group, comprised of EPA, EPA's technical support contractor
for this effort (Battelle) and stakeholders representing Transit Authority's then ranked the
identified equipment based upon criteria pertinent to the equipment's use during a biological
remediation within a subway system. The three highest pieces of identified pieces of equipment
(MM Sprayers, AOF, and Dust Boss sprayers) were subjected to durability tests, to evaluate their
compatibility with pAB. These tests included 100 hours of operation with pAB using smaller
proxy equipment designed in consultation with the vendors of the equipment. Based on
durability assessment two pieces of equipment (AOF and Dust Boss sprayers) were further
down-selected to participate in a field scale demonstration at a subway platform/tunnel at Fort
A.P.	Hill. The equipment was placed atop a flatbed railcar and water was sprayed through the
subway platform/tunnel at a speed of 1.2 mph while video and leaf wetness data (5 locations)
were collected. Based on the leaf wetness data, review of video, and observer input (Appendix
D), a single piece of equipment was selected (AOF) to perform field-scale efficacy tests using
B.g.	spores as a surrogate for B. anthracis.
Efficacy testing was conducted within Battelle's ABT testing facility that permitted full-scale
implementation as the internal dimensions of this facility were representative of many existing
subway tunnels. Decontamination efficacy of operationally sprayed pAB against B.g. was
evaluated at target delivery speeds of 1.2 and 2.4 mph, target temperature of 10 °C, uncontrolled
RH ranging from 59 to 98%, coupon orientations simulating floor and column (right side) and
contact times ranging from 30 min to 12 hours (overnight) for a total of 5 tests. Ceramic tile
resulted in > 6 LR at each condition tested (tests 1-3). Unpainted concrete resulted in LRs
ranging from 1.62 to 2.34 and 1.32 to 3.02 at locations 1 and 2, respectively. Since concrete was
more challenging, Test 4 utilized concrete only with
repeat applications (2 and 3) with 30 min contact
times between applications. This resulted in
increased LR ranging from 2.95 to 4.70. Actual
operational parameters as measured were within
acceptable ranges and are detailed in Section 5. The
detailed decontamination efficacy results are found
in Appendix A.
7.1 Durability Testing
Results comparing performance of the three down-
selected equipment from the market survey (MM
Sprayer, AOF, and Dust Boss sprayers) operated
with pAB are shown in Figures 7-3 and 7-4. After
four hours of operation both the MM Sprayer and Figure 7-1. MM sprayer failure.
Dust Boss pAB test equipment were found to have
failed while the control equipment performed as anticipated. Further investigation found that the
MM sprayer diaphragm pump had failed as noted by lack of operational pressure when found
due to a ruptured seal as well as the evacuation of pump oil from the reservoir as shown in
Figure 7-1. The vendor was consulted and provided replacement seal part numbers. The Dust
20
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Boss sprayer had also failed as noted by a sharp increase in liquid flow rate as shown in Figure
7-3. Upon investigation, it was found that the pAB had a corrosive effect on the brass nozzle and
had increased the orifice size as shown in Figure 7-2. The vendor was consulted and determined
that a stainless-steel version (part # 64-000131 A) of the provided nozzle would likely result in
increased durability, though this nozzle was not used for this testing.
Figure 7-2. Dust Boss nozzle failure. From left: control, test 1 test 2 and test 3 nozzles.
7000
6000
5000
c
1 4000
E
| 3000
IZ
2000
1000
0
Durability Flow Rate
,/
Dust Boss Nozzle Failure 1-3
/
AOF Nozzle 1 Failure
AOF Nozzle 2 Failure
AOF Nozzle 3 Failure

MM Sprayer Pump Failure 1-3
0 S 9 15 18 22 28 34 40 47 54 61 67 74 81 88 95 100
Hours
Figure 7-3. Durability test flow rate.
Durability Pressure
-DB Control
-DB-1
-DB-2
DB-3
-MM Control
MM-1
MM-2
MM-3
-AOF Control
-AOF-1
-AOF-2
AOF-3
40
10
0

Dust Boss Nozzle Failure 1-3
AOF Nozzle 2 Failure
V
^AOF Nozzle 1 Failure
^ AOF Nozzle 3 Failure
MM Sprayer Pump Failure 1-3
Figure 7-4. Durability test pressure.
-DB Control
DB-1
DB-3
-DB-2
-MM Control
-MM-1
MM-2
MM-3
-AOF Control
-AOF-1
AOF-2
AOF-3
0 5 9 15 18 22 28 34 40 47 54 61 67 74 81 88 95 100
Hours
21
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The AOF system performed the
longest with the first nozzle
failure occurring after 15 hours
of operation followed by nozzle 2
failure at 22 hours and nozzle 3
failure at 67 hours as noted by
decreased pressure and increased
flow rate. Upon investigation of
each nozzle failure it was found
that the plastic nozzle orifice
housing had become brittle and
broken off as seen if Figure 7-5.
Testing was continued out to 100
hours with the AOF pump only
which resulted in no observable
deterioration of flow rate or
pressure. The vendor was
consulted and determined that
using a standard ceramic disc and
core nozzle would eliminate the observed failure, though this nozzle was not used for this
testing. Further details on the durability test parameter results are found in Appendix B.
7.2 Field Scale Demonstration
Results comparing water deposition rate performance of the AOF and Dust Boss equipment
operated with water at a speed of 1.2 mph are shown in Figures 7-6 and 7-7. The AOF sprayer
delivered approximately 908 L of water over the entire distance of the subway system (-197 m)
in approximately 6 min at a flow rate of 151 1pm. The radial placement of the nozzles in
addition to the added down-facing spray bar resulted in 100% max coverage of all wetness
sensors as noted in Table 7-1. Video collected during testing showed the wicking effect of the
applied liquid into the concrete walls. Over the course of approximately 12 min large portions of
the walls changed from visually dark color (saturated) to a much lighter color indicating a drying
effect.
The Dust Boss sprayer delivered approximately 189 L of water over the entire distance of the
subway system in approximately 6 min at a flow rate of 32 1pm. The design of the Dust Boss
sprayer resulted in a more focused application of liquid and was only able to achieve 100%
coverage at one location. While an individual DB30 system may not be adequate for full
coverage of a subway tunnel and platform, adding multiple DB30 system may result in the
desired coverage. The temperature and relative humidity during testing ranged from 16.7 to 18.2
°C and 75 to 93% respectively.
Figure 7-5. AOF nozzle failure. From left: control, test 1,
test 2, and test 3 nozzles.
22
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Table 7-1. Fort A.P. Hill Subway Percent Wetness
Equipment

Railing
Tunnel Wall
Column (Back)
Ceiling
Tracks
Air-O-Fan
Average
56.7
34.3
93.4
95.6
90.1
Max
100.0
100.0
100.0
100.0
100.0
Dust Boss
Average
83.2
20.9
42.1
27.3
25.4
Max
100.0
37.1
68.8
47.7
48.8
AOF FAPH % Wetness
120
100
80
60
40
20
0
/

f V
/
VI V ,

r

M
l

I,
U
	
v/1
i j


JJi
II u
1	j
l/v/
*H*H(N(N(Y>r0^-^-L/lL/lUDUDr^r^00 00CT>CT>OO*H*H(N(Nr0r0^-
L/l L/l L/l L/l L/l L/l L/l L/l L/l L/l L/l L/l L/l L/l L/l L/l L/l L/l O O O O O O O O O
OOOOOOOOOOOOOOOOOO^H^H^H^H^H^H^H^H^H
railing
•tunnel wall
•column back
ceiling
•tracks
Time
Figure 7-6. AOF sprayer Fort A.P. Hill (FAPH) demonstration % wetness.
Dust Boss FAPH % Wetness
120
railing
¦wall
¦column back
ceiling
¦tracks
Time
Figure 7-7. Dust Boss sprayer Fort A.P. Hill (FAPH) demonstration % wetness.
23
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7.3 Efficacy Testing
The decontamination efficacy of pAB delivered via the AOF orchard sprayer against B.g. was
evaluated on ceramic tile and unpainted concrete at two locations, contact times of 30 min or
overnight, delivery speeds of 1.2 and 2.4 mph, and repeat applications of 1, 2 and 3 with a 30
min contact time between applications. A target temperature of 10 °C was used to represent the
ambient environmental conditions that would be expected in underground subway platforms and
tunnels.
Results are organized by test condition in Figures 7-8 through 7-11 to visualize the effect of
contact time, operational speed, and repeat application of pAB and water, respectively. Figure 7-
8 indicates little to no difference exists between contact times of 30 min and 12 hours
(overnight). Similarly, as shown in Figure 7-9, little difference is seen between spray delivery
speeds of 1.2 and 2.4 mph. Both tests show significant differences between Ceramic Tile and
Unpainted Concrete with average LR of 6.8 and 1.9, respectively.
Contact Time Comparison
^ 7
ox
»n r
ON o
-H
a 5
o
r
3. 3
O 1
-I
~	30 Min Contact
~	Overnight Contact
Column
Ceramic Tile
Floor
Ceramic Tile
Column	Floor
Unpainted Concrete Unpainted Concrete
Figure 7-8. 30 min (Test 1) vs overnight contact time (Test 2).
24
-------
Operational Speed Comparison
u
5? 6
*n
5
-H 3
C
.2 4
tj
3
-------
1.2 mph Mechanical Removal on Concrete
10
9
-------
against B.g. was evaluated at target delivery speeds of 1.2 and 2.4 mph, target temperature of 10
°C, uncontrolled RH ranging from 59 to 98%, coupon orientations simulating floor and column
(right side), and contact times ranging from 30 min to 12 hours (overnight) for a total of 4 tests.
Since efficacy was not affected by speeds up to 2.4 mph (25 min/mile) nor contact times longer
than 30 minutes, it can be estimated that one AOF unit could deliver three applications of pAB
(151 1pm) per mile of subway tunnel/platform in 2 hours and 45 minutes (25 min application
time, 30 min contact time, per application). These conditions achieved complete inactivation on
ceramic tile and ~4 LR on unpainted concrete during testing. This application regimen would
use 11,325 L of decontaminant per mile of track.
Over the course of the study all testing conducted with ceramic tile resulted in > 6 LR of B.g.,
while no conditions were found that resulted in > 6 LR of B.g on unpainted concrete. It was
found that neither decontamination delivery speeds of 1.2 or 2.4 mph, nor increased contact
times greater than 30 min resulted in a significant effect on LR. Repeat applications of pAB up to
three each resulted in increased efficacy.
This work provides several candidate technologies that could be useful during remediation
following a wide area release of B. anthracis, specifically in a subway environment. This study
also provides information on the efficacy of pAB as delivered by the top selected equipment (
AOF) against surrogate B.g. spores for decontamination of common subway materials that could
become contaminated with B.a. spores. Such results may be useful in the development of
guidance to aid in deployment of sporicidal liquid after a wide-area release of B.a. spores in a
subway environment.
Note: See Appendix E for additional results from add-on testing.
27
-------
8.0 References
1.	U. S. Environmental Protection Agency. Determining the Efficacy of Liquids and Fumigants
in Systematic Decontamination Studies for Bacillus anthracis Using Multiple Test Methods.
Research Triangle Park, NC: U.S. Environmental Protection Agency. US EPA Report 600/R-
10/088, December 2010.
2.	Calfee MW, Ryan SP, Wood JP, Mickelsen L, Kempter C, Miller L, Colby M, Touati A,
Clayton M, Griffin-Gatchalian N, McDonald S, Delafield R, Laboratory evaluation of large-
scale decontamination approaches. J. Applied Microbiology, May 2012; 112(5): 874-82
28
-------
Appendix A
Detailed Test Results
Efficacy Results
The detailed decontamination efficacy results for pH amended bleach (pAB) sprayed with Air-O-Fan
(AOF) against Bacillus atrophaeus on Two material types are shown in Table A-l. Colony-forming
units (CFU) were observed on all procedural blanks indicating mechanical removal and redeposition
of spores.
Table A-l. Inactivation of Bacillus atrophaeus Spores Using pH Amended Bleach3
Mean Recovered B. atrophaeus
Test
Number







Positive Control15
Test Couponc






Ceramic Tile LI

2.49 ± 0.40 x 107
5.67 ±9.81 xlO2
6.31 ±2.11
1
pAB
AOF
0.5
10
Ceramic Tile L2
Unpainted Concrete LI
Unpainted Concrete L2
1.99E+08
9.42 ±2.51 x 106
7.93 ± 1.03 x 105
5.69 ± 7.55 x 106
0.00 ±0.00
1.99 ± 0.78 xlO4
5.90 ± 6.09 xlO3
>6.96 ±0.12
1.62 ±0.20
3.02 ±1.16





Ceramic Tile LI

3.13 ±0.71 x 106
0.00 ±0.00
>6.49 ±0.12
2
pAB
AOF
12
10
Ceramic Tile L2
Unpainted Concrete LI
Unpainted Concrete L2
8.80E+07
4.65 ± 0.40 x 106
2.53 ± 1.31 x 105
5.06 ± 0.45 x 105
0.00 ±0.00
6.12 ± 4.00 xlO3
2.76 ± 3.03 xlO4
>6.67 ±0.04
1.66 ±0.55
1.48 ±0.63





Ceramic Tile LI

1.35 ± 0.38 x 107
0.00 ±0.00
>7.12 ±0.14
3
pAB
AOF
12
10
Ceramic Tile L2
Unpainted Concrete LI
Unpainted Concrete L2
8.80E+07
1.09 ±0.11 x 107
1.64 ± 0.36 x 105
1.17 ± 0.53 x 105
0.00 ±0.00
2.92 ± 3.68 xlO3
8.34 ± 6.45 xlO3
>7.04 ±0.05
2.34± 1.37
1.32 ±0.73
4a
pAB
AOF
0.5
10
Unpainted Concrete LI
Unpainted Concrete L2
9.90E+07
2.98 ± 1.86 x 106
8.43 ± 2.06 x 105
1.46 ± 2.26 xlO3
3.22 ±2.71 xlO2
3.78 ± 1.13
3.51 ±0.41
4b
pAB
AOF
0.5
10
Unpainted Concrete LI
Unpainted Concrete L2
9.90E+07
3.21 ± 2.80 x 106
1.10 ± 0.50 x 106
3.00 ±4.33 102
8.34 ± 7.54 xlO2
4.32 ±0.92
3.95 ±2.03
4b
pAB
AOF
0.5
10
Unpainted Concrete LI
Unpainted Concrete L2
9.90E+07
1.62 ± 0.66 x 106
1.19 ± 0.25 x 106
1.67 ± 0.85 xlO3
4.30 ± 7.45 xlO3
2.95 ±0.36
4.70 ± 2.69
a Data are expressed as the mean (± SD) of the logs of the number of spores (CFU) observed on three 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
-------
Appendix B
Detailed Durability Results
Durability Results
The detailed durability results for pH-amended bleach (pAB) sprayed with MM Sprayers, Dust Boss sprayer, and AOF proxy systems are
shown in Tables B-l and B-2.
Table B-l. Durability Testing Pressure
Pressure Measurements (psi)
Equipment
5hr
9hr
15hr
18hr
22hr
28hr
34hr 40hr 47hr
54hr
61hr
67hr
74hr
81hr
88hr
95hr
lOOhr
DB Control
53
51















DB-1
54
42'















DB-2
54
42'















DB-3
53
40'















AOF Control
27
27
27
27
27 26
27 27
27 26
27 27
27 27 27 27 27 27
26 26
26 26
26 26
26 26
26 26
26 26
26 26
26 26
AOF -1
27
26
26
26
26 24'












AOF-2
27
27
26
26
26 24
24 25
24 24'










AOF-3
27
26
26
26
26 24
24 24
24 24
26+ 26
27 26 26 26 26 26
26 26
26 26
26 26'
26 26
26 26
26 26
26 26
26 26
MM Control
57
58















MM-1
57
0'















MM-2
57
0'















MM-3
57
0'















*indicates equipment found failed.
^pressure adjusted to maintain operational conditions with reduced number of functional nozzles.
B-l
-------
Table B-2. Durability Testing Flow
Flow Rate Measurements (mL/min)
Equipment

5hr

9hr

15hr

18hr
22hr
28hr
34hr
40hr
47hr
DB Control
260
320











DB-1
240
5770'











DB-2
240
4950'











DB-3
190
4770'











AOF Control
1420
1500
1480
1450
1460
1440
1480
1460
1480 1460
1460 1460
1460 1480
1480 1480
1500 1480
AOF-1
1440
1500
1500
1520
1470
4200'
4260
4650
4680




AOF-2
1690
1610
1540
1560
1540
1540
1620
1630
1600 4000'




AOF-3
1420
1440
1430
1430
1400
1380
1380
1340
1320 1380
1450 1480
1440 1460
1500 1460
1440 1460
MM Control
1660
1690











MM-1
1690
0'











MM-2
1780
0'











MM-3
1780
0'











*indicates equipment found failed.
Table B-2. Durability Testing Flow (Continued)
Flow Rate Measurements (mL/min)
Equipment	54hr	61hr	67hr	74hr	81hr	88hr	95hr	lOOhr
DB Control	
DB-1	
DB-2	
DB-3	
AOF Control	1500 1480 1420 1440 1460 1460 1460 1480 1450 1460 1480 1480 1480 1460 1460 1480
AOF-1	
AOF-2	
AOF-3	1460 1460 1440 1480 1480 4820' 4820 4850 4900 4880 4850 4880 4920 4890 4900 4940
MM Control	
MM-1	
MM-2	
MM-3	
*indicates equipment found failed.
B-2
-------
Appendix C
Detailed Market Survey Results
Table C-l. Commercial Equipment for Subway Decontamination
Company Model# Flow Rate
Engine/ Diameter Number Spray
PTO
of fan of fans Distance
Weight
Width X
Length
Horse	Nozzle
Height Power/Fuel Tank Material Info/Droplet
Tank Size	size (micron)
Price	Unique Features
Country of
Origin
Nelson Hardie
Mfg, Co., Inc.
engine and Dual 34"
10-25 GPM PTO fan PTO 2
available drive
500 Gal.
3,5001bs,
lOOOGal.
4,1001bs.
70-100PTP	t-jet ceramic
12 ga., Type 304
102x241" 75-85" HP/540 RPM 6 ™ , disk nozzle/50-
stainless steel
max	200
radial spray pattern,
myers pump,
Yuba City, CA,
USA
Nelson Hardie
Mfg, Co., Inc.
Super 92
engine and
10-25 GPM PTO
available
Dual 46"
fan, 325
HP engine
drive
9,080 Lbs.
(Empty)
150 Gallon 12 ga., Type 304
102x322" 75-85"	s ™	50-200
tank 325Hp stainless steel
100-120K radial spray pattern
Yuba City, CA,
USA
M.K. Rittenhouse PRM1500-
Radial Fan
Sprayers
37 GPM (140.1
LPM), 725 PSI PTO
(50 bar)	
35.5" (90
cm) Fan
30-40' 12001bs.
unknown t-
395 gal polyethylene jet ceramic
disk nozzle
$10,250.00
14 double-sided
flipover nozzles
Buffalo, NY,
USA
, 40" steel
engine and
Air-O-fan D-40R 1-100 GPM at ^	axial flow
PTO
Products Corp. 1,000 Gallon 200 PSI, Yes	"reverse"
available
tan
pounds
105x246" 62-72"
156 HP stainless steel
60-100K radial spray pattern
Reedley, CA,
USA
MM Sprayers MM LG
USA	400
engine and
PTO
available
583 lbs
Dry
45 x 100"	54"	13 HP 106 gal poly	t-jet
9,437.00 radial spray pattern
Lynden, WA,
USA
single fan
with
directed
hoses
1 unknown 281-5701bs
300-4000 L
69x192"	69"	fiberglass	varies
resists chemicals used
S428-S36082 for pesticide
treatments
Molinella, Italy
Oktopus yes 81 to 110
P/T, various	L/l'
single fan
with
directed
hoses
1 unkown 281-5701bs 63x142" 79-142" 1500 L polyethylene tank varies S8539-S14834 Self cleaning filters Molinella, Italy
Vinetech
equipment
Air Directed "
Sprayers
Quantum
Mist Citrus
Sprayer
1.2 to 20 GPM
per fan
PTO 15 or 20" scaleable 10'
NA - fans
15 and 20" fans configured hydraulic
available on cusstom drive
frame
50-80 microns
S1480-S1680
per fan
Prosser, WA,
USA
Nightstar	electric 22"
1901 0.5 - 4.1 GPH 115V 19 DIAMETE
ULV/LVM	AMP R FAN
220 LBS
treats up EMPTY /
toeOKft2 252 LBS
FULL
ELECTRIC polyethylene,
81" MOTOR, 1 FLUSH TANK
HP, 3 gal ISHDPE
$7,300.00 Wetted Parts material
PLUS	HDPE, BRASS
SHIPPING & AND VITON
HANDLING	SEALS
East Moline, IL,
USA
Air velocities
range from 528 electric
fpmto 113 (to 120V 5A
140') fpm	
1051b
(47.6 kg)
Blades: High
96" when	*
96" round	No tank performance —10 microns
vertical
polyamide nylon
Lexington, KY,
USA
Easy 4000 260 LPM
engine
372 kg
40-100K	sprays foams
Mooresville, NC,
USA
C-l
-------
Table C-2. Commercial Equipment for Subway Decontamination (Continued)
Company Model# Flow Rate
Engine/ Diameter Number Spray
PTO of fan of fans Distance
Weight
Width X
Length
Height Power/Fuel
Tank Size
Nozzle
Tank Material Info/Droplet
size (micron)
Unique Features
Country of
Origin
custom
custom
,, . ,, , fabricated Requires 65
Electrostatic Electrostatic	yes, 2.88-5.03	sprayers	fabricated based
100SR	PTO	1 unknown 600 lbs dry	based on HP tractor
Sprayers Spraying Systems	LPM	use blowers	on specific	. .
specific minimum
needs.
needs.
40 micron $29,000 to	St Watkinsville,
Stainless Steel	electrostatic nozzles
droplet size $31,000	GA,USA
Dust Boss DB-30 1.4-2.8GPM Electric 9200 CFM 1	100' 800 pounds 63x 102"
Does not have a
tank
Anywhere
from 50 to 200
microns
Rental of a
DB-30 would
be 875 per
week
0-70° or 0 to 359°
oscillation
Peoria, IL,USA
Average of	1800
Dust Boss DB-45	, 6	Electnc 1800 CFM 1	150'	,	72x76"
around 12 GPM	pounds
Does not have a
tank
Anywhere
from 50 to 200
microns
Rental of a
DB-45 would
be 975 per
week
0-70° or 0 to 359°
oscillation
Peoria, IL,USA
Dust
Suppression
Equipment
Dust Boss DB-100 17-39 GPM Electric
3200
pounds
Rental of a
Anywhere
Does not have a „	DB-100 would
from 50 to 200
tank	be 1,600 per
microns
week
0-70° or 0 to 359°
oscillation
Peoria, IL,USA
12-26.7 gal/min.
„ ., ,	somewhere
0.5 GPM fluid electric or
DB-60 „	. , around 30 1
flow at 55 psi air motorized
inches
pressure
4,500
328'	, 106x238"
pounds
Does not have a
tank
Anywhere
from 15 to 30
microns
Rental of an
OB-60G
would be
1,600 per
week
0-70° or 0 to 359°
oscillation
Peoria, IL,USA
Major
NA 19041 Kg 104x326"
132" 14093 Litres
Stainless Steel
3100 Gal.
Varies
Ballyhaunis, Co.
Mayo Ireland
Chief Tain CVT 11500 varies
NA 4400kg 102x266" 113" 11500 Litres
Ballyhaunis, Co.
_^Ma^ofrelan^_
Dyndafog (may
not be suitable for 1200	120 gal/hr engine NA various
Foggers all chemicals)
467 to 620
NA	43 x 60"
lbs
42"	55 e
thermal fogger, but
can order optional
cold fog kit
Houston, TX,
USA
VectorFog TU100 60-90 LPH engine
NA
NA 1.5 KG 35x51"
36"	40 s
cold fogger Miami, FT,, USA
De-Icing
Equipment
hydraulic
Dultmeir DU1A045 yes, varies pump or NA
	engine	
only
2500 lbs 83 x 182"
73"	NA
1800 gal polly
14,978.00 flooding nozdes Omaha, NE, USA
1300
Central Equipment 	 yes, 200 gal/min
H 1	Skidded J	°	engine NA	NA
LLC	max	^
Sprayer
only
1100 lbs
dry 15000 68x 138"
lbs wet
1300 gal high
density poly
all wetted parts are Port Byron, NY,
plastic 90" spray bar	U SA
C-2
-------
Appendix D
Mod 5 Add-on Test Results
Efficacy Results
The detailed decontamination efficacy results for 5 additional tests funded through contract modification 5 of pH-amended bleach (pAB)
and 2% diluted bleach sprayed with AOF sprayer against Bacillus atrophaeus (B.g.) on two material types are shown in Table D-l. Colony
forming units (CFU) were observed on all procedural blanks indicating mechanical removal and redeposition of spores.
Table D-l. Add-on Inactivation of Bacillus atrophaeus Spores using pH-Amended and dilute 2% Bleach3
Test
Number







Positive Control
(unsprayed)
Positive Control1* (water
sprayed)
Test Couponc (Decon
sprayed)

M5-1
pAB
AOF
10
0.5
10
Ceramic Tile LI
Ceramic Tile L 2
Unpainted Concrete LI
Unpainted Concrete L2
9.10E+07
6.39 ± 0.74 xlO7
1.45 ± 0.42 xlO6
6.41 ± 0.80 x 107
6.49 ± 1.04 x 107
2.01 ± 1.45 x 106
3.29 ±2.62x10®
3.68 ± 2.05 x 107
0.00 ±0.00
2.71 ± 1.11 x 105
3.71 ± 2.88 x 104
0.35 ±0.37
>7.81 ±0.07
0.82 ±0.33
1.94 ±0.40
M5-2
pAB
AOF
5
0.5
10
Ceramic Tile LI
Ceramic Tile L 2
Unpainted Concrete LI
Unpainted Concrete L2
9.53E+07
7.75 ± 2.05 x 107
5.51 ± 8.21 x 106
6.65 ± 1.00 xlO7
7.85 ± 2.86 xlO7
1.51 ±0.45x10®
2.10 ± 1.38 x 106
8.35 ± 1.79 x 104
0.00 ±0.00
8.95 ± 4.76 x 104
7.83 ± 2.65 x 103
3.99 ±0.94
>7.88 ±0.12
1.35 ±0.46
2.37 ±0.30
M5-3
2%
NaOCl
AOF
2.4
0.5
10
Ceramic Tile LI
Ceramic Tile L 2
Unpainted Concrete LI
Unpainted Concrete L2
9.87E+07
6.28 ± 0.62 xlO7
1.17 ±0.46x10®
6.05 ± 1.53 x 107
1.15 ± 0.62 x 107
1.29 ±0.43x10®
1.99 ±0.82x10®
0.00 ±0.00
0.00 ±0.00
1.05 ± 0.46 x 104
0.00 ±0.00
>7.77 ±0.09
>7.22 ±0.19
2.11 ±0.23
>6.27 ±0.14
M5-4
2%
NaOCl
AOF
2.4
0.5
10
Unpainted Concrete LI
Unpainted Concrete L2
9.17E+07
1.07 ±0.35x10®
1.37 ±0.46x10®
5.62 ±3.23x10®
1.66 ± 1.02 x 103
0.00 ±0.00
3.47 ± 1.31
>6.67 ±0.27






Unpainted Concrete LI


3.82 ±0.83x10®
4.40 ± 6.69 x 102
4.95 ± 1.33
a Data are expressed as the mean (± SD) of the logs of the number of spores (CFU) observed on three 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).
eLl and L2 represent (Ll=column, L2=floor)
D-l
-------
Comparison of pH Amended and 2% Dilute Bleach
The decontamination efficacy of pH Amended (pAB) and two percent diluted bleach delivered
via the AOF orchard sprayer against B.g. was evaluated on ceramic tile and unpainted concrete at
two locations, contact times of 30 min, delivery speeds of 2.4 mph, and repeat applications of 1,
2 and 3 with a 30 min contact time between applications. A target temperature of 10 °C was used
to represent the ambient environmental conditions that would be expected in underground
subway platforms and tunnels.
Results are organized by test condition in Figures D1 through D4 to visualize the effect of
decontaminate, operational speed, and repeat application respectively. Figure El indicates a
significant difference with two percent dilute bleach resulting in a higher log reduction (LR) for
concrete at the floor location.
pAB vs 2% Bleach at 2.4 mph Comparison
ox
T,
ON
-H
e
0
•-&
1
O 2
hJ
1
0
T
-E—

rh







|-i-1






































ph



















~	pAB (Test 3)
~	2% Bleach (Test M5-3)
Column
Ceramic Tile
Floor	Column	Floor
Ceramic Tile Unpainted Concrete Unpainted Concrete
Figure Dl. pH amended bleach (pAB) vs 2% dilute bleach at 2.4 mph.
Similarly, as shown in Figure D2, little difference is seen between spray delivery speeds of 1.2
and 2.4 mph however when increasing speed to 5 and 10 mph a reduction of LR is observed at
the column location while floor location remains similar. This finding is further supported by
measurement of mass deposition of water as shown in Figure D4. At speeds of 1.2 and 2.4 mass
deposition of water remains consistent, however with increased speeds of 5 and 10 mph a
reduced deposition of liquid was observed.
D-2
-------
Operational Speed Comparison
° 7
*n r
ON O
-H
C 5
o
i:
CD 3
W) 0
o ^
hJ
1
£i
lil
~	1.2 mph(Test2)
~	2.4 mph (Test 3)
~	5 mph (Test M5-2)
~	10 mph (Test M5-1)
Column
Ceramic Tile
Floor
Ceramic Tile
Column	Floor
Unpainted Concrete Unpainted Concrete
Figure D2. Efficacy of pH amended bleach (pAB) at varied application speeds.
Water Deposition per m2
600.00
500.00
400.00
^ 300.00
200.00
100.00
0.00
¦Ceramic Column
Ceramic Floor
¦Concrete Column
¦Concrete Floor
1.2
2.4
10
mph
Figure D3. Mass deposition of water at various delivery speeds.
Finally, the repeat application (1, 2, and 3 applications) of two percent bleach with 30 minute
contact times between each application resulted in similar LR when compared to pAB at the
column location as seen in Figure D4. However, at the floor location, a significant increase in
LR was noted using two percent bleach resulting in complete inactivation after just one
application.
D-3
-------
Repeat Application Comparison (Concrete Only)
6
ox
T,
* 5
c
.2 4
t3
3
-------
Appendix E
Technology Demonstration Feedback Results
E-l
-------
I nderground Transport Ki'st oration (ITR)
Operational Totiuiolog} Demonstration Observer Form
DATE: (J Ujy^	
<0r^
ORGANIZATION. T T F - Cj		
ROLF- IN THE ORGANIZATION ( flfh/ Pl«„A,/
Organizational l-ccdbnek (ii vou've alrcad\ completed this section on another day. please skip)
1.	What responsibilities docs your organization have in the event of a biological
contamination event? fit	,	^
W|Y'yU f0 O^jJ
S fi if ej
2.	What response capabilities does your organization currently have (e.g. decontamination
technologies, survey instrumentation, etc.)
H % e/(»Af\cvr h CUhJ C citA J) Hf\f V A	Qci\
3.	Does your organization have a detailed response plan in the ease of a wide-area
decontamination event? . .
Not mf 7* > }pj,
4.	What are the most important factors in considering purchase of and planning for use of
wide area decontamination technologies? rrr
L r  e/j ^ f f flu ^ c *jf
5.	Does your organization stockpile decontamination technologies for a large contamination
event? Why or why not? r- t	f
J. f	t*ro„r.	^
6.	Describe any current situations your organization is facing requiring biological mitigation
and response? What technologies arc you considering?
7.	Please describe any other aspects of your organization you'd like us to know about.
8.	Would you like us to contact you for detailed discussion?
-------
Technology Demonstration Feedback
Demonstrated Technology Air-( )~Fan
1.	What arc the observed benefits of this technology?
C J i AA M\
2.	What are the observed limitations?
/f^i 11 r~y.	*a vy ' f ^ ^1 ^ f
3.	Why or why not would this technology be helpful to your organization?
4.	What additional tools/support would you need to implement use of this technology?
5.	What improvements could you envision making to this technology to make it more
useful?	/	|	r	>
C-^uc,	C,„r Cfl)/ rf^r( ^UJ
6.	Why or why not would von consider stockpiling this technology for response situations?
7.	Upon generation of secondary aqueous waste, does your organization generally plan or
prefer to contain or treat the waste?
T/&rs\frf vo f'fA-
8.	Please provide any other comments
9. Would you like us to contact you for detailed discussion?
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Demonstrated Technology Dust Boss
1.	What are the observed benefits of this technology"?
2.	What are the observed limitations?
f Q t)	i	c /It f -o- T ^
3.	Why or why not would this technology be helpful to your organization?
4.	What additional tools/support would you need to implement use of this technology?
5.	What improvements could you envision making to this technology to make it more
useful?
J^-y	^ <^(<*.>1 f j .(/t >s-y j
6.	Why or why not would you consider stockpiling this technology for response situations?
7.	Upon generation of secondary aqueous waste, does your organization generally plan or
prefer to contain or treat the waste?
f/oAj /V fcpA
8.	I'ieiise provide any other comments
9. Would you like us to contact you for detailed discussion?
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Underground Transport Restoration (l TR)
Operational Tecluiolo"} Demonstration Obsor\cr Form
DATE: Qo-t- 7„n l,L
NAME: t\< ca*s - tar \ ^	\j) Af
ORGANIZATION 		 JT*-- cs.			
ROLE IN THE ORGANIZATION	. CJjgN ftonnef L^i
Organizational Feedback (if\ou'\c alrcad\ completed this section on anuthcr dav, please skip)
1.	What responsibilities does your organization have in the event of a biological
contamination event'.' ___	C	haJ br
H+if s/ mi	&>^A O ix\ \ V;) , oo^a 5^ ') -y, ^ P «
5.	Does your organization stockpile decontamination technologies for a large contamination
event? Why or why not?	v ,	, ,	.
c)<- > C 
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Technology Demonstration Feedback
Demonstrated Technology Air-O-Fan
1,	What arc the observed benefits of this technology?
——	j
2,	What are the observed limitations?
— Ia.	^ flM	^ x -
3.	Why or why not would this technology be helpful to your organization?
fs)^, tyi4 U> \ \ I
(M. Ccn Op-€/o.+Tx] £
4.	What additional tools'support would you need to implement use of this technology?
hv^Z^-
5. What improvements could you envision making to this technology to make it more
useful?
—— v i cl-e * vvu i l^r;
6.. Why or why not would you consider stockpiling this technology for response situations'?
"Wwn' ^ n^jOi re/Mi* fKctvf U< ca, I i
7.	Upon generation of secondary aqueous waste, does your organization generally plan or
prefer to contain or treat the waste?
Oo
8.	Please provide any other comments
9, Would you like us to contact you for detailed discussion?
-------
Dcmoiisirateil Technology Dust Boss
1. What arc the observed benefits of this technology'.'
2. What are the observed limitations?
3, Why or why not would this technology be helpful to your organization?
4. What additional tools/support would you need to implement use of this technology?
5, What improvements could you envision making to this technology to make it more
useful?
6, Why or why not would you consider stockpiling this technology for response situations?
7. Upon generation of secondary aqueous waste, does vour organization generally plan or
prefer to contain or treat the waste?
8. Please provide any other comments
9. Would you like us to contact you for detailed discussion?
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I'ndcr'ground Transport Restoration (I TR)
Operational Technology Demonstration Observer Form
PATH: I1	- i
\ A M1 -:	'
ORGANIZATION	^ _ 		
ROLE IN THE ORGANIZATION^	1J ¦ ' "L VL'T
O ruanizational I'ccdhack (ii \\ >u"vc already completed this section on an other day, please skip)
1.	What responsibilities does your organization have in the event of a biological
contamination event?
	¦' u ov V
2.	What response eapahilities does your organization currently have (e.g. decontamination
technologies, survey instrumentation, etc.)
3.	Does your organization have a. detailed response plan in the case of a wide-area
decontamination event?
4. What are the most important factors in considering purchase of and planning for use of
wide area decontamination technologies?
5.	Does your organization stockpile decontamination technologies for a large contamination
event? Whv or whv not?
'
6.	Describe any current situations your organization is facing requiring biological mitigation
and response? What technologies are you considering?
7. Please describe any other aspects of your organization you'd like us to know about.
X. Would you like us to contact you for detailed discussion?
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Technology Demonstration 1-cod hack
Demonstrated Technology . lir-O-Tan
1.	What arc the observed benefits ofthis technology?
1 ' " DC
2.	What are the observed limitations?
'V	ri	
3.	Why or why not would this technology be helpful to your organization?
^CW<-£'S "W A^^^T -T
4.	What additional tools/support would you need to implement use ofthis technology?
5.	What improvements could you envision making to this technology to make it more
useful?
6.	Why or why not would you consider stockpiling this technology for response situations '
7,	Upon generation of secondary aqueous waste, docs your organization generally plan or
prefer to contain or treat the waste?
~\V1[	To rOAJ'WAl
8.	Please provide any other comments
9. Would you like us to contact you for detailed discussion?
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PcmonsiriKccf Technology Dust Boss
1.	What are the observed benefits of this technology'.'
vis, \ J l.- \\Sj '•-} \ j~-;i	\j\ /.J X;-{j lC...
2.	What are the observed limitations?
	rv""^ 1	(V-C - KiSG L4	ivc-ao	x r Cue ^	
3.	Why or why not would this technology be helpful to your organization?
\ , 1 1 A'Vl /O x i-C ^ ^	
4.	What additional tools-support would you need to implement use of this technology?
5.	What improvements could you envision making to this technology to make it more
useful?
A4ut;n^:	'T o t
6.	Why or why not would you consider stockpiling this technology for response situations?
7.	Upon generation of secondary aqueous waste, does your organization generally plan or
prefer to contain or treat the waste?
To C 0 OTv AJ
8.	Please provide any other comments
9. Would you like us lo contact you for detailed discussion?
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lTndcr«round Transport Restoration (I'TR)
Operationn! Technology Demonstration Observer Form
I) AT I-.: / '5 OC,t IU __
NAMii: rUr:s
ORGAMZA'I ION	A) / fH	_
ROLE IN THE QRCi ANIZ A'l ION	C t) ^CO 	
Organization:!) Feedback (if you've aircad\ completed this section on another dav. please skip)
1.	What responsibilities does your organization have in the event of a biological
contamination event? Quf	WWp*r recuse* i.
2.	What response capabilities does your organization currently ha\e (e.g. decontamination
technologies. surve\ instrumentation, etc.) * 4 * u ,	r- tu. ^ /t-l
"	-	[r^ C V\tn/C w\o%T iff I "-t 5\J\ i\
ept1 cjjes /, A - p, L\viL[L «/ Wa 5uivip Wj	("Jtffy
3.	Does your organization have a detailed response plan in the ease of a wide-area
decontamination event'? ^ fypfcftlta ^ 0pfrrc*ft i«a $mntl
) ctiv^v of- ?-	'	"
4.	What are the most important factors in considering purchase of and planning for use of
wide area decontamination technologies?
5.. Does your organization stockpile decontamination technologies for a large contamination
event? Why or why not?
6. Describe any current situations your organization is facing requiring biological mitigation
and response? What technologies are you considering?
7.	Please describe any other aspects of your organization you'd like us to know about.
\jJt o/ L	frui^ ry £\clc*|>r <>W w\sVi oof Sojj^ OA ^€lw\
J* hoi *aMOH'-j ji' &
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Technology Demonstration lecdhnck
Demonstrated Technology Air-O-l 'an
1.	What are the observed benefits of this technology?	«	,	.
Oef i^iWAy	[W.	a f /
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Demonstrated Hclmo/agy Dust Boss
1, What arc the observed benefits of this technology?
2. What are the observed limitations?
3. Why or why not would this technology be helpful to your organization?
4. What additional tools/support would you need to implement use of this technology?
5. What improvements could you envision making to this technology to make it more
6. Why or why not would you consider stockpiling this technology for response situations?
7. Upon generation of secondary aqueous waste, docs your organization generally plan or
prefer to contain or treat the waste?
8. Please provide any other comments
9. Would you like us to contact you for detailed discussion?
useful?
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I ndergrounci Transport Restoration (I'TR)
Operational I ethnology Demonstration Observer !• orni
ORGANIZATION
N C&- \(kmLC^

()ruani/ational I;ecdhack (if vou vc ativ;ttlv completed this sect ion on another dav. p 1 ca.se skip|
1. What responsibilities docs your organization have in the event of a biological
contamination event?
2. What response capabilities does your organization currently have (e.g. decontamination
technologies, survey instrumentation, etc.)
3, Does your organization have a detailed response plan in the case of a wide-area
decontamination event?
4. What are the most important factors in considering purchase of and planning for use of
wide area decontamination technologies'1
5.	Does your organization stockpile decontamination technologies for a large contamin m m
event? Why or why not?
6.	Describe any current situations your organization is facing requiring biological mitigation
and response? What technologies are you considering?
7, Please describe any other aspects of your organization you'd like us to know about.
8. Would you like us to contact you for detailed discussion?
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Tcchnoloav Demonstration Kecdhack
Demonstrated Technology Air-O-J an
1. What arc the observed benefits of this technology?
, t are the observed limitations?	*	I
/^b Igtb4 jAn
)
3. Why or why not would this technology be helpful to your organization?
4. What additional tools/support would you need to implement use of this technology?
5.	What improvements eoukl you envision making to ibis technology to make it more
cWwy Hxaaj- u-mA «^r
6.	Why or wfty not would you consider stockpiling this technology for response situations?
7.. Upon generation of secondary aqueous waste, does your organization generally plan of
prefer to contain or treat the waste?
8. Please provide any other comments
9. Would you like us to contact you for detailed discussion?
-------
Ih'mniisiratcd Tec hnolngy DustJIoss
1, What arc the observed benefits of this technology?
unserved nenetits ot tins technology.	j g 
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rnticrsiiound Transport Restoration (ITR)
Operational Technology Demonstration Obser\er form
PATH: Cb Oj {{fi 	
NAME;
ORGANIZATION	(fl) Amy
ROLE IN THE ORGANIZATION C-^}ll¥ SpeC.iUil
Organizational I'OctHiack (il you \e already completed this .section un another da v. please skip}
1.	What responsibilities docs wur organization have in the event of a biological
eonlaininiiliiin e\ont?	&mp/zS /DecQn k»C. if OCMuS
' I	I
2.	What response capabilities docs your organization currently have (e.g. decontamination
5C&lT.CS*Qn'cyJ^m^1^ion*ctc,)^^ 6oc^/he( &- 0
3.	Does your organization have a detailed response plan in the case of a wide-area
decontamination cvent?^/£ J
4.	What are the most important factors in considering purchase of and planning for use of
wide jtvn dcainuimimtlion tcchnokiyiuN"' Coif ,J ,f ftyi- filce^'Ac^
IW fbMzr, 4 ; j- ,5 ScLa_	'	T7
5.	Does your organization stockpile decontamination technologies for a large contamination
event'.' Why or why not'.' Yd$ /	^ec& -k> ^ /A]of
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Technolou\ Demons!ration liidhack
Demonstrated Technology Air-O-Fan
}. What are the observed benefits of"this technology?
jfeepiA^	t	ej	jcr pecpi^ b
Co^h^.vt/cd
2. What.are the otwerved limijatior
(MT effceJ'y u/oJi rtfi

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3.	Why orivhy not would this technology be helpful to your organization?
Our liQ ,> fAotX o'C$c.H /So CaS^~(J'	jL Sfrlpp'Of wuU
LSrcd-	1
4,	What additional tools/support would you need to implement use of this technology?
JOr"^	fo	f'r*
5.	What improvements could you envision making to this technology to make it more
TTfT OM>	1"^ 'ny& Zr h~k<
/WT<7 k&<$ So ihc-f dw'f tWvtt
6.	\Vh> or why not would you consider stockpiling thkjeehnology for response situations?
of- *¦ eL spfcxr i.lC ~Hz*cK
1. Upon generation of secondary aqueous waste, does your organization generally plan or
prefer to contain or treat the waste?
^S-
8, Please provide any other comments
n/fl
9. Would you like us to contact you for detailed discussion?
-------
Dcinonslratccl Technology Dust Boss
1. What arc the observed benefits of this technology?
fzc*'^«y m'tU«£ Cjr pwSScJfc;
3. Why or why not wcnild this technology be helpful to your organization?
5ACo {>[ /
4. 1	;hnoIogy?
5.	" 1 '	'	' "	jlogy to make it more
6, Why or why not would you consider stockpiling this technology lor response situations?
dectir fozlr^	CC^icr* />ci	c^~
7. Upon generation of secondary aqueous waste, docs your organization generally plan or
~ r to contain or treat the waste?
8,	wide any other comments
9 "r --->J yon like us to contact you for detailed discussion?
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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
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